The CRISPR-Cas9 system is now thought to be the method for gene editing therapy in humans with minimal side-effects. However, significant off-target effects were until recently believed to be just insertions and deletions (indels) of less than 20 base pairs. Therefore, the CRISPR-Cas9 system is assumed to be very specific making it a useful tool for gene editing in cells. However, this approach may not be as straightforward as it sounds.

Figure 1: Crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5'-NGG-3' PAM (Anders et al. 2014).  

Ihry et al. recently reported that p53 inhibits the CRISPR-Cas9 system in human pluripotent stem cells (hPSCs). The research group found that double-strand breaks induced by Cas9 are toxic and kill most hPSCs.

Next, Kosicki et al. recently showed that DNA breaks introduced by single-guide RNA-Cas9 frequently resolved into deletions extending over many kilobases. This research group used long-read sequencing and long-range PCR genotyping for the study of DNA break repair mechanisms. Additionally, the researcher observed lesions distal to the cut site and crossover events as well. As a result, Kosicki et al. reason, the observed genomic damage in mitotically active cells caused by CRISPR–Cas9 editing may have pathogenic consequences.

A third study (Haapaniemi et al. 2018) reported that genome editing by CRISPR-Cas9 induced a p53-mediated DNA damage response. The cell cycle was arrested in immortalized human retinal pigment epithelial cells. However, inhibition of p53 prevented the damage response and increased the rate of homologous recombination from a donor template.

As a result, more research is needed since correcting somatic mutations in human cells using CRISPR-Cas9 may cause more harm than doing good. Employing bridged nucleic acids (BNAs) for more selective gene editing may also improve the selectivity of the CRISPR-Cas9 system.

Reference

Carolin Anders, Ole Niewoehner, Alessia Duerst, and Martin Jinek; Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014 Sep 25; 513(7519): 569–573.

Haapaniemi, Emma, Botla, Sandeep, Persson, Jenna, Schmierer, Bernhard, Taipale, Jussi; 2018. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nature Medicine 24, 927-930 (2018).

Ihry, Robert J., Worringer, Kathleen A., Salick, Max R., Frias, Elizabeth, Ho, Daniel, Theriault, Kraig, Kommineni, Sravya, Chen, Julie, Sondey, Marie, Ye, Chaoyang, Randhawa, Ranjit, Kulkarni, Tripti, Yang, Zinger, McAllister, Gregory, Russ, Carsten, Reece-Hoyes, John, Forrester, William, Hoffman, Gregory R., Dolmetsch, Ricardo, Kaykas, Ajamete;  p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells. Nature Medicine 24, 939-946 (2018).

Kosicki, Michael, Tomberg, Kärt, Bradley, Allan; Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nature Biotechnology 2018/07/16/online, 36, 765-771 (2018).

http://dx.doi.org/10.1038/nbt.4192. https://www.nature.com/articles/nbt.4192#supplementary-information

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PCR based methods can be used for the detection of single point mutations (SNPs) present in genetic disorders.


The detection of mutations in the genome or transcriptome is of great importance for the diagnosis of genetic disorders, as well as pre-symptomatic testing, conformational diagnosis as well as forensic identity testing. To detect genetic syndromes, two groups of tests are available, molecular and cytogenetic tests.

After the identification and definition of mutations, diagnostic methods or tests can be used to find them using techniques such as allele-specific oligonucleotide hybridization, allele-specific amplification, ligation, primer extension and the artificial introduction of restriction sites.

PCR allows mutation detection, however, PCR itself does not detect the actual mutation. PCR generates an amplicon that is then analyzed by some other method to find possible variations within the amplicon. PCR based methods only detect mutations that have been previously identified by some other techniques if now sequencing step is added.

PCR based methods

Real-Time PCR

Real-time PCR using nonspecific DNA-binding dyes

Real-time PCR using labeled probes

• Cleavage-based or hydrolysis or dual-labeled probes

• Molecular Beacons

• FRET probes

• Scorpion probes
  

Nonspecific DNA binding dyes only allow the determination of the presence or absence of an amplicon. No information on the precise nature of the product can be determined. The dye STBR Green I emits fluorescence when bound to double-stranded DNA. The amount of intercalated dye will increase as the number of DNA molecules increases. The emitted fluorescence is directly proportional to the copy number. Nonspecific DNA binding dyes allow measuring gene expression levels and the detection of pathogens. Unfortunately, intercalation dyes detect accumulation of both specific and nonspecific PCR products. 

The solution structure of the fluorescent bis-intercalator dye which contains a benzothiazole moiety similar as present in SYBR Green I illustrates the nature of the binding to the DNA helix.

                       TOTO               Models of the TOTO-dsDNA complex (PDB ID 108D)
  
In the structure of the DNA complex with the fluorescent bis-intercalator,  bis-intercalating dye 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene) bis[4-(3-methyl -2,3- dihydro benzo-1,3-thiazolyl-2-methylidene) quinolinium] tetraiodide (TOTO) in the benzothiazole ring system is twisted relative to the quinoline in the uncomplexed TOTO molecule. TOTO is selective for the CTAG-CTAG  double-helix which is explained by its ability to adapt to the base pair propeller twist of dsDNA which optimizes stacking and the hydrophobic interaction between the thymidine methyl group and the benzothiazole ring. A 3000-fold fluorescence enhancement occurs upon binding of TOTO to dsDNA. In the complex, the nucleobase forms a clamp around the benzothiazole ring and the quinolinium ring of TOTO such that it can no longer rotate around the cyanine methane bonds. The clamping prevents rotation of the dye, and the chromophore loses excitation energy by fluorescence instead.

Reference

PDP ID 108d : Spielmann HP, Wemmer DE, Jacobsen JP.; Solution structure of a DNA complex with the fluorescent bis-intercalator TOTO determined by NMR spectroscopy. Biochemistry. 1995 Jul 11;34(27):8542-53.] https://www.ncbi.nlm.nih.gov/Structure/pdb/108D 

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The trials and tribulations of peptide vaccines development.

Learning from past errors.

Dante J. Marciani, Sc.D., Ph.D.

Vaccination or immunization against pathogenic agents has been one of the most successful developments to prevent disease in the history of medicine. Indeed, vaccination goes back as early as the XV century, when in China immunization against smallpox was carried out by inhaling the dry scabs from those affected by the disease. Subsequent developments by Jenner, Pasteur, and others, stablished vaccination as an effective method to prevent some infectious diseases. Moreover, as the understanding of immune protection against infectious agents increased, new refinements to vaccine development were used; like the introduction of the sub-unit vaccines, which frequently avoids immune responses leading to allergies and even autoimmunity. Indeed, the developments that allowed the creation of sub-unit vaccines, also benefited basic research, when the use of antibodies against specific antigens started to be used in biochemistry, microbiology, and others areas. An approach that lead to the development of the monoclonal antibodies by Milstein and Köhler, for which they received the Nobel Prize in Medicine and Physiology in 1984. Hence, antibodies have become an important tool in research; i.e. the initial results derived from basic research have frequently encouraged the development of vaccines against infectious agents and cancer. Hence, due to their relevance in basic research, it is not surprising that sub-unit vaccines, composed of an antigen plus an adjuvant, are the most commonly used. In fact, the antigen directs the immune system to induce a specific response against itself, while the adjuvant decides the nature and magnitude of the immune response against that antigen.

In fact, vaccine development until two decades ago was sort of an empirical process, where the adjuvant component was frequently a mixture of ill-defined immune stimulators, like the complete Freund’s adjuvant (CFA), which contains whole bacteria. A situation that changed with the discovery of innate immunity, its receptors and ligands; suddenly, a series on well-defined compounds, typically derived from pathogens, were found to induce an immune response by binding to specific cell receptors called Toll-like receptors, or TLRs. This type of response, i.e. innate immunity, has been well-conserved during evolution, with an additional refinement in vertebrates, where innate immunity triggers a more specific, long-term one, called acquired immunity. But, different TLRs induced different kinds of acquired immune responses. Also, TLRs can interact with each other, a situation that may lead to synergic effects, which result in a better immune response, or in other cases in an antagonistic effect, leading to inhibition of the immune response. A result of the induction of immunity via TLRs, is that the acquired response is a pro-inflammatory Th1/Th17 immunity; immunities that are effective in fighting pathogens, but detrimental when they are directed against self-antigens, leading to a new type of increasing diseases, known as autoimmune conditions.

The increasing popularity of antibodies as protective agents and research tools, has created a frequently chaotic situation, where vaccines or immunization strategies have induced an undesirable response, rather than the beneficial one expected. A consequence of a variety of errors made during the design of those “vaccines,” which may include the immunogen or antigen, as well as the adjuvant, has been the significant number of vaccine failures at the development phase or even at the clinical phase. Mistakes that could have been prevented by a critical evaluation of the vaccine’s different components and a rational approach to their design. Here, we would discuss some of those errors, which have led in many occasions to unexpected and unexplained failures, which frequently have been blamed on the scientific bases supporting a vaccine, rather than the defective design of those vaccines.

