FANA antisense oligonucleotides, also called FANA ASOs, are synthetic single-stranded modified oligonucleotides that allow modulation of gene expression by enzymatic degradation of a target RNA. FANA refers to the 2′-deoxy-2′-fluoro-D-arabinonucleic acid analog. Through complementary Watson–Crick base pairing, FANA ASOs can recognize and bind specific RNA sequences, including mRNAs, miRNAs, and long noncoding RNAs.

Unlike siRNAs, FANA ASOs utilize RNase H-mediated RNA cleavage for gene silencing.

After FANA ASOs bind to their RNA target, the endogenous enzyme Ribonuclease H (RNase H) recognizes the FANA/mRNA duplex and cleaves the RNA. After cleavage, the fragmented mRNA is degraded further by nucleases. The cell recycles the FANA ASOs, and the degradation of multiple mRNA copies by a single FANA ASO increases the silencing efficiency and lowers the dose required.

FANA ASOs have a phosphorothioate backbone and modified flanking nucleotides. A fluorine atom replaces the 2'-OH group, which increases the resistance of the FANA ASO to degradation and enhances binding to targeted mRNA.

Studying DNA and RNA structures and functions has attracted researchers across diverse scientific fields. The last decades saw significant developments in the field of therapeutic oligonucleotides. 

The difficulty of delivering oligonucleotides to target organs and tissues limited the wide application of oligonucleotide therapy. Different research groups have extensively leveraged modified nucleic acids to unravel structural and chemical biology scientific inquiries.

2′-deoxy-2′-fluoroarabinonucleic acid (FANA) has emerged as a significant DNA-like nucleic acid with unique attributes adaptable for various biological applications (Figure 1). It contains the more electronegative and less bulky fluorine at the 2’ position, mimicking the DNA: RNA hybrid structure and supporting RNase H activity.

Figure 1: Structure of DNA and FANA oligonucleotides

Application of FANA-Antisense Oligonucleotides (FANA ASOs) oligonucleotides

• FANA ASO can target Citrus Greening Disease, known as Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas). Sandoval-Mojica et al. reported the effective suppression of CLas transmission and reduction of its impact on citrus trees, paving the way for innovative strategies in managing agricultural pathogens and safeguarding citrus cultivation.

• The chemokine CCL3 is pivotal in immune cell activation and recruitment in the injured spinal cord. A study conducted by Pelisch. et al.  employed specifically designed FANA ASOs to inhibit CCL3 expression in a murine contusive SCI model. Results revealed the successful penetration of self-delivering FANA ASO molecules into the spinal cord lesion site, effectively suppressing CCL3 transcript expression

• FANA ASOS were evaluated in vitro to target the highly conserved regions in the HIV-1 genome, offering a multifaceted approach to combat HIV-1 infection. The studies of Kalot et al. and Takahashi et al. suggest that FANA ASOs are promising antisense oligonucleotide for antiretroviral therapeutics. The ability of FANA ASOs to target RNA by recruiting RNase H1 and sterically blocking RNA dimerization further enhances their therapeutic potential.

The continuous exploration of FANA in diverse scientific domains highlights its potential for groundbreaking advancements in therapeutics, genetics, and nanotechnology, contributing significantly to the evolving landscape of nucleic acid science.

Reference

Kalota A, Karabon L, Swider CR, Viazovkina E, Elzagheid M, Damha MJ, Gewirtz AM. 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing. Nucleic Acids Res. 2006 Jan 18;34(2):451-61. [PMC]

Pelisch N, Rosas Almanza J, Stehlik KE, Aperi BV, Kroner A. Use of a Self-Delivering Anti-CCL3 FANA Oligonucleotide as an Innovative Approach to Target Inflammation after Spinal Cord Injury. eNeuro. 2021 Mar 11;8(2):ENEURO.0338-20.2021. [PMC]

Sandoval-Mojica AF, Hunter WB, Aishwarya V, Bonilla S, Pelz-Stelinski KS. Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem. Sci Rep. 2021 Feb 2;11(1):2760. [PMC]

Abs: Absorption = Peak excitation wavelength [nm]

Em: Emission = Peak emission wavelength [nm]

Ext. Coeff.: Molare extinctions coefficient [M-1cm-1]

MW: Molecular weight [g/mol] of the dye labelled on 3' and 5' end of an oligonucleotide. For internal labelling 279 g/mol for the Amino linker needs to be added.

Synthetic oligonucleotide-based therapeutics, including antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), and modified oligonucleotides, require high-quality products at high quantities and free of impurities.

Phosphorylated oligonucleotides such as 5′-triphosphates are necessary biochemical and therapeutic tools. The 5'-triphosphate modification is an essential nucleic acid modification found in all living organisms, participating in metabolic processes and providing energy to drive many processes in living cells.

The 5-triphosphate group in nucleotides plays a fundamental role in energy transfer, nucleic acid synthesis, transcription, signaling, and enzymatic reactions. Its presence provides chemical properties and molecular interactions required for these crucial biological processes.

5'-triphosphate oligonucleotides can act as antiviral and anticancer inhibitors, as substrates for polymerase chain reactions, and nucleic acids ligation reactions, allowing structural and mechanistic studies. Also, 5'-triphosphate oligonucleotides can stimulate an immune response and are intermediates in the enzymatic synthesis of m7G-5′-ppp capped RNAs.

Oligonucleotides containing a 5'-triphosphate group are short chains of nucleotides modified with a triphosphate group at their 5'-end. Various molecular biology and biotechnology applications utilize 5'-triphosphate oligonucleotides, particularly in nucleic acid-based therapeutics.

The 5'-triphosphate (5'-TP) group plays a crucial role in the function of nucleotides and nucleic acids. 5’-TP refers to three phosphate groups attached to the 5'-carbon of a nucleotide.

The triphosphate moiety is responsible for several essential functions associated with nucleic acids:

High Energy Activated Molecules: The presence of the triphosphate group in nucleotides, such as adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), provides a high-energy bond. Hydrolysis of the terminal phosphate group releases energy, which cells can harness for various cellular processes, including muscle contraction, active transport of ions, DNA and RNA synthesis, and enzymatic reactions.

Nucleic Acid Synthesis: During DNA and RNA synthesis, the triphosphate group is critical for adding nucleotides to the growing nucleic acid chain. In DNA replication, for example, the 5'-TP group of a deoxyribonucleoside triphosphate (dNTP) is a substrate for DNA polymerase enzymes. The formation of a phosphodiester bond between the 5'-phosphate of the incoming dNTP and the 3'-hydroxyl group of the growing DNA chain is catalyzed by a polymerase, resulting in the elongation of the DNA strand.

RNA Transcription: Transcription requires the triphosphate group to initiate and elongate RNA chains. The RNA polymerase enzyme recognizes the triphosphate group at the 5'-end of the incoming ribonucleoside triphosphate (rNTP) and incorporates it into the growing RNA molecule during transcription. The 5'-TP group stabilizes the RNA polymerase binding and facilitates proper transcription initiation and elongation.

Signaling Molecules: Certain nucleotides with a triphosphate group, such as GTP and ATP, function as important signaling molecules within cells. GTP is a molecular switch in G-protein signaling pathways and several cellular processes, including cell growth, differentiation, and intracellular signaling cascades. In addition to its energy-carrying role, ATP acts as a signaling molecule in cell signaling, muscle contraction, and enzymatic reactions.

Enzymatic Reactions: The triphosphate group can participate in enzymatic reactions as a phosphate donor or acceptor. Kinases, for example, transfer a phosphate group from ATP to target molecules, regulating their activity. Similarly, phosphatases remove phosphate groups, modulating the function of their target molecules.

Some key features and applications of 5'-TP oligonucleotides are:

Immunostimulatory Properties: 5'-triphosphate (5'-TP) oligonucleotides can interact with pattern recognition receptors, Toll-like receptors 3 (TLR3), TLR7, TLR8, and TLR9 to activate the immune system. This activation leads to the induction of cytokines and other immune response mediators, making them useful in immunotherapy and vaccine development.

The cytosolic pattern recognition receptor RIG-I detects negative-stranded RNA viruses that do not have double-stranded RNA but contain panhandle blunt short double-stranded 5′-triphosphate RNA in their single-stranded genome. In 2009, Schlee et al. reported that synthetic single-stranded 5′-triphosphate oligoribonucleotides could not bind and activate RIG-I. However, the addition of the synthetic complementary strand resulted in optimal binding and activation of RIG-I.

Antiviral Activity: Certain 5'-TP oligos containing specific sequences of nucleotides can exert antiviral effects by targeting viral nucleic acids or proteins. They can interfere with viral replication and trigger immune responses against viral infections.

RNA Interference (RNAi): 5'-TP oligonucleotides as part of small interfering RNA (siRNA) molecules trigger RNA interference. RNAi silences or knocks down specific genes. The presence of the triphosphate group at the 5' end enhances the efficiency of siRNA uptake and subsequent gene silencing.

Gene Editing and Genome Engineering: In the gene-editing technique CRISPR-Cas9, 5'-TP oligonucleotides can guide the Cas9 nuclease to specific genomic targets. 5'-TP oligonucleotides serve as the template for repairing or modifying DNA sequences and enable precise gene editing and genome engineering. Here, 5΄-phosphate mimics stabilize the 5΄-end of the guide strand. The modification protects the guide RNA from phosphatase degradation and 5΄- to 3΄-exonucleases. This modification significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. 5΄-(E)-vinylphosphonate stabilizes the 5΄-phosphate group, enabling systemic delivery and silencing in the kidney and heart.

Diagnostic and Therapeutic Applications: Diagnostic and therapeutic approaches utilize 5'-TP oligonucleotides as probes in the polymerase chain reaction (PCR) or in-situ hybridization (ISH).

Modification: 5'-TP oligonucleotides can be modified with various functional groups or conjugated to other molecules, for example, targeting ligands or therapeutic agents for enhanced specificity and efficacy in therapeutic applications.

Synthetic 5'-TP oligonucleotides are typically chemically synthesized and may require modifications to enhance stability, cell penetration, or target specificity, depending on the desired application.

Reference

ATP [Adenosine_triphosphate]

Bare, G. A. L., Horning, D. P. Chemical Triphosphorylation of Oligonucleotides. J. Vis. Exp. (184), e63877, doi:10.3791/63877 (2022). [jove]

Rig-I [RIG-I]

Thillier Y, Decroly E, Morvan F, Canard B, Vasseur JJ, Debart F. Synthesis of 5' cap-0 and cap-1 RNAs using solid-phase chemistry coupled with enzymatic methylation by human (guanine-N⁷)-methyl transferase. RNA. 2012 Apr;18(4):856-68. [PMC]

Schlee M, Roth A, Hornung V, Hagmann CA, Wimmenauer V, Barchet W, Coch C, Janke M, Mihailovic A, Wardle G, Juranek S, Kato H, Kawai T, Poeck H, Fitzgerald KA, Takeuchi O, Akira S, Tuschl T, Latz E, Ludwig J, Hartmann G. Recognition of 5' triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus. Immunity. 2009 Jul 17;31(1):25-34. [PMC]

Zlatev I, Lackey JG, Zhang L, Dell A, McRae K, Shaikh S, Duncan RG, Rajeev KG, Manoharan M. Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA. Bioorg Med Chem. 2013 Feb 1;21(3):722-32. [article]

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 " Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including 5'-triphosphate and back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides".

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2'-O, 4'-C-Ethylene-bridged Nucleic Acids (ENA) are promising modified nucleic acids in which ethylene is bridged at the furanose sugar ring at the 2'-O and 4'-C ends to form a less strained six-membered ring with C3'-endo conformation. ENA gapmers effectively recruited RNase H, and ENA act as splice modulators such as exon-skipping antisense oligonucleotides and antigene oligonucleotides. Also, clinical trials are currently being conducted using ENA oligonucleotide to treat Duchenne muscular dystrophy (DMD). The aim of the treatment is to skip the normal exon 45 of the dystrophin gene to prevent the occurrence of a stop codon in exon 45. This will restore the functional dystrophin. This technology is owned by Daiichi Sankyo, and they are conducting a Phase II clinical study in Japan.^[1]

Background: Koizumi et al. developed the building block for bicyclo 2′-O, 4′-C-ethylene-bridged nucleic acid (ENA) and compared the properties of 2’,4’-BNA/LNA, a third-generation nucleic acid. Also, further development on synthetic methods to improve the productivity of the monomer has been reported.^[2] Interestingly, apart from high thermodynamic stability, ENA showed enhanced nuclease resistance while retaining the binding affinity strength of other 2’,4’-BNA/LNA. ENA restricts the single-strand flexibility of RNA by forcing the North conformation of sugar puckering within nucleosides due to the 2′-O,4′-C-ethylene-linkage like 2’,4’-BNA/LNA. Studies have shown that 2′,4′-bridged nucleic acids can improve the ability of complementary strands to bind to duplexes. However, a recent report by J. Kawakami et al discovered that not all 2′,4′-bridged nucleic acids can lead to duplex stabilization. When comparing the properties of 2’,4’-C-bridged 2’-deoxynucleotide (CRN) with ENA, it was found that the introduction of one CRN into DNA/RNA duplexes destabilized them. ^[3,4]

Figure1: Structure of 2’,4’-BNA/LNA, ENA and their C3’- endo form

What makes ENA a better choice?

1. Binding affinity: ENA exhibits higher binding affinity to complementary RNA and can match that of 2′,4′-BNA/LNA (ΔTm=+3∼5 °C per modification).^[5]

2. Nuclease resistance: ENAs exhibited greater nuclease resistance than natural DNA and 2′,4′-BNA/LNA. Including one ENA in oligonucleotides significantly enhances their resistance to exonucleases, surpassing that of 2′,4′-BNA/LNA.^[6]

3. Thermal stability: ENA exhibits high thermal stability. A single modification of 2’O, 4’-C-ethyleneguanosines resulted in a 10-fold increase in the binding constant of the DNA/RNA duplex.^[7]

4. Chimeric RNA and ENA show greater efficacy compared to phosphorothioate oligodeoxynucleotides in causing exon 19 skipping in dystrophin mRNA.^[8]

5. ENA exhibits a great ability to form a triplex structure with double-stranded DNA.^[6]

These results indicate that ENA is more suitable as an antisense oligonucleotide and is expected to have better antisense activity than 2′,4′-BNA/LNA. These remarkable characteristics distinguish it from other nucleic acids and render it an attractive choice for developing antisense therapies. Considering its exceptional chemical stability, ENA holds significant potential for developing oligonucleotides that are resistant to nuclease degradation, thereby enhancing their efficacy and promoting their clinical utility. The unique properties of ENA make it a compelling area of research for those involved in the field of antisense therapeutics.

References

1. M. Matsuo et al., Genes, 2017, 8, 67.

2. M. Michida et al., Organic Process Research and Development, 2022, 26, 1289-1307.

3. J. Kawakami et al., Nucleosides, Nucleotides & Nucleic acids, 2024, 1, 57-64.

4. M. Koizumi, Biological and Pharmaceutical Bulletin, 2004, 27, 453-456.

5. M. Koizumi et al., Bioorganic & Medicinal Chemistry Letters, 2002, 12, 73–76.

6. M. Koizumi et al., Bioorganic & Medicinal Chemistry, 2003, 11, 2211–2226.

7. M. Koizumi et al., Nucleic Acids Symposium Series, 2005, 49, 171-172.

8. M. Matsuo et al., Oligonucleotides, 2004, 14, 33-40.

In the endogenous regulatory process called RNA interference (RNAi), short double-stranded RNA oligonucleotides cause sequence-specific posttranscriptional silencing of genes. Small interference RNA (siRNA) has now emerged as a promising therapeutic strategy for the development of novel drugs. However, for successful systemic delivery of siRNA therapeutics, siRNAs must penetrate cellular barriers to reach their targets in the cytoplasm.

The commonly utilized intravenous injection of siRNA poses a significant challenge. After intravenous injection, the blood circulation distributes siRNAs to organs where, at the same time, the siRNA undergoes elimination. When arriving in an organ, siRNA enters the interstitium by leaving the intravascular space within a blood vessel. From the tissue interstitium, siRNA is transported across the interstitial space to target cells. The target cell internalizes siRNA via endocytosis. This process encapsulates siRNA in endocytic vesicles that fuse with endosomes. After entering the cell, siRNA must escape from the endosomes into the cytosol for RISC loading.

Among other approaches, cell-penetrating peptides enable systemic siRNA delivery. Utilizing a cell-penetrating peptide recently, Tönges et al. developed a more straightforward method to deliver siRNA into rat neurons.

A cell-penetrating peptide modified with a stearyl moiety known as stearyl-R8 enables peptide-mediated transfection of cells and possible organelles.

Tönges et al. 2006 showed that a stearylated octaarginine peptide (stearyl-R8) and artificial virus-like particles facilitate the transfection of siRNA into primary rat neuron cells.

The arginine peptide-siRNA complex shuttles the oligonucleotide through the cell membrane into the cell. Complexing siRNA oligonucleotides with stearyl-R8 removes the need for the covalent attachment of the siRNA to the arginine carrier peptide.

Tönges et al. found that peptide-mediated transfection with stearyl-R8 and the polymer/lipid-based cellular delivery with artificial virus-like particles are both easy to handle. Only pipet-based mixing of siRNA with the selected transfection reagent is needed. No further chemical modifications of the siRNA are required.

For an efficient transfection experiment, the research group used synthetic Stearyl-R8 to prepare the Stearyl-R8:siRNA mixture with a cation-to-anion charge ratio 2:1.

This approach may also enable specific targeting of organelles, for example, mitochondria, lysosomes, the endoplasmic reticulum (ER), and the Golgi apparatus. All these organelles are enclosed with membranes or have specialized membrane-engulfed compartments embedded within the cytoplasm. Since they all perform specific roles in the cell, their dysfunction can lead to various pathophysiologies.

Reference

Geng J, Wang J, Wang H. Emerging Landscape of Cell-Penetrating Peptide-Mediated Organelle Restoration and Replacement. ACS Pharmacol Transl Sci. 2023 Jan 16;6(2):229-244. [PMC]

Tönges, L, Lingor, P, Egle, R, Dietz, GP, Fahr, A and Bahr, M (2006). Stearylated octaarginine and artificial virus-like particles for transfection of siRNA into primary rat neurons. RNA 12: 1431-1438. [PMC]

Wang J, Lu Z, Wientjes MG, Au JL. Delivery of siRNA therapeutics: barriers and carriers. AAPS J. 2010 Dec;12(4):492-503. [PMC]

https://www.biosyn.com/tew/MITO-Porters-Enable-Delivery-of-Antisense-Drugs-to-Mitochondria.aspx

https://www.biosyn.com/tew/Cell-penetrating-peptides-for-the-delivery-of-siRNA-into-cells.aspx

https://www.biosyn.com/tew/cell-penetrating-or-trojan-peptides-cpps.aspx

https://www.biosyn.com/tew/cell-penetrating-peptides.aspx

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 " Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including 5'-triphosphate and back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides".

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The recent approval of Nedosiran (RIVFLOZATM) by the FDA was another landmark moment in the field of hyperoxaluria. Hyperoxaluria is increased hepatic oxalate production, leading to recurrent calcium oxalate kidney stones.

Primary hyperoxaluria is an inherited defect of oxalate metabolism that can lead to recurrent renal stones, nephrocalcinosis, and eventually end-stage renal disease.

The small interfering RNA (siRNA) therapy for treating primary hyperoxaluria was developed by Dicerna Pharmaceuticals, now a Novo Nordisk company. In the US, Nedosiran received its first approval on 29 September 2023.

Nedosiran lowers urinary oxalate levels in children aged nine and younger than nine years and adults with primary hyperoxaluria type 1 (PH1) and relatively preserved kidney function.

PH is a family of autosomal recessive genetic disorders characterized by excessive oxalate synthesis in the liver. Excess oxalate leads to insoluble calcium oxalate crystals in the kidneys and the development of kidney stones.

The liver enzyme lactate dehydrogenase (LDH) encoded by the LDHA gene catalyzes the conversion of glyoxylate to oxalate in liver cells. This reaction is the last step of oxalate formation in the liver. Reducing LDH by inhibiting LDHA with RNA interference therapies is a promising treatment strategy for all subtypes of PH.

Following the approval of Lumasiran, Nedosiran is the second siRNA approved for the treatment of PH1. Unlike Nedosiran, Lumasiran inhibits glycolate oxidase to decrease glyoxylate overproduction in peroxisomes.  

Current synthesis technologies allow the production of short oligonucleotide complexes 21 to 27 nucleotides in size. In addition, the synthesis of therapeutic siRNAs containing specific chemical modifications is now also possible. Once synthesized, siRNAs are delivered and evaluated for their activities and drug properties in vitro and in vivo models.

The first therapeutic siRNA approved by the FDA in August 2018 was Patisiran. Patisiran is a therapeutic for the treatment of hereditary transthyretin amyloidosis. In this autosomal dominant disease, the accumulation of misfolded TTR protein throughout the body results in progressive neuropathy, cardiomyopathy, and ophthalmic disease, among other end‐organ effects.

Reference

Bhasin B, Ürekli HM, Atta MG. Primary and secondary hyperoxaluria: Understanding the enigma. World J Nephrol. 2015 May 6;4(2):235-44.[PMC]

Febina, M B. Nedosiran: The rescue from hyperoxaluria. Amrita Journal of Medicine 20(1):p 44-46, January-March 2024.  [AJM]

Forbes TA, Brown BD, Lai C. Therapeutic RNA interference: A novel approach to the treatment of primary hyperoxaluria. Br J Clin Pharmacol. 2022 Jun;88(6):2525-2538. [PMC]

National Kidney Foundation [Kidney.org]

Syed YY. Nedosiran: First Approval. Drugs. 2023 Dec;83(18):1729-1733. doi: 10.1007/s40265-023-01976-4. Erratum in: Drugs. 2024 Jan 22. [PMC]

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 " Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including 5'-triphosphate and back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides".

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Antisense oligonucleotides (ASOs) are short, synthetic, single-stranded oligodeoxynucleotides. ASOs can alter RNA and reduce, restore, or modify protein expression through several mechanisms. ASO-mediated therapies target the source of the pathogenesis, thereby having a higher chance of success than therapies targeting downstream pathways. An improved understanding of antisense pharmacology enabled the translation of these therapeutics into the clinic. Several ASO-mediated therapies have now received approval from the US Food and Drug Administration. However, enabling successful ASO therapies in the clinic requires optimizing ASO delivery, target engagement, and safety profiles. 

Synthetic antisense oligonucleotides can modulate RNA function, influencing gene expression levels, exon skipping, and epitranscriptomics. However, understanding the function of various RNAs and the proteins they interact with can take time and effort. 

The ASO technology can potentially change the therapeutic landscape for many neurological and non-neurological conditions soon. Antisense technology promises to deliver therapeutics for treating diseases by targeting RNA. 

Modifications in approved oligonucleotide-based drugs are mainly based on a few sugar and backbone modifications. Modifications use in earlier ASO drugs are 2’-fluoro (2’-F), 2’-O-Methyl (2’-O-Me), phosphorothioate (PS) chemistries and 2’-O-methoxyethyl (2’-O-MOE) RNA and neutral phosphorodiamidate morpholino oligomer (PMO) backbone analogs.

Bio-Synthesis offers a comprehensive suite of technologies to enable your RNA research and help you to answer these critical questions.

Selected References

ASO Insights

Egli, M., Manoharan, M.; Chemistry, structure and function of approved oligonucleotide therapeutics, Nucleic Acids Research, Volume 51, Issue 6, 11 April 2023, Pages 2529–2573, NAR

Rinaldi, C., Wood, M. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat Rev Neurol 14, 9–21 (2018). Nature

Table 1: Approved Antisense Drugs

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 " Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including 5'-triphosphate and back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides".

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CpG oligonucleotides (CpG ODNs or CpG) are short single-stranded DNA oligonucleotides containing a cytosine guanine (CpG) motif within their sequence. The “p” in this motif refers to the phosphodiester group linking the two nucleotides.

Oligonucleotides containing unmethylated CpG motifs act as immunostimulants for the innate immune system. Immune-stimulating oligonucleotides (ISOs) activate or stimulate the innate immune system via their interaction with pattern recognition receptors, encode immunostimulatory proteins or peptides, or silence specific genes to block negative regulators of the immune system. Several specific nucleic acids and oligonucleotides can act as immunostimulants.

During viral detection, cellular antiviral defenses sense foreign nucleic acids among abundant self-nucleic acids. This mechanism is known as “immune sensing.” However, an effective antiviral defense requires a balanced process of sensing foreign nucleic acids and ignoring self-nucleic acids.

This balance is accomplished by a multilevel system combining the immune sensing of pathogen-specific nucleic acid structures with specific labeling of self-nucleic acids and nuclease-mediated degradation.

Sensing nucleic acids released from pathogens and damaged or malignant cells is the primary mechanism by which innate immune cells recognize “foreign” substances and activate signaling pathways to initiate their antimicrobial and proinflammatory functions.

TLR9 recognizes unmethylated CpG regions and, when stimulated, activates B cells and human plasmacytoid dendritic cells. The activation results in a potent T helper-1 (Th1)-type immune response and an antitumor response in mouse tumor models and patients. Mammals mainly express TLR9 in subsets of Dendritic Cells and B cells. TLR9 receptors recognize different CpG motifs. Optimal sequences are GTCGTT and GACGTT for human TLR9.

Unmethylated CpG DNA containing CpG-dinucleotides is more common in bacterial genomes than in vertebrate genomes. Methylation at the CG sites generally inhibits the activity of CpG dinucleotides. The CpG motif stimulates immune cells via the toll-like receptor 9 signaling pathway.

Krieg et al. (1995) iteratively determined that the immunostimulatory activity of DNA sequences is restricted to a stretch of 12 to 20 base pairs containing CpG dinucleotides with selective flanking bases with the motif 5′-Pu-Pu-CpG-Pyr-Pyr-3′ as being biologically active.

The diverse functions of nucleic acids and oligonucleotides are critical components in vaccine development and cancer immunotherapy. For example, vaccination of the mucous membranes, the inner lining tissue cells of the nose, mouth, lungs, and stomach can initiate enhanced systemic and mucosal humoral and cellular immune responses, resulting in protection against pathogens, even at a different mucosal site. Also, combining CpG ODNs with radiation and chemotherapy can be effective. For example, treating tumors with CpG ODNs combined with radiation and docetaxel enhances the response and improves the cure rate of murine tumors.

Unfortunately, the instability, poor pharmacokinetics profile, non-specific biodistribution, and difficulty in accessing intracellular targets of CpG oligonucleotides make it challenging to develop CpG oligonucleotide-based therapeutics.

CpG 1018 is an adjuvant used in Heplisav-B vaccine made up of CpG motifs. When CpG 1018 is included in vaccines, it increases the body’s immune response.

Reference

Bauer M., Heeg K., Wagner H. DNA activates human immune cells through a CpG sequence-dependent manner. Immunology. 1999;97:699–705.

CpC 1018 CDC info: https://www.cdc.gov/vaccinesafety/concerns/adjuvants.html

Chen W, Jiang M, Yu W, Xu Z, Liu X, Jia Q, Guan X, Zhang W. CpG-Based Nanovaccines for Cancer Immunotherapy. Int J Nanomedicine. 2021 Aug 5;16:5281-5299.  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8352601/

CpG oligonucleotides (https://en.wikipedia.org/wiki/CpG_oligodeoxynucleotide).

Jahrsdörfer B, Weiner GJ. CpG oligodeoxynucleotides as immunotherapy in cancer. Update Cancer Ther. 2008 Mar;3(1):27-32. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2390897/

Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 1995;374:546.

Lin Y-X, Wang Y, Blake S, Yu M, Mei L, Wang H, Shi J, RNA Nanotechnology-Mediated Cancer Immunotherapy, Theranostics, 10 (2020) 281–299. 

Meng F, Wang J, Yeo Y. Nucleic acid and oligonucleotide delivery for activating innate immunity in cancer immunotherapy. J Control Release. 2022 May;345:586-600. doi: 10.1016/j.jconrel.2022.03.045. Epub 2022 Mar 26. PMID: 35351528; PMCID: PMC9133138. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9133138/ )
 

Schlee M, Hartmann G, Discriminating self from non-self in nucleic acid sensing, Nature Reviews Immunology, 16 (2016) 566–580.
 

Shen T, Zhang Y, Zhou S, Lin S, Zhang X-B, Zhu G, Nucleic Acid Immunotherapeutics for Cancer, ACS Appl Bio Mater, 3 (2020) 2838–2849. 

Weiner GJ, Liu HM, Wooldridge JE, Dahle CE, Krieg AM. Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10833-7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23500/
 

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CpG oligonucleotides (CpG ODNs or CpG) are short single-stranded DNA oligonucleotides containing a cytosine guanine (CpG) motif within their sequence. The “p” in this motif refers to the phosphodiester group linking the two nucleotides.

Oligonucleotides containing unmethylated CpG motifs act as immunostimulants for the innate immune system. Immune-stimulating oligonucleotides (ISOs) activate or stimulate the innate immune system via their interaction with pattern recognition receptors, encode immunostimulatory proteins or peptides, or silence specific genes to block negative regulators of the immune system. Several specific nucleic acids and oligonucleotides can act as immunostimulants.

During viral detection, cellular antiviral defenses sense foreign nucleic acids among abundant self-nucleic acids. This mechanism is known as “immune sensing.” However, an effective antiviral defense requires a balanced process of sensing foreign nucleic acids and ignoring self-nucleic acids.

This balance is accomplished by a multilevel system combining the immune sensing of pathogen-specific nucleic acid structures with specific labeling of self-nucleic acids and nuclease-mediated degradation.

Sensing nucleic acids released from pathogens and damaged or malignant cells is the primary mechanism by which innate immune cells recognize “foreign” substances and activate signaling pathways to initiate their antimicrobial and proinflammatory functions.

TLR9 recognizes unmethylated CpG regions and, when stimulated, activates B cells and human plasmacytoid dendritic cells. The activation results in a potent T helper-1 (Th1)-type immune response and an antitumor response in mouse tumor models and patients. Mammals mainly express TLR9 in subsets of Dendritic Cells and B cells. TLR9 receptors recognize different CpG motifs. Optimal sequences are GTCGTT and GACGTT for human TLR9.

Unmethylated CpG DNA containing CpG-dinucleotides is more common in bacterial genomes than in vertebrate genomes. Methylation at the CG sites generally inhibits the activity of CpG dinucleotides. The CpG motif stimulates immune cells via the toll-like receptor 9 signaling pathway.

Classes of CpG oligonucleotides and their effects  (wiki/CpG_ODNs)

Krieg et al. (1995) iteratively determined that the immunostimulatory activity of DNA sequences is restricted to a stretch of 12 to 20 base pairs containing CpG dinucleotides with selective flanking bases with the motif 5′-Pu-Pu-CpG-Pyr-Pyr-3′ as being biologically active.

The diverse functions of nucleic acids and oligonucleotides are critical components in vaccine development and cancer immunotherapy. For example, vaccination of the mucous membranes, the inner lining tissue cells of the nose, mouth, lungs, and stomach can initiate enhanced systemic and mucosal humoral and cellular immune responses, resulting in protection against pathogens, even at a different mucosal site. Also, combining CpG ODNs with radiation and chemotherapy can be effective. For example, treating tumors with CpG ODNs combined with radiation and docetaxel enhances the response and improves the cure rate of murine tumors.

Unfortunately, the instability, poor pharmacokinetics profile, non-specific biodistribution, and difficulty in accessing intracellular targets of CpG oligonucleotides make it challenging to develop CpG oligonucleotide-based therapeutics.

CpG 1018 is an adjuvant used in Heplisav-B vaccine made up of CpG motifs. When CpG 1018 is included in vaccines, it increases the body’s immune response.

Reference

Bauer M., Heeg K., Wagner H. DNA activates human immune cells through a CpG sequence-dependent manner. Immunology. 1999;97:699–705. [PMC]

CpC 1018 CDC info

Chen W, Jiang M, Yu W, Xu Z, Liu X, Jia Q, Guan X, Zhang W. CpG-Based Nanovaccines for Cancer Immunotherapy. Int J Nanomedicine. 2021 Aug 5;16:5281-5299. [PMC]

CpG oligonucleotides.

Jahrsdörfer B, Weiner GJ. CpG oligodeoxynucleotides as immunotherapy in cancer. Update Cancer Ther. 2008 Mar;3(1):27-32. [PMC]

Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 1995;374:546. [PubMed]

Lin Y-X, Wang Y, Blake S, Yu M, Mei L, Wang H, Shi J, RNA Nanotechnology-Mediated Cancer Immunotherapy, Theranostics, 10 (2020) 281–299. [PMC]  

Meng F, Wang J, Yeo Y. Nucleic acid and oligonucleotide delivery for activating innate immunity in cancer immunotherapy. J Control Release. 2022 May;345:586-600. [PMC]

Schlee M, Hartmann G, Discriminating self from non-self in nucleic acid sensing, Nature Reviews Immunology, 16 (2016) 566–580.

Shen T, Zhang Y, Zhou S, Lin S, Zhang X-B, Zhu G, Nucleic Acid Immunotherapeutics for Cancer, ACS Appl Bio Mater, 3 (2020) 2838–2849. 

Weiner GJ, Liu HM, Wooldridge JE, Dahle CE, Krieg AM. Immunostimulatory oligodeoxynucleotides containing the CpG motif are effective as immune adjuvants in tumor antigen immunization. Proc Natl Acad Sci U S A. 1997 Sep 30;94(20):10833-7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC23500/
 

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What is the cause of asthma?

Reduced exposure in early life to microbes and microbial products is thought to cause asthma. People who were raised on farms and who attended out-of-home daycare earlier have fewer and less severe atopic disorders. The notion of “atopic disorder” describes a wide range of genetically mediated allergic diseases. The list includes allergic rhinitis, asthma, and atopic dermatitis (AD) associated with heightened T-helper type 2-driven inflammatory responses to common allergens, especially inhaled and food allergens.

The exact causes of asthma are not yet known. Many different reasons may contribute to asthma. Many environmental factors may trigger asthma in people sensitive to this atopic disorder.

(Source: https://simple.wikipedia.org/wiki/Asthma).

Bacterial DNA, which differs from mammalian DNA in the frequency of cytosine-guanine (CpG) dinucleotides, is an immune-stimulating substance. CpG oligonucleotides (CpG ODNs) induce type 1 T helper (Th1) cells to produce Th1 cytokines (interferon-γ, interleukin-2, and tumor necrosis factor-β) to activate macrophages. Macrophages are responsible for cell-mediated immunity and phagocyte-dependent protective responses.

Scientists have speculated that CpG ODNs may help prevent or reverse the white blood cell-linked (eosinophilic) inflammation of atopic asthma. Inducing the regulatory-type responses of T cells and antigen-presenting cells with CpG ODNs is thought to protect against atopic diseases such as asthma. Ongoing clinical trials examine CpG ODNs used alone and as adjuvants for immunotherapy in human populations with atopic diseases.

Bacterial and viral DNA oligodeoxynucleotides containing unmethylated cytosine-guanine (CpG) dinucleotides are found much more frequently than in vertebrates. These strongly immune-active CpG ODNs induce the proliferation and activation of B cells. 

In recent decades, research scientists identified additional specific immune responses to CpG dinucleotide DNA in molecular pathways linking recognition of the CpG ligand with its effects. A limited number of human immune cells, most notably plasmacytoid DCs and B cells, constitutively express the receptor for CpG DNA, TLR-9.

CpG DNA enters cells through a process called endocytosis, in which cells absorb external substances by engulfing them with the cell membrane. After entering the cell, TLR-9 recognizes CpG oligonucleotides and translocate them to the nucleus, where they activate the nuclear factor-κB.

The early effects of exposure primarily lead to increased innate immune responses. Activated B cells and plasmacytoid DCs release IL-10, type-I IFNs, IL-12, IFN-inducible protein-10, and other cytokines and chemokines, inducing a regulatory/Th1-oriented inflammatory milieu. 

Natural killer cells, T cells, and others that amplify and modulate the immune response react to these molecules. The induction of costimulatory receptors, immunoglobulin isotype switching by B-cells, and the activation of a cascade of cellular responses promoting adaptive immune responses are later effects.

Reference

Akira S. TLR signaling. Curr Top Microbiol Immunol 2006;311:1–16. [PubMed]

Bellanti JA, Settipane RA. The atopic disorders and atopy … "strange diseases" now better defined! Allergy Asthma Proc. 2017 Jul 1;38(4):241-242. [PMC]

Berger A. Th1 and Th2 responses: what are they? BMJ. 2000 Aug 12;321(7258):424. [PMC]

Creticos PS, Schroeder JT, Hamilton RG, Balcer-Whaley SL, Khattignavong AP, Lindblad R, Li H, Coffman R, Seyfert V, Eiden JJ, et al. Immunotherapy with a ragweed-Toll-like receptor 9 agonist vaccine for allergic rhinitis. N Engl J Med 2006;355:1445–1455. [PubMed]

Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000;408:740–745. [PubMed] 

Kline JN. Eat dirt: CpG DNA and immunomodulation of asthma. Proc Am Thorac Soc. 2007 Jul;4(3):283-8. [PMC]

Obermeier F, Strauch UG, Dunger N, Grunwald N, Rath HC, Herfarth H, Scholmerich J, Falk W. In vivo CpG DNA/toll-like receptor 9 interaction induces regulatory properties in CD4+CD62L+ T cells which prevent intestinal inflammation in the SCID transfer model of colitis. Gut 2005;54:1428–1436. [PMC] [PubMed]

Romagnani S. Th1/Th2 cells. Inflamm Bowel Dis. 1999 Nov;5(4):285-94. [PubMed]

Zeng, G., Zhang, G. & Chen, X. Th1 cytokines, true functional signatures for protective immunity against TB?. Cell Mol Immunol 15, 206–215 (2018). [CMI], [nature]

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Gapmers are short deoxyribonucleic acid-based antisense oligonucleotides. Antisense oligonucleotides are chemically modified to enhance the pharmacological properties of RNase H1-antisense oligonucleotides (ASOs). Common modifications of gapmer antisense oligonucleotides include a phosphorothioate (PS) backbone and different 2’-modifications. Usually, each end of an ASO gapmer contains 2’-modifications. The PS backbone is a common component of therapeutic nucleic acids.

Generally, gapmers are designed to hybridize or bind to target ribonucleic acid (RNA) sequences to silence a gene through induced cleavage of the targeted sequence by RNase H.

Intracellular proteins are known to bind antisense oligonucleotides (ASOs) containing phosphorothioate bonds. PS-ASOs are known to bind to plasma proteins, including albumin and α2-macroglobulin, that prevent ASOs from being rapidly discarded through urinary excretion. Compared to PO-ASOs, PS-ASOs enhance cellular uptake both in vitro and in vivo.

Therapeutic PS-ASOs enter cells mainly through endocytic pathways and are released from endocytic particles into the cytosol and nucleus to bind to complementary RNAs via base-pairing. 2′-modifications also affect ASO activity, often by increasing the binding affinity of ASOs to RNA targets.

Liang et al. have identified and characterized more than 50 intracellular proteins interacting with ASOs. However, the mechanism of how ASOs interact with these proteins remains largely unknown.

MALAT PS MOE DNA gamer ASO in double stranded form (PDB ID 7ZVN).

2’-MOE PS nucleotides are shown in cyan. PS DNA gapmer in black. All cytidines in the ASO are methylated (5meC). Location of mismatches are highlighted with red dots.

In 2016, Liang et al. studied the protein-binding effects of ASOs containing phosphorothioate (PS) in the backbone and 2′-modifications in the ribose. The researchers utilized affinity selection and competition assays to show that the PS backbone modification dominates protein binding. The study found that compared to a PO-backbone, PS-modified ASOs bind more proteins more tightly. Also, the number of PS-modified nucleotides affects the binding of many proteins to ASOs. ASOs containing less than 10 PS-modified nucleotides interacted with proteins significantly weaker than ASOs with greater PS numbers, and the 2′-modifications in the wings of gapmer ASOs affected protein binding as well.

According to Liang et al., the Hsp90 protein interacts more strongly with PS-ASOs containing locked-nucleic acid (LNA) or constrained-ethyl-bicyclic-nucleic acid ((S)-cEt) modifications than with 2′-O-methoxyethyl (MOE). Also, ASOs bind to the mid-domain of the Hsp90 protein. ASOs containing the hydrophobic 2′-modifications (S)-cEt or LNA in the 5′-wing interact with Hsp90.

Reduction of the Hsp90 protein decreased the activity of PS-ASOs with 5′-LNA or 5′-cEt wings but not with 5′-MOE wings. This study by Liang et al. indicates that the Hsp90 protein enhances the activity of ASOs modified with PS and LNA moieties or PS and (S)-cEt moieties, suggesting that different chemical modifications can improve the therapeutic efficacy of PS-ASOs.

In 2020, Hyjek-Składanowska reported a crystal structure-based model of the DNA-binding domain of the PC4 protein in complex with a PS 2′-OMe DNA gapmer. In the structure, each PC4 dimer contains two DNA-binding interfaces. The 5′-terminal 2′-OMe PS flank is bound by one interface. The regular PS DNA central part is bound in opposite polarity as a hairpin-like structure by the other interface. Binding to the ASO induces the formation of a dimer of dimers of PC4. The base pairing between regions of the ASO stabilizes the binding to each PC4 dimer. The PS nucleic acid interacts with the protein through electrostatic and hydrophobic contacts. NanoBRET binding assay confirmed these contacts.

Human Transcription Cofactor PC4 DNA-binding domain in complex with full phosphorothioate 5-10-5 2'-O-methyl DNA gapmer antisense oligonucleotide [6YCS].

Hyjek-Składanowska et al. suggest that this structure provides insights into the molecular forces governing the interactions of PS ASOs with cellular proteins. Furthermore, it provides a potential model for how these interactions can cause cellular toxicity. Pandey et al. (2021) state that the interaction between human positive coactivator 4 (PC4) and the tumor suppressor protein p53 is crucial in initiating apoptosis. PC4 is an abundant nuclear protein. Since PC4 assisted-p53-dependent apoptosis may play a central role in certain neurodegenerative diseases, disrupting p53-PC4 interactions may be a promising drug target for specific disease pathologies.

More recently, in 2023, Hyjek-Składanowska et al. reported the crystal structure of human Annexin A2 in complex with a phosphorothioate 5-10 2”-MOE DNA gapmer ASO. The research group co-crystallized the C-terminal domain (CTD) core of annexin A2 (AnxA2) with a phosphorothioate ( PS) 2′-methoxyethyl (MOE) DNA gapmer ASO. The crystal structure revealed that unique hydrophobic interactions between PS groups, lysine and arginine residues account for the enhanced affinity of PS ASOs to the protein surface. These results demonstrate that this interaction mechanism appears to be a general phenomenon observed not only for nucleic acid-binding proteins but may also explain interactions of ASOs proteins.

Crystal structure of human Annexin A2 in complex with full phosphorothioate 5-10 2'-methoxyethyl DNA gapmer antisense oligonucleotide solved at 1.87 Å resolution [7ZVN].

The annexin A (ANXA) protein family is a well-known tissue-specific multigene family encoding calcium (Ca2⁺) phospholipid-binding proteins. ANXA proteins play essential roles in cancer progression, proliferation, invasion, and metastasis in several diseases.

Reference

Crooke ST, Vickers TA, Liang XH. Phosphorothioate modified oligonucleotide-protein interactions. Nucleic Acids Res. 2020 Jun 4;48(10):5235-5253. [PMC]

Hyjek-Składanowska M, Vickers TA, Napiórkowska A, Anderson BA, Tanowitz M, Crooke ST, Liang XH, Seth PP, Nowotny M. Origins of the Increased Affinity of Phosphorothioate-Modified Therapeutic Nucleic Acids for Proteins. J Am Chem Soc. 2020 Apr 22;142(16):7456-7468. [PubMed][pdb/6ycs]

Hyjek-Składanowska M, Anderson BA, Mykhaylyk V, Orr C, Wagner A, Poznański JT, Skowronek K, Seth P, Nowotny M. Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding. Nucleic Acids Res. 2023 Feb 22;51(3):1409-1423. [PMC]

Liang XH, Shen W, Sun H, Kinberger GA, Prakash TP, Nichols JG, Crooke ST. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity. Nucleic Acids Res. 2016 May 5;44(8):3892-907. [PMC]

Pandey B, Dev A, Chakravorty D, Bhandare VV, Polley S, Roy S, Basu G. Insights on the disruption of the complex between human positive coactivator 4 and p53 by small molecules. Biochem Biophys Res Commun. 2021 Nov 12;578:15-20. [PubMed]

Zhang H, Zhang Z, Guo T, Chen G, Liu G, Song Q, Li G, Xu F, Dong X, Yang F, Cao C, Zhong D, Li S, Li Y, Wang M, Li B, Yang L. Annexin A protein family: Focusing on the occurrence, progression and treatment of cancer. Front Cell Dev Biol. 2023 Mar 3;11:1141331. [PMC] 

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 " Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including 5'-triphosphate and back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides".

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Enhancer RNAs add a crucial regulatory layer to the genome. Kim et al. (2010) identified a new class of RNAs called enhancer RNAs or eRNAs. Enhancers are cis-regulatory gene elements and are crucial for controlling temporal and cell-type specific patterns of gene expression.

Several classes of non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs (ncRNAs), play diverse roles in post-transcriptional regulation of mRNA stability and epigenetic control of chromatin activities.

Enhancer RNA or eRNA are RNAs transcribed by RNA polymerase II (RNAPII) from the domain of transcription enhancers. They are a class of short, non-coding RNAs transcribed from DNA enhancer regions, 50 to 2,000 nucleotides in length. Enhancer regions are bidirectionally transcribed into enhancer RNAs (eRNAs). As a response to signaling pathways, intergenic DNA elements known as enhancers regulate target gene transcription. Enhancers interact with promotors over large genomic distances. Enhancers contain binding sites for transcription factors promoting RNA polymerase II (RNAPII) recruitment and activation. Enhancers carry unique epigenetic marks, distinguishing them from promoters.

These regulatory elements also have an open chromatin conformation that increases accessibility to transcription factors and RNAPII. Rahman et al. recently reported that eRNAs are localized exclusively in the nucleus. The induction of eRNAs occurs with similar kinetics as that of target mRNAs. eRNAs are beginning to develop mostly as enhancers. The steady-state levels of eRNAs remain lower than those of their related mRNAs and RNAs at the single-allele level. Also, eRNAs are rarely co-expressed with their target loci. However, active gene transcription does not require continuous transcription of eRNAs or the accumulation of eRNAs at enhancers.

Recently developed genome-wide sequencing methods allow studying stimulus-dependent enhancer functions in tissue cells. Kim et al. (2010) found that the level of eRNA expression at neuronal enhancers positively correlates with the level of mRNA synthesis at nearby genes. These observations suggest that eRNA synthesis occurs specifically at enhancers that actively promote mRNA synthesis and that a widespread mechanism of enhancer activation involves RNAPII binding and eRNA synthesis.

Kim et al. suggested that establishing and maintaining the chromatin landscape at enhancers requires eRNA synthesis for enhancer function. It is also possible that the eRNA transcripts are functionally crucial by themselves. A typical gene transcribed by RNA polymerase II has a promotor that extends upstream from the site where transcription is initiated (Figure 1). 

Figure 1: Overview of a typical gene transcribed by RNA polymerase II.

The promotor contains several short sequence elements that bind transcription factors, <10 base pairs (bp) in length, and promotors are dispersed over a sequence region >200 bp. Enhancers contain a more closely packed array of elements that bind transcription factors. Enhancer regions appear to be located at a distance of several kilobases (kb). The DNA duplex may be coiled or rearranged such that transcription factors at the promotor and the enhancer interact to form a large protein-DNA complex. 

Enhancers contain bidirectional elements that allow assisting initiation. The presence of a promotor increases its activity. The enhancer is located distinct from the promotor, and its position relative to the promotor can vary substantially. An enhancer can stimulate any promotor placed in its vicinity. Enhancers often show redundancy in function. DNA must be able to form a loop structure if proteins bound at an enhancer several kb distant from a promotor interact directly with proteins bound in the vicinity of the starting point such that the enhancer and promotor are near to each other. Enhancers may function by bringing proteins into the area of the promotor.

Methods that allow analysis of eRNAs:

• Reverse transcription-PCR (RT-PCR)
• RNA fluorescence in situ hybridization (RNA-FISH)
• RNA polymerase II chromatin immunoprecipitation coupled with high-throughput sequencing (RNAPII ChIP–seq)
• Global run-on sequencing (GRO-seq)
• 5′GRO-seq or GRO-cap
• BruUV-seq
• RNA-seq (total)
• RNA-seq (poly(A))
• Cap analysis of gene expression (CAGE) followed by deep sequencing
• Chromatin- bound RNA-seq
• RNA-Seq in isolated 'transcription factories'
• Native elongating transcript sequencing (NET-seq)
• RNA capture sequencing (CaptureSeq)
• Chromatin isolation by RNA purification (ChIRP-seq)

Reference

Bose DA, Berger SL. eRNA binding produces tailored CBP activity profiles to regulate gene expression. RNA Biol. 2017 Dec 2;14(12):1655-1659. [PMC]

Jourdain AA, Koppen M, Rodley CD, Maundrell K, Gueguen N, Reynier P, Guaras AM, Enriquez JA, Anderson P, Simarro M, Martinou JC. A mitochondria-specific isoform of FASTK is present in mitochondrial RNA granules and regulates gene expression and function. Cell Rep. 2015 Feb 24;10(7):1110-21. [Cell Reports]

Kim, T.-K., Hemberg, M., Gray, J. M., Costa, A. M., Bear, D. M., Wu, J., … Greenberg, M. E. (2010). Widespread transcription at neuronal activity-regulated enhancers. Nature, 465(7295), 182–187. [Nature]

Lewin, Benjamin; Genes VII chapter 20, 2000. 

Li, W., Notani, D. & Rosenfeld, M. Enhancers as non-coding RNA transcription units: recent insights and future perspectives. Nat Rev Genet 17, 207–223 (2016). [Nature Reviews genetics]

Rahman, S., Zorca, C. E., Traboulsi, T., Noutahi, E., Krause, M. R., Mader, S., & Zenklusen, D. (2017). Single-cell profiling reveals that eRNA accumulation at enhancer–promoter loops is not required to sustain transcription. Nucleic Acids Research, 45(6), 3017–3030. [NAR]

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Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including 5'-triphosphate and back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides".

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Therapeutic peptides have emerged as promising drugs for treating various medical conditions, including type 2 diabetes and weight loss. 

The chronic metabolic disorder Type 2 diabetes is distinguished by insulin resistance and impaired insulin secretion, resulting in elevated blood sugar levels. Obesity often coexists with type 2 diabetes and worsens its complications, making weight management a crucial aspect of diabetes treatment.

Peptides play significant roles in various physiological processes within the body. In the context of type 2 diabetes and weight loss, therapeutic peptides offer several advantages over traditional medications, including high specificity, low toxicity, and reduced risk of adverse effects.

One of the most well-known therapeutic peptides for type 2 diabetes treatment is a glucagon-like peptide-1 (GLP-1) agonist. The intestine secretes the hormone peptide GLP-1 in response to food intake. GLP-1 induces insulin secretion from pancreatic beta cells, inhibits glucagon secretion, slows gastric emptying, and promotes satiety. Synthetic GLP-1 agonists, such as exenatide, liraglutide, and dulaglutide, mimic the actions of endogenous GLP-1. In patients with type 2 diabetes, GLP-1 helps to regulate blood sugar levels. Additionally, GLP-1 agonists are associated with weight loss due to their effects on appetite suppression and delayed gastric emptying.

In recent years, the commercial approval of peptide agonists of the glucagon-like peptide-1 (GLP-1) receptor for type II diabetes treatment has marked a significant milestone in the field. The FDA's approval of liraglutide (Victoza®; Novo Nordisk), semaglutide (Ozempic®, Rybelsus®; Novo Nordisk), and dulaglutide (Trulicity®; Eli Lilly), and semaglutide (Wegovy®; Novo Nordisk) for weight loss has not only expanded the range of treatment options but also sparked a new wave of interest in this type of peptide.

Another class of therapeutic peptides used in treating type 2 diabetes are amylin analogs. Amylin is a hormone co-secreted with insulin by pancreatic beta cells. It regulates glucose metabolism by suppressing glucagon secretion, slowing gastric emptying, and promoting satiety. Pramlintide, the synthetic analog of amylin, allows the treatment of patients for whom other medications have failed. Pramlintide helps to reduce glucose levels after a meal, which can result in weight loss by reducing food intake.

In addition to targeting glucose metabolism, therapeutic peptides for weight loss aim to regulate appetite and energy expenditure. One example is the melanocortin-4 receptor (MC4R) agonist setmelanotide. MC4R is involved in regulating food intake and energy balance. Setmelanotide stimulates MC4R signaling, leading to reduced appetite and increased energy expenditure. Setmelanotide allows the treatment of rare genetic disorders associated with severe obesity, such as pro-opiomelanocortin (POMC) deficiency and leptin receptor (LEPR) deficiency.

Despite their therapeutic potential, therapeutic peptides for type 2 diabetes and weight loss may have limitations, including the need for injection and high cost. Also, there are potential side effects, such as nausea, vomiting, and hypoglycemia. However, a significant goal of ongoing research is to overcome these challenges by developing novel peptide-based therapies with improved efficacy, safety, and convenience of administration. 

Overall, therapeutic peptides represent promising drugs to manage type 2 diabetes and obesity, offering targeted approaches to address underlying metabolic dysregulation and promote better health outcomes.

Weight loss peptides targeting the glucagon-like peptide-1 (GLP-1) receptor interact with multiple organs and tissues (See Figure 1).

Figure 1: Physiological actions of glucagon-like peptide-1 (GLP-1) 

(Source: wikimedia commons)

Therapeutic peptide drugs are pharmaceuticals that selectively target biological molecules. The FDA has approved over 80 peptide drugs, and many more are enrolled in preclinical studies and clinical development. Moreover, the versatility of therapeutic peptides is now being harnessed in a wide range of medical areas, from cancer and metabolism to respiratory, cardiovascular, urology, autoimmune, pain, and antimicrobial applications. This broad spectrum of applications not only demonstrates the adaptability of therapeutic peptides but also underscores their immense potential in revolutionizing modern medicine, inspiring a new era of treatment possibilities.

Drugs to treat type 2 diabetes and chronic obesity target the glucagon-like peptide-1 receptor (GLP-1R). Therapeutic peptides such as glucagon, liraglutide, and semaglutide are peptide hormone mimetics targeting GLP-1R. The introduction of side chain modifications avoids degradation and increases the pharmacokinetics of the peptides.

Standard automated solid-phase peptide synthesis allows timely synthesis of these peptide analogs in mg to gram amounts. However, cost-efficient methods for larger quantities are now available for combining solid-phase synthesis with liquid-phase synthesis. Proprietary synthetic approaches developed by Biosynthesis Inc. now allow the synthesis of these peptides ranging from mgs to kgs.

Table 1: Therapeutic GLP-1 Peptide Analogs

Reference

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Serinol nucleic acids (SNAs), a unique class of synthetic nucleic acid analogs, are designed to mimic the structure and function of natural nucleic acids (DNA and RNA). They stand out in the realm of synthetic polymers, known as xeno-nucleic acids (XNAs), due to their distinct properties. With their ability to expand the chemical diversity of nucleic acids, XNAs, including SNAs, hold immense potential in diverse fields such as biotechnology and medicine. Oligonucleotides modified with SNAs hybridize to both DNA and RNA.

Structure and Composition

The backbone structure of SNAs differs from the natural deoxyribose (in DNA) or ribose (in RNA) sugar backbone structure. In SNAs, the backbone contains serinol (2-amino-1,3-propanedial) units. Serinol is a molecule derived from serine, an amino acid, and consists of an amine and an alcohol group attached to a two-carbon chain. This structural modification results in distinct properties of SNAs, including increased stability and binding affinity.

Figure 1: Structures of PNA, SNA, DNA, and RNA. Serinol (2-amino-1,3-propanediol) building blocks are linked via normal phosphodiester linkages giving them a negative charge. SNAs can be used in siRNAs, molecular beacon and ASOs.

Key Features and Advantages

Enhanced Stability: Compared to natural nucleic acids, SNAs are more stable, making them more resistant to enzymatic degradation. This allows for various applications in biological systems.

High Binding Affinity: The altered backbone structure of SNAs can result in more robust and specific binding to complementary nucleic acid sequences, which is helpful for applications in diagnostics and therapeutics, where precise targeting is essential.

Resistance to Nucleases: Synthetic SNAs are not easily recognized and degraded by nucleases, enzymes that typically break down natural nucleic acids. This extends the functional lifespan of SNAs in biological environments.

Applications

Gene Therapy: Oligonucleotides modified with SNAs enable the delivery of therapeutic genes or oligonucleotides into cells. Their stability and binding properties enhance the efficiency and effectiveness of gene delivery.

Diagnostics: In diagnostic assays, SNAs can be employed as probes to detect specific nucleic acid sequences. Their high specificity and resistance to degradation improve the accuracy and reliability of these tests.

Antisense and RNAi Therapies: Well-designed SNA oligonucleotide can selectively bind to mRNA transcripts and modulate gene expression. This application is particularly relevant for developing treatments for genetic diseases and viral infections.

Research Tools: SNAs can serve as valuable tools in molecular biology research, enabling the study of nucleic acid interactions and the development of new biotechnological methods.

Development of SNAs

Ramasamy et al. (1996) and Benhida et al. (1998) synthesized SNA phosphoramidites to incorporate SNAs into oligonucleotides and found that oligonucleotide duplexes containing small numbers of SNAs are less stable than natural duplexes. One single substitution with an SNA reduced melting temperatures by 0.5 to 12 °C depending on the sequence and location of the substitution. Many years later, Kashida et al. (2011) observed that oligonucleotides fully modified with SNAs can hybridize with SNAs, RNA, and DNA in an antiparallel fashion to form relatively stable right-handed helices. In these SNA oligonucleotides, sequence designs allow for the control of their chirality, and reversing their sequences inverses their chirality. However, Le et al. (2017) years later, reported that SNAs bind RNA more strongly than DNA and showed that SNAs are very stable to nucleases, even to snake venom phosphodiesterase.

Similar to serinol nucleic acids, Sakata & Fujii (2000) synthesized acyclic adenosine and thymidine analogs containing L- and D-threonine. Using the phosphoramidites of these nucleic acids allowed their incorporation into oligonucleotides using automated solid-phase synthesis. Incorporating threonine nucleic acid analogs into oligonucleotide duplexes did not destabilize hybrid duplexes; however, the analogs' stereochemistry influenced the resulting duplexes' stability.

Le et al. (2017) synthesized an SNA antisense oligonucleotide (AO) and investigated its potential to induce exon-skipping, a process where specific exons in a gene's DNA sequence are 'skipped' during the process of gene expression. The study found that the SNA-based AO can effectively induce Dmd exon-23 skipping in vitro. Its related PNA analog showed very high RNA binding affinity but failed to cause significant exon-23 skipping due to poor transfection efficiency with lipid-based transfection reagents.

The phosphodiester linkage between SNA monomers is a key factor that allows for the complexation of SNAs with lipid-based transfection reagents, leading to a superior cellular uptake capability. Moreover, SNA-modified AO demonstrated exceptionally high resistance to nuclease degradation. In 2017, SNA chemistry was a relatively new and unexplored territory for therapeutic applications.

Kamiya et al. (2020) solved the crystal structures of RNA hybridizing with SNA and L-αTNA. The heteroduplexes have unwound right-handed helical structures. The structure revealed that base pairs in the heteroduplexes align perpendicularly to the helical axes. The observed unwound helical structures originate from interactions between nucleobases and neighboring backbones of L-aTNA and SNA through CH–O bonds. In addition, SNA and L-αTNA form a triplex structure via C:G*G parallel Hoogsteen interactions with RNA.

In summary, serinol nucleic acids represent a promising advancement in synthetic biology. Their unique properties make them suitable for a range of applications in medicine and biotechnology. Their enhanced stability, high binding affinity, and resistance to enzymatic degradation are vital features that differentiate them from natural nucleic acids and other synthetic analogs. As biomedical research progresses, SNAs will likely play an increasingly important role in developing innovative therapeutic and diagnostic solutions.

Reference

Benhida, R., Devys, M., Fourrey, J.L., Lecubin, F., and Sun, J.S.; Incorporation of serinol derived acyclic nucleoside analogues into oligonucleotides: Influence on duplex and triplex formation. Tetrahedron Letters, Volume 39, Issue 34, 1998, 6167-6170. [Science Direct]

Kamiya Y, Satoh T, Kodama A, Suzuki T, Murayama K, Kashida H, Uchiyama S, Kato K, Asanuma H. Intrastrand backbone-nucleobase interactions stabilize unwound right-handed helical structures of heteroduplexes of L-aTNA/RNA and SNA/RNA. Commun Chem. 2020 Nov 6;3(1):156. [PMC]

Kashida H, Nishikawa K, Shi W, Miyagawa T, Yamashita H, Abe M, Asanuma H. A helical amplification system composed of artificial nucleic acids. Chem Sci. 2021 Jan 12;12(5):1656-1660. [PMC]

Le, B. T., Murayama, K., Shabanpoor, F., Asanuma, H., and Veedu, R. N.; Antisense oligonucleotide modified with serinol nucleic acid (SNA) induces exon skipping in mdx myotubes. RSC Adv., 2017, 7, 34049-34052.

Murayama K, Kashida H, Asanuma H. The Use of Serinol Nucleic Acids as Ultrasensitive Molecular Beacons. Methods Mol Biol. 2019;1973:261-279. [PubMed]

Ramasamy, K.S., and Seifert, W.; Amino acid nucleic acids: Synthesis and hybridization properties of a noval class of antisense oligonucleotides. Bioorg Med Chem Lett, 1996, 6, 1799-1804.

Sakata M, Fujii M. Synthesis and properties of novel acyclic nucleotides. Nucleic Acids Symp Ser. 2000;(44):47-8. [PubMed]

SNA-RNA structure: https://www.ncbi.nlm.nih.gov/Structure/pdb/7BPG

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A gene’s function can be defined by reducing or completely disrupting its normal expression. Since its discovery, interference RNA (RNAi) has offered a magic bullet to disrupt gene expression in many organisms. However, in recent years, new biotechnological tools have been developed for knocking down a gene thereby increasing the array of methods now available to a researcher.  

Gene knockdown methods are powerful tools that help medical scientists better understand gene function and regulation. This approach can lead to significant advancements in biology, medicine, and biotechnology.

Knocking down a gene is not merely a scientific process; it's a practical solution with real-world applications. It allows us to dissect gene function, deepen our understanding of disease mechanisms, validate therapeutic targets, and improve agricultural practices. This controlled method of studying the consequences of reduced gene expression provides invaluable insights across various research fields.

Knockdown methods, with their ability to target a gene in a cell, tissue, or organism, are versatile tools. They find extensive use in genetic research, allowing us to probe into the function of genes by observing the outcomes of their reduced expression.

Here is a list of some knockdown methods

1. RNA Interference (RNAi)

Small Interfering RNA (siRNA)

Design and Synthesis: Design siRNA sequences complementary to the selected target mRNA. The use of automated chemical synthesis ensures the production of modified and unmodified siRNA duplexes.

Transfection: This step involves introducing the siRNA into the cells using transfection protocols. These protocols, which utilize lipofection or electroporation, are used in molecular biology and genetics research for their effectiveness in gene expression regulation.

Incorporation: After transfection, the siRNA is incorporated into the RNA-induced silencing complex (RISC).

mRNA Degradation: RISC utilizes the siRNA to guide the degradation of the target mRNA, thereby significantly reducing gene expression. This method ensures productive results.

Short Hairpin RNA (shRNA)

Like RNA interference, this method involves the use of short hairpin RNA to regulate gene expression.

Vector Construction: Clone shRNA sequences into plasmid or viral vectors (like lentivirus) for vector construction.

Transduction/Transfection: Introduce the vectors into the cells. Viral vectors can integrate into the genome, providing stable and long-term knockdown.

Processing and Incorporation: The shRNA is processed into siRNA by the cellular machinery and incorporated into RISC.

mRNA Degradation: Like siRNA, the RISC complex degrades the target mRNA.

2. Morpholino Oligonucleotides

Design: Design morpholino oligonucleotides complementary to the target mRNA sequence.

Microinjection: Inject morpholinos into embryos or cells. Typically, morpholino antisense oligonucleotides are used in early developmental stages.

Translation Blockage: Morpholinos bind to the target mRNA and block its translation.

3. CRISPR Interference (CRISPRi)

Guide RNA Design: Design guide RNAs (gRNAs) that target the promoter region of the gene of interest.

dCas9-Repressor Fusion: Use a dead Cas9 (dCas9) protein fused to a transcriptional repressor domain (e.g., KRAB).

Transfection/Transduction: Introduce the gRNA and dCas9-repressor into cells via plasmid vectors or viral delivery systems.

Transcriptional Repression: The dCas9-repressor complex binds to the target DNA and inhibits transcription without cutting the DNA.

4. Antisense Oligonucleotides (ASOs)

Design: Design antisense oligonucleotides complementary to the target mRNA.

Delivery: Deliver the ASOs into cells using various transfection methods.

Binding and Inhibition: ASOs bind to the target mRNA, preventing translation or promoting degradation.

5. Transcription Factor Decoys

Design: Create synthetic oligonucleotides that mimic the binding sites of specific transcription factors.

Delivery: Introduce these decoys into cells.

Binding: The decoys bind to transcription factors, preventing them from binding to the target gene’s promoter and reducing gene expression.

Steps for Performing Gene Knockdown Experiments (A General Procedure)

Select the Target Gene: Choose the gene to be knocked down based on the research goal.

Precision is Key in the design of Knockdown Reagents. It's essential to design siRNA, shRNA, morpholinos, ASOs, or gRNAs specific to the targeted gene to ensure the accuracy of the knockdown process.

Synthesis/Cloning: Synthesize the oligonucleotides or clone the shRNA/gRNA into appropriate vectors.

Delivery into Cells: Introduce the knockdown reagents into cells using transfection (e.g., lipofection, electroporation) or transduction (e.g., lentivirus, adenovirus).

Validation is Essential: Confirmation of Knockdown. It is crucial to validate the reduction of gene expression using techniques like quantitative PCR (qPCR) for mRNA levels, Western blotting for protein levels, or functional assays to ensure the reliability of the results.

Analysis of Phenotypic Effects: To understand the gene's role, assess the biological effects of gene knockdown on cell function, viability, and phenotype.

Each method has specific protocols and optimization steps depending on the cell type, target gene, and experimental conditions. Proper controls and validation are crucial for ensuring the specificity and efficiency of gene knockdown.

Applications For Gene Knockdown Methods

Understanding Gene Function:

Functional Analysis: By reducing the expression of a specific gene, researchers can study the resulting phenotypic changes. Reducing gene expression allows researchers to determine a gene's role in various biological processes.

Gene Interaction Studies: Knockdown experiments can reveal how genes interact, providing insights into genetic pathways and networks.

Modeling Diseases:

Disease Mechanisms: Many diseases are caused by or associated with the overexpression or malfunction of specific genes. Knocking down these genes in model organisms or cell lines helps unravel the underlying mechanisms of these diseases.

Identification of Essential Genes: Cells need essential genes for survival and proliferation. Cancer cells or pathogens, in particular, need specific genes to thrive. Knocking down such genes can help identify potential therapeutic targets.

Drug Discovery and Validation:

Target Identification and Validation: Knocking down genes suspected to be involved in disease processes allows researchers to test whether these genes are viable drug targets.

Mechanism of Action Studies: Determining how drugs affect cellular processes by observing changes in phenotype after gene knockdown.

Functional Genomics:

High-Throughput Screening: Large-scale gene knockdown studies like RNAi screens help identify genes involved in specific cellular functions or disease states.

Gene Redundancy Studies: Understanding compensatory mechanisms where knocking down one gene can reveal the roles of related or redundant genes.

Developmental Biology:

Embryonic Development: Studying the effects of gene knockdown during various stages of development can reveal the roles of genes in growth, differentiation, and morphogenesis.

Tissue and Organ Development: Understanding how specific genes contribute to the formation and function of tissues and organs.

Pathway Analysis:

Dissecting Signaling Pathways: By knocking down genes involved in signaling pathways, researchers can identify the roles of individual components and how they interact within the pathway.

Agricultural Research:

Improving Crop Traits: Knocking down genes that negatively affect crop yield, disease resistance, or stress tolerance can lead to the development of better-performing plant varieties.

Pest and Disease Management: It is important to identify and manipulate genes that endow crops with resistance to pests and diseases.

Synthetic Biology:

Engineering Gene Networks: In synthetic biology, gene knockdown allows the removal or reducing the activity of endogenous genes to create space for synthetic gene circuits or to study the function of synthetic networks.

Studying Compensatory Mechanisms:

Genetic Redundancy: Knocking down a gene can help uncover how other genes compensate for its loss, providing insights into genetic robustness and redundancy.

Regenerative Medicine:

Tissue Repair and Regeneration: Knocking down genes involved in tissue repair and regeneration can help understand the genetic basis of these processes, which can aid in developing regenerative therapies.

References

Boettcher M, McManus MT. Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR. Mol Cell. 2015 May 21;58(4):575-85. [PMC]

“This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools.”

Mondal, M., Peter, J., Scarbrough, O. et al. Environmental RNAi pathways in the two-spotted spider mite. BMC Genomics 22, 42 (2021).[Bmc Genomics]

“Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design.”

Senapati, D., Patra, B. C., Kar, A., Chini, D. S., Ghosh, S., Patra, S., & Bhattacharya, M. (2019). Promising approaches of small interfering RNAs (siRNAs) mediated cancer gene therapy. Gene.719, 2019,144071. [Gene]

Suzuki T, Nunes MA, España MU, Namin HH, Jin P, Bensoussan N, Zhurov V, Rahman T, De Clercq R, Hilson P, Grbic V, Grbic M. RNAi-based reverse genetics in the chelicerate model Tetranychus urticae: A comparative analysis of five methods for gene silencing. PLoS One. 2017 Jul 12;12(7):e0180654.[PMC]

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The fluorescent analog of deoxycytidine (C), pyrrole-deoxycytidine (pyrrole-dC, PdC), specifically base pairs with deoxyguanosine (G) and is a valuable tool for probing the structures of nucleic acid or protein-nucleic acid complexes. 

Pyrrolo-dC and pyrrolo-C base analogs code as cytidine (C), making them ideal probes for DNA structure and dynamics studies: Pyrrolo-dC and pyrrolo-C base analogs base pair like the standard dC nucleotide. The small size of these analogs does not perturb the structure of the DNA helix and is also well tolerated by several DNA and RNA polymerases. The analogs are highly fluorescent, and their red-shifted excitation and emission spectra eliminate or reduce background fluorescence originating from proteins.

Pyrrolo-dC (PydC) and pyrrolo-C (PyC) are structurally modified fluorescent deoxycytidine and cytidine analogs that maintain a proper Watson-Crick hydrogen bonding. The fluorescence cytidine analog Pyrrolo-C (PC), or 3-[β-D-2-ribofuranosyl]-6-methylpyrrolo [2,3-d] pyrimidin-2(3H)-one, can base-pair with guanosine (G) and can be selectively excited in the presence of natural nucleosides. Figure 1 shows the chemical structure of the pyrollo-dC analog.

Figure 1: Pyrrolo-dC [6-methyl-3-(2-deoxy-β-d-ribo-furano-syl)-3H-pyrrolo[2,3-d]pyrimidin-2-one)].

Pyrrolo-dC or PdC {(6-Methyl-3-(β-D-2-deoxyribofuranosyl) pyrrolo [2,3-d] pyrimidine-2-one, pyrrol-2-ylcarbonyl) deoxycytidine, {6-methyl-3-(2-deoxy-b-Dribofuranosyl)-3H-pyrrolo [2,3-d] pyrimidin-2-one)} is a base analog that fluoresces when not base-paired and exhibits little effect on DNA stability, structure, and O6-alkyl-guanine DNA alkyltransferase (AGT) repair. Incorporating pyrrolo-dC into oligonucleotides allows measurement of base flipping by human O6-alkylguanine DNA alkyltransferase.

Pyrrolo-dC based probes

Incorporation of the pyrrolo-cytosine analog into oligonucleotides allows the design and synthesis of site-specific probes useful for the study of RNA structure and dynamics. In a probe strand, the fluorophore is placed opposite a SNP site. Hybridization to a perfectly matched target sequence causes the fluorophore to stack between the surrounding nucleobases, resulting in a light-down or OFF response. However, if a mismatch is present, the probe sequence cannot achieve a perfectly stacked conformation, and a light-up reaction or ON is observed. 

Applications

Pyrrolo-dC, a nucleoside with unique properties, is a key player in various biochemical applications, particularly in research and drug development. Its ability to synthesize nucleic acid derivatives for the study of DNA and RNA structures is unparalleled. Moreover, its potential to inhibit cancer cell growth makes it also a promising candidate for anticancer drug development. Figure 2 illustrates the chemical structures of Pyrrolo-dC, its phosphoramidite and the respective base pairs.

Figure 2: Upper panel: Chemical structures of pyrrolo-dC and its phosphoramidite. Lower panel: Base pairing between guanine (G) and pyrrolo-deoxycytidine (pyrrolo-dC) and between G and C.

Mammalian genomes contain cytosine-guanine (CG) dinucleotide clusters, and CG dinucleotides are the target of DNA methyltransferases. After methylation, the spontaneous deamination of methylcytosine to thymine (mC to T) makes methylated cytosines unusually susceptible to mutation and consequent depletion. CpG islands are loci in which CG dinucleotides remain relatively enriched.

In a groundbreaking study, Woo et al. (1996) replaced deoxyguanosine (dG) and deoxycytidine (dC) with deoxyinosine (dI) and 3-(2'-deoxy-beta-D-ribofuranosyl) pyrrolo-[2,3-d]-pyrimidine-2-(3H)-one (dP) to synthesize modified oligodeoxyribonucleotides (ODNs). These modified ODNs demonstrated unique hybridization properties, exhibiting selective complementary binding and the ability to form stable, sequence-specific hybrids with unmodified nucleic acid strands. 

Liu and Martin (2001) site-specifically introduced fluorescent probes into DNA to map melted regions of the DNA directly in a functionally paused elongation complex. 

The pyrrolo-dC probe allows investigating of the transcription bubble in the T7 RNA polymerase elongation complexes. Pairing pyrrolo-dC with complementary guanine quenches the fluorescence of this base analog but enhances its fluorescence in single-stranded DNA. Hence, the melting or opening of DNA strands increases fluorescence intensity.

The fluorescence intensity of the pyrrole-dC probe, which substitutes for cytosine bases, is sensitive to its environment; the fluorescence is quenched in duplex DNA relative to its fluorescence in single-stranded DNA. This probe provides direct information on the local melting of the DNA. Placement of this new probe at specific positions in the non-template strand shows that the elongation bubble extends about eight bases upstream of the pause site.

Building on Woo et al.’s results, Berry et al. (2004) incorporated pyrrolo-dC into oligodeoxyribonucleotides using standard automated oligonucleotide synthesis, showcasing its potential as a fluorescent analog of deoxycytidine.

Dash et al. (2004) made a significant breakthrough by demonstrating the use of pyrrolo-dC in an oligonucleotide-based probe to study RNA/DNA hybrids containing the human immunodeficiency virus type-1 3′-polypurine tract. This novel application of pyrrolo-dC showcases its versatility and potential in various research areas. Additionally, Zang et al. (2005) utilized pyrrolo-dC incorporated into oligonucleotides to study base flipping by human O6-alkylguanine DNA alkyltransferase (AGT) involved in DNA repair, further expanding the scope of its applications.

Zhang and Wadkins (2009) investigated the structural effects of pyrrolo-dC incorporated in DNA hairpins. The researchers also studied how pyrrolo-dC influenced the binding of the hairpins by a fluorescent analog of the drug Actinomycin D. The study revealed that there is very little perturbation of the DNA structure upon incorporation of pyrrolo-dC and that the analog allows studying DNA secondary structures. Further, this study extended the use of pyrrolo-dC for analyzing DNA secondary structures, such as DNA hairpins. Using pyrrolo-dC in oligonucleotides enables the differentiation of paired and unpaired bases in secondary structures. Similarly, this analog is expected to enable studies of related secondary structures such as cruciforms and quadruplexes.  Further, the study revealed that pyrrolo-dC can act as a fluorescence resonance energy transfer donor for the fluorescent drug 7-aminoactinomycin D.

Li et al. (2010) developed signal-on flurescent aptasensor based on unlabeled aptemers and pyrrolo-dC for selective and sensitive target detection. 

Ming and Seela (2012) reported the synthesis of pyrrolo-dC click adducts useful for SNP detection in long DNA targets.

Also in 2012, Ming et al. reported the synthesis of oligonucleotides containing a G-clamp or a pyrrolo-dC. This study revealed that a pyrrolo-dC derivative behaved like dC but the a G-clamp formed a more stable base pair with 2′-deoxyisoguanosine in DNA with parallel chain orientation than with 2′-deoxyguanosine in aps DNA.  

Noe et al. (2012) described the synthesis and photophysical properties of four fluorescent nucleoside analogs, related to pyrrolo-C (PyC) and pyrrolo-dC (PydC) through the conjugation or fusion of a thiophene moiety. The research group reported the results of their photophysical analysis of the nucleosides compared to PyC.

Tor Y. (2024) reported that isomorphic emissive nucleosides and nucleotides, particularly purine analogs, can serve as substrates for diverse enzymes. For example, emissive analogs interacting with metabolic and catabolic enzymes can open optical windows into the biochemistry of nucleosides and nucleotides as metabolites, coenzymes, and second messengers. Real-time fluorescence-based assays for adenosine deaminase, guanine deaminase, and cytidine deaminase have been fabricated and used for inhibitor discovery. The synthesis of emissive cofactors, coenzymes, and second messengers, together with xyNTPs and native enzymes, allows for fluorescence-based monitoring of biotransformation in real time.

Reference 

Berry, D. A.; Jung, K.; Wise, D. S.; Sercel, A. D.; Pearson, W. H.; Mackie, H.; Randolf, J. B.; Somers, R. L. Tetrahedron Lett 2004, 45, 2457-2461. Tetrahedron Letters

Dash C., Rausch J.W., Le Grice S.F. Using pyrrolo-deoxycytidine to probe RNA/DNA hybrids containing the human immunodeficiency virus type-1 3′ polypurine tract. Nucleic Acids Res. 2004; 32:1539-1547. [PMC]

Fluorescent Base Analogs: https://www.biosyn.com/tew/Fluorescent-Base-Oligo-Modification.aspx

Glass JL, Thompson RF, Khulan B, Figueroa ME, Olivier EN, Oakley EJ, Van Zant G, Bouhassira EE, Melnick A, Golden A, Fazzari MJ, Greally JM. CG dinucleotide clustering is a species-specific property of the genome. Nucleic Acids Res. 2007;35(20):6798-807. [PMC]

Li, T., Fu, R., Park, H.G. Pyrrolo-dC based fluorescent aptasensors for the molecular recognition of targets. Chem. Commun. (2010), 46: 3271-3273. - Pyrrolo-dC aptamer. [ChemCom]

Liu C, Martin CT. Fluorescence characterization of the transcription bubble in elongation complexes of T7 RNA polymerase. J Mol Biol. 2001 May 4;308(3):465-75. [PubMed]

Ming, X.; Seela, F. A nucleobase-discriminating pyrrolo-dC click adduct designed for DNA fluorescence mismatch sensing. Chem. Eur. J. 2012, 18, 9590–9600. [PubMed]

Ming, X.; Ding, P.; Leonard, P.; Budow, S.; Seela, F. Parallel-stranded DNA: Enhancing duplex stability by the ‘G-clamp ’ and a pyrrolo-dC derivative. Org. Biomol. Chem. 2012, 10, 1861–1869. [RSC]

Noé MS, Ríos AC, Tor Y. Design, synthesis, and spectroscopic properties of extended and fused pyrrolo-dC and pyrrolo-C analogs. Org Lett. 2012 Jun 15;14(12):3150-3. [PMC]

Preetham, Habbanakuppe D., Sharath Kumar, Kothanahally S., Kandaswamy, Aravind, Rangappa, Shobith, Gatasheh, Mansour K., Muddegowda, Umashankara, Rangappa, Kanchugarakoppal S.; Alternative Approach to Access 5-Hydroxy-1H-pyrrol-2-(5H)-ones from Base-Induced Tandem Intramolecular Cyclization of Sulfur Ylide with Ketones and 1,3-Hydroxy Rearrangement. ACS Omega 2023, 8, 50, 48251-48257. American Chemical Society. [PMC]

Seela F, Peng X. Synthesis and properties of 7-substituted 7-deazapurine (pyrrolo[2,3-d]pyrimidine) 2'-deoxyribonucleosides. Curr Protoc Nucleic Acid Chem. 2005 Jul;Chapter 1:Unit 1.10. [PubMed]

Seela, F.; Peng, X.; Budow, S. Advances in the synthesis of 7-deazapurine-pyrrolo[2,3-d]pyrimidine-2 ′-deoxyribonucleosides including D- and L-enantiomers, fluoro derivatives and 2′,3′-dideoxyribonucleosides. Curr. Org. Chem. 2007, 11, 427–462. [ingenta]

Seela F, Sirivolu VR. Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple bonds: synthesis, base pairing and fluorescent dye conjugates prepared by the azide-alkyne "click" reaction. Org Biomol Chem. 2008 May 7;6(9):1674-87. [RSC]

Tessmer I, Fried MG. Insight into the cooperative DNA binding of the O⁶-alkylguanine DNA alkyltransferase. DNA Repair (Amst). 2014 Aug;20:14-22. [PMC]

Tor Y. Isomorphic Fluorescent Nucleosides. Acc Chem Res. 2024 May 7;57(9):1325-1335. [PMC]

Woo J, Meyer RB Jr, Gamper HB. G/C-modified oligodeoxynucleotides with selective complementarity: synthesis and hybridization properties. Nucleic Acids Res. 1996 Jul 1;24(13):2470-5. [PMC]

Yamada K, Tsunoda H, Ohkubo A, Seio K, Sekine M. Synthesis and hybridization properties of oligonucleotides having 4-N-(pyrrol-2-ylcarbonyl)deoxycytidine. Nucleic Acids Symp Ser (Oxf). 2009;(53):115-6. [PubMed]

Zang H, Fang Q, Pegg AE, Guengerich FP. Kinetic analysis of steps in the repair of damaged DNA by human O6-alkylguanine-DNA alkyltransferase. J Biol Chem. 2005 Sep 2;280(35):30873-81. [JBC]

Zhang, X., Wadkins, R.M. DNA hairpins containing the cytidine analog pyrrolo-dC: structural, thermodynamic, and spectroscopic studies. Biophys. J. (2009), 96: 21884-1891. [PMC]

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What is a G-clamp?

The G-clamp, a unique molecular structure or motif, is specifically designed to interact with guanine-rich sequences in DNA or RNA oligonucleotides. It is a tricyclic analog of cytosine, the 9-(2-aminoethoxy)-phenoxazine analog known as the G-clamp. However, the 9-(3-aminopropyl)-phenoxazine analog is known as the propyl-G-clamp. The phenoxazine-derived tricyclic G-clamp strongly binds to guanine (G) through additional interactions, including π-stacking, the electrostatic attraction of a positively charged amine, and hydrogen bonding at the Hoogsteen face.

G-clamps, with their ability to selectively bind to guanine bases, find applications in various research and therapeutic settings. They are instrumental in the design of molecular probes, drugs, and other biotechnological tools. Their value in biotechnology lies in their ability to enhance the performance of oligonucleotides in a wide range of applications.

In the context of modified oligonucleotides, a "G-clamp" refers to a modified nucleic acid analog designed to increase the binding affinity and specificity for sequences containing guanine (G) bases. The "G" in G-clamp refers to guanine, one of the four nucleotides that make up DNA and RNA. The G-clamp modification enhances the stability of the hybridization between an oligonucleotide and its complementary DNA or RNA strand at sites where the guanine base is present.

In specifically designed nucleic acid analogs, a "G-clamp" modifies the affinity or specificity for guanine bases, thereby "clamping" onto guanine-containing sequences more effectively. In the antisense paradigm of drug discovery, G-clamps play a crucial role. Chemically modified oligonucleotides (ONs) are used as therapeutic agents, and G-clamps, with their improved binding to RNA sequences and stability to nucleases, contribute to their effectiveness. The identification of more potent ONs, facilitated by G-clamps, allows for the development of novel antisense-based human therapeutics that can be effective at lower doses.

G-clamps exhibit improved selective binding to guanosine (G) via (1) improved stacking interactions of the extended aromatic ring system, (2) formation of a fourth Hoogsteen-type hydrogen bond (H-bond) between a tethered amino group and O(6) of G. The G-clamp approach typically works by introducing an additional interaction, such as a hydrogen bond or a stacking interaction, with the guanine base strengthening the overall binding and making the oligonucleotide more effective in its role, whether in diagnostics, therapeutics, or research applications.
 

Key Applications of G-clamps are:

Antisense Oligonucleotides 

In therapeutic settings, G-clamp modifications can be used to improve the efficacy of antisense oligonucleotides by increasing their binding affinity to target RNA sequences, leading to more effective gene silencing.

Probes and Primers

G-clamp modifications can enhance the sensitivity and specificity of molecular probes and primers used in techniques like PCR, qPCR, and in situ hybridization.

Gene Editing

In some gene editing technologies, G-clamp-modified oligonucleotides can help increase the precision of targeting specific DNA sequences, improving the efficiency of the editing process.
 

A brief history of G-Clamping

Lin & Matteucci (1998) reported a tricyclic aminoethyl-phenoxazine deoxycytidine, the so-called “G-clamp,” which tightly binds to a guanine (G) base through the formation of four hydrogen bonds in addition to a solid π-stacking effect of the tricyclic phenoxazine ring (see Figure 1).

Figure 1: Chemical structures of cytosine, 5-methyl-cytosine, the cytosine G-clamp analog reported by Lin & Matteucci and the model of G-clamp-guanine interactions.

Rajeev et al. (2002) reported the synthesis of the tricyclic cytosine analogs phenoxazine, 9-(2-amino-ethoxy)-phenoxazine (G-clamp), and 9-(3-aminopropoxy)-phenoxazine (propyl-G-clamp) and their incorporation into PNA oligomers (Figure 2) using Boc-chemistry based solid-phase synthesis. PNAs with a single G-clamp modification showed a significantly enhanced affinity toward RNA and DNA targets compared to unmodified PNA with mismatch discrimination.

Figure 2: Chemical structure of a G-clamp PNA (Rajeev et al. 2002).

Mair et al. (2002) investigate the nuclease resistance of model oligomers containing a G-clamp utilizing a snake venom phosphodiesterase-based in vitro assay as the hydrolytic enzyme. The study revealed that a single incorporation at the 3'-terminus completely protected the oligonucleotides from degradation.

Wilds et el. (2003) developed an oligonucleotide analog containing a heterocycle, the guanidinium G-clamp, designed to form five H-bonds to guanosine (see Figure 3). The research group introduced the guanidinium group post-synthetically by treatment of the deprotected oligonucleotide containing a free amino group with a solution of 1H-pyrazole-1-carboxamidine. The result was that a single incorporation of this modification into an oligodeoxynucleotide sequence increased duplex stability by 13º and 16º per modification in RNA and DNA oligonucleotides.

Figure 3: H-Bonding scheme of a) G-clamp and b) guanidinium G-clamp to guanosine (Wilds et el. 2003). The introduction of G-clamp into PNA significantly improved the thermodynamic stability of the PNA/DNA duplex.

Figure 4: Structure of a DNA duplex with a single guanidinium G-clamp. (Left) PDB 1KGK: Cytosine Analog that Forms Five Hydrogen Bonds to Guanosine: the Guanidino G-Clamp. (Wilds et al. 2003). (Right) DNA duplex [d(GC*GTATMOEACGC)]₂ with single guanidinium G-clamp (C*) and 2’-O-methoxyethylthymine (TMOE) modifications per strand.

Holmes et al. (2003) reported the synthesis of a 2′-O-methyl (OMe) riboside phosphoramidite derivative of the G-clamp tricyclic base and its incorporation into a series of small steric-blocking OMe oligonucleotides targeting the apical stem-loop region of human immunodeficiency virus type 1 (HIV-1) trans- activation-responsive (TAR) RNA. This study showed that an OMe G-clamp has RNA-binding enhancement abilities. The effectiveness of the oligonucleotide steric block inhibition of Tat-dependent trans-activation both in vitro and in cells is governed by factors more complex than RNA-binding strength alone. This complexity presents a fascinating challenge for further research in the field of nucleic acid chemistry and molecular biology.

Lopez-Tena et al. (2003) reported pseudo-complementary analogs of the G:C base pair leveraged on steric and electrostatic repulsion between the cationic phenoxazine analog of cytosine (G-clamp, C+) and N-7 methyl guanine (G+), both cationic. The researchers found that while complementary peptide nucleic acids (PNA) form a more stable homoduplex than the PNA:DNA heteroduplex, oligomers based on pseudo-C:G complementary PNA favor PNA:DNA hybridization. These properties enable dsDNA invasion at physiological salt concentration. The researchers obtained stable invasion complexes with low equivalents of PNAs (2–4 equiv).
This high yield of dsDNA invasion allowed the detection of RT-RPA amplicon using a lateral flow assay (LFA), which also allowed the discrimination of two strains of SARS-CoV-2 to single nucleotide resolution. Adding a G-clamp to a PNA resulted in a stronger binding affinity. However, the addition of a G-clamp resulted in a significant reduction in hybridization to N7-G but a higher affinity to unmodified G, suggesting that probes can be designed to detect this natural post-transcriptional modification by competitive binding of oligomers bearing or not a G-clamp.

Wojciechowski & Hudson (2008) developed a [bis-o-(aminoethoxy)phenyl]-pyrrolo-cytosine analog (boPhpC) designed for tight binding affinities to the guanine base and sensitive fluorescent properties. These analogs exhibit a significant increase in target-binding affinities of oligonucleotides and allow the development of sensitive fluorophore oligonucleotides.

Kuhn et al. (2010) modified a γ-PNA oligomer with a guanidinium G-clamp to enhance specific targeting of double-stranded DNA (dsDNA). The researcher group designed γ-PNAs that allow strand-invasion of dsDNA of a size length ranging from 15 to 20 base pairs. The study measured binding rates for dsDNA strand invasion by a pentadecameric γ-PNA oligomer, including targeting of matching and mismatching target sequences. The study found that synthetic γ-PNA oligomers allow selective isolation of dsDNA fragments or supercoiled plasmid DNA from DNA mixtures utilizing affinity capture protocols. The potential of γ-PNA oligomers in diagnostic assays and their versatility in other tools in molecular biology, genomics, and biotechnology opens exciting new possibilities for future research. 

Also, in 2012, Ming et al. reported the synthesis of oligonucleotides containing a G-clamp or a pyrrolo-dC. This study revealed that a pyrrolo-dC derivative behaved like dC but that a G-clamp formed a more stable base pair with 2′-deoxyguanosine in DNA with parallel chain orientation than with 2′-deoxyguanosine in aps DNA.

Yamada et al. (2014) synthesized oligonucleotides with 4-N-(1H-pyrrol-2-ylcarbonyl)-deoxycytidine (dCPyc) and related derivatives. Synthetic oligodeoxynucleotides containing dCPyc hybridized with a higher affinity to DNA and RNA than the unmodified oligodeoxynucleotides. Molecular dynamic simulation studies revealed that the CPyc residue can form four hydrogen bonds with the opposite G nucleobase by keeping a more planar structure than the CInc residue where the Pyc group was replaced with a 1H-indol-2-ylcarbonyl group.

Wilds et al. (2020) also reported a "guanidino G-clamp" that can form a base pair with G using two additional hydrogen bonds.

Knizhnik et al. (2023) compared three types of such antiviral candidates: (i) locked nucleic acids (LNA), (ii) LNA–DNA gapmers, and (ii) G-clamp-containing phosphorothioates (CPSs) complementary to pseudoknot (PK) stems for their ability to unwind pseudoknot structures. The researchers found that modified antisense oligonucleotides (ONs) CPS-3 with two G-clamp insertions and total phosphorothioate backbone modifications showed only a moderate PK unfolding potential and was inferior to G54-LNA in the luciferase assays. However, the G-clamp eventually outperformed G54-LNA in cells exposed to the live virus. This result may be because, unlike LNA, CPS ONs can activate RNase H, highlighting the advantages of dual-effect antivirals.

Das et al. (2023) synthesized chlorophosphoramidate morpholino monomers containing the tricyclic cytosine analogs phenoxazine, G-clamp, and G8AE-clamp and incorporated these into 12-mer oligonucleotides. The resulting phosphorodiamidate morpholino oligomers, containing a single G-clamp, exhibited a higher affinity for complementary RNA and DNA compared to the unmodified oligomers under neutral and acidic conditions. The researchers found that duplexes of RNA and DNA with G-clamp-modified oligomers adopt a B-type helical conformation, and their binding affinities are sequence and position-dependent.

Matsubayashi et al. (2024) reported that ASOs with a G-clamp modification exhibit a high binding affinity to complementary RNA sequences. G-clamp ASOs showed efficient gene silencing effects on complementary RNAs, including MALAT1 RNA and Mapt mRNA, in vitro. This research group's in vitro studies demonstrate the promising potential of G-clamp ASOs in gene silencing, offering a positive outlook for the future of gene therapy. Also, G-clamp modifications do not alter the binding properties of proteins in cerebrospinal fluid.

López-Tena and Winssinger (2024) recently studied the impact of charges on the hybridization kinetics and thermal stability of PNA duplexes. Adding a G-clamp to a PNA oligomer results in a cationic PNA, improving the strength and stability of a PNA:DNA duplex.

Lukina et al. (2024) recently studied the recognition of 8-oxoguanosine paired with an 8-oxoG-clamp by the Human 8-oxoguanine-DNA glycosylase, the enzyme removing oxidative DNA lesions. The study showed that the oxoG-clamp fluorophore, initially proposed for the detection of oxidized purines in the DNA, impedes the removal of 8-oxoG by 8-oxoguanine-DNA-glycosylase (OGG1). OGG1 neither hydrolyzed the N-glycosidic bond in oxoguanine nor cut the sugar-phosphate backbone of the oxoG–clamp–containing DNA duplex. The oxoG-clamp residue prevents the oxoG base from protruding into the active center pocket of OGG1. Also, it hinders cleavage of abasic sites (AP: apurinic/apyrimidinic sites, the most prevalent DNA lesion sites), even if the oxidized base has already been removed. The oxoG-clamp in the complementary strand destabilizes the duplex. An OxoG-clamp presence in the opposite strand affects the thermodynamic and structural characteristics of the oligonucleotide. It changes the features of enzyme-substrate complex formation and the processing of repair enzymes and other DNA-binding proteins.

Reference

8-Oxo-G-Clamp

AP-dC-CE phosphoramidite

AP-dC = G-clamp

Das A, Ghosh A, Kundu J, Egli M, Manoharan M, Sinha S. Synthesis and Biophysical Studies of High-Affinity Morpholino Oligomers Containing G-Clamp Analogs. J Org Chem. 2023 Nov 3;88(21):15168-15175. doi: 10.1021/acs.joc.3c01658. Epub 2023 Oct 16. PMID: 37843026. [PubMed]

Efthymiou T, Gong W, Desaulniers JP. Chemical architecture and applications of nucleic acid derivatives containing 1,2,3-triazole functionalities synthesized via click chemistry. Molecules. 2012 Oct 26;17(11):12665-703. [PMC]

Flanagan WM, Wolf JJ, Olson P, Grant D, Lin KY, Wagner RW, Matteucci MD. A cytosine analog that confers enhanced potency to antisense oligonucleotides. Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3513-8. [PMC]

Guanidino G-Clamp PDB 1KGK

Haaima G., Hansen H.F., Christensen L., Dahl O., Nielsen P.E. Increased DNA binding and sequence discrimination of PNA oligomers containing 2,6-diaminopurine. Nucleic Acids Res. 1997;25:4639–4643. doi: 10.1093/nar/25.22.4639. [PMC] [PubMed]

Holmes SC, Arzumanov AA, Gait MJ. Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2'-O-methyl G-clamp ribonucleoside analogues. Nucleic Acids Res. 2003 Jun 1;31(11):2759-68. [PMC]

Knizhnik E, Chumakov S, Svetlova J, Pavlova I, Khodarovich Y, Brylev V, Severov V, Alieva R, Kozlovskaya L, Andreev D, Aralov A, Varizhuk A. Unwinding the SARS-CoV-2 Ribosomal Frameshifting Pseudoknot with LNA and G-Clamp-Modified Phosphorothioate Oligonucleotides Inhibits Viral Replication. Biomolecules. 2023 Nov 17;13(11):1660. [PMC]

Kuhn H, Sahu B, Rapireddy S, Ly DH, Frank-Kamenetskii MD. Sequence specificity at targeting double-stranded DNA with a γ-PNA oligomer modified with guanidinium G-clamp nucleobases. Artif DNA PNA XNA. 2010 Jul;1(1):45-53. [PMC]

Lin, K.Y. and Matteucci, M.D., A cytosine analogue capable of clamp-like binding to guanine in helical nucleic acids. J. Am. Chem. Soc. 1998, 120, 33, 8531–8532. [ACS]

López-Tena M, Farrera-Soler L, Barluenga S, Winssinger N. Pseudo-Complementary G:C Base Pair for Mixed Sequence dsDNA Invasion and Its Applications in Diagnostics (SARS-CoV-2 Detection). JACS Au. 2023 Feb 1;3(2):449-458. [PMC]

López-Tena M, Winssinger N. Impact of charges on the hybridization kinetics and thermal stability of PNA duplexes. Org Biomol Chem. 2024 Jul 17;22(28):5759-5767. [PMC]

Lukina MV, Zhdanova PV, Koval VV. Structural and Dynamic Features of the Recognition of 8-oxoguanosine Paired with an 8-oxoG-clamp by Human 8-oxoguanine-DNA Glycosylase. Curr Issues Mol Biol. 2024 Apr 29;46(5):4119-4132. [PMC]

Maier MA, Leeds JM, Balow G, Springer RH, Bharadwaj R, Manoharan M. Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine ("G-clamp") and origins of their nuclease resistance properties. Biochemistry. 2002 Jan 29;41(4):1323-7. [PubMed]

Matsubayashi T, Yoshioka K, Lei Mon SS, Katsuyama M, Jia C, Yamaguchi T, Hara RI, Nagata T, Nakagawa O, Obika S, Yokota T. Favorable efficacy and reduced acute neurotoxicity by antisense oligonucleotides with 2',4'-BNA/LNA with 9-(aminoethoxy)phenoxazine. Mol Ther Nucleic Acids. 2024 Mar 18;35(2):102161. [PMC]

Mikame Y, Yamayoshi A. Recent Advancements in Development and Therapeutic Applications of Genome-Targeting Triplex-Forming Oligonucleotides and Peptide Nucleic Acids. Pharmaceutics. 2023 Oct 23;15(10):2515. [PMC]

Ming, X.; Ding, P.; Leonard, P.; Budow, S.; Seela, F. Parallel-stranded DNA: Enhancing duplex stability by the ‘G-clamp ’ and a pyrrolo-dC derivative. Org. Biomol. Chem. 2012, 10, 1861–1869. [RSC]

Rajeev KG, Maier MA, Lesnik EA, Manoharan M. High-affinity peptide nucleic acid oligomers containing tricyclic cytosine analogues. Org Lett. 2002 Dec 12;4(25):4395-8. [PubMed]

Wahba AS, Esmaeili A, Damha MJ, Hudson RH. A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity. Nucleic Acids Res. 2010 Jan;38(3):1048-56. [PMC] FRET

Wilds CJ, Maier MA, Tereshko V, Manoharan M, Egli M. Direct observation of a cytosine analogue that forms five hydrogen bonds to guanosine: guanidino G-clamp. Angew Chem Int Ed Engl. 2002 Jan 4;41(1):115-7. [PubMed]

Wilds, C.J., Maier, M.A., Manoharan, M., and Egli, M., Structural Basis for Recognition of Guanosine by a Synthetic Tricyclic Cytosine Analogue: Guanidinium G-Clamp. Helvetica 2003, 86, 966-978. [pdf]

Yamada K, Masaki Y, Tsunoda H, Ohkubo A, Seio K, Sekine M. A new modified cytosine base capable of base pairing with guanine using four hydrogen bonds. Oorg Bimol Chem. 2014 Apr 14;12(14):2255-62. [RSC]

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We at Bio-Synthesis Inc. value our customers' privacy, and we are dedicated to protecting any personal data. This Policy is designed to provide you with a clear understanding of how Bio-Synthesis Inc. collects, uses, shares, transfers, and processes any data from or about our customers.

Personal Data

Any "Personal Data" refers to information directly or indirectly identifying or linked to an individual. This data type can include information such as name, address, telephone number, credit card details, email address, ID number, individual Internet Protocol ("IP") address of an electronic device, or any other identifying code. It's important to note that statistical and non-identifiable metric data, which cannot be linked to an individual, are not considered Personal Data, providing you with an added layer of security and peace of mind.

SCOPE

Bio-Synthesis's Policy underscores our unwavering commitment to privacy, detailing how we collect, process, or disclose Personal Data about our customers and how we process the data by exercising applicable legal rights. This Policy applies to online and offline information collection, including our website and any mobile applications when we provide products and services.

Our Policy also defines how Bio-Synthesis notifies customers about prospective information and products of interest and how our customers interact with us in person, by telephone, or by mail where this Policy is posted or referenced. 

There may be occasions where you have been provided with a circumstance-specific privacy notice that is separate from this Policy, such as privacy notices for specific activities like Recruitment. To the extent that a customer is provided with a different notice, those notices apply and govern our interactions with you as a customer.

When you provide Personal Data about parties other than yourself, you play a crucial role in ensuring they understand how we will process their data and, where applicable, obtain any necessary consent required in advance. Your proactive approach in this matter is highly appreciated.

We at Bio-Synthesis Inc. are committed to processing Personal Data by complying with all applicable laws. This ensures that your data is handled in a manner that respects your rights and privacy.

Please note that if you do not wish to provide your Data to us, some products or services may become unavailable to you. Your use of any or all our products indicates you are aware of our data collection, their use, data transfer, and disclosure of your information as described in this Policy to the extent permitted by applicable law. We at Bio-Synthesis Inc. are committed to processing Personal Data by complying with applicable laws, and we appreciate your understanding and cooperation in this matter. 

YOUR INFORMATION

We communicate with our customers for different reasons. Those interactions may result in Bio-Synthesis Inc. directly or indirectly gaining access to personal Data. The table below illustrates how Bio-Synthesis may collect, process, and use your Data. Our Policy also governs the legal basis for processing information, and the potential recipients of this information. Rest assured, we are committed to complying with all privacy laws to ensure your data is secure and protected. However, not everything may apply to all circumstances.

GENERAL CATEGORIES OF PERSONAL DATA COLLECTED

The table below lists categories and data sources that Bio-Synthesis may process under our Policy for the purpose and legal bases for such processing. Please note that items in this table may be shared, received, or processed by Bio-Synthesis and persons with the legal right to access Personal Data and Parties during potential business transactions.

AGGREGATED, ANONYMIZED AND DE-IDENTIFIED DATA

Bio-Synthesis Inc. may process anonymized/de-identified data. This is data for which the characteristics that can identify a customer, directly or indirectly, have been removed such that a customer is no longer identifiable, and this information is no longer considered Personal Data under data protection laws. We rely on our legitimate business interest, scientific or historical research and/or statistical purposes, consent or other purposes that may be required or allowed by law as the legal basis to anonymize Personal Data.

Bio-Synthesis may also obtain and use certain types of combined data sets such as demographic data for any purpose (“Aggregated Data”). Aggregated Data may be derived from a customers personal data but does not directly or indirectly reveal the identity. For example, we may aggregate certain information technology-related data of a customer with others’ data to calculate the percentage of users accessing a specific feature on our website. We may use Aggregated Data for any purpose without restriction. However, if we re-combine or re-connect Aggregated Data with personal data so that it can directly or indirectly identify a customer, we treat the combined data as personal data which will be used in accordance with this Policy.

COMBINING INFORMATION

Bio-Synthesis may combine collected information on the website with information we receive from a customer, by email, or by other forms of communication. We also combine information customers provide with information we obtain from third parties, service providers, publicly available sources, or related companies.

INFORMATION COLLECTED FROM CHILDREN

Our sites and applications are meant for adults. We do not knowingly collect Personal Data from children 17 years old or younger without permission from a parent or legal guardian. If you are a parent or legal guardian and think your child has given us information, you can email or write to us using the details in the ‘Contact Us’ section below.

INFORMATION STORAGE

We may transfer, process, and store information from customers to the US, Canada, India, European Union member states, the United Kingdom, or other countries. Our site and business may be subject to US laws, which may not afford the same level of protection as those in your country.

CROSS BORDER DATA TRANSFERS

We may transfer Personal Data to recipients in countries other than the country in which Personal Data was originally collected. When we transfer any Personal Data in such a manner, we take steps to protect data consistent with the laws and requirements in the specific country, including the requirements that apply to cross-border data transfers. We implement appropriate technical and organizational measures to provide a level of security appropriate to the risk of protecting Personal Data against accidental or unlawful destruction, loss, alteration, unauthorized disclosure, or access. As is the case with all websites, applications, products, and services, we unfortunately cannot guarantee the security of the data always collected.

SALE OR TRANSFER OF DATA

If we are involved in a sale or transfer of all or some of our business assets or operations via a share or asset transaction, personal data may be transferred to the acquiring organization who will be required to take at least the same or higher standards of care in the treatment of any Personal Data. Should such a sale or transfer occur, if required by law, we will inform our customers about this and may withdraw consent to or, as applicable, instigate any other legally available rights as detailed in the “Rights and Choices” section of this Policy with regards to the processing and use of your Personal Data by the transferee.

COOKIES, WEB BEACONS, AND OTHER TRACKING TOOLS

As outlined in the table above, customer’s interaction with our websites is an additional source for collecting information. Bio-Synthesis Inc. may use “cookies”, web beacons, and other technologies to help evaluate and improve the content or functions of products or services we provide. We collect information through several methods:

Web beacons, Pixels, Tags, Tracking Cookies, Marketing Cookies, Analytic Cookies, Social Media Cookies

Our Cookie Policy provides more detailed information about this topic and how we use cookies to enhance your experience and better serve customers.

THIRD PARTY LINKS AND TOOLS

We may link to other sites or applications on our platforms that we do not control. If you click on a third-party link, you will be taken to a platform we do not control. This policy does not apply to the privacy practices of that website or platform. Read other companies’ privacy policies carefully. We are not responsible for these third parties. Our site may also serve third party content that contains their own cookies or tracking technologies. We do not control the use of those technologies.

DATA RETENTION

We will retain Personal Data for as long as is necessary to carry out the purposes Personal Data was collected for or for the period prescribed by applicable laws, whichever is longer. In considering how long to retain your Personal Data the following are considered:

• The potential risk of harm if the data was subject to unauthorized use or disclosure,
• The volume and sensitivity of the Personal Data,
• Applicable legal requirements, and
• If circumstances have changed such that the purposes for which the Personal Data was collected can be achieved by other means.

When the retention of your Personal Data is no longer required, we will delete or anonymize the data according to the details provided above.

YOUR RIGHTS AND CHOICES

Some jurisdictions such have provided individuals with rights in relation to the processing of their Personal Data. These rights are not available to everyone, and they do not necessarily apply in all contexts.

Depending on the applicable law or the legal basis, you may have the right to:

• Object to the processing of your Personal Data
• Request access to your Personal Data.
• Request correction of your Personal Data should your Personal Data be inaccurate, incomplete, or obsolete.
• Request erasure/deletion of your Personal Data.
• Withdraw your consent to future processing where we processed Personal Data on the basis of your consent.
• Request restrictions on the processing of your Personal Data, including restricting the sale of or sharing of your Personal Data.
• Request the transfer of your Personal Data to yourself or a third party.
• Opt-out of certain transfers to third parties.

To exercise a right that you believe you may be entitled to under applicable law you can contact us directly by submitting a request through info@biosyn.com. We may need to verify your identity before we fulfil your request or, under applicable law, we may be unable to action your submission. We shall notify you in a timely manner of such decisions or requirements as necessary.

Filing a Complaint. If you are not able to resolve a problem directly with us and wish to make a formal complaint, you can contact your local data protection authority or other enforcement authority.

CONTACT US

If you have any questions about this Policy or our data practices, you can write to us at:

Bio-Synthesis Inc.

800 Mario Court

Lewisville, Texas 75057

Alternatively, you can email us directly at info@biosyn.com.

POLICY UPDATES

From time to time, we may change our privacy policies. The most updated copy will be found on our website. Please check our site periodically for updates.

An Artificially Expanded Genetic Information System (AEGIS) is a transformative concept in biotechnology and genetic engineering. It involves creating or modifying genetic systems to expand their capabilities beyond natural limits, offering hope for revolutionary advancements. Modifications may include [1] the additon of new genes, [2] designing novel genetic pathways, or [3] engineering organisms to produce new types of proteins or metabolites.

Applications for the AEGIS system are:

[1] Synthetic Biology: AEGIS in synthetic biology promises enhancements in the design and construction of new biological building blocks. Also, novel devices and systems, such as redesigning existing natural biological systems for other purposes, will be possible.

[2] Genetic Engineering: Genetic engineering aims to enhance or modify the genetic makeup of organisms to improve their performance, yields, or capabilities. An example is a genetically modified crop with new traits like drought resistance.

[3] Biotechnology Applications: The development of organisms with new or improved functionalities enabling applications in medicine, agriculture, or industrial processes.

The idea behind AEGIS is to push the boundaries of what biological systems can do by expanding their genetic information and capabilities artificially. This can lead to innovative solutions in various fields, but it also underscores the importance of considering safety, ethics, and environmental impact, urging us to be responsible and mindful in our pursuits.
The Benner group developed the artificially expanded genetic information system (AEGIS). Like a DNA polymer, the AEGIS system utilizes a biopolymer with twelve (12) building blocks. AEGIS allows the attachment of functional groups for the development of enhanced artificial polymer systems with binding and catalytic functions.

In AEGIS, nucleotides utilize Watson–Crick pairs by combining hydrogen bond donor and acceptor groups to form additional orthogonal nucleobase pairs. These newly developed nucleic acids resemble natural nucleotides in size, shape, and pairing geometries. However, they are independently replicable and do not interfere with DNA double helix structures.

The heterocycles used in the AEGIS system are instrumental in the creation of novel base pairs. They rearrange hydrogen donors and acceptors of standard bases to implement additional hydrogen bonding patterns, as demonstrated by the Z:P and S:B base pairs (Figure 2). These novel base pairs are orthogonal to C:G and A:T base pairs, a significant achievement of the AEGIS system.

Recently, Benner’s research group solved the structure of the E. coli multi-subunit cellular RNA polymerase (RNAP) recognizing unnatural nucleobases in a six-letter expanded genetic system (Figure 4).

This structure validates the design philosophy for AEGIS unnatural base pairs. The solved structure highlighted the importance of Watson-Crick complementarity needed for the design of AEGIS base pair recognition.

Selected References

Benner SA, Hutter D, Sismour AM. Synthetic biology with artificially expanded genetic information systems. From personalized medicine to extraterrestrial life. Nucleic Acids Res Suppl. 2003;(3):125-6. [PubMed]

Benner, S.A. (2004). Chemistry. Redesigning genetics. Science 306, 625–626.

Benner SA, Ricardo A, Carrigan MA. Is there a common chemical model for life in the universe? Curr Opin Chem Biol. 2004 Dec;8(6):672-89. [PubMed]

Benner, S.A., Kim, H.-J., Merritt, K.B., Yang, Z., McLendon, D.C., Hoshika, S., and Hutter, D. (2015). Next-generation DNA in pathogen detection, surveillance, and CLIA-waivable diagnostics. SPIE Digital Library 9490, 94900K-94900K – 6.

Biondi, E., Lane, J.D., Das, D., Dasgupta, S., Piccirilli, J.A., Hoshika, S., Bradley, K.M., Krantz, B.A., and Benner, S.A. (2016). Laboratory evolution of artificially expanded DNA gives redesignable aptamers that target the toxic form of anthrax protective antigen. Nucleic Acids Res. 44, 9565–9577. [PMC]

Biondi E, Benner SA. Artificially Expanded Genetic Information Systems for New Aptamer Technologies. Biomedicines. 2018 May 9;6(2):53. [PMC]

Karalkar N.B., Hoshika S., Laos R., Shaw R.W., Matsuura M., Fajardo D., Moussatche P. Alternative watson-crick synthetic genetic systems. Cold Spring Harbor Perspect. Biol. 2016;8 doi: 10.1101/cshperspect.a023770. [PMC] 

Oh J, Shan Z, Hoshika S, Xu J, Chong J, Benner SA, Lyumkis D, Wang D. A unified Watson-Crick geometry drives transcription of six-letter expanded DNA alphabets by E. coli RNA polymerase. Nat Commun. 2023 Dec 12;14(1):8219. [PMC, pdb/8SY7]

Richards NGJ, Georgiadis MM. Toward an Expanded Genome: Structural and Computational Characterization of an Artificially Expanded Genetic Information System. Acc Chem Res. 2017 Jun 20;50(6):1375-1382. [PMC]

Sismour AM, Benner SA. Synthetic biology. Expert Opin Biol Ther. 2005 Nov;5(11):1409-14. [PubMed]

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 " Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including artificial nucleic acids, 5'-triphosphate and back-bone modifications, conjugation to fatty acids, biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides".

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First reported in humans in Africa in 1970 the disease can be transmitted to humans through direct contact with infected animals, such as rodents or primates, or through contact with bodily fluids, lesions, or contaminated materials. It can also spread from person to person through respiratory droplets or contact with contaminated materials.

Symptoms of monkeypox include fever, rash, and swollen lymph nodes. The rash often starts as flat, red spots (macules) on the face, trunk, and limbs, which then develop into raised bumps (papules), filled with clear fluid (vesicles), and then pus (pustules) before scabbing over. The illness usually lasts between 2 to 4 weeks.

Considered less severe than smallpox, but monkeypox it can still be serious, especially in young children, pregnant women, and individuals with weakened immune systems. Vaccination against smallpox has been shown to provide some protection against monkeypox, and there is also a specific vaccine for monkeypox that has been developed.

Monkeypox PCR Primers

Monkeypox PCR primers allow detection of the monkeypox virus. Specifically designed primers match sequences unique to the monkeypox virus genome.

These primers allow:

Detection and Diagnosis: PCR primers amplify viral DNA in patient samples and help confirm the presence of monkeypox infection.

Research: They are used in research settings to study the virus, track its spread, or evaluate the effectiveness of treatments and vaccines.

Surveillance: These primers help monitor outbreaks and understand epidemiological patterns.

Table 1: Primer Sets according to the CDC.