In the context of DNA and RNA sequencing, sequence terminators play a crucial role as specific molecules that indicate the end of a of oligonucleotide sequence. For instance, in Sanger sequencing, terminator bases are instrumental in halting the DNA polymerization process at specific points, thereby enabling the determination of the DNA sequence. However, next-generation sequence terminators are chemical or biological molecules used in modern next-generation sequencing (NGS) technologies. NGS, also known as high-throughput sequencing, allows for the rapid sequencing of genomes or targeted genomic regions of interest.

Chemistry of Terminators in NGS

The first-generation sequencing (FGS) which included Maxam Gilbert methods and Sanger sequencing opened a new door into diagnostics of genetic diseases since 1977. In the traditional Sanger sequencing, chain terminators, known as dideoxynucleotides (ddNTPs), were the norm. These terminators, lacking a hydroxyl group on the 3'-carbon of the sugar molecule, when incorporated into oligonucleotides during DNA synthesis, terminate polymerization. The advent of next-generation sequencing technologies has seen a significant evolution of this concept in several ways. General structures of deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs) are shown below as well as the DNA elongation and termination reactions catalyzed by DNA polymerase.

Reversible Terminators

Next generation sequencing (NGS) utilizes sequencing by synthesis (SGS). Older versions called second sequencing (SGS) are [1] Roche/454 sequencing, [2] Ion torrent; proton/PGM sequencing; [3] Illumina (Solexa) sequencing, and [4] SOLID sequencing. Modern NGS platforms often use reversible terminators. Reversible terminators are nucleotide analogs that, like ddNTPs, stop DNA synthesis at each step but can be chemically removed to allow the addition of the next nucleotide. Since these analogs allow reversion of the terminating reactions, they are called "reversible terminators." Illumina's sequencing-by-synthesis (SBS) platform uses this approach.

Third-generation sequencing (TGS) methods utilize single molecular and real-time sequencing technologies. For example, the Pacific Biosciences platform uses a single-molecule real-time technology. Since a closed and circular ssDNA template can be replicated automatically during DNA library preparation, no PCR is required.During sequencing, fluorescence signals are activated by a laser as soon as a labeled dNTPs is incorporated into DNA. The color and duration of the emitted light is recorded by a camera system in real time in the flow cell equipped with zero mode waveguides. Because the time of the base incorporation is longer as the base is modified, the time called “interpulse duration” indicates the DNA modification event. However, nanopore sequencing utilizes a nanopore inserted in an electrical resistant membrane. The potential applied across the membrane results in a current flowing only through nanopore. During sequencing, a characteristic disruption in the electrical current is measured as a nucleotide passes through the nanopore, identifying the nucleotide.

Specifics of NGS Technologies

Illumina Sequencing as an Example

The Illumina sequencing platform uses reversible terminator chemistry. In this approach, multiple single template DNA fragments are attached to a flat surface, and nucleotides are added one by one during sequencing by synthesis. Each incorporated nucleotide is tagged with a fluorescent reversible terminator deoxyribonucleotide. After the incorporation of a nucleotide, the dye is read or imaged to determine the base, and chemical removal of the terminator allows the next sequencing cycle to proceed.

(For more information see https://www.biocompare.com/Editorial-Articles/590720-Guide-to-NGS-Platforms/)

Advantages of Reversible Terminators

Higher Accuracy

Reversible terminators help achieve high accuracy in sequencing by allowing precise control over the sequencing process and reducing errors.

Longer Reads

Although shorter than PacBio or Oxford Nanopore technologies, NGS technologies using reversible terminators can achieve reasonably long read lengths compared to older methods, making next-generation sequence terminators crucial components of modern sequencing technologies, allowing for rapid and accurate sequencing of large amounts of DNA. [Read length information:  https://support.illumina.com/ko-kr/bulletins/2020/04/maximum-read-length-for-illumina-sequencing-platforms.html]

The reversible termination sequencing-by-synthesis approach amplifies the sequence of a template by stepwise primer elongation. It is known as a second-generation-sequencing technology on the Illumina platform. The general reversible termination sequencing process involves

(i) immobilizing the sequencing templates and primers on a solid support;

(ii) primer extension by one base plus termination;

(iii) recognizing the color of the fluorophore carried by the extended base to identify the incorporated nucleotide after washing away the unincorporated nucleotides;

(iv) removal of the fluorescent tag and the 3′-O blocking group;

(v) washing again and repeating the former steps

(ii–iv). The whole process can be summarized as extension–termination–cleavage–extension cycle.

Structure schematics of irreversible and reversible terminators utilized in sequencing technologies.

Reversible terminators have their advantages and disadvantages.

The 3’-reversible blocking group of the 3’-O-blocked reversible terminator should result in a better termination effect. However, the 3-unblocked reversible terminator is more easily accepted by DNA polymerases due to the missing modified moiety on the 3’-OH. Polymerases discriminate between ribonucleoside triphosphates and 2’-deoxyribonucleotide triphosphates by inspecting the 2’- and 3—positions more closely. The missing oxygen atom at the 3’-position will prevent DNA polymerases from further catalytic elongation of additional nucleosides.

Commercially available reversible terminators with a blocking group at 3’-OH.

All three reversible terminators shown below showed a good performance in their reversible termination function, nearly achieving 100% of 3’-O blocking efficiency and fluorescent label group cleavage after primer extension termination.

Phosphoramidites of chain terminators allows their use in automated solid phase oligonucleotide synthesis.

Unblocked reversible terminators

Reference

Oligo Chain Terminator and Modifications

Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, Boutell JM, Bryant J, Carter RJ, Keira Cheetham R, Cox AJ, Ellis DJ, Flatbush MR, Gormley NA, Humphray SJ, Irving LJ, Karbelashvili MS, Kirk SM, Li H, Liu X, Maisinger KS, Murray LJ, Obradovic B, Ost T, Parkinson ML, Pratt MR, Rasolonjatovo IM, Reed MT, Rigatti R, Rodighiero C, Ross MT, Sabot A, Sankar SV, Scally A, Schroth GP, Smith ME, Smith VP, Spiridou A, Torrance PE, Tzonev SS, Vermaas EH, Walter K, Wu X, Zhang L, Alam MD, Anastasi C, Aniebo IC, Bailey DM, Bancarz IR, Banerjee S, Barbour SG, Baybayan PA, Benoit VA, Benson KF, Bevis C, Black PJ, Boodhun A, Brennan JS, Bridgham JA, Brown RC, Brown AA, Buermann DH, Bundu AA, Burrows JC, Carter NP, Castillo N, Chiara E Catenazzi M, Chang S, Neil Cooley R, Crake NR, Dada OO, Diakoumakos KD, Dominguez-Fernandez B, Earnshaw DJ, Egbujor UC, Elmore DW, Etchin SS, Ewan MR, Fedurco M, Fraser LJ, Fuentes Fajardo KV, Scott Furey W, George D, Gietzen KJ, Goddard CP, Golda GS, Granieri PA, Green DE, Gustafson DL, Hansen NF, Harnish K, Haudenschild CD, Heyer NI, Hims MM, Ho JT, Horgan AM, Hoschler K, Hurwitz S, Ivanov DV, Johnson MQ, James T, Huw Jones TA, Kang GD, Kerelska TH, Kersey AD, Khrebtukova I, Kindwall AP, Kingsbury Z, Kokko-Gonzales PI, Kumar A, Laurent MA, Lawley CT, Lee SE, Lee X, Liao AK, Loch JA, Lok M, Luo S, Mammen RM, Martin JW, McCauley PG, McNitt P, Mehta P, Moon KW, Mullens JW, Newington T, Ning Z, Ling Ng B, Novo SM, O'Neill MJ, Osborne MA, Osnowski A, Ostadan O, Paraschos LL, Pickering L, Pike AC, Pike AC, Chris Pinkard D, Pliskin DP, Podhasky J, Quijano VJ, Raczy C, Rae VH, Rawlings SR, Chiva Rodriguez A, Roe PM, Rogers J, Rogert Bacigalupo MC, Romanov N, Romieu A, Roth RK, Rourke NJ, Ruediger ST, Rusman E, Sanches-Kuiper RM, Schenker MR, Seoane JM, Shaw RJ, Shiver MK, Short SW, Sizto NL, Sluis JP, Smith MA, Ernest Sohna Sohna J, Spence EJ, Stevens K, Sutton N, Szajkowski L, Tregidgo CL, Turcatti G, Vandevondele S, Verhovsky Y, Virk SM, Wakelin S, Walcott GC, Wang J, Worsley GJ, Yan J, Yau L, Zuerlein M, Rogers J, Mullikin JC, Hurles ME, McCooke NJ, West JS, Oaks FL, Lundberg PL, Klenerman D, Durbin R, Smith AJ. Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 2008 Nov 6; 456 (7218):53-9. [PMC]

Bowers J., Mitchell J., Beer E., Buzby P.R., Causey M., Efcavitch J.W. Virtual terminator nucleotides for next-generation DNA sequencing. Nat Methods. 2009;6:593–595. [PMC] 

Chen F, Dong M, Ge M, Zhu L, Ren L, Liu G, Mu R. The history and advances of reversible terminators used in new generations of sequencing technology. Genomics Proteomics Bioinformatics. 2013Feb;11(1):34-40. [PMC]

Chen F., Gaucher E.A., Leal N.A., Hutter D., Havemann S.A., Govindarajan S. Reconstructed evolutionary adaptive paths give polymerases accepting reversible terminators for sequencing and SNP detection. Proc Natl Acad Sci U S A. 2010;107:1948–1953. [PMC]

Gardner A.F., Wang J., Wu W., Karouby J., Li H., Stupi B.P. Rapid incorporation kinetics and improved fidelity of a novel class of 3′-OH unblocked reversible terminators. Nucleic Acids Res. 2012;40:7404–7415. [PMC] 

Green and Sambrok. Molecular Cloning. A laboratory Manual. 4^th edition. CSH Press 2012, 736-737.

Guo J., Xu N., Li Z., Zhang S., Wu J., Kim D.H. Four-color DNA sequencing with 3′-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proc Natl Acad Sci U S A. 2008; 105:9145–9150. [PMC]

Hutter D., Kim M.J., Karalkar N., Leal N., Chen F., Guggenheim E. Labeled nucleoside triphosphates with reversibly terminating aminoalkoxyl groups. Nucleosides Nucleotides Nucleic Acids. 2010; 29:879–895. [PMC] 

Ju, J, Kim DH, Guo J, Meng Q, Li Z, Cao H, et al. DNA sequencing with non-fluorescent nucleotide reversible terminators and cleavable label modified nucleotide terminators. PCT Int Appl Publ 2008; WO2009054922.

Li Z., Bai X., Ruparel H., Kim S., Turro N.J., Ju J. A photocleavable fluorescent nucleotide for DNA sequencing and analysis. Proc Natl Acad Sci U S A. 2003;100:414–419. [PMC]

Litosh V.A., Wu W., Stupi B.P., Wang J., Morris S.E., Hersh M.N. Improved nucleotide selectivity and termination of 3′-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates. Nucleic Acids Res. 2011;39:e39. [PMC] 

Pelletier et al. 1996: 7ICG [7ICG]

Pelletier H, Sawaya MR, Wolfle W, Wilson SH, Kraut J. A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase beta. Biochemistry. 1996 Oct 1;35(39):12762-77. [ACS]

Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H., & Kraut, J. (1996) Biochemistry 35, 12742−12761.

Stupi B.P., Li H., Wang J., Wu W., Morris S.E., Litosh V.A. Stereochemistry of benzylic carbon substitution coupled with ring modification of 2-nitrobenzyl groups as key determinants for fast-cleaving reversible terminators. Angew Chem Int Ed Engl. 2012;51:1724–1727. [PMC]

van Dijk EL, Jaszczyszyn Y, Naquin D, et al. The Third Revolution in Sequencing Technology. Trends Genet 2018;34:666-81. [PubMed]

Wu J., Zhang S., Meng Q., Cao H., Li Z., Li X. 3’-O-modified nucleotides as reversible terminators for pyrosequencing. Proc Natl Acad Sci U S A. 2007;104:16462–16467. [PMC] 

Wu W., Stupi B.P., Litosh V.A., Mansouri D., Farley D., Morris S. Termination of DNA synthesis by N⁶-alkylated, not 3′-O-alkylated, photocleavable 2′-deoxyadenosine triphosphates. Nucleic Acids Res. 2007;35:6339–6349. [PMC] 

Xiao T, Zhou W. The third generation sequencing: the advanced approach to genetic diseases. Transl Pediatr. 2020 Apr;9(2):163-173. [PMC]

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Self-avoiding molecular recognition systems (SAMRS) prevent the binding or interaction of similar molecular analogs with each other.

Because of the enormous diversity of pathogens, pathogen detection methods are needed to identify multiple genetic targets simultaneously. Recent research in synthetic biology produced a variety of non-standard nucleic acid analogs thought to circumvent obstacles encountered when scaling up multiplexed PCR amplification to larger reaction sets.

In 2005, Benner& Sismour reported examples of alternative nucleobases as parts of DNA nucleobases that can be used as interchangeable building modules. The Benner group developed these alternative nucleobases further into AEGIS and SAMRS analogs.

In 2008 and 2010 Hoshika et al. introduced the “Self-Avoiding Molecular Recognition Systems” or SAMRS, which uses DNA analogs that can bind to natural DNA but not other SAMRS analogs. The SAMRS concept utilizes the DNA analogs 2-aminopurine-2'-deoxyriboside (A*), 2'-deoxy-2-thiothymidine (T*), 2'-deoxyinosine (G*), and N4-ethyl-2'-deoxycytidine. These SAMRS DNA analogs bind to their natural DNA targets but not to other SAMRS analogs. SAMRS is very useful for multiplexed polymerase chain reactions.

The SAMRS concept can be applied in supramolecular chemistry, drug delivery, and materials science.

Key Features of SAMRS are:

Selective Binding: SAMRS typically have a specific target molecule they recognize and bind to while avoiding others with similar structural features.

Self-Assembly: Also, SAMRS can self-assemble into larger structures, like nanomaterials or functional materials, without forming undesired aggregates.

Chemical Design: The molecular architecture of SAMRS is not a result of chance. It is carefully designed, using steric hindrance or specific functional groups that favor interactions with the target while minimizing interactions with similar molecules. 

Applications: SAMRS has potential uses in sensors, catalysis, drug design, and targeted delivery systems, where selective interaction is crucial for functionality and effectiveness.

Dynamic Behavior: SAMRS are not static entities. They can adapt their interactions in response to environmental changes, enhancing their specificity and efficiency.

Self-avoiding molecular recognition systems are a fascinating area of research combining principles from chemistry, biology, and materials science to create functional and selective molecular interactions.

Chemical Structures of Standard Watson Crick Pairs 

Three (3) hydrogen bonds.                                    Two hydrogen bonds.

The SAMRS Concept

SAMRS bases, indicated with a *, can base pair with standard bases in the target but not with other SAMRS bases. The non-standard nucleic acids from the Self-Avoiding Molecular Recognition Systems (SAMRS: A*, T*, G*, and C*) are structurally modified versions of the standard DNA nucleobases (A, T, G, and C) (Hoshika 2008). SAMRS nucleobases maintain the ability to base pair with standard DNA nucleobases but not with their SAMRS complement. Primers modified with SAMRS components anneal to and amplify natural DNA and RNA.

However, SAMRS:SAMRS pairs (T*:A* and C*:G*) are thermodynamically disfavored.

Sharma et al. (2014) showed that most of the undesired side products of recombinase polymerase amplification (RPA) can be avoided if primers contain components of SAMRS suggesting that SAMRS‐RPA may become a powerful tool within the range of amplification techniques available to scientists. RPA is used at low temperatures and does not force the detection system to recreate base‐pairs following Watson–Crick rules, hence it produces undesired products that can impede the amplification of desired products, complicating analysis.

Wang et al. (2020) showed that the use of water-soluble graphene oxide and SAMRS primers significantly improved the specificity of recombinase polymerase amplification (RPA) detection.

Yang et al. (2020) utilized SAMRS nucleobases for the elimination of primer dimers for improved SNP detection.

Babar et al. (2024) used computational methods for the study of modified DNA nucleobase sensing on monolayers of MoS₂ and MoSSe. The results of this study indicated that MoSSe (Se side) monolayers are a promising platform for the selective detection of DNA bases.

Kawabe et al. (2024) utilized SAMRS together with the artificially expanded genetic information system (AEGIS) and next-generation sequencing for amplicon identification to avoid primer dimer formation in specific multiplexed PCR assays. Using this approach, Kawabe et al. showed that a low-cost, portable sequencing platform allows high throughput analysis of wastewater, soil, and human stool samples.

Reference

Babar V, Sharma S, Shaikh AR, Oliva R, Chawla M, Cavallo L. Detecting Hachimoji DNA: An Eight-Building-Block Genetic System with MoS2 and Janus MoSSe Monolayers. ACS Appl Mater Interfaces. 2024 May 1;16(17):21427-21437. [PMC]

Benner SA, Sismour AM. Synthetic biology. Nat Rev Genet. 2005 Jul;6(7):533-43. [PMC]

Hoshika S.; Chen F.; Leal N. A.; Benner S. A. Self-Avoiding Molecular Recognition Systems (SAMRS). Nucleic acids symp. ser. 2008, 52 (1), 129–130. 10.1093/nass/nrn066. [PubMed]

Hoshika S, Chen F, Leal NA, Benner SA. Artificial genetic systems: self-avoiding DNA in PCR and multiplexed PCR. Angew. Chem. Int. Ed. Engl. 2010;49:5554–5557. [PMC] [PubMed]

Kawabe H, Manfio L, Pena SM, Zhou NA, Bradley KM, Chen C, McLendon C, Benner SA, Levy K, Yang Z, Marchand JA, Fuhrmeister ER. Harnessing non-standard nucleic acids for highly sensitive icosaplex (20-plex) detection of microbial threats. medRxiv [Preprint]. 2024 Sep 10:2024.09.09.24313328. [PMC]

SAMRS [Firebirdbio]

Sharma N, Hoshika S, Hutter D, Bradley KM, Benner SA. Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS) Chembiochem. 2014;15:2268–2274. doi: 10.1002/cbic.201402250. [PMC] [PubMed]

Wang Y., Jiao W. W., Wang Y., Wang Y. C., Shen C., Qi H., et al.. (2020). Graphene oxide and self-avoiding molecular recognition systems-assisted recombinase polymerase amplification coupled with lateral flow bioassay for nucleic acid detection. Mikrochim Acta 187, 667. doi:  10.1007/s00604-020-04637-5 [PubMed]

Yang Z, Le JT, Hutter D, Bradley KM, Overton BR, McLendon C, Benner SA. Eliminating primer dimers and improving SNP detection using self-avoiding molecular recognition systems. Biol Methods Protoc. 2020 Feb 10;5(1):bpaa004. [PMC]

Patent EP2321332A1: Granted 2017-03-27, active, anticipated expiration 209-08-19. [EP2321332A1]

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Modifications of nucleic acids can alter their physical properties, stability, interaction potential, and biological function. Naturally modified nucleic acids regulate vital cellular processes, including gene expression, DNA repair, and protein synthesis. Artificial modifications have become essential tools in biotechnology, drug development, and therapeutic strategies like gene editing and RNA-based therapies in recent decades.

Many of these modifications occur naturally. Presently there are 143 known modified ribonucleosides. Examples are methylation and acetylation. The artificial introduction of nucleic acid modifications can stabilize oligonucleotides and make them more resistant to nucleases.

Understanding the effect of modifications on nucleic acid properties is essential for insights into genetic regulation, biotechnology, and therapeutic applications.

Structural Changes

Nucleic acid modifications often alter the three-dimensional structure of DNA or RNA, affecting how these molecules fold and interact.

Examples are:

Methylation: Added methyl groups, such as 5-methylcytosine in DNA, can change the flexibility and conformation of the DNA helix. Methylation makes DNA less accessible to transcription factors, which silences gene expression.

Phosphorothioate Modification: A phosphorothioate is a common non-natural modification in which a sulfur atom replaces a non-bridging oxygen atom in the phosphate backbone. This modification enhances the resistance of nucleic acids against nucleases, making it useful in therapeutic oligonucleotides like antisense molecules.

Base Modifications: Modified RNA bases such as pseudouridine (Ψ) can improve base-pairing stability, influence RNA secondary structures, and enhance protein interactions.

Changes in Stability

Nucleic acid modifications can either enhance or reduce the stability of the modified molecules, affecting their lifespan and functionality:

Methylation of DNA (for example, in CpG islands): Methylation can protect DNA from degradation. Methylation is important in epigenetic regulation. Methylation at CpG sites can prevent recognition by specific proteins, such as transcription factors, while promoting binding by others, such as methyl-CpG-binding proteins.

RNA Modifications: Various modifications are available for RNA molecules, such as adding a 2'-O-methyl group, which increases their resistance to degradation by exonucleases and stabilizes the RNA structure. Modified mRNAs, like those used in mRNA vaccines, often have pseudouridine instead of uridine to increase stability and decrease immune recognition.

Modifications Affecting Base Pairing and Hybridization

Nucleic acid modifications may influence the hydrogen bonding patterns and base-pairing properties of oligonucleotides:

Base Methylation: Methylation of bases often alters Watson-Crick hydrogen bonding, potentially interfering with base pairing and affecting replication and transcription fidelity.

Modified Bases in tRNA: In transfer RNA (tRNA), modifications like inosine or queuosine at certain positions enhance the flexibility of base pairing, which is crucial for the accurate and efficient translation of the genetic code.

Bridged or Locked Nucleic Acids (BNAs, LNAs): Modifications like BNA and LNAconstrain the ribose ring via a methylene-based bridge, significantly increasing the melting temperature of DNA/RNA duplexes and improving the affinity and stability of hybridization.

Impact on Gene Expression and Regulation

Epigenetic modifications like DNA methylation and histone modifications can regulate gene expression:

Gene Silencing: DNA methylation of promoter regions, particularly in CpG islands, often leads to the repression of gene transcription. DNA methylation is a significant mechanism in gene regulation and cellular differentiation.

Histone Modifications: While not directly modifying DNA, histone modifications, usually acetylation or methylation, alter the accessibility of DNA by loosening or tightening the DNA-histone interaction, thereby modulating the transcriptional activity of nearby genes.

Effects on Enzymatic Processes

Enzymes involved in nucleic acid metabolism, such as polymerases, endonucleases, and ligases, can be sensitive to modifications:

DNA Replication and Repair: DNA modifications such as 5-methylcytosine can interfere with recognizing and repairing DNA mismatches, impacting mutation rates.

RNA Splicing and Translation: Modifications in pre-mRNA or mRNA, for example, an added 5’-cap and poly-A tail, are needed for proper splicing, transport, and translation. Additionally, mRNA modifications like N6-methyladenosine (m6A) affect splicing efficiency and translation rates, influencing gene expression.

Therapeutic and Biotechnological Applications

Chemical modifications of nucleic acids enable drug development in biotechnology and therapeutic applications:

Antisense Oligonucleotides and siRNA: Phosphorothioate backbones or 2'-O-methyl modifications increase resistance to nuclease degradation in therapeutic oligonucleotides.

CRISPR/Cas9: Modifications added to guide RNAs (gRNAs) increase their stability and efficiency in directing Cas9 to specific genomic locations.

Immune Response Modulation

Nucleic acid modifications are also critical in immune system recognition:

Avoidance of Immune Detection: Modified RNA, such as mRNA with pseudouridine, reduces innate immune activation, essential in therapeutic applications like mRNA vaccines to prevent rapid degradation and reduce inflammatory responses.

Immunostimulatory Effects: Certain modifications, like CpG oligodeoxynucleotides, are known to stimulate the immune system and are being explored as adjuvants in cancer immunotherapy.

Modifications for siRNAs, ASOs, AMOs, and gapmers. 

• Inserting mismatches into oligonucleotides will decrease a duplex's melting temperature (Tm) and prevent hybridization or polymerization.

• A higher Tm value correlates with improved binding affinity and results in a more robust duplex. More energy is required to destabilize the connection between both molecules.

• The sugar ring and the backbone are the targets for most modifications.
• The C2′ position is the selected site for modifications. The C2′ position defines the conformation of the sugar ring. Many introduced changes at this position shift the conformation of the sugar moiety from a C2′-endo (southern conformation, typical of DNA duplexes) to a C3′-endo sugar pucker (northern conformation, typical of RNA duplexes), improving the binding affinity of ASOs and AMOs for RNA complements.

• Also, in this conformation, the 2′-modification is closer to the 3′-phosphate group, conferring higher nuclease resistance to the oligonucleotide.

• Modification at the 2′-carbon of the ribose, for example, 2′-OMe, 2′-MOE and 2′-F, increase binding affinity in the following order of increased potency: 2′-OMe ≅ 2′-MOE < 2′-F).

• These substitutions can be combined to improve potency. For example, MOE/LNA, 2′-OMe/LNA, or 2′-F/MOE are examples of oligonucleotide mixmers with enhanced binding affinity compared to oligonucleotides containing only one type of substitution.

Table 1: Effects of Modifications on Melting Temperature

Reference

BNAs

Davis S, Lollo B, Freier S, et al.; Improved targeting of miRNA with antisense oligonucleotides. Nucleic Acids Res. 2006; 34:2294–304.  [PMC] [PubMed]

Egli M, Minasov G, Tereshko V, et al.. Probing the Influence of Stereoelectronic Effects on the Biophysical Properties of Oligonucleotides:  Comprehensive Analysis of the RNA Affinity, Nuclease Resistance, and Crystal Structure of Ten 2′-O-Ribonucleic Acid Modifications. Biochemistry. 2005; 44:9045–57.  [PubMed]

Freier S. The ups and downs of nucleic acid duplex stability: structure-stability studies on chemically-modified DNA:RNA duplexes. Nucleic Acids Res. 1997; 25:4429–43. [PMC] [PubMed]

Ishiguro H, Kimura M, Takeyama H. Role of microRNAs in gastric cancer. World J Gastroenterol. 2014 May 21;20(19):5694-9. [PMC]

LNAs

Lennox K a, Behlke M a. A direct comparison of anti-microRNA oligonucleotide potency. Pharm Res. 2010;27:1788–99. [PubMed]

Lennox K, R Owczarzy, Thomas DM, et al.. Improved Performance of Anti-miRNA Oligonucleotides Using a Novel Non-Nucleotide Modifier. Mol Ther Nucleic Acids. 2013;2:e117. [PMC] [PubMed]

Lima JF, Cerqueira L, Figueiredo C, Oliveira C, Azevedo NF. Anti-miRNA oligonucleotides: A comprehensive guide for design. RNA Biol. 2018 Mar 4;15(3):338-352. [PMC]

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The bioconjugation field continues to grow, making bioconjugation technics important and essential in biotechnology, molecular biology, medicine, and materials science. Bioconjugation methods enable the attachment of biomolecules such as proteins, nucleic acids, antibodies, and carbohydrates to other molecules and surfaces.

Bioconjugation is vital in biological and biomedical applications because it can modify and manipulate biological systems to produce bioconjugates for therapeutic development.

Bioconjugation Services: Bio-Synthesis Inc. offers comprehensive support for nucleic acid delivery of DNA, RNA and peptide therapeutics. Our innovative bioconjugation approach harness the specificity of targeting molecules such as antibody drug conjugates (ADC), antibody oligonucleotide conjugates (AOC), or cell-penetrating peptide conjugates (CPPs), as well as others, with the functional variety of oligonucleotides. 

Bioconjugations enables the development of powerful tools for research, diagnostics and therapeutic applications. 

Basics in Bioconjugation Chemistry: Basic facts about bioconjugation chemistry can be reviewed in “Basic Bioconjugation Chemistry of Reactive Groups in Biomolecules." Click here!

Drug Delivery: Bioconjugation enables targeted drug delivery by attaching therapeutic agents to biomolecules, such as antibodies or peptides, which can specifically bind to targets in diseased cells. For example, antibody-drug conjugates (ADCs) deliver chemotherapy directly to cancer cells, minimizing side effects.

Drug Carriers

Drug Conjugates 

Paclitaxel Fluorescein DNA conjugates

Cell Delivery

The cellular delivery of drugs refers to drugs delivered at the cellular level. Cell delivery is essential in pharmaceutical research and biotechnology. Cell delivery focuses on designing systems or vehicles that enable transporting therapeutic agents directly to specific cells or tissues in the body, enhancing efficacy while reducing side effects. There are several strategies and technologies for drug delivery to cells, depending on the nature of the drug and the target:

Cell Delivery and Uptake

Cell-Penetrating Peptides (CPPs) - Delivery of proteins into cells. 

CRISPR and Gene Editing Tools

Delivery across the Blood Brain Barrier 

Exosomes

Hydrogels and Microneedles (Hydrogels; Microneedles) 

Nanoparticles (Liposomes, Polymeric nanoparticles, Gold nanoparticles, Carbon nanotubes, Dendrimers).

Oligo Modifications for Cell Delivery and Uptake  

pH-Responsive and Stimuli-Responsive Systems

Prodrugs

Targeted Delivery (Ligand-receptor targeting; Folate receptor targeting)

Viral Vectors (Adenoviruses, Lentiviruses, Adeno-associated viruses (AAV))

Antibody Drug Conjugates (ADC), what are they? 

ADC QC

ADC Linker Design

ADCs to Treat Cancer

Antibody Modification, Labeling, and Conjugation

Diagnostics: Bioconjugation allows for the development of sensitive diagnostic tools. For example, conjugating fluorescent dyes or enzymes to antibodies is fundamental for ELISA or immunohistochemistry.

Imaging: By attaching imaging agents such as radioactive isotopes or fluorescent markers to specific biomolecules, bioconjugation enables real-time visualization of biological processes or disease states utilizing PET scans or fluorescence microscopy.

Therapeutics

Vaccines: Bioconjugation allows the creation of conjugate vaccines, where weak antigens, for example, polysaccharides, are linked to solid protein carriers. This vaccination improves the immune system's ability to recognize and respond to specific pathogens, such as in vaccines for Haemophilus influenza type b (Hib).

Gene Therapy: Conjugating DNA or RNA molecules to vectors, drugs, nanoparticles, proteins, or peptides, can improve the delivery and uptake of genetic material into target cells, potentially allowing the treatment of genetic disorders by correcting or silencing faulty genes.

Protein, Peptide and Enzyme Engineering: Site-Specific Labeling: Bioconjugation techniques enable the precise attachment of labels or functional groups to specific sites on proteins, enabling the study of protein structure, function, and interactions, which are essential for drug development and understanding disease mechanisms.

Peptide conjugation: Peptide conjugation enables the attachment of chemical moieties to peptides to enhance their properties for drug and diagnostic use. 

Peptide conjugation can result in

Improved drug properties: 

Peptide conjugation can improve circulation stability and targeting of drugs in vivo as well as reduce toxic side effects.

Improved solubility: Conjugating DNA, RNA, or drugs to peptides can increase their solubility. 

Improved cell permeability: Peptide conjugation can facilitate cell permeability. 

Extended plasma half-life: Peptide conjugation is an effective strategy for extending plasma half-life. 

Promoting oral absorption: Peptide conjugation plays a crucial role in drug development by promoting oral absorption. This practical benefit addresses a common challenge in drug delivery. 

Noval peptide constructs: Peptide conjugation allows the creation of novel chimeric molecules by conjugating peptides to nonbiological molecules such as polyethylene glycol (PEG) or biological molecules such as DNA, RNA, lipids, sugars, and proteins. 

Peptide Amino Acid Conjugates

Enzyme Immobilization: Bioconjugation can immobilize enzymes on solid supports, enhancing their stability and reusability in industrial processes such as biocatalysis to increase the efficiency of processes in industries like pharmaceuticals and food production.

Biosensor Development: Bioconjugation is critical in developing biosensors that detect specific molecules or pathogens. These devices can identify and quantify biomolecules in environmental monitoring, food safety, and medical diagnostics by linking biological recognition elements such as antibodies or aptamers to a sensor surface.

HRP Conjugates

Lanmodulin Binding peptide

Methylen Blue Labeling

Nanotechnology and Material Science: Nanoparticle Functionalization: By conjugating biomolecules to nanoparticles, researchers can create materials with unique properties for applications such as drug delivery, bioimaging, or catalysis.

Smart Biomaterials: Bioconjugation techniques help develop materials that respond to biological stimuli. These materials can change their properties in response to environmental changes like pH or temperature, making them valuable for tissue engineering, wound healing, and drug release systems.

Biomarker Discovery and Analysis: Bioconjugation is essential in proteomics and genomics for identifying and analyzing biomarkers. By tagging specific molecules, researchers can track and study the behavior of biomolecules in complex biological systems, aiding in disease diagnosis and therapeutic target identification.

Biophysical and Structural Biology Studies: Bioconjugation allows researchers to label biomolecules for structural and biophysical studies (e.g., X-ray crystallography, NMR spectroscopy, or cryo-electron microscopy), providing insights into the mechanisms of biological processes and aiding in the design of new therapeutic agents.

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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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Morpholino Types for Therapeutic Oligonucleotides

Morpholinos are synthetic molecules utilized in antisense morpholino-based oligonucleotides designed to bind to specific RNA sequences to block biological processes, such as mRNA translation into protein. Unlike regular DNA or RNA oligonucleotides, morpholinos have a modified backbone of morpholine rings linked by non-ionic phosphorodiamidate bonds. This modification makes PMOs highly stable and resistant to degradation by cellular enzymes, such as nucleases. Morpholinos have been used in model organisms such as zebrafish, Xenopus (frogs), and mice for research purposes.

In the context of oligonucleotide chemistry, morpholinos and thiomorpholinos contain backbone modification allowing the design of chimeras to modify the stability, binding affinity, and cellular uptake properties of therapeutic oligonucleotides.

Building Block Structures of DNA/RNA, Phosphorodiamidate Morpholino, and Thiomorpholino Oligonucleotides.

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Features of morpholinos

Mechanism of action: PMOs can block access to RNA molecules including mRNA by binding to specific sequences, inhibiting translation, or modifying splicing.

Advantages: PMOs are stable, do not elicit immune responses, and are highly specific, reducing off-target effects.

Applications: Morpholinos are widely used in developmental biology, gene knockdown experiments, and therapeutic approaches to silence disease-causing genes.

Limitations: However, the delivery into cells can be challenging. Hence, unique delivery methods like electroporation, microinjection, or specific carrier molecules are often required.

Types of Morpholinos

There are several types of morpholinos, each designed for specific applications.

Phosphorodiamidate morpholinos (PMOs)

Phosphorodiamidate morpholino oligomers (PMOs) are synthetic analogs of DNA or RNA, designed for use in antisense therapy. Unlike natural nucleic acids, PMOs have a modified backbone where the sugar-phosphate structure is replaced by a phosphoramidate linkage and the ribose is replaced by a morpholine ring, enhancing the molecule's stability and resistance to nucleases, making PMOs highly stable in biological environments. Phosphorodiamidate morpholinos are commonly synthesized using a linear synthetic method.

Thiomorpholinos (TMOs)

Thiomorpholinos (TMOs) are sulfur-containing analogs of morpholine, where a ribose is rplaced by a morpholine ring and one of the non-bridging oxygen atoms on the phosphor atom is replaced by sulfur. This modification often introduces differences in chemical properties, such as increased lipophilicity and different reactivity patterns, which can be useful in drug design, especially in improving membrane permeability and modifying metabolic stability.

TMOs are morpholino nucleotides with a thiophosphoramidate inter-nucleotide linkage instead of phosphorodiamidate in PMO. Standard solid phase synthesis using phosphoramidite chemistry allows the synthesis of TMOs from the 3' - to the 5’-end.

Due to this greater flexibility in synthesis, TMOs can target a broader range of RNAs, compared to PMOs, by incorporating different RNA/DNA modifications. Also, TMOs have enhanced chemical stability, making them more resistant to nucleases. The chemical stability prolongs their activity in biological systems. Compared to PMOs, the thiol modifications in TMOs can lead to stronger binding to target RNA, potentially increasing their effectiveness in gene regulations. The design of TMOs allows for more specific targeting, minimizing off-target effects and unintended interactions with non-target sequences.

TMOs allow for regulating exon skipping activities, for example, in Marfan Syndrome and Duchenne Muscular Dystrophy, and in regulating TUG 1 RNA.

TMOs perform well at lower concentrations for exon 23 skipping and exhibit excellent potency compared to PMOs and other modifications.

Compared to MOE modifications, a TMO gapmer has a higher allele selective knockdown of the "fused in sarcoma" (FUS) gene.

Standard Morpholinos

Standard morpholinos are unmodified morpholinos used primarily for research purposes. They bind to complementary RNA sequences, blocking translation or splicing by steric hindrance.

Standard morpholinos are often used in:

  Gene knockdown studies: Preventing the production of specific proteins by inhibiting mRNA translation.

  Splice-modifying studies: Blocking the splicing of pre-mRNA to study the role of certain splice variants.

Cell-penetrating peptide–morpholinos PPMOs

Cell-penetrating peptide–morpholinos are a type of morpholino oligonucleotide that are covalently conjugated with an arginine-rich peptide also known as vivo-morpholino.

Vivo-Morpholinos

Vivo-morpholinos are designed for delivery into tissues or whole organisms. Vivo-morpholino oligonucleotide contain an octa-guanidine dendrimer attached via a triazine core. Vivo-morpholinos can be peptide conjugates to enhance cell permeability and uptake.

Vivo-morpholinos are used in:

  In vivo gene knockdown: Used in animal models, these morpholino conjugates provide better uptake than standard morpholinos.

  Therapeutic applications: Vivo-morpholinos potentially allow the treatment of diseases like Duchenne Muscular Dystrophy (DMD) by altering splicing or blocking harmful RNA transcripts.

Photo-morpholinos

Photo-morpholinos contain a photo-cleavable group that inactivates the molecules until exposed to light, allowing spatial and temporal control over gene knockdown or RNA blocking. Photo-morpholinos are used in:

  Precise gene knockdown: Light activation allows researchers to control when and where gene silencing occurs in tissues or embryos.

  Developmental biology studies: Photo-morpholinos offer precise control during critical stages of embryonic development.

Lipid-conjugated Morpholinos

Lipid-conjugated morpholinos are coupled with lipophilic groups (e.g., cholesterol) to improve cellular uptake and stability. These are ideal for:

  Systemic delivery: Lipid-conjugated morpholinos are used for in vivo applications where efficient tissue penetration is needed.

  Increased stability: Lipid-conjugated morpholinos are tend to be more stable in biological fluids, enhancing their effectiveness in live organisms.

End-modified Morpholinos

End-modified Morpholinos have chemical modifications at their 3’- or 5’-ends, improving stability, binding affinity, or cellular uptake. End-modified Morpholinos are used in cases where standard morpholinos may degrade or have low efficacy.

Antisense Morpholinos

Antisense morpholinos target and block complementary RNA sequences, preventing the translation of specific genes. These are mainly used in research to:

  Study gene function.

  Validate potential therapeutic targets.

The type of morpholino is selected based on the selected application, whether it be for research, in vivo studies, or potential therapeutic development.

To investigate more, follow these links:

Custom-Morpholinos

Thiomorpholino-Oligonucleotides-or-TMOs

Thiomorpholino Oligonucleotides

Reducing-off-target-effects-of-the-sirna-therapeutics

A-Collection-of-Approved-Antisense-Therapeutic-Drugs-2024

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Bio-Synthesis provides custom morpholinos including Phosphorodiamidate Morpholino, and Thiomorpholino Oligonucleotides.

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 inter-nucleotide linkages.

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

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Peptide-Oligonucleotide conjugates (POCs) are synthetic molecules, in which a peptide is conjugated to an oligonucleotide, for example, a linear or cyclic peptide linked by a covalent bond or linker to an oligonucleotide or its analogs.

Peptide-oligonucleotide conjugates (POCs) combine peptides with oligonucleotides through a covalent bond or a linker molecule, combining the unique properties of each to enhance biological or therapeutic applications. This combination leverages the binding specificity of oligonucleotides with the diverse functionality of peptides to improve cellular targeting, cellular uptake, and stability.

Peptide   -   Linker  -  Oligonucleotide

Peptide Design, Synthesis, and Conjugation

Linear or cyclized peptides can be designed and synthesized via solid-phase peptide synthesis. Non-standard amino acids can be incorporated during synthesis or post-synthesis as desired. In general peptide range between 5 to 25 amino acids in length.

Linkers and conjugation

A variety of activated linkers is commercially available for conjugation. Alternatively, standard bioconjugation techniques allow the synthesis of simple and complex conjugates. Non-cleavable, cleavable, and self-immolating linkers allow the design of specific complex POCs.

Oligonucleotide Design and Synthesis

Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) can be designed based on target genes. Single and double-stranded RNA or DNA oligonucleotides can be used for conjugation.

Modifications

Optimized synthesis and conjugation methods allow the incorporation of base, sugar, and backbone modifications for increased stability and efficiency.

Fluorescence and Dye Labeling

In addition, selected conjugation methods, such as click chemistries, enable the addition of fluorescent or dye moieties to POCs.

Synthesis

The synthesis of POCs generally involves the chemical conjugation of a peptide to an oligonucleotide through a variety of linkers. These linkers can be stable or cleavable under certain conditions, such as pH-sensitive or enzymatically degradable linkers, to release the oligonucleotide within specific cellular compartments.

Potential use of POCs

Enhancing Targeting and Delivery: Specific peptides can target specific cell types or tissues, improving the delivery of the oligonucleotide component to desired locations. For example, cell-penetrating peptides (CPPs) like TAT or R9 are often used to shuttle oligonucleotides across cell membranes.

Improving Stability and Bioavailability

Conjugation of oligonucleotides to peptides stabilizes and protects them from nucleases in the bloodstream or cellular environments, extending the conjugate's half-life, especially beneficial for therapeutic applications.

Broader Therapeutic Applications

Various therapeutic uses, including gene silencing, gene editing, and cancer therapy, demand specific designs of POCs. POCs enable targeting particular genes or molecular targets by linking therapeutic oligonucleotides, such as antisense oligonucleotides (ASOs), siRNAs, or aptamers, with targeting peptides.

Diagnostic and Research Tools

POCs are powerful probes for molecular imaging and diagnostics. Conjugating fluorescently labeled peptides with oligonucleotides can help visualize molecular interactions, monitor gene expression, or detect specific nucleic acid sequences within cells.

Customizable Design

The synthesis of designed sequences, with controlled lengths of both the peptide and oligonucleotide components, allows customization to fit various biochemical and cellular conditions.

Reference and Links

Fàbrega C, Aviñó A, Navarro N, Jorge AF, Grijalvo S, Eritja R. Lipid and Peptide-Oligonucleotide Conjugates for Therapeutic Purposes: From Simple Hybrids to Complex Multifunctional Assemblies. Pharmaceutics. 2023 Jan 18;15(2):320. [PMC]

Klabenkova K, Fokina A, Stetsenko D. Chemistry of Peptide-Oligonucleotide Conjugates: A Review. Molecules. 2021 Sep 6;26(17):5420. [PMC]

Bioconjugation

Dna-Peptide-Conjugates

Peptide-conjugated-Antisense-Oligonucleotides-for-Exon-Skipping-Therapeutics

Therapeutic-Antisense-Oligonucleotides-(ASOs)-and-their-Modifications

How-to-label-oligonucleotides-internally

siRNA-Peptide-Conjugates

Enzyme-Bioconjugation

Backbone-Modified-Oligonucleotide-Synthesis

Effects-of-Modifications-on-Nucleic-Acid-Properties

Does-the-pH-influence-the-stability-of-double-stranded-DNA

What-are-Threofuranosyl-Nucleotides-or-TNAs

FDA-approves-first-radiopharmaceutical-peptide-drug-conjugate-somatostatin-based-octreoscan-for-imaging-1994-and-lutathera-for-therapy-2018-of-neuroendocrine-tumors

A-triplex-forming-bridged-nucleic-acid-oligonucleotide-peptide-conjugate-increases-transfection-efficiency-by-targeting-the-nucleus

Oligo-Antibody-Conjugation

Peptide-Conjugation 1

Peptide-Conjugation 2

Streptavidin-oligonucleotide-conjugation

What-is-the-difference-between-post-synthetic-labeling-and-labeling-during-synthesis

Functional-group-modification-for-conjugation

A-new-characterization-method-for-cyclic-and-stapled-peptides

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Bio-Synthesis provides custom morpholinos including Phosphorodiamidate Morpholino, and Thiomorpholino Oligonucleotides.

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 inter-nucleotide linkages.

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

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DNA-Alanyl-PNA chimeras are oligomers combining peptide nucleic acids (PNAs) and DNA oligonucleotides for the creation of new molecules with improved properties and a potential to be beneficial in DNA diagnostics. 

Roviello et al. (2010) reported the synthesis of mono-methoxy-trityl/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras. The derivatives enabled the synthesis of alanyl-PNA/DNA chimeras following the molecular beacon concept via standard phosphoramidite DNA synthesis.

Hybrid DNA-Alanyl-PNA chimeras combine a DNA oligonucleotide segment with a peptide nucleic acid (PNA) segment linked by an alanyl bridge. PNAs are oligonucleotide analogs with a pseudo peptide skeleton instead of a sugar-phosphate backbone that binds to DNA and RNA with high specificity and selectivity. 

The image below shows an example of an Alanyl-PNA/DNA/alanyl-PNA-chimera composed of alanyl amino acids carrying linked nucleobase in β-position of the chain combined with a linker and a DNA segment:

Design of a DNA-PNA chimera

Select DNA Segment: The DNA segment contains natural nucleotides, allowing it to hybridize specifically with complementary DNA or RNA strands through Watson-Crick base pairing.

Select PNA Segment: The PNA segment is a sequence of synthetic nucleic acid analogs, where the sugar-phosphate backbone is a neutral peptide-like backbone. This modification makes PNAs highly stable, resistant to enzymatic degradation, and binding firmly to complementary DNA or RNA.

Select Linker: The alanine-based linker connects the DNA and PNA segments, preserving structural integrity and flexibility. The alanyl linker provides a bridge that enables the chimera to retain both DNA-like and PNA-like properties. Other linker types maybe used for the design of similar chimeric molecules.

-> Combine and synthesize.

DNA-PNA chimeras are valuable in research because of their unique hybridization properties, which make them useful in diagnostics, gene targeting, and molecular biology studies. Their stable backbone and sequence-specific affinity can improve target specificity and resistance to nucleases, making them promising tools in therapeutic and diagnostic applications.

Synthesis: Solid phase assembly enables the synthesis of DNA/alanyl-PNA chimeras, oligomers with a mixed oligonucleotide/peptide backbone. Chimeric oligomers with a mixed oligonucleotide/peptide backbone are synthesized using DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids contained acid labile monomethoxytrityl (MMT) groups for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. The resulting Boc/acyl intermediates, during deprotection, are compatible with the standard phosphoramidite DNA synthesis strategy.

The oligomerization of nucleo-amino acids results in rigid and well-defined double strands based on a linear topology-based base pair recognition, stacking, and solvation. Base pairs stacking occurs at a distance of about 3.5 A°, resulting in an extended peptide backbone. However, introducing glycines in opposite positions in a molecular beacon makes intercalation possible.

Properties: DNA/alanyl-PNA chimeras have improved aqueous solubility and cellular uptake compared to pure PNAs and bind exclusively in the antiparallel orientation under physiological conditions. 

Biological functions: DNA/alanyl-PNA chimeras can assume biological functions, such as a primer for DNA polymerases. In addition, they can also stimulate the cleavage of target RNA through RNase H. 

Potential use: DNA/alanyl-PNA chimeras have the potential to be beneficial in DNA diagnostics. 

Reference

Roviello GN, Gröschel S, Pedone C, Diederichsen U. Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras. Amino Acids. 2010 May;38(5):1301-9. doi: 10.1007/s00726-009-0324-x. Epub 2009 Jul 24. Erratum in: Amino Acids. 2010 May;38(5):1311-2. [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, chimeric molecules, as well as base, sugar and inter-nucleotide linkages.

Bio-Synthesis provides custom morpholinos including Phosphorodiamidate Morpholino, and Thiomorpholino Oligonucleotides.
Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.

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Non-linking phosphorus backbone modifications are chemical changes made to the phosphodiester backbone of oligonucleotides to modify their stability, binding affinity, and resistance to enzymatic degradation without affecting the overall backbone linkage. These modifications introduce structural variations in the backbone phosphate groups of DNA or RNA without disrupting sequential linkages between nucleotides.

Incorporation of these modifications into the backbone of oligonucleotides enables the design of enhanced therapeutic oligonucleotides, such as antisense oligonucleotides, siRNAs, or aptamers, where increased stability and altered cellular uptake is desirable.

Kumar and Caruthers (2020) developed a series of novel oligonucleotides where one or both nonbridging oxygens in the phosphodiester backbone are replaced with an atom or molecule that introduces enhanced molecular properties beneficial for the development of therapeutic oligonucleotides with unique biological activity.

Kumar and Caruthers utilized two complementary approaches: Phosphoramidites that can act directly as synthons for the solid phase synthesis of oligonucleotide analogs. However, this approach was only sometimes feasible due to the instability of various synthons toward the reagents used during the synthesis of oligonucleotides. Therefore, the researchers selected a complementary approach to develop phosphoramidite synthons that allowed incorporation into oligonucleotides with minimum changes in the solid phase DNA synthesis protocols. This approach also enabled the introduction of functional groups for generating appropriate analogs post-synthetically. Oligonucleotides containing an alkyne group linked to phosphorus in the backbone allow attaching molecules such as amino acids and peptides.

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Phosphorothioates (PS): In a PS modification, one of the non-bridging oxygen atoms in the phosphate is replaced with sulfur. This modification retains a negative charge but increases resistance to exonucleases and endonucleases, enhancing stability in biological systems.

Methylphosphonates: In this modification, one of the non-bridging oxygen atoms on the phosphate is replaced with a methyl group. This modification is neutral, as it lacks a negative charge, making it resistant to nucleases and improving cell permeability.

Borane phosphonates:  Borane phosphonate is a chemical that involves replacing a non-bridging oxygen atom in the phosphate backbone with a borane group (BH₃).

Phosphonoacetates: Phosphonoacetates contain a phosphonate group (-PO₃²⁻) attached to an acetate moiety (-CH₂CO₂⁻) used in various chemical and biological contexts due to its unique structure and reactivity and solubility in water.

Phosphonoformates: Here the backbone is modified with a phosphonoformate group altering the properties of nucleic acids, influencing stability, charge, and biological activity of resulting oligonucleotides.

Triazoylphosphonates: In triazoylphosphonate modified oligonucleotides a triazole ring is linked to a phosphonate group. The 1,2,3-triazole ring is often synthesized via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry allowing the attachment of functional groups.

Phosphotriesters: Phosphotriesters can act as intermediates in synthetic and biological applications. The R group can be selected for specific chemical and biological properties.

Phosphoramidates: Here, an amino group replaces the non-bridging oxygen. This modification improves cellular delivery and binding characteristics, though it’s less commonly employed than other backbone modifications.

Pyridiniumboranephosphonates: In oligonucleotides modified with pyridiniumboranephosphonates a borane group (BH₃) is attached to a phosphonate moiety. These compounds are of interest in synthetic and medicinal chemistry due to their unique properties.

Reference

Kumar P, Caruthers MH. DNA Analogues Modified at the Nonlinking Positions of Phosphorus. Acc Chem Res. 2020 Oct 20;53(10):2152-2166. [Pubmed]

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Cancer is a complex tissue containing a multitude of stromal cells as well as extracellular matrix. Hence, targeting the microenvironment of a tumor or cancer cell may allow destroying the malignant tumor cells. Peptides derived from natural sources or synthetic screens exhibit high specificity, are flexible and have low antigenicity. Tumor targeting or homing peptides bind to specific sites in the vasculature allowing targeting of organs or pathological tissues. When conjugated to drugs or oligonucleotides, these peptides can increase the uptake of the conjugates by tumors with reduced accumulation at non-targeted sites.

Tumor homing or tumor targeting peptides are short chains of amino acids with usually fewer than 50 residues designed or naturally occurring to deliver molecules, nanoparticles, or drugs to specific targets in cells, tissues, or organs. These peptides are often used in research, diagnostics, and therapeutic applications because they enhance the precision of delivery, reducing the risk of 'off-target effects'-unintended interactions with non-diseased cells or tissues that can lead to side effects.

Key Characteristics of Targeting Peptides

Specificity: Targeting peptides bind selectively to receptors, proteins, or other molecular targets predominantly on specific cells or tissues. Examples include tumor-specific peptides that target cancer cells or peptides that bind to inflamed tissues.

Small Size: The compact structure of targeting peptides, which is a result of their short chain of amino acids, allows for efficient penetration into tissues and cells. This compactness enables them to navigate through biological barriers and reach their intended targets more effectively.

Customizable:Modifications of targeting peptides enhance their stability, binding affinity, or resistance to enzymatic degradation.

Types of Targeting Peptides

Tumor-Homing Peptides: Tumor-homing peptides target cancer cells by binding to markers like integrins, such as RGD peptides for αvβ3 integrins.

Cell-Penetrating Peptides (CPPs): CPPs facilitate cellular uptake of large or impermeable molecules, such as TAT peptide, derived from HIV.

Organ-Targeting Peptides: Organ-targeting peptides enable the delivery of therapeutic agents to specific organs, such as brain-targeting peptides that cross the blood-brain barrier.

Inflammation-Targeting Peptides: These peptides target inflamed or damaged tissues and are often used in autoimmune or wound-healing therapies.

Antimicrobial Peptides: Antimicrobial peptides bind to microbial membranes for targeted action against bacteria, fungi, or viruses.

Applications of Targeting Peptides

Drug Delivery: Targeting peptides attached to drugs or nanoparticles allow for precise delivery to diseased cells.

Imaging: Conjugation of targeting peptides to imaging agents for diagnostic purposes allow detection of tumors.

Therapeutics: Targeting peptides when used as therapeutic agents can block disease pathways.

Biosensing: Incorporation of targeting peptides in biosensors enable detection of specific biological markers.

Targeting peptides are a vital component of modern biomedicine. Their ability to improve efficacy and safety in various therapies, from drug delivery to imaging and therapeutics, is significant.

Targeting Peptides

Reference

Le Joncour V, Laakkonen P. Seek & Destroy, use of targeting peptides for cancer detection and drug delivery. Bioorg Med Chem. 2018 Jun 1;26(10):2797-2806. [Sciencedirect]

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As research progresses, circular RNAs (circRNAs, also known as cyclic RNAs) have the potential to revolutionize diagnostics, therapeutics, and biotechnology. Small circRNAs are non-coding RNAs characterized by their covalently closed-loop structure, which makes them resistant to exonucleases. Unlike linear RNAs, they lack free 5'- and 3'-ends. CircRNAs are generated primarily through back-splicing, where a downstream splice donor site joins with an upstream splice acceptor site within pre-mRNA. CircRNAs have emerged as critical players in cellular biology, with diverse applications and roles. Their unique properties, such as stability, abundance, and tissue-specific expression, make them intriguing molecules for both basic research and translational applications. Small Circular RNAs are typically less than 1,000 nucleotides long and are a subset of circRNAs that can vary significantly in size. Their circular nature enhances their stability in cells compared to linear RNAs.

Structures of circular or cyclic adenosines, cA₄ and cA₆

Known roles of circRNAs

miRNA sponges: Some circRNAs can bind and sequester microRNAs, preventing them from interacting with their mRNA targets.

Protein interactions: CircRNAs may interact with RNA-binding proteins, influencing their activity.

Translation: Although most circRNAs are non-coding, some small circRNAs can code for proteins under specific conditions.

Biogenesis: CircRNAs arise from precursor mRNA (pre-mRNA) during splicing, involving the canonical spliceosomal machinery, but in a non-linear arrangement.

Type III CRISPR system: Cyclic oligoadenylate (cOA) molecules are second messengers in the type III CRISPR system. cOAs activate promiscuous ancillary nucleases that indiscriminately degrade host and viral DNA/RNA. Type III CRISPR-Cas systems provide adaptive immunity against foreign mobile genetic elements through RNA-guided interference. Sequence-specific recognition of RNA targets by the type III effector complex triggers the generation of cOA second messengers that activate ancillary effector proteins, reinforcing the host immune response. Cyclic tetra-AMP (cA4) activates the ancillary nuclease Can2.

Biological Relevance

CircRNAs are involved in diverse cellular processes, expressed in a tissue- and developmental-stage-specific manner. Dysregulated circRNAs have been linked to diseases such as cancer, neurological disorders, and cardiovascular diseases.

Roles of circRNAs in Cellular Biology

MicroRNA (miRNA) Sponges: CircRNAs can act as competitive endogenous RNAs (ceRNAs), binding to miRNAs and preventing them from interacting with their mRNA targets, for example, CDR1as (ciRS-7) with multiple binding sites for miR-7, modulating miR-7's downstream effects.

Protein Interaction Scaffolds: CircRNAs can bind and modulate the activity of RNA-binding proteins (RBPs), acting as molecular sponges or scaffolds influencing processes like transcription, splicing, or protein localization.

Translation into Functional Peptides: Some circRNAs contain internal ribosome entry sites (IRESs) or N6-methyladenosine (m6A) modifications, enabling translation into proteins or peptides. These peptides can have regulatory or functional roles in cells.

Regulation of Gene Expression: CircRNAs can influence gene transcription or post-transcriptional processing by interacting with the transcriptional machinery or splicing factors.

Epigenetic Modulation: Emerging evidence suggests circRNAs play roles in modifying chromatin structure or function indirectly through miRNA and RBP interactions.

Applications of circRNAs

Biomarkers for Disease Diagnosis

CircRNAs' stability in body fluids, such as blood, saliva, and cerebrospinal fluid, makes them ideal non-invasive disease biomarkers.

In Cancer: Altered circRNA expression profiles have been linked to tumorigenesis and metastasis. circRNAs are associated with bladder cancer and hepatocellular carcinoma occurrence and development.

In Neurological Disorders: Specific circRNAs are dysregulated in diseases like Alzheimer's and Parkinson's.

In Cardiovascular Diseases: CircRNAs play roles in heart development, myocardial infarction, and atherosclerosis.

Therapeutic Targets

Targeting of circRNAs may restore normal cellular function.

miRNA Inhibition: Blocking oncogenic circRNAs can prevent them from acting as miRNA sponges.

Gene Editing: CRISPR/Cas systems enable disruption or modification of circRNAs.

Drug Delivery Vehicles: Correctly engineered circRNAs could allow the delivery of RNA-based therapeutics, such as siRNAs, antisense oligonucleotides, or ribozymes. Their circular structure provides a stable platform for extended activity.

Protein and Peptide Therapeutics: Synthetic circRNAs encoding therapeutic peptides could allow targeting diseases where protein supplementation or functional peptides are needed.

Synthetic Biology and Biotechnology: CircRNAs may allow the design of stable RNA-based systems for gene expression in synthetic biology or as molecular scaffolds in various biotechnological applications.

Challenges and Future Directions

Biogenesis Control: Engineering circRNAs for specific applications requires precise control over their formation and properties.

Delivery Systems: Efficient methods to introduce circRNAs into cells or tissues are still needed and under development.

Functional Annotation: Many circRNAs remain uncharacterized and have unknown biological relevance.

Safety and Immunogenicity: CircRNA-based therapies must avoid triggering immune responses.

Synthesis of modified circRNA

References

https://www.biosyn.com/circular-oligonucleotide.aspx

https://www.biosyn.com/tew/Synthetic-long-single-stranded-and-circular-DNA.aspx

https://www.biosyn.com/tew/what-are-circular-oligonucleotides.aspx

https://www.biosyn.com/faq/How-stable-are-circular-oligonucleotides.aspx

https://www.biosyn.com/tew/enzymatic-synthesis-of-circular-oligonucleotides.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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Tricyclo-DNA (tcDNA) is a synthetic nucleic acid analog modified with a rigid tricyclic structure in the sugar backbone. This modification enhances the properties of nucleic acids. Due to its structural modification, tcDNA has many applications in therapeutics, diagnostics, and molecular biology. TcDNA analogs are result of a search for novel oligonucleotide analogs for use in therapeutics and DNA-based diagnostics. This analog is part of a series of analogs exhibiting interesting biological properties useful for enhanced oligonucleotides. The solved crystal structure of a DNA duplex with tricyclo-DNA residues explains the increased RNA affinity of tcDNA relative to DNA. The resistance of tcDNA to nucleases can also be gleaned from the structure. The Leumann group at the University of Bern studied the effect of restricted conformational flexibility on the pairing of nuclei acids (Leumann 2001). Modeling studies suggest that the cyclopropane ring in tcDNA causes an unfavorable steric interaction at exo- and endonuclease active sites resulting in a higher protection against degradation. The incorporation of one tricyclo-thymidine residue in the center of the self-complementary dodecamer duplex (d(CGCGAATtCGCG), t = tricyclothymidine) resulted in a strongly stabilized monomolecular hairpin loop structure compared to that of the corresponding pure DNA dodecamer with a ΔTm = +20 ºC. This result indicated the effect of tcT on the (tetra)loop-stabilizing properties of this rigid nucleoside analog.

Structures of tricyclo-DNA compared with DNA

Applications

Therapeutics Antisense Oligonucleotides (ASOs): ASOs can use tcDNA in various ASO-based treatments for the downregulation of gene expression by binding to mRNA and preventing translation, for example, for treating Duchenne Muscular Dystrophy (DMD) via exon skipping. tcDNA is helpful for the development of antisense therapeutics targeting mRNA. Its high binding affinity and nuclease resistance allow downregulation of gene expression.

Gene Silencing and Editing: Oligonucleotides modified with tcDNA exhibit improved delivery and stability of CRISPR guide RNAs or other RNA-interfering molecules, increasing gene-editing precision.

RNA Modulation and Splicing Correction: Oligonucleotides modified with tcDNA can restore proper gene function by modulating pre-mRNA splicing, making it suitable for treating diseases caused by splicing mutations.

Exon Skipping in DMD (Duchenne Muscular Dystrophy): tcDNA can induce exon skipping in pre-mRNA to restore the reading frame of defective genes, especially in genetic disorders like DMD.

RNA Targeting in Central Nervous System (CNS) Disorders: The ability of tcDNA to cross the blood-brain barrier makes it a promising candidate for central nervous system (CNS) therapies for effectively delivering therapeutic nucleic acids for treating neurological diseases like Huntington's or ALS.

Molecular Probes and Diagnostics

Increased Stability: Molecular probes modified with tcDNA have enhanced stability in biological environments, making them ideal for diagnostics and molecular probes for studying biological systems, including qPCR and hybridization-based technologies.

Biomarker Detection: tcDNA incorporated into molecular probes enhance the sensitivity and specificity of nucleic acid-based biomarker detection in complex biological samples.

High Binding Specificity: tcDNA's structural modifications allow precise hybridization with complementary DNA or RNA strands, reducing off-target effects in diagnostic applications.

Drug Delivery 

Nucleic Acid Delivery

Improved Cellular Uptake: tcDNA modifications can enhance the delivery of oligonucleotides into cells without the need for additional transfection agents. To enhance delivery into specific tissues or cells, conjugation of tcDNA-modified oligonucleotides to targeting moieties such as lipids, for example, cholesterol or peptides, is possible.

Lipid Nanoparticles and Conjugates: Formulation of conjugated tcDNA-based oligonucleotides into lipid nanoparticles (LNPs) enhances therapeutics' delivery more effectively while ensuring stability.

Tools for Research and Development

Gene Function Studies: Inhibiting or modifying the expression of specific RNA transcripts in cellular and animal models with the help of tcDNA-based molecular probes allows exploring gene functions.

Structural Studies: The rigid structure of tcDNA enales detailed studies of nucleic acid-protein interactions hopefully contributiong ot our understanding of fundamental biological processes.

Study of Nucleic Acid-Protein Interactions: The rigid structure of tcDNA provides insights into nucleic acid-protein interactions, aiding in the development of novel drugs.

CRISPR Modifications: Oligonucleotides modified with tcDNA may enhance the stability and delivery of guide RNAs in CRISPR systems.

Benefits of tcDNA

Blood-Brain Barrier Penetration: Expands application to CNS-related disorders.

Enhanced Nuclease Resistance: Protects from degradation in biological systems.

Good Pharmacokinetic Profile: Allows for better therapeutic distribution.

High Binding Affinity: Improved hybridization to target RNA/DNA.

Low Toxicity: Reduces adverse effects in therapeutic applications.

Reduced Immunogenicity: tcDNA incorporated into therapeutic oligonucleotides minimizes immune response.

References

Aupy P, Echevarría L, Relizani K, Goyenvalle A. The Use of Tricyclo-DNA Oligomers for the Treatment of Genetic Disorders. Biomedicines. 2017 Dec 22;6(1):2 [PMC]. The authors discuss the potential of tricyclo-DNA oligomers as a therapeutic approach for genetic diseases, particularly focusing on their application in antisense therapy strategies to target and correct genetic mutations at the RNA level.

Aupy P, Echevarría L, Relizani K, Zarrouki F, Haeberli A, Komisarski M, Tensorer T, Jouvion G, Svinartchouk F, Garcia L, Goyenvalle A. Identifying and Avoiding tcDNA-ASO Sequence-Specific Toxicity for the Development of DMD Exon 51 Skipping Therapy. Mol Ther Nucleic Acids. 2020 Mar 6;19:371-383 [PMC]. The authors discuss the potential of tricyclo-DNA oligomers as a therapeutic splice-switching application for the treatment of Duchenne muscular dystrophy (DMD).

Bizot F, Fayssoil A, Gastaldi C, Irawan T, Phongsavanh X, Mansart A, Tensorer T, Brisebard E, Garcia L, Juliano RL, Goyenvalle A. Oligonucleotide Enhancing Compound Increases Tricyclo-DNA Mediated Exon-Skipping Efficacy in the Mdx Mouse Model. Cells. 2023 Feb 23;12(5):702 [PMC]. The authors demonstrated the normalization of cardiac function in mdx mice after a 12-week-long treatment with a combined ASO + OEC therapy. The findings indicate that compounds facilitating endosomal escape can significantly improve the therapeutic potential of exon-skipping therapeutics offering promising perspectives for the treatment of DMD.

Bizot F, Tensorer T, Garcia L, Goyenvalle A. Impact of the Inhibition of Organic Anion Transporter on Tricyclo-DNA-Mediated Exon Skipping in the <i>mdx</i> Mouse Model. Nucleic Acid Ther. 2023 Dec;33(6):374-380. [PubMed]. The authors discuss how OAT inhibition does not improve the therapeutic potential of ASO-mediated exon-skipping approaches for the treatment of DMD.

Blitek M, Phongsavanh X, Goyenvalle A. The bench to bedside journey of tricyclo-DNA antisense oligonucleotides for the treatment of Duchenne muscular dystrophy. RSC Med Chem. 2024 Jul 19;15(9):3017-3025. [PubMed]. The authors review the bench to bedside journey of tricyclo-DNA-ASOs from their early preclinical evaluation as fully phosphorotiated-ASOs to the latest generation of lipid-conjugated-ASOs and the remaining challenges of ASO-mediated exon-skipping therapy for DMD and future perspectives for this promising chemistry of ASOs.

Doisy M, Vacca O, Fergus C, Gileadi T, Verhaeg M, Saoudi A, Tensorer T, Garcia L, Kelly VP, Montanaro F, Morgan JE, van Putten M, Aartsma-Rus A, Vaillend C, Muntoni F, Goyenvalle A. Networking to Optimize Dmd exon 53 Skipping in the Brain of  Dmdx52 Mouse Model. Biomedicines. 2023 Dec 7;11(12):3243. [PMC]. The authors discuss the difficulty of exon 53 skipping in mdx52 mice when using a combination of multiple ASOs simultaneously to reach substantial levels of exon 53 skipping, regardless of their chemistry (tcDNA, PMO, or 2′MOE). 

Doisy M, Vacca O, Saoudi A, Goyenvalle A. Levels of Exon-Skipping Are Not Artificially Overestimated Because of the Increased Affinity of Tricyclo-DNA-Modified Antisense Oligonucleotides to the Target Dmd Exon. Nucleic Acid Ther. 2024 Oct;34(5):214-220. [PubMed]. The authors discuss that the use of tricyclo-DNA for exon skipping therapeutics does not inflate the observed levels of exon skipping due to the AON binding too strongly to the target exon in the DMD gene, which could potentially lead to inaccurate results in assessing the effectiveness of exon skipping therapy.

Dugovic B, Wagner M, Leumann CJ. Structure/affinity studies in the bicyclo-DNA series: Synthesis and properties of oligonucleotides containing bc(en)-T and iso-tricyclo-T nucleosides. Beilstein J Org Chem. 2014 Aug 12;10:1840-7. doi: 10.3762/bjoc.10.194. PMID: 25161745; PMCID: PMC4142851. [PMC]. The authors discuss how the incorporation of modified thymine nucleosides, specifically "bc(en)-T" (a bicyclo[2.2.1]heptene-modified thymine) and "iso-tricyclo-T" (an isomeric tricyclic thymine derivative) impact the stability and binding properties of the resulting oligonucleotide duplexes. 

Echevarría L, Aupy P, Relizani K, Bestetti T, Griffith G, Blandel F, Komisarski M, Haeberli A, Svinartchouk F, Garcia L, Goyenvalle A. Evaluating the Impact of Variable Phosphorothioate Content in Tricyclo-DNA Antisense Oligonucleotides in a Duchenne Muscular Dystrophy Mouse Model. Nucleic Acid Ther. 2019 Jun;29(3):148-160. [PubMed]. The authors discuss how varying the amount of phosphorothioate (PS) modifications on the backbone of tricyclo-DNA antisense oligonucleotides (tcDNA-ASOs) affects their ability to induce exon skipping in a mouse model of Duchenne Muscular Dystrophy (DMD), assessing the optimal level of PS modification for achieving efficient therapeutic outcomes while minimizing potential toxicities associated with high PS content.

Egli M, Pallan PS. Insights from crystallographic studies into the structural and pairing properties of nucleic acid analogs and chemically modified DNA and RNA oligonucleotides. Annu Rev Biophys Biomol Struct. 2007;36:281-305. [PubMed]. The review provides insights into the structural changes and pairing behaviors of modified nucleic acids, particularly when studying analogs with altered sugar moieties or base modifications. These types of studies can reveal how modifications impact the overall structure of the nucleic acid duplex, its stability, and its interaction with other molecules, allowing for rational design of new therapeutic agents with desired properties.

Egli M, Pallan PS. Crystallographic studies of chemically modified nucleic acids: a backward glance. Chem Biodivers. 2010 Jan;7(1):60-89. [PMC]. A scientific review looking back at research using X-ray crystallography for the analysis of nucleic acid structures (DNA and RNA) that have been chemically altered, providing insights into how these modifications affect their molecular shape and potential applications in fields like drug development and gene therapy.

Ezzat K, Aoki Y, Koo T, McClorey G, Benner L, Coenen-Stass A, O'Donovan L, Lehto T, Garcia-Guerra A, Nordin J, Saleh AF, Behlke M, Morris J, Goyenvalle A, Dugovic B, Leumann C, Gordon S, Gait MJ, El-Andaloussi S, Wood MJ. Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides. Nano Lett. 2015 Jul 8;15(7):4364-73. [PMC]. This research paper reports that for therapeutic antisense oligonucleotide (ASO) drugs to be effectively taken up by cells through receptor-mediated endocytosis, they need to self-assemble into nanoparticles, they must spontaneously form tiny particles through molecular interactions, which significantly enhances their cellular uptake compared to single, free ASOs

Gerber AB, Leumann CJ. Synthesis and properties of isobicyclo-DNA. Chemistry. 2013 May 27;19(22):6990-7006. doi: 10.1002/chem.201300487. Epub 2013 Apr 23. PMID: 23613358. [PubMed]. This study reports the chemical synthesis of isobicyclo-DNA building blocks, which are modified DNA nucleotides featuring a unique bicyclic structure, along with an investigation into their biophysical and biological properties; essentially creating a new type of DNA with altered structural features for potential applications in research related to nucleic acid chemistry and biology.

Goyenvalle A, Griffith G, Avril A, Amthor H, Garcia L. Un nouvel outil pour le traitement de la myopathie de Duchenne : les tricyclo-ADN [Functional correction and cognitive improvement in dystrophic mice using splice-switching tricyclo-DNA oligomers]. Med Sci (Paris). 2015 Mar;31(3):253-6. French. [medisci]

Goyenvalle A, Griffith G, Babbs A, El Andaloussi S, Ezzat K, Avril A, Dugovic B, Chaussenot R, Ferry A, Voit T, Amthor H, Bühr C, Schürch S, Wood MJ, Davies KE, Vaillend C, Leumann C, Garcia L. Functional correction in mouse models of muscular dystrophy using exon-skipping tricyclo-DNA oligomers. Nat Med. 2015 Mar;21(3):270-5. [PubMed]. This report demonstrated the physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice making tcDNA-AON chemistry attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.

Goyenvalle A, Leumann C, Garcia L. Therapeutic Potential of Tricyclo-DNA antisense oligonucleotides. J Neuromuscul Dis. 2016 May 27;3(2):157-167. [PMC]. This research paper demonstrated the potential of tricyclo-DNA ASOs for treating a range of diseases, particularly neuromuscular disorders like Duchenne muscular dystrophy (DMD), due to their unique pharmacological properties, including the ability to be taken up by various tissues after systemic administration, allowing for exon-skipping and splicing correction at the genetic level, offering a promising avenue for therapeutic intervention in genetic diseases affecting the muscles.

Guncay A, Yokota T. Antisense oligonucleotide drugs for Duchenne muscular dystrophy: how far have we come and what does the future hold? Future Med Chem. 2015;7(13):1631-5. [PubMed]. This research paper discusses the use of ASOs for the treatment of Duchenne muscular dystrophy.

Hari Y, Dugovič B, Istrate A, Fignolé A, Leumann CJ, Schürch S. The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes. J Am Soc Mass Spectrom. 2016 Jul;27(7):1186-96. [ACS]. This research paper discusses how the gas-phase behavior of tcDNA duplexes impact the activation entropy on the fragmentation kinetics in tandem mass spectrometric experiments, indicating that this type of analysis may not be suited to determine the relative stability of different types of nucleic acid duplexes.

Hollenstein M, Leumann CJ. Synthesis and biochemical characterization of tricyclothymidine triphosphate (tc-TTP). Chembiochem. 2014 Sep 5;15(13):1901-4. [PubMed]. This research paper reported that Tc-TTP is a substrate for the Vent (exo−) DNA polymerase, a polymerase that allows for multiple incorporations of tc-T nucleotides under primer extension reaction conditions. The substrate acceptance was found to be rather low, as also observed for other sugar-modified analogues. However, Tc-TTP and tc-nucleotide-containing templates do not sustain enzymatic polymerization under physiological conditions indicating that tc-DNA-based antisense agents will not enter natural metabolic pathways that can lead to long-term toxicity.

Imbert M, Blandel F, Leumann C, Garcia L, Goyenvalle A. Lowering Mutant Huntingtin Using Tricyclo-DNA Antisense Oligonucleotides As a Therapeutic Approach for Huntington's Disease. Nucleic Acid Ther. 2019 Oct;29(5):256-265. doi: 10.1089/nat.2018.0775. Epub 2019 Jun 11. PMID: 31184975.

Istrate A, Johannsen S, Istrate A, Sigel RKO, Leumann CJ. NMR solution structure of tricyclo-DNA containing duplexes: insight into enhanced thermal stability and nuclease resistance. Nucleic Acids Res. 2019 May 21;47(9):4872-4882. doi: 10.1093/nar/gkz197. PMID: 30916334; PMCID: PMC6511864.

Istrate A, Katolik A, Istrate A, Leumann CJ. 2'β-Fluoro-Tricyclo Nucleic Acids (2'F-tc-ANA): Thermal Duplex Stability, Structural Studies, and RNase H Activation. Chemistry. 2017 Aug 1;23(43):10310-10318. doi: 10.1002/chem.201701476. Epub 2017 Jun 13. PMID: 28477335.

Istrate A, Medvecky M, Leumann CJ. 2'-Fluorination of tricyclo-DNA controls furanose conformation and increases RNA affinity. Org Lett. 2015 Apr 17;17(8):1950-3. doi: 10.1021/acs.orglett.5b00662. Epub 2015 Apr 2. PMID: 25837683.

Ittig D, Gerber AB, Leumann CJ. Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes. Nucleic Acids Res. 2011 Jan;39(1):373-80. doi: 10.1093/nar/gkq733. Epub 2010 Aug 17. PMID: 20719742; PMCID: PMC3017593.

Ittig D, Liu S, Renneberg D, Schümperli D, Leumann CJ. Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: a comparison of tricyclo-DNA with LNA. Nucleic Acids Res. 2004 Jan 15;32(1):346-53. doi: 10.1093/nar/gkh187. PMID: 14726483; PMCID: PMC373297.

Ittig D, Luisier S, Weiler J, Schümperli D, Leumann CJ. Improving gene silencing of siRNAs via tricyclo-DNA modification. Artif DNA PNA XNA. 2010 Jul;1(1):9-16. doi: 10.4161/adna.1.1.11385. PMID: 21687522; PMCID: PMC3109438.

Ivanova G, Arzumanov A, Gait MJ, Reigadas S, Toulmé JJ, Andreola ML, Ittig D, Leumann C. Comparative studies of tricyclo-DNA- and LNA-containing oligonucleotides as inhibitors of HIV-1 gene expression. Nucleosides Nucleotides Nucleic Acids. 2007;26(6-7):747-50. doi: 10.1080/15257770701490928. PMID: 18066894.

Ivanova G, Reigadas S, Ittig D, Arzumanov A, Andreola ML, Leumann C, Toulmé JJ, Gait MJ. Tricyclo-DNA containing oligonucleotides as steric block inhibitors of human immunodeficiency virus type 1 tat-dependent trans-activation and HIV-1 infectivity. Oligonucleotides. 2007 Spring;17(1):54-65. doi: 10.1089/oli.2006.0046. PMID: 17461763.

Karuppasamy M, Alexander MS. Restoration of brain dystrophin using tricyclo-DNA ASOs restores neurobehavioral deficits in DMD mice. Mol Ther Nucleic Acids. 2023 May 25;32:841-842. doi: 10.1016/j.omtn.2023.04.007. PMID: 37273785; PMCID: PMC10238456.

Leumann CJ. Conformationally Restricted Oligonucleotide Analogues 'Made in Bern': A Mini Review. CHIMIA 55, 12(2001). https://doi.org/10.2533/chimia.2001.1041

Leumann CJ. Sugar modification as a means to increase the biological performance of oligonucleotides. Nucleic Acids Symp Ser (Oxf). 2006;(50):55-6. doi: 10.1093/nass/nrl167. PMID: 17150814.

Lietard J, Leumann CJ. Synthesis, pairing, and cellular uptake properties of C(6')-functionalized tricyclo-DNA. J Org Chem. 2012 May 18;77(10):4566-77. doi: 10.1021/jo300648u. Epub 2012 May 7. PMID: 22551389.

Lundin KE, Gissberg O, Smith CIE, Zain R. Chemical Development of Therapeutic Oligonucleotides. Methods Mol Biol. 2019;2036:3-16. doi: 10.1007/978-1-4939-9670-4_1. PMID: 31410788.

Medvecky M, Istrate A, Leumann CJ. Synthesis and properties of 6'-fluoro-tricyclo-DNA. J Org Chem. 2015 Apr 3;80(7):3556-65. doi: 10.1021/acs.joc.5b00184. Epub 2015 Mar 20. PMID: 25767996.

Morin A, Stantzou A, Petrova ON, Hildyard J, Tensorer T, Matouk M, Petkova MV, Richard I, Manoliu T, Goyenvalle A, Falcone S, Schuelke M, Laplace-Builhé C, Piercy RJ, Garcia L, Amthor H. Dystrophin myonuclear domain restoration governs treatment efficacy in dystrophic muscle. Proc Natl Acad Sci U S A. 2023 Jan 10;120(2):e2206324120. doi: 10.1073/pnas.2206324120. Epub 2023 Jan 3. PMID: 36595689; PMCID: PMC9926233.

Omairi S, Hau KL, Collin-Hooper H, Montanaro F, Goyenvalle A, Garcia L, Patel K. Link between MHC Fiber Type and Restoration of Dystrophin Expression and Key Components of the DAPC by Tricyclo-DNA-Mediated Exon Skipping. Mol Ther Nucleic Acids. 2017 Dec 15;9:409-418. doi: 10.1016/j.omtn.2017.10.014. Epub 2017 Oct 26. PMID: 29246319; PMCID: PMC6114118.

Pallan PS, Ittig D, Héroux A, Wawrzak Z, Leumann CJ, Egli M. Crystal structure of tricyclo-DNA: an unusual compensatory change of two adjacent backbone torsion angles. Chem Commun (Camb). 2008 Feb 21;(7):883-5. doi: 10.1039/b716390h. Epub 2007 Dec 21. PMID: 18253536. [pdb|2RF3|B Chain B, Crystal Structure of Tricyclo-DNA: An Unusual Compensatory Change of Two Adjacent Backbone Torsion Angles. CGCGNATTCGCG] https://pubs.rsc.org/en/content/articlelanding/2008/cc/b716390h

Petkova MV, Stantzou A, Morin A, Petrova O, Morales-Gonzalez S, Seifert F, Bellec-Dyevre J, Manoliu T, Goyenvalle A, Garcia L, Richard I, Laplace-Builhé C, Schuelke M, Amthor H. Live-imaging of revertant and therapeutically restored dystrophin in the Dmd^EGFP-mdx mouse model for Duchenne muscular dystrophy. Neuropathol Appl Neurobiol. 2020 Oct;46(6):602-614. doi: 10.1111/nan.12639. Epub 2020 Jul 27. PMID: 32573804.

Relizani K, Echevarría L, Zarrouki F, Gastaldi C, Dambrune C, Aupy P, Haeberli A, Komisarski M, Tensorer T, Larcher T, Svinartchouk F, Vaillend C, Garcia L, Goyenvalle A. Palmitic acid conjugation enhances potency of tricyclo-DNA splice switching oligonucleotides. Nucleic Acids Res. 2022 Jan 11;50(1):17-34. doi: 10.1093/nar/gkab1199. PMID: 34893881; PMCID: PMC8754652.

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Relizani K, Goyenvalle A. Use of Tricyclo-DNA Antisense Oligonucleotides for Exon Skipping. Methods Mol Biol. 2018;1828:381-394. doi: 10.1007/978-1-4939-8651-4_24. PMID: 30171555. 

Relizani K, Griffith G, Echevarría L, Zarrouki F, Facchinetti P, Vaillend C, Leumann C, Garcia L, Goyenvalle A. Efficacy and Safety Profile of Tricyclo-DNA Antisense Oligonucleotides in Duchenne Muscular Dystrophy Mouse Model. Mol Ther Nucleic Acids. 2017 Sep 15;8:144-157. doi: 10.1016/j.omtn.2017.06.013. Epub 2017 Jun 22. PMID: 28918017; PMCID: PMC5498286.

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Renneberg D, Leumann CJ. Exploring Hoogsteen and reversed-Hoogsteen duplex and triplex formation with tricyclo-DNA purine sequences. Chembiochem. 2004 Aug 6;5(8):1114-8. doi: 10.1002/cbic.200400069. PMID: 15300836.

Renneberg D, Leumann CJ. Watson-Crick base-pairing properties of tricyclo-DNA. J Am Chem Soc. 2002 May 29;124(21):5993-6002. doi: 10.1021/ja025569+. PMID: 12022832.

Robin V, Griffith G, Carter JL, Leumann CJ, Garcia L, Goyenvalle A. Efficient SMN Rescue following Subcutaneous Tricyclo-DNA Antisense Oligonucleotide Treatment. Mol Ther Nucleic Acids. 2017 Jun 16;7:81-89. doi: 10.1016/j.omtn.2017.02.009. Epub 2017 Mar 14. PMID: 28624227; PMCID: PMC5415958.

Saoudi A, Barberat S, le Coz O, Vacca O, Doisy Caquant M, Tensorer T, Sliwinski E, Garcia L, Muntoni F, Vaillend C, Goyenvalle A. Partial restoration of brain dystrophin by tricyclo-DNA antisense oligonucleotides alleviates emotional deficits in <i>mdx52</i> mice. Mol Ther Nucleic Acids. 2023 Mar 21;32:173-188. doi: 10.1016/j.omtn.2023.03.009. PMID: 37078061; PMCID: PMC10106732.

Saoudi A, Fergus C, Gileadi T, Montanaro F, Morgan JE, Kelly VP, Tensorer T, Garcia L, Vaillend C, Muntoni F, Goyenvalle A. Investigating the Impact of Delivery Routes for Exon Skipping Therapies in the CNS of DMD Mouse Models. Cells. 2023 Mar 15;12(6):908. doi: 10.3390/cells12060908. PMID: 36980249; PMCID: PMC10047648.

Scheidegger SP, Leumann CJ. Strand invasion properties and serum stability of alpha-tricyclo-DNA. Nucleic Acids Symp Ser (Oxf). 2008;(52):139-40. doi: 10.1093/nass/nrn071. PMID: 18776292.

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Stauffiger A, Leumann CJ. Synthesis of the bicyclo-[4.3.0]-thymidyl-nucleoside via pd(II)-mediated ring expansion chemistry. Nucleosides Nucleotides Nucleic Acids. 2007;26(6-7):615-9. doi: 10.1080/15257770701490407. PMID: 18066866.

Steffens, Ralph; Leumann, Christian J. (2016). Synthesis and Thermodynamic and Biophysical Properties of Tricyclo-DNA. ACS Publications. Collection. https://doi.org/10.1021/ja983570w

Zarrouki F, Relizani K, Bizot F, Tensorer T, Garcia L, Vaillend C, Goyenvalle A. Partial Restoration of Brain Dystrophin and Behavioral Deficits by Exon Skipping in the Muscular Dystrophy X-Linked (mdx) Mouse. Ann Neurol. 2022 Aug;92(2):213-229. doi: 10.1002/ana.26409. Epub 2022 Jun 13. PMID: 35587226; PMCID: PMC9544349.
 

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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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