The adjuvant    

A widespread believe in vaccine development, is the wrong assumption that all adjuvants are created equal; which may have been acceptable more than twenty years ago, before the discovery of innate immunity its receptors and ligands. Indeed, it is common to see papers where an antigen after being found to be immunogenic by using CFA, is then combined with well-defined adjuvants, like QS-21, monophosphoryl lipid A (MPL), and others, expecting to have a similar immune response. A situation that usually results in disappointments and reaching the wrong conclusions, as well as causing delays in the development of critically needed vaccines and frequently abandoning their development. Unfortunately, a frequent explanation for these disappointments, has been to blame the science rather than the poor vaccine design. Indeed, several vaccines that when tested in animals using CFA, induced a strong immune response as determined by in vitro assays, failed in vivo testing. A situation that may be worsened by the fact that in some diseases, the CFA induced immune response may potentiate the disease that it is supposed to prevent and/or treat. A finding that usually led to the wrong conclusion that a vaccine against that disease is impossible; a speculation frequently proven to be wrong, when an adjuvant that induces the right immune response is used to develop an effective vaccine. Hence, while adjuvants like CFA may be practical to elicit the production antibodies against an antigen, it may not be advisable to consider those initial methods as stepping stones to develop a vaccine.  

To complicate the issue more, the fact is that there are three types of acquired immunity, i.e. Th1 and Th17, which are pro-inflammatory, and Th2 that is anti-inflammatory. Yet, the most common immune responses are Th1 and Th2, with the strongly inflammatory Th17 being less common, but more damaging, as many autoimmune diseases show. Paradoxically, in many papers it is indicated that the optimal immune response must have both pro-and anti-inflammatory immunities, which conveys the impression that a pro-inflammatory immunity, Th1 or Th17, may exist alone and without the anti-inflammatory Th2 immunity, the latter being responsible for the humoral immune response that induces antibody production. Thus, many articles strive to reach that optimal adjuvant that induces both immunities rather than only the pro-inflammatory one. The fact is that there is no adjuvant or agent that can induce a sole pro-inflammatory immunity, Th1 or Th17, since both of them are always followed by the anti-inflammatory Th2 immunity; a relatively newcomer from the evolutionary point of view. Indeed, the milder Th2 immunity always follows the Th1 and/or Th17 immunities, likely as a repair mechanism to ameliorate the damage in the host caused by the pro-inflammatory immunities. Of interest is that different from Th1 and Th17 immunities, Th2 can exist by itself by inhibiting the pro-inflammatory immunities; a property that offers potential therapeutic uses in autoimmunity.

Hence, as a result of the advances in immunology, derived from the use of genetic as well as biochemistry, medicinal chemistry and other methods to study the immune response, the adjuvant component has evolved from being a “little dirty secret” to a collection of structurally well-defined agonists that by interacting with the appropriate cell-receptors, promote a useful immune response. Interestingly, as indicated before, different immune stimulatory ligands interact with specific receptors to induce an immune response following distinctive pathways; pathways that may be synergistic or antagonistic, depending of the adjuvants. Therefore, a deliverate adjuvants’ selection, can increase and modify the immune response well beyond the limits of that attained with single adjuvants; a strategy that is seldom exploited in vaccine development. Hence, the rational design of effective vaccines, requires a knowledge of the mechanisms of action (MOA) of the different adjuvants, in order to select those that induce an optimal response. Actually, the MOA for several innate immunity ligands, e.g. MPL, CpG oligonucleotides and others ligands, acting on antigen-presenting-cells (APCs), have been elucidated. A more complicated situation is that of the MOA for saponin-based adjuvants like QS-21, where this glycoside acts independently in a coordinate but differential manner, on both T-cells and APCs, to induce a strong pro-inflammatory response. However, while QS-21 is a potent pro-inflammatory adjuvant, a potential drawback is its instability, that results in a change in its immune stimulatory properties, from being a pro-inflammatory to a sole anti-inflammatory adjuvant. A severe problem in vaccines against pathogens or cancer, where a pro-inflammatory immunity is crucial for their efficacy. Saponin-based adjuvants, because of its physicochemical properties and MOA, are sensitive to a vaccine’s formulation; in fact, the addition of non-ionic detergents to QS-21, results in a significant increase in its Th1 immune stimulatory activity. An increase that results in damaging inflammatory effects, like those observed during the AN-1792 vaccine clinical studies against Alzheimer’s disease, which led to terminating this vaccine’s studies. Hence, a rational vaccine design must consider besides the adjuvant and antigen, the nature of the excipients and the possibility that unexpected interactions may lead to unwanted side effects.

Several strategies have been tried to achieve stabilization of QS-21, most of them using formulations in which this glycoside is sequestered, limiting the exposure of its ester bond between its fucosyl residue and fatty acids to the aqueous environment, to prevent this way its hydrolysis. A protection that can be somewhat achieved by incorporating QS-21 into liposomes, this way removing the ester bond from contact with water; while this approach reduces the hydrolytic process, it does not abolish it, and its effectiveness may be affected by excipients, temperature and other factors. Another strategy has been to replace the labile ester bond by a stable covalent bond, an approach that unfortunately failed to recognize the role of the different chemical structures from QS-21. As a consequence, the final products while reminiscent of QS-21, lack some chemical groups essential for immune stimulation. While these new derivatives induce an antibody immune response, it is unknown which type of immunity, pro- or anti-inflammatory, is being induced; a critical requirement in vaccine design. Another alternative to achieve a stable immune stimulatory glycoside, has been to replace the original acyl group with a new lipophilic chain linked by a stable covalent bond, at a different location in the glycoside, but preserving the groups critical for adjuvanticity. This new compound, GPI-0100, induces a pro-inflammatory immunity, albeit at higher doses than QS-21, but it is stable in an aqueous environment, regardless of the temperature, pH and other factors. Moreover, the immune stimulatory properties of GPI-0100, may be enhanced by changing the vaccines’ formulations. Thus, some effective options have been developed to overcome the instability of the adjuvant QS-21, while retaining most of its unique immune stimulatory properties.     

An adjuvant commonly used is alum, traditionally presumed to be a sole Th2 adjuvant, is the oldest adjuvant allowed in human vaccines. While its MOA was a mystery for many decades, it has been elucidated; a complex process where uric acid plays the role of an endogenous danger signal, which triggers a complex cascade of events leading to an immune response. Although alum induced response has the characteristics of Th2 immunity, it also elicits some responses associated with pro-inflammatory immunity, e.g. complement activation, induction of IFN-γ production and monocytes activation, all responses that indicate that alum may not be a sole Th2 adjuvant. In fact, that alum has been successfully used for years in infectious disease vaccines where a pro-inflammatory immunity is desirable, points to a potentially not too obvious pro-inflammatory activity. But, alum in contrast to adjuvants like QS-21, does not elicit the production of high affinity or avidity antibodies, characteristic of an effective immune response; which may explain the alum-containing vaccines’ poor performance in the elderly, a classic example being the influenza vaccine. Hence, while safe, alum would be a dubious first choice in view of the new and more effective adjuvants currently available. Hence, it is relevant to identify the type of immune response desired, the need for a long-term memory, production of cytotoxic T-lymphocytes (CTLs), antibody isotype, and other factors, in order to select the best suited adjuvant or combination of adjuvants to fulfill those needs.  

The immunogen – T-dependent antigens   

The other vaccine’s critical component is the immunogen, i.e. the antigen, which is supposed to induce an immune response directed against itself, this way providing the antigen-specific response required from vaccines. Hence, it is evident that immunogens are very diverse and their number quite large. Broadly speaking, immunogens can be classified into two groups: T-dependent and T-independent antigens, depending on the need for helper T-cells; the first group is composed of protein antigens while the second is made up of non-protein ones, like polysaccharides and nucleic acids, with lipids being a special group. The best-characterized group is the T-dependent made up of proteins; hence, the present article will be focused on protein and peptide antigens, with a subsequent article dealing with T-independent antigens.

Proteins, because of their complex structures have a large variety of antigenic determinants or epitopes, which are protein regions recognized by the immune system, i.e. B- and T-cells. Thus, epitopes may be linear, i.e. peptide amino acid sequences, or conformational, which are composed of various discontinuous amino acid sequences which form a 3-dimensional structure, the same that is recognized by the immune system. Actually, conformational epitopes play a crucial role in the interactions with various cellular receptors; hence, these epitopes are important targets for neutralizing antibodies, which by blocking the interactions of these 3D-epitopes with their receptors, interfere with infectivity and processes initiated by stimulatory compounds like growth factors and cytokines. In contrast, the linear epitopes are usually T-cell epitopes, that induce T-cell mediated immunity with secretion of inflammatory cytokines and CTL production. Yet, the initial T-cell immunity will be always followed by an antibody response against that linear epitope. Therefore, it is evident that conformational epitopes would include both B- and T-cell epitopes, to allow the polypeptide chain’s proper folding, in order to achieve the right conformation. Yet, these epitopes may be also formed by discontinuous amino acid residues from a linear polypeptide chain; like in the case where an epitope is formed by contiguous amino acid residues on the surface of an α-helix. In this case, the contiguous amino acids on the coiled peptide, form a linear epitope, amino acids that are surrounded by a diversity of amino acid residues. Hence, it is apparent that peptides have a significant potential to be used as effective immunogens; but, in general the attempts to develop effective vaccines based on peptides have delivered mostly disappointing results.

A close examination of the principles on which peptide vaccines are founded, shows a series of systematic errors and wrong assumptions that have contributed to those failures.  Peptide vaccines, due to their size and lack of T-helper epitopes, elicit a weak immunity with a poor immunological memory; hence, it has been customary to conjugate the relevant peptides to a carrier protein, like serum albumin, tetanus toxoid and KLH, to provide the necessary T-helper epitopes. However, these proteins cause a phenomenon known as “carrier-induced epitope-specific suppression” or CIESS, where the specific anti-peptide antibody response is suppressed, because the carrier protein induces an antibody response that competes with the anti-peptide response. A situation that results in a weak immune response that frequently is different from the expected anti-peptide. Hence, a new trend is the use of well-defined T-helper epitopes, derived from other proteins, or an artificially designed one, like PADRE. Yet, the carrier problem is often exacerbated by the use of certain cross-linkers, to covalently link the peptide to the carrier protein. For instance, one of the most common linkers used are the heterobifunctional ones carrying both maleimidyl and succinimidyl groups, popular because of their commercial availability and their easy reactivity with the thiol group from cysteine, an amino acid residue added at one of the ends of a peptide of interest. However, due to the high immunogenicity of the maleimide group, the immune response is often directed against that chemical group rather than the peptide, a situation that leads to the suppression of the antipeptide immune response and the researcher’s emotional distress. An unusual situation, since this undesirable effect of the maleimide group has been known for over two decades. Yet, these undesirable effects are not limited to maleimide, but they include other groups with aromatic character, like benzyl and some heterocyclic aromatic compounds.

Paradoxically, many immunogenic preparations, including some vaccines undergoing clinical studies, has the maleimide group in their conjugated antigens in combination with carrier proteins like KLH. Constructs that may interfere with the production of an effective immune response against the targeted peptide. Thus, the CIESS phenomenon may explain some of the peptide vaccines’ poor clinical results and why their progress has been so far, limited at best, despite of their promise. Still, these negative results are not limited to T-dependent antigens, i.e. proteins and peptides, but they also extend to T-independent antigens, a topic that will be the subject of a future article.

The excipients

By definition, an excipient must be inert, i.e. it should not alter the properties of the adjuvant or the immunogen; a situation that frequently is not fulfilled in vaccine development, which leads to unsatisfactory results. A typical example would be that of the AN-1792 vaccine to treat Alzheimer’s disease; a vaccine formulated with the potent pro-inflammatory adjuvant QS-21. Although the selection of that adjuvant to treat a neurodegenerative proteopathy was unwise, the patients receiving the initial vaccine formulation did not show any side-effects in Phase I. But, during the Phase II of such clinical studies and using a vaccine formulation that was modified by addition of a non-ionic detergent, many patients had meningoencephalitis, a damaging brain inflammation; a development that lead to termination of the study. Evidently, those designing this vaccine, were unaware that QS-21 was a potent pro-inflammatory adjuvant, and that the formulation used during Phase I was being altered by the addition of a non-ionic detergent. An apparently innocuous change in formulation, which induced a large increase in the pro-inflammatory activity of QS-21; an alteration that would have discovered in Phase I, if the protocols governing clinical studies would have been followed. Regrettably, those damaging effects due to shortcomings in the vaccine formulation, caused significant damage to that vaccine’s development. Yet, there are cases where an excipient, accidentally may contribute in a positive or negative way to a vaccine’s efficacy.

A group of excipients that it has been assumed to be inert and stable, is the one composed of various non-ionic detergents, like the detergent used in the AN-1792 vaccine; compounds that are usually used to prevent aggregation of the vaccines’ antigens. Nonetheless, several infectious disease vaccines are formulated with oil-water emulsions that contain non-ionic detergents, as adjuvants. But, a concern that is usually ignored, is that non-ionic detergents oxidize with production of aldehyde groups, which are immunologically active and that can deliver alternative co-stimulatory signals to T-cells, leading to their activation. Therefore, it is likely that those aldehyde groups by delivering a co-stimulatory signal, may contribute to the efficacy of vaccine requiring a pro-inflammatory immunity; yet, it may cause damaging side-effects in cases where the immune response must be an anti-inflammatory one. While it is apparent that those working in vaccine development are largely unaware of the implications of these oxidation products on vaccines’ performance, scientists working with biologics are well aware of those effects. In fact, many therapeutic proteins, like monoclonal antibodies cytokines and other products, suffer changes induced by the reaction of aldehydes with the proteins’ amino groups.

Hence, during sub-unit vaccine development all of the possible interactions between the different components must be considered, to prevent undesirable and/or unexpected results, which may confuse the subject. But, these comments should also apply to the development of the immunogenic formulas used in basic research; particularly because frequently those initial findings progress to become therapeutic products.

Relevant publications

Akira, S. et al. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2001. 2:675-680

Allen JE, and Wynn TA. Evolution of Th2 immunity: A rapid repair response to tissue destructive pathogens. PLoS Pathog. 2011. 7(5): e1002003

Bergmann-Leitner ES and Leitner WW. (2014) Adjuvants in the driver’s seat: how magnitude, type, fine specificity and longevity of immune responses are driven by distinct classes of immune potentiators. Vaccines 2014. 2:252–296.

Boeckler C, et al. Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes. J Immunol Methods. 1996. 191:1-10. 

del Guercio MF, et al. Potent immunogenic short linear peptide constructs composed of B cell epitopes and PAN DR T helper epitopes (PADRE) for antibody responses in vivo. Vaccine 1997. 15, 441–448.

Genito CJ, et al. Liposomes containing monophosphoryl lipidA and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013. Vaccine 2017. 35:3865-3874

Ghosh M, et al. Carrier protein influences immunodominance of a known epitope: implication in peptide vaccine design. Vaccine, 2013. 31:4682-4688

Jegerlehner A, et al. Carrier induced epitopic suppression of antibody responses induced by virus-like particles is a dynamic phenomenon caused by carrier-specific antibodies. Vaccine. 2010. 28:5503-5512.

Kooijman S, et al. Novel identified aluminum hydroxide-induced pathways prove monocyte activation and pro-inflammatory preparedness. J Proteomics. 2018. 175:144-155

Kornbluth RS and Stone GW. Immunostimulatory combinations: designing the next generation of vaccine adjuvants. J Leukoc Biol. 2006. 80:1084-1102.

Li W, et al. Peptide vaccine: Progress and challenges. Vaccines, 2014. 2:515-536

Maggio ET. Polysorbates, peroxides, protein aggregation, and immunogenicity – a growing concern. J Excipients and Food Chem. 2012. 3:45-53

Marciani DJ, et al. Fractionation, structural studies, and immunological characterization of the semi-synthetic Quillaja saponins derivative GPI-0100. Vaccine. 2003. 21:3961-3971

Marciani DJ. Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity. Drug Discov. Today. 2003. 8:934–945.

Marciani DJ. A retrospective analysis of the Alzheimer’s disease vaccine progress – The critical need for new development strategies. J. Neurochem. 2016. 137:687-700.

Marciani, DJ. Elucidating the mechanisms of action of saponin derived adjuvants. Trends Pharmacol Sci. 2018. 39:573-585

Rhodes J.et al. Therapeutic potentiation of the immune system by costimulatory Schiff-base-forming drugs. Nature. 1995. 377:71-75.

Skwarczynski M and Toth I. Peptide-based synthetic vaccines. Chem Sci. 2016. 7:842-854

Yamaguchi Y. et al. Guidance for peptide vaccines for the treatment of cancer. Cancer Sci. 2014. 105:924-931

Extracellular vesicles, including exosomes and microvesicles, allow early diagnosis, prognosis and potentially targeted treatments of cancer. Circulating exosomes are a source of stable RNAs including mRNAs, microRNAs, and lncRNAs.

Exosomes are cell-derived vesicles present in body fluids such as blood, urine, as well as in cell culture mediums. Exosomes are tiny vesicles released from plasma membranes of different cell types. Exosomes are cellular protein complexes that contain enzymes degrading nuclear and cytoplasmic RNA.

In contrast, endosomes are membrane-bound vesicles present in the cytoplasm formed during endocytosis, the process that transports molecules into the cell by engulfing them with its membrane.

Apparently all cell types produce extracellular vesicles (EVs). EVs deliver a variety of biomolecules such as lipids, proteins, DNA, mRNA, microRNA as well as long non-coding RNAs (lncRNAs), into cells, dynamically and bidirectionally.

Classification and definition of extracellular vesicles

(i)    Small nano-sized exosomes are formed within the cytoplasm by inward budding of endosomes pooled into multivesicular bodies (MVBs). The molecular transporting machinery called “the endosomal sorting complex” is involved in sorting and incorporation of the molecular material into multivesicular bodies (MVBs). MVBs fuse with the cell membrane and release exosomes into the extracellular space,

(ii)    Nano- to micro-sized vesicles or microvesicles shed from the plasma membrane, and

(iii)    Micro-sized vesicles are created as byproducts of cell death or apoptotic bodies.

Long non-coding RNAs (lncRNAs)

Long non-coding RNAs (lncRNAs) are a heterogeneous group of non-coding transcripts localized in different cell compartments and are usually longer than 200 nucleotides. lncRNAs are non-protein coding RNAs distinct from housekeeping RNAs such as tRNAs, rRNAs, and snRNAs, and independent from small RNAs with a specific molecular processing machinery, for example, micro- or piwi-RNAs.

lncRNAs are classified into intragenic (intronic or antisense) and intergenic lncRNAs. Some of these are stable, highly expressed and conserved, while others have a high turnover, often are barely detectable and poorly conserved. lncRNAs have diverse functions which they exert by interacting with DNA, RNA and proteins in a sequence-specific and conformational manner, where they can act as a scaffold, a decoy or as enhancer RNA. However, the exact role of exosomal lncRNAs is not at all clear. Dragomir et al. assume that lncRNAs could be a loading vehicle for miRNAs, mRNAs, and other complex molecules into the exosome but this hypothesis will need to be confirmed experimentally. Synthetic lncRNAs may allow more detailed functional studies of these RNA species.

The structural model for the SINEB2 element of the long non-coding RNA activator of translation AS Uchl1 solved by NMR is an excellent example for a long non-coding RNA (Figure 1. Podbevšek et al. 2018). The invSINEB2/183 RNA folds into a structure with mostly helical secondary structure elements. SINE elements have been hypothesized to act as portable domains in lncRNAs contributing to their biological functions.

Figure 1: NMR based model of the structure for the SINEB2 element of the long non-coding RNA activator of translation AS Uchl1 (PDB ID: 5LSN). A: A cartoon model of the structure is shown. B: The surface of the SINEB2 element is shown.

SINEUPs are a new class of natural, synthetic antisense long non-coding RNAs that activate translation. Zuchelli et al. in 2015 reported the discovery of a new functional class of natural and synthetic antisense lncRNAs that stimulate translation of sense mRNAs. The research group named these molecules SINEUPs since for them to function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. The existence of natural SINEUPs suggests that embedded Transposable Elements may represent functional domains in long non-coding RNAs.

Also, the Zuchelli et al. argue that the design of synthetic SINEUPs to target antisense sequences of mRNAs of choice allows for a scalable increase of protein synthesis of potentially any gene of interest.

Examples of the categorization for non-coding RNAs

(Source: Ernst and Morton, 2013). 

Examples of lncRNAs showing diversity of functions

 (Source: Kashi et al. 2016).

Many lncRNAs are found protected in human body fluids within circulating tumor cells or EVs. These lncRNAs are quite stable and can be extracted from blood or other body fluids as potential biomarkers for disease diagnosis, prognosis and therapy. The expression of lncRNAs can be blocked with antisense oligonucleotides (ASOs), aptamers, hammerhead ribozymes, siRNAs, or small specific molecules. Since some lncRNAs appear to form scaffolds to bind and recruit protein complexes to specific genomic loci, targeting them may open up new ways to treat certain cancers. ASOs as well as CRISPR based method allow studying unknown functions of lncRNA.

Also, Naderi-Meskin et al. suggested that exosome analysis can be used as a strategy for cancer diagnostics and monitoring dynamic changes during cancer development and therapy. Exosomal lncRNAs as potential diagnosis biomarkers and prognosis indicators in cancer.

Exosomal lncRNAs as potential diagnosis biomarkers and prognosis indicators in cancer 

 (Source: Dragomir et al. 2018).

Reference

Dragomir, M., Chen, B., & Calin, G. A. (2018). Exosomal lncRNAs as new players in cell-to-cell communication. Translational Cancer Research, 7(Suppl 2), S243–S252.

Ernst, C., & Morton, C. C. (2013). Identification and function of long non-coding RNA. Frontiers in Cellular Neuroscience, 7, 168.

Hewson C, Morris KV; Form and Function of Exosome-Associated Long Non-coding RNAs in Cancer. Curr Top Microbiol Immunol. 2016;394:41-56. doi: 10.1007/82_2015_486.

Hojjat Naderi-Meshkin, Xin Lai, Raheleh Amirkhah, Julio Vera, John E J Rasko, Ulf Schmitz; Exosomal lncRNAs and cancer: connecting the missing links, Bioinformatics, , bty527.

Kaori Kashi, Lindsey Henderson, AlessandroBonetti, PieroCarninci;  Discovery and functional analysis of lncRNAs: Methodologies to investigate an uncharacterized transcriptome. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, Volume 1859, Issue 1, January 2016, Pages 3-15. 

Podbevšek, P., Fasolo, F., Bon, C., Cimatti, L., Reißer, S., Carninci, P., … Gustincich, S. (2018). Structural determinants of the SINE B2 element embedded in the long non-coding RNA activator of translation AS Uchl1. Scientific Reports, 8, 3189. http://doi.org/10.1038/s41598-017-14908-6.

Zhang, J., Li, S., Li, L., Li, M., Guo, C., Yao, J., & Mi, S. (2015). Exosome and Exosomal MicroRNA: Trafficking, Sorting, and Function. Genomics, Proteomics & Bioinformatics, 13(1), 17–24. http://doi.org/10.1016/j.gpb.2015.02.001.

Zucchelli, S., Cotella, D., Takahashi, H., Carrieri, C., Cimatti, L., Fasolo, F., … Gustincich, S. (2015). SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation. RNA Biology, 12(8), 771–779. 

 
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Guanine rich oligonucleotides allow detection of potassium ions

Oligonucleotides containing sequences from human telomere DNA or a thrombin binding aptamer can form tetraplex structures upon binding of potassium ions. Structural changes associated with the formation of the tetraplex fold allow the development of potassium-sensing oligonucleotide (PSO) probes or sensors.

In a PSO, two fluorescent dyes are attached to both terminal ends of the oligonucleotide. Combining fluorophores with a potassium sensing oligonucleotides that allow for fluorescence resonance energy transfer (FRET) or excimer emission based detection methods upon binding of the K⁺ ion enabling monitoring of K+ ions in biological systems.

A flexible single-stranded DNA oligonucleotide with a guanine-rich sequence folds into a tetraplex structure in the presence of potassium ions. The single-stranded oligonucleotide, 5’-GGTTGGTGTGGTTGG-3’, when immobilized onto a graphene surface acts as a probe allowing detection of potassium ions in a graphene-based biosensor devise (Lui et al. 2018).

The potassium ion is essential in biological systems such as the human body such as the maintenance of transmembrane potential and hormone secretion. Therefore, a selective and specific detection and quantification of potassium ions in biological systems would be quite significant. Methods such as fluorescent, electrochemical, and electrical methods allow the selective detection and recognition of K⁺ ions but maybe not sensitive enough to work for biological systems. Therefore, Lui et al. recently reported the development of a guanine-rich DNA aptamer as a highly sensitive and selective biosensor for the detection of K⁺ ions. The formation of guanine-quadruplexes from guanine-rich oligonucleotides that have a strong affinity for capturing K⁺ ions is the basis for this biosensor. 

Figure 1 shows the structural models for the aptamer-potassium complex as well as for the thrombin-aptamer-potassium complex.

Figure 1: Structural model of a thrombin-potassium aptamer complex (PDB ID 4DII, left) and the potassium binding aptamer (right) as reported by Krauss et al. in 2012.

Liu et al. in 2018 used this aptamer for the fabrication of a Hall-effect-based biosensor using single-layer graphene for the detecting of the K⁺ ion. The biosensor was prepared over wafer-scale areas by a catalytic growth technique of chemical vapor deposition (CVD) employing the Van der Pauw technique which allows monitoring multiple electrical properties of graphene films during the detection process.

Other potassium sensing oligonucleotides are also possible. For example, Nojima et al. in 2002 synthesized a potassium sensing oligonucleotide with high selectivity for K⁺ ions as a FRET probe. The researchers connected the fluorophores 6-carboxyfluorescein (6-FAM) and 6-carboxy-tetramethylrhodamine (6-TAMRA) to the oligonucleotide at the 5’- and 3’-terminal ends, respectively, using the DNA sequence GGG TTA GGG TTA GGG TTA GGG for the synthesis. Oligonucleotides carrying this sequence, or a similar one can fold into a tetraplex structure or fold that contains a cavity in which a K⁺ ion can fit in. The presence of the K⁺ ion stabilizes the tetraplex structure of the PSO and FRET can occur between the two fluorophores. Circular dichroism (CD) revealed that the two fluorophores are not close to each other in the absence of the potassium ion. This PSO is also reported to detect sodium ions but with a much lower sensitivity, ~43,000-times for K⁺ over Na⁺ ions, which suggests that the structure of the tetraplex differs between K⁺ and Na⁺. 

Reference

Russo Krauss I, Merlino A, Randazzo A, Novellino E, Mazzarella L, Sica F. High-resolution structures of two complexes between thrombin and thrombin-binding aptamer shed light on the role of cations in the aptamer inhibitory activity. Nucleic Acids Res. 2012;40(16):8119-28.

Xiangqi Liu, Chen Ye, Xiaoqing Li, Naiyuan Cui, Tianzhun Wu, Shiyu Du, Qiuping Wei, Li Fu, Jiancheng Yin, and Cheng-Te Lin; Highly Sensitive and Selective Potassium Ion Detection Based on Graphene Hall Effect Biosensors. Materials 2018, 11, 399.

Nojima T, Ueyama H, Takagi M, Takenaka S.; Potassium sensing oligonucleotide, PSO, based on DNA tetraplex formation. Nucleic Acids Res Suppl. 2002;(2):125-6.

Takenaka S, Juskowiak B.; Fluorescence detection of potassium ion using the G-quadruplex structure. Anal Sci. 2011;27(12):1167-72.

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As reported by Oshima et al. in 2018, inserting bridged nucleic acids (BNAs) into oligonucleotide probes increase the melting temperature (T_m) of the probes leading to an increase of the delta Tm (Δ Tm) between m⁶A-containing RNA and unmethylated RNA as compared with DNA probes. The insertion of artificial nucleic acids such as BNAs into oligonucleotides increases both binding affinities to the target nucleic acid and sensitivity for detecting a single base mismatch.

A technique called northeastern blotting allows measuring m⁶A modifications in RNA. But this method is less efficient, however recent technological developments allow for a more accurate and comprehensive analysis of these modifications in RNA.

Oshima et al. found that the insertion of bridged nucleic acids (BNAs) into DNA probes increases the difference in melting temperature between N⁶-methyladenosine (m⁶A)-containing RNA and unmethylated RNA. This approach allowed the researcher to quantify methylation efficiency at m6A sites in E. coli 23S rRNA with high accuracy. The design of the fluorescent BNA probes is illustrated in figure 1.

Figure 1: Fluorescent BNA probes for m⁶A RNAs as designed by Oshima et al. (2018).

N6-Methyladenosine (m6A) modifications are relatively abundant modifications in RNA molecules in ribosomal RNA (rRNA), small nuclear RNA (snRNA) and messenger RNA (mRNA) of various species. N6-Methyladenosine (m6A) modifications of RNA occur in eukaryotes in mRNA, rRNA, tRNA, and microRNA and appear to be reversible since demethylation enzymes are known to be present in various species. RNA modifications affect RNA splicing, translation, degradation, and localization. Modified RNAs have significant roles in transcriptional control, but their role in the regulation of diverse physiological pathways is still unclear. The structure of a RNA duplex containing m6A is illustrated in figure 2.

Figure 2: Structure of methylated RNA duplex as reported by Roost et al. in 2015. m⁶A is oriented anti in a paired duplex, with the methyl group in the anti conformation as well. (A) Structure of the 10 bp duplex with methylated adenosines. (B) Structural model of N⁶-Methyladenosine as found in RNA. (C) Base pairs alone of m⁶A/U as found in the duplex.

m⁶A modifications influence many physiological processes such as circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity. m⁶A modifications are thought to control RNA degradation, localization, and splicing. However, their exact role in pathophysiological states is still unclear. Therefore, one can reason that the investigation of m⁶A modifications are essential to provide insights into molecular physiology and molecular pathways influenced by this modification.

.

Reference

Roost C, Lynch SR, Batista PJ, Qu K, Chang HY, Kool ET. Structure and thermodynamics of N6-methyladenosine in RNA: a spring-loaded base modification. J Am Chem Soc. 2015;137(5):2107-15.

Oshima T , Ishiguro K , Suzuki T , Kawahara Y .; Quantification of methylation efficiency at a specific N6-methyladenosine position in rRNA by using BNA probes. Chem Commun (Camb). 2018 Aug 23;54(69):9627-9630. doi: 10.1039/c8cc03713b.

Wang CY, Lin MH, Su HT. A Method for Measuring RNA N 6-methyladenosine Modifications in Cells and Tissues. J Vis Exp. 2016;(118):54672. Published 2016 Dec 5. doi:10.3791/54672

Applications for Bridged Nucleic Acids (BNAs)

BNA Clamping

BNA clamp oligonucleotides inhibit DNA primer extension on a single-stranded template and also arrest reverse transcription on a single-stranded RNA template. Clamp oligonucleotides are useful tools for sequence-specific control of gene expression as well as for a therapeutic agent. Clamping oligonucleotides are single-stranded nucleic acids recognized by a complementary (antisense) oligonucleotide to form a short double helix.

Oligopyrimidine-oligopurine helical formations are recognized by a third clamping oligonucleotide forming triple helices. Two oligonucleotides can be linked to each other such that they form a unique clamp with the target sequence on the single-stranded template (Giovannangeli et al. 1991).

Mutations on the epidermal growth factor receptor (EGFR) cause a variety of cancers including breast and lung cancers. The single mutation T790M on tyrosine kinase domain of EGFR signifies the response to the popular cancer drug gefitinib, which leads to the development of resistance to gefitinib. Detecting the mutation thus guide effective therapeutical options for patients who are in need of cancer drug treatments. BNANC clamping using real-time PCR enables effective detection of the EGFR single mutation T790M. Read the BSI Poster (Detection Method for the EGFR Single Mutation T790M).

Reference

Giovannangeli et al., Single-Stranded DNA as a Target for Triple-Helix Formation, J. Am. Chem. Soc., 113, 7775-7777 (1991).

BNA^NC Gapmers

BNA^NC Gapmers revert splicing and reduce RNA Foci in myotonic dystrophy type 1 (DM1) cells. DM1 is a multisystemic disease caused by an expanded CTG repeat in the 3’-untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) gene. Gapmers targeting within the repetitive region of DMPK preferentially degrades the mutant allele. 

Myotonic dystrophy is the most common type of muscular dystrophy in which the expanded CUG repeat RNA (CUG_exp RNA) is retained in the nucleus where it forms RNA foci leading to defects in regulated alternative splicing events during development. However, if the development of CUG expanded repeat RNA foci could be prevented, the disorder will not materialize. Manning et al. used the antisense BNA gapmer strategy to degrade CUG expanded repeat (CUG_exp) RNA in immortalized human TeloMyoD fibroblast cell lines expressing telomerase and containing a tetracycline-inducible MyoD to promote the myogenic program in response to growth. Visualization of RNA was achieved using a FISH probe targeting the repeat RNA of the RNA foci in the untreated and treated cells.

BNA^NC gapmers targeting within the repetitive region of DMPK preferentially degrade the mutant allele thereby decreasing RNA foci. Manning et al. were able to show that antisense BNA^NC gapmers could be used to potentially revert splicing defects in myotonic dystrophy type 1 (DM1) cells.

Reference

BNA Gapmers

Daguenet E, Dujardin G, Valcárcel J. The pathogenicity of splicing defects: mechanistic insights into pre-mRNA processing inform novel therapeutic approaches. EMBO Rep. 2015;16(12):1640-55.

Manning KS, Rao AN, Castro M, Cooper TA. BNA^NC Gapmers Revert Splicing and Reduce RNA Foci with Low Toxicity in Myotonic Dystrophy Cells. ACS Chem Biol. 2017;12(10):2503-2509. 

Antisense BNA-Peptide Conjugates

Antisense BNA/DNA-peptide-conjugates allow the treatment of drug-resistant infections.

The opportunistic patogen, Acinetobacter baumannii, primarily found in hospital-acquired infections has developed multi-resistance. The presence of the aac(6’ )-Ib gene give the pathogen resistance to amikacin and other aminoglycosides wich severely limits the effectiveness of these antibiotics.

Lopez et al. designed an antisense oligodeoxynucleotide (ODN4) that binds to a duplicated sequence on the aac(6’)-Ib mRNA, overlapping the initiation codon, which efficiently inhibited translation in vitro. A nuclease-resistant hybrid oligomer composed of 2’, 4’ -bridge nucleic acid-NC(BNANC) residues and deoxynucleotides (BNA-NC/DNA) conjugated to the permeabilizing cell-penetrating peptide (RXR)4XB (CPPBD4) inhibited translation in vitro at the same levels observed when testing ODN4.

Reference

Lopez, Christina, Arivett, Brock A., Actis, Luis A., and Tolmasky, Marcelo E.; Inhibition of AAC(6’)-Ib-Mediated Resistance to Amikacin in Acinetobacter baumannii by an Antisense Peptide-Conjugated 2’ ,4’ -Bridge Nucleic Acid-NC (BNA-NC)/DNA Hybrid Oligomer. doi:10.1128/AAC.01304-15. Antimicrobial Agents and Chemotherapy.

CRISPR-Cas9 based Gene-Editing = CRISPR-BNAs

Adding BNAs (2′,4′-BNA^NC[N-Me]) to CRISPR-RNAs (crRNAs) dramatically improves the accuracy in CRISPR-based gene-editing. The replacement of natural crRNA molecules with synthetic crRNAs containing bridged nucleic acids, or BNAs, enhances the binding affinity to crRNAs.

Off-target effects or off-target cutting and the generation of additional mutations remain a significant barrier for using Cas9-based gene editing methods. BNA modified crRNAs improve the specificity of the CRISPR-Cas9 system and illustrate the power of recently developed synthetic nucleic acid technologies to solve problems in enzyme specificity as well.

Reference

Cromwell, Christopher R., Sung, Keewon, Park, Jinho, Krysler, Amanda R., Jovel, Juan, Kim, Seong Keun, and Hubbard, Basil P.; 2018. Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity. Nature Communications 9, 1448.

BNAs as Molecular Tools

Molecular diagnostics is essential for drug development. Therefore, molecular tools are needed for identifying infections, screening of cancer or tumors, hepatitis, genetic disorders, and tissue screening to minimize the risks of tissue rejection.

Also, to allow utilization of available sequence information from genomic data efficiently, molecular tools are needed for the investigation of the molecular biology governing metabolic pathways in various species, including humans. Molecular diagnostic tools are particularly valuable for the detection of bacterial bloodstream infections. BNAs lend themselves as building blogs for the development of next-generation molecular tools.

BNAs as molecular tools.

Molecular diagnostic tools for bacterial bloodstream infections.

Tools for RNA targeting.

Genomic and Proteomic Tools.

Reference

Jean Pierre Rutanga and Therese Nyirahabimana, “Clinical Significance of Molecular Diagnostic Tools for Bacterial Bloodstream Infections: A Systematic Review,” Interdisciplinary Perspectives on Infectious Diseases, vol. 2016, Article ID 6412085, 10 pages, 2016. https://doi.org/10.1155/2016/6412085.

Insertion of bridged nucleic acids (BNAs) into DNA probes increases the difference in melting temperature between N6-methyladenosine (m6A)-containing RNA and unmethylated RNA. This approach allows quantification of methylation efficiency at m6A in RNAs with high accuracy.

Inserting bridged nucleic acids (BNAs) into oligonucleotide probes increase the melting temperature (Tm) of the probes leading to an increase of the delta Tm (Δ Tm) between m6A-containing RNA and unmethylated RNA as compared with DNA probes. 

Reference

Oshima T , Ishiguro K , Suzuki T , Kawahara Y .; Quantification of methylation efficiency at a specific N6-methyladenosine position in rRNA by using BNA probes. Chem Commun (Camb). 2018 Aug 23;54(69):9627-9630. doi: 10.1039/c8cc03713b.

Rapid Mutation Detection with BNAs

The enzyme DNA methyltransferase 3A (DNMT3A) methylates DNA by catalyzing the transfer of methyl groups to specific CpG regions in DNA. DNMT3A is point mutated in many myeloid malignancies, but the mutation frequency varies between different entities. For example, in adult acute myeloid leukemia (AML) DNMT3A mutations are found in 14–34% of cases from different series, 5–15% of MDS cases, 10% of chronic myelomonocytic leukemia (CMML) patients, 5.7% of primary myelofibrosis (PMF) patients, 12% of cases with systemic mastocytosis, and in approximately 18% of T cell acute lymphoblastic leukemia (T-ALL) cases.

Shivarov et al. in 2014 showed for the first time that BNA(NC)-modified probes can be used for the quantitative detection of DNMT3A R882 mutations using bead-based suspension assays. This assay can be successfully implemented in the diagnostics for patients with myeloid malignancies, as it is rapid, and reliable regarding specificity and sensitivity.

Reference

Shivarov V, Ivanova M, Naumova E (2014) Rapid Detection of DNMT3A R882 Mutations in Hematologic Malignancies Using a Novel Bead-Based Suspension Assay with BNA(NC) Probes. PLoS ONE 9(6): e99769. https://doi.org/10.1371/journal.pone.0099769.

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In Drosophila,  A’-form RNA helices drive microtubule-based mRNA transport

A cell uses microtubule-based mRNA transport to restrict protein expression to a specific location within the cell. The exact location of proteins in cells defines cell polarity, axis determination, and neuronal function. RNA localization coupled to translational repression creates structural and functional asymmetry within cells. Cytoskeletal elements such as microtubules and actin filaments associated with mechanoenzymes mediate the transport of a significant fraction of transcripts. The best-studied model systems of RNA localization are the oocyte and the early embryo in Drosophila melanogaster.

In eukaryotes, asymmetric localization of mRNAs plays widespread roles in protein targeting important for cellular processes such as patterning of embryonic axes, polarized cell functions, and synaptic plasticity. To understand the structural basis of recognition of cis-acting mRNA location signals by motor complexes Bullock et al. in 2010 used NMR spectroscopy for the determination of the tertiary structure of an RNA element responsible for mRNA transport. 

Figure 1: Surface model of the solution structure of K10 TLS RNA.

According to Bullock et al. the K10 TLS RNA adopts a stem-loop structure capped by an octanucleotide loop (5′-A(18)UUAAUUC(25)-3′) displaying a compact fold. The helical part of the TLS can consist of three regions: an upper helix composed of seven Watson-Crick A-U or U-A base pairs, a middle helix of three Watson-Crick base pairs, flanked at each end by single nucleotide bulges on the 3′ side, and a lower helix consisting of a G-U and six Watson-Crick base pairs. The two unpaired bases adopt different orientations relative to the helices, and the base moiety of C33 resides in the major groove maintains the helical twist between the adjacent base pairs. The base of A37 is stacked in between the middle and the lower helix and increases the helical twist between the adjacent base pairs.

Reference

Bullock SL, Ringel I, Ish-Horowicz D, Lukavsky PJ. A'-form RNA helices are required for cytoplasmic mRNA transport in Drosophila. Nat Struct Mol Biol. 2010;17(6):703-9. 

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Incorporation of EdU or 5-ethynyl-2′-deoxyuridine allows the sensitive detection of DNA synthesis in cells using “click chemistry."

In 2008, Salic and Mitchinson reported the EdU based method for a fast and sensitive detection of DNA synthesis in vivo.  No sample fixation or DNA denaturation is required and biological structures are preserved as well.

As reported, the process works in cultured cells, the intestine, and brain of whole animals in which the cells incorporate 5-ethynyl-2'-deoxyuridine (EdU) into replicating DNA. After incorporation, the terminal alkyne group is available for the reaction with organic azides using Cu(I)-catalyzed click reactions.

Figure 1: Schematics of the copper (I) catalyzed click chemistry.

Salic and Mitchinson found that EdU incorporated into the DNA of proliferating mammalian cells, for example, into DNA replicated in vitro in Xenopus egg extracts by addition to the cycling extract preparations. Varying ascorbic acid and fluorescent azide concentrations allow controlling the amount of the fluorescent signal generated during EdU detection.

Salic and Mitchinson also indicate that the EdU-labeling method is well suited for optical imaging of cellular DNA at a nanometer resolution.

In organisms, genetic mutations can arise in fertilized eggs as well as in subsets of cells. Somatic mosaicism refers to the presence of two genetically distinct populations of cells within an individual originating from postzygotic mutations. In humans, these somatic mutations are not transmitted to children but can affect different genomic sizes ranging from single nucleotides to entire chromosomes. Medical researchers now think that somatic mosaicism can cause cancer, neurodegenerative, as well as monogenic, and complex diseases.

Figure 1. C99. C99 is the transmembrane carboxyl-domain of the amyloid precursor protein (APP) cleavaged by ϒ-secretase releasing amyloid-beta polypeptides (Barrett et al. 2012). 

Reference

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RNAi using 2’,4’-BNA^NC[NH] nucleic acid analogues

RNA interference (RNAi) or gene silencing by double-stranded RNA, was discovered in 1998 and in 2006 the Nobel Prize in Physiology or Medicine was awarded to Andrew Z. Fire and Craig C. Mello for this discovery. Since then it has emerged as a vital and powerful molecular biological tool for the regulation of gene expression and is now considered to have enormous potential for the treatment of a range of diseases. Since advancements in bioengineering and nanotechnology have led to improved control of delivery and release of siRNA therapeutics, several RNAi-based therapeutics are now in Phase II and Phase III clinical trials.

RNAi is thought to be a natural defense mechanism that has evolved for the protection of organisms from RNA viruses. Cells can recognize double-stranded RNA (dsRNA) as an intruder. When this happens, the enzyme Dicer is recruited to cut the foreign RNA into smaller pieces called siRNA. These RNA pieces consist of approximately 22 nucleotides in length. One strand of the siRNA then binds to a target viral mRNA in a sequence-specific manner creating a signal for the destruction of the mRNA. The result is the interference with the further production of the viral proteins needed for a virus to replicate so that the RNA interference mechanism interferes with the expression of a particular gene that shares a sequence with the dsRNA that is homologous to that gene. 

Mechanism of RNA interference.

RNAi technologies utilize short double-stranded RNA (dsRNA) of approximately 21 base pair length with a two nucleotide (nt) 3’-overhang for the silencing of genes. These dsRNAs are generally called small interfering RNA (siRNA). siRNA 12 to 22 nucleotides in length are the active agent in RNAi. The siRNA duplex serves as a guide for mRNA degradation. Upon siRNA incorporation into the RNA-induced silencing complex (RISC) the complex interacts with a specific mRNA and ultimately suppresses the mRNA signal. The sense strand or passenger strand of siRNA is typically cleaved at the 9th nucleotide downstream from the 5’-end of the sense strand by Argonaute 2 (Ago2) endonuclease. The activated RISC complex containing the antisense strand or guide strand binds to the target mRNA through Watson–Crick base pairing causing degradation or translational blocking of the targeted RNA.

However, the in-vivo use of RNAi or siRNA as a drug has remained difficult due to obstacles encountered such as low biostability and unacceptable toxicity possibly caused by off-target effects. Various types of chemical modifications to improve the pharmacokinetics and to overcome bio-instability problems have been investigated over the years to improve the stability and specificity of the RNAi duplexes. In some cases, the chemical modification in siRNAs has improved the serum stability of siRNAs. However, often RNAi activity was lost, but the careful placement of some specific modified residues enables enhanced siRNA biostability without loss of siRNA potency. Some of these particular modifications have reduced siRNA side effects, such as the induction of recipient immune responses and inherent off-targeting effects and have even enhanced siRNA potency. Various chemically modified siRNAs have been investigated, among them were bridged nucleic acids such as 2’,4’-methylene bridged nucleic acid 2’,4’-BNAs, also known as locked nucleic acid or LNA. Some of these modified siRNAs showed promising effects.

BNA nucleic acid analogues are useful tools for gene silencing using RNAi

Rahman et al. in 2010 investigated the effect of both 2’,4’-BNA and 2’,4’-BNA-NC modifications in gene silencing experiments using RNAi technology.

The scientists observed that 2’,4’-BNAs (LNAs) and 2’,4’-BNA-NCs are equally effective in RNAi activity if incorporated appropriately, especially in the sense strand of siRNA.

Design examples of BNA nucleic acid analogs useful for gene silencing by RNAi to inhibit the expression of firefly luciferase in CHO-luc cells. Modifications of the sense strand consecutively up to five BNA residues might be tolerable.

Native sense strand:

5’-CTTACGCUGAGUACUUCGATT-3’ 

Native antisense strand:

3’-TTGAAUGCGACUCAUGAAGCU-5’

Duplexes formed:

  5’-CTTACGCUGAGUACUUCGATT-3’

3’-TTGAAUGCGACUCAUGAAGCU-5’

  5’-GCUGAGUACUUCGAAAUGUTT-3’

3’-TTCGACUCAUGAAGCUUUACA-5’     

Modifications with BNAs that worked the best

  5’-CTTACGCUGAGUACUUCGATT-3’ Tm = 83 °CSense Strand

3’-TTGAAUGCGACUCAUGAAGCU-5’           Antisense Strand

  5’-CTTACGCUGAGUACTUCGATT-3’ Tm = 87 °C

3’-TTGAAUGCGACUCAUGAAGCU-5’

  5’-CTTACGCUGAGTACTUCGATT-3’ Tm = 86 °C

3’-TTGAAUGCGACUCAUGAAGCU-5’

  5’-GCTGAGTACUTCGAAAUGTTT-3’ Tm = 86 °C

3’-TTCGACUCAUGAAGCUUUACA-5’

  5’-GCTGAGTACUUCGAAATGUTT-3’ Tm = 86 °C

3’-TTCGACUCAUGAAGCUUUACA-5’

Design Rules

The design rules or guidlines for the design of highly effective RNAi indicate that dsRNA used for RNAi should follow the following four design parts at the same time:

(i)     A/U at the 5’-end of the antisense strand,

(ii)    G/C at the 5’-end of the sense strand,

(iii)   AU-richness in the 5’-terminal third of the antisense strand, and

(iv)   Absence of any GC stretch over 9 base pairs in length. (See figure below).

(v)    Incorporation of BNAs at strategic positions.

These guidelines appear related to the molecular mechanism of RISC assembly as reported by Ui-Tei et al. in 2004. The first three guidelines together with adding BNAs to the sense strand may allow the 5’-end of the antisense strand of siRNA to be situated at or near the thermodynamically less stable siRNA duplex end. 

Structures of highly effective siRNA

Please also review the following papers for more details.

Reference

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.; Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature. 1998 Feb 19;391(6669):806-11.

S. M. Abdur Rahman, Hiroyuki Sato, Naoto Tsuda, Sunao Haitani, Keisuke Narukawa, 

RNAi: RISC Gets Loaded

Ui-Tei,K., Naito,Y., Takahashi,F., Haraguchi,T., Ohki-Hamazaki,H., Juni,A., Ueda,R. and Saigo,K. (2004) Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference. Nucleic Acids Res., 32, 936–948.

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In Drosophila,  A’-form RNA helices drive microtubule-based mRNA transport

A cell uses microtubule-based mRNA transport to restrict protein expression to a specific location within the cell. The exact location of proteins in cells defines cell polarity, axis determination, and neuronal function. RNA localization coupled to translational repression creates structural and functional asymmetry within cells. Cytoskeletal elements such as microtubules and actin filaments associated with mechanoenzymes mediate the transport of a significant fraction of transcripts. The best-studied model systems of RNA localization are the oocyte and the early embryo in Drosophila melanogaster.

In eukaryotes, asymmetric localization of mRNAs plays widespread roles in protein targeting important for cellular processes such as patterning of embryonic axes, polarized cell functions, and synaptic plasticity. To understand the structural basis of recognition of cis-acting mRNA location signals by motor complexes Bullock et al. in 2010 used NMR spectroscopy for the determination of the tertiary structure of an RNA element responsible for mRNA transport. 

Figure 1: Surface model of the solution structure of K10 TLS RNA.

According to Bullock et al. the K10 TLS RNA adopts a stem-loop structure capped by an octanucleotide loop (5′-A(18)UUAAUUC(25)-3′) displaying a compact fold. The helical part of the TLS can consist of three regions: an upper helix composed of seven Watson-Crick A-U or U-A base pairs, a middle helix of three Watson-Crick base pairs, flanked at each end by single nucleotide bulges on the 3′ side, and a lower helix consisting of a G-U and six Watson-Crick base pairs. The two unpaired bases adopt different orientations relative to the helices, and the base moiety of C33 resides in the major groove maintains the helical twist between the adjacent base pairs. The base of A37 is stacked in between the middle and the lower helix and increases the helical twist between the adjacent base pairs.

Reference

Bullock SL, Ringel I, Ish-Horowicz D, Lukavsky PJ. A'-form RNA helices are required for cytoplasmic mRNA transport in Drosophila. Nat Struct Mol Biol. 2010;17(6):703-9. 

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In organisms, genetic mutations can arise in fertilized eggs as well as in subsets of cells. Somatic mosaicism refers to the presence of two genetically distinct populations of cells within an individual originating from postzygotic mutations. In humans, these somatic mutations are not transmitted to children but can affect different genomic sizes ranging from single nucleotides to entire chromosomes. Medical researchers now think that somatic mosaicism can cause cancer, neurodegenerative, as well as monogenic, and complex diseases.

Figure 1. C99. C99 is the transmembrane carboxyl-domain of the amyloid precursor protein (APP) cleavaged by ϒ-secretase releasing amyloid-beta polypeptides (Barrett et al. 2012). 

Reference

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The Hepatitis B virus (HBV) causes liver infection, but for some people, it is just an acute or short-term infection, however, for others, it can become a long-term, chronic infection called chronic hepatitis.

Chronic Hepatitis B can lead to serious health issues. Vaccination against HBV is the best way to prevent infections with HBV. However, infections with HBV remain a top health problem worldwide. In 2013 alone, HBV infections caused approximately 600,000 deaths. According to Papastergiou et al., more than 350 million people are chronically infected with HBV.

Detection and measurements of HBV DNA levels are now routinely used for the identification of infectious, chronic carriers and the prediction and monitoring of efficacies of antiviral treatment.

Real-time PCR (RT PCR) is now widely accepted as the gold standard for quantification of viral nucleic acids. RT PCR allows quantification of HBV DNA with improved speed of detection, high sensitivity, and reproducibility, as well as a low risk of contamination. Several real-time PCR assays have been developed usually using two pairs of primers and probes often based on the conserved S and C regions of the HBV genome.

Primers used for the amplification of HBV complete genomes 
 

^aSuffix “F” indicates forward primer, whereas suffix “R” indicates reverse primer.

^bWith reference to NC_003977. https://www.ncbi.nlm.nih.gov/nuccore/NC_003977Hepatitis B virus (strain ayw) genome

^cAn annealing temperature of 54°C was used during sequencing cycling to ensure a high rate of success of sequencing.

The innate immune system discriminates between self and non-self mainly based on receptors recognizing non-self molecules present in pathogens, but not present in the host. Recent observations showed that inosine present in the epitranscriptome and the adenosine deaminases acting on RNA (ADAR1) protein establishes innate immune tolerance for endogenous host double-stranded RNA. So far over a hundred (100) million adenosine to inosine editing sites have been identified in the human transcriptome.

The term “transcriptome” refers to the sum of all the messenger RNA molecules expressed from the genes of an organism. However, the term “epitranscriptome” refers to all biochemical modifications of RNA within a cell. 

ADARs are editing enzymes converting adenosine (A) to inosine (I) in duplex RNA. This modification has wide-ranging consequences on RNA function.

Structure of hADAR2d E488Q bound to the Bdf2-C RNA duplex. Matthews et al. 2016. Matthews et al. in 2016 reported four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures help explaining the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. Studying these structures may help to increase our understanding of the effects of ADAR mutations associated with human disease.


Inosine sites are mostly found in embedded Alu sequences which form potentially innate immune-stimulating dsRNA hairpins in transcripts. The nucleotide inosine is commonly found in tRNAs and is formed when hypoxanthine attaches to a ribose ring via a β-N9-glycoside bond.

Viral nucleic acids are detected by innate immune sensors that act as readers of epitranscriptome RNA modifications. 'A-to-I editing' can alter a protein's coding sequence. In humans, this is crucial for keeping the innate immune response in check.

Inosine can pair with any natural base allowing the design of oligonucleotide primers spanning target sequences containing single nucleotide polymorphisms (SNPs). The use of inosine-containing amplification primers allows the amplification of a specific DNA fragments from different alleles.

Because inosine pairs preferentially with cytidine (C) its introduction to RNA via the action of ADARs destabilizes double-stranded RNA by changing AU base-pairs to IU mismatches.

Reference

PCR based methods as well as in-situ hybridization multiplexing methods when used for pathogen detection allow the simultaneous analysis of multiple nucleic acid sequences in a single reaction. Multiplexing can potentially save a lot of time, cost and labor but requires precise work and often involves many complicated steps. The design of general primers enables the development of universal methods for the detection of multiple species during a single analysis run.

Real-time PCR (quantitative PCR, qPCR) is now a very well-established detection, quantification, and typing method for different microbial agents in a variety of diagnostic settings. However, to make PCR successful, there are specific issues in qPCR that developers and users of this technology must address. Kralik and Ricchi in 2017 published an essential guide that describes the use of the correct terminology and definitions, the understanding of the principle of PCR, difficulties with interpretation and the presentation of data, as well as the limitations of qPCR in different areas of microbial diagnostics and parameters necessary for qPCR performance.

Oligonucleotides modified with bridged nucleic acid (BNAs) residues have stronger affinities for their complementary targets than natural nucleic acid bases and are known to avoid dimer formation and mismatch hybridization. The use of BNAs achieving enhanced and efficient priming.

Barghouthi SA in 2011 reported the development of a universal method allowing the detection of 101 tested isolates by producing one or more PCR products from each isolate. Sequencing of strains and aligning the resulting sequences using BLAST enabled the identification of the detected bacterial species.

To enable detection of microbes in the gut of animals and possibly humans, Guillen et al. in 2016 showed that 16S primer sets enabled the analysis of bacteria in the colon of rats. The use of selected universal primers provided knowledge of the abundance of microorganisms and the bacterial diversity in rat colon biopsies.

Sun et al. in 2008 showed that oligonucleotide pentamer primer pairs modified with BNAs enabled the amplification of genomic DNA demonstrating that the use of bridged nucleic acid pentamers as universal primer pairs together with suspension array genotyping allowed the identification of multiple distinct genes or species with a single amplification procedure. Sun et al. thereby demonstrated that BNA based modified pentamer-based PCR are very useful for pathogen detection and identification. Furthermore, Levin et al. in 2006 were able to show that the incorporation of BNAs at the 5’-prime end of PCR primers increased the Phred Q30 score during DNA sequencing dramatically.

Biosynthesis Inc. offers BNA modified oligonucleotides.

Reference

Barghouthi SA. A universal method for the identification of bacteria based on general PCR primers. Indian J Microbiol. 2011;51(4):430-44. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3209952/

Guillen IA, Camacho H, Tuero AD, et al. PCR Conditions for 16S Primers for Analysis of Microbes in the Colon of Rats. J Biomol Tech. 2016;27(3):105-12. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4920503/

Kralik P, Ricchi M. A Basic Guide to Real Time PCR in Microbial Diagnostics: Definitions, Parameters, and Everything. Front Microbiol. 2017;8:108. Published 2017 Feb 2. doi:10.3389/fmicb.2017.00108. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5288344/

Joshua D. Levin, Dean Fiala, Meinrado F. Samala, Jason D. Kahn, Raymond J. Peterson; Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers, Nucleic Acids Research, Volume 34, Issue 20, 1 November 2006, Pages e142, https://doi.org/10.1093/nar/gkl756

Lopez I, Ruiz-Larrea F, Cocolin L, et al. Design and evaluation of PCR primers for analysis of bacterial populations in wine by denaturing gradient gel electrophoresis. Appl Environ Microbiol. 2003;69(11):6801-7. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC262258/

Sun Z, Chen Z, Hou X, Li S, Zhu H, Qian J, et al. (2008) Locked Nucleic Acid Pentamers as Universal PCR Primers for Genomic DNA Amplification. PLoS ONE 3(11): e3701. https://doi.org/10.1371/journal.pone.0003701. https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0003701

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Toehold probes utilize the thermodynamic properties of specifically designed oligonucleotides for specific detection of a target sequence. Correctly designed toehold probes selectively detect the correct target and prevent the non-specific detection of target sequences that differ by a single non-matching nucleotide.

DNA oligonucleotides containing bridged nucleic acids (BNAs) or locked nucleic acids (LNAs) are known to bind selectively to target DNA at room temperature. These oligonucleotides include a toehold sequence binding at the end of the target allowing for selective enrichment and purification of DNA fragments.

Force spectroscopy revealed that probe-target hybrids are sufficiently stable to withstand forces over 65 pN allowing determination of force-extension curves. However, the incorporating of BNA nucleotides into toehold probes has limitations since this is sequence dependent thereby limiting the number of BNAs per oligonucleotide probe sequence.

Also, depending on the sequence, oligonucleotides with a high BNA content are prone to self-hybridization. Therefore the trend for self-hybridization of the oligonucleotides will need to be checked with an oligonucleotide design tool found on several websites.

Reference

Toeholds:  https://www.ncbi.nlm.nih.gov/pmc/?term=toehold

Hermans, Nicolaas; Huisman, Juriën Jori; Brouwer, Thomas Bauke; Schächner, Christopher; van Heusden, G. Paul H.; Griesenbeck, Joachim; van Noort, John; Toehold-enhanced LNA probes for selective pull down and single-molecule analysis of native chromatin. (2017) Scientific Reports 7(1) 16721.  https://www.nature.com/articles/s41598-017-16864-7

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qPCR is a very useful method for the detection and measurement of bacterial abundance in environmental samples. Using internal standards allows comparison of detected amounts of target amplicon with the internal standards. Internal standards can be prepared either from cloned bacterial DNA or as synthetic DNA blocks using automated solid phase oligonucleotide synthesis. This approach circumvents the problem of differential amplification when using dissimilar targets and standard amplicons. The small subunit ribosomal RNA (SSU rRNA) genes used as target sequences enable the unique identification of bacteria in a sample. Different internal standards are needed to allow estimation of amounts of different bacteria.

In general, PCR typically results in a 105 to 106-fold amplification of target DNA when 20 or 30 PCR cycles are used allowing the design of very sensitive detection and quantification method. The use of general primers amplifies almost all SSU rRNA sequences. However, if unique PCR primers are chosen, only a single species or a small group of bacterial SSU rRNA sequences in the sample will be amplified allowing for selective detection of bacterial species.

PCR is very sensitive which in theory is capable of detecting a single target molecule in samples, but amplification usually saturates during the later cycles. Therefore, the amount of specific sequences in the final amplification product is not always proportional to the amount of the source DNA. Since different sequences can be amplified to different extents, their abundance in the final PCR product does not correlate with their abundance in the original sample; hence quantitative PCR (qPCR) methods are required for the determination of specific SSU rDNA sequences in real samples. Also, the 16S rRNA genes have been used extensively for detection and identification of bacterial species; however, if sequence information on the 23S rRNA gene and the internal transcribed spacer (ITS) region that lies between the 16S and 23S rRNA genes is available, these sequence stretches can also be used as target sequences.

To overcome limitations of sequence discrimination, for example, to prevent the amplification of host sequences when amplifying bacterial DNA, primers containing bridged nucleic acids (BNAs) at specific locations are useful for enhanced amplification of specific target DNA sequences. Another approach is the use of PCR clamping using BNA oligonucleotides for improved and selective amplification of species-specific target DNA.

Molecular probes and primer sets enable the qPCR method. FRET-probes that contain a reporter and a quencher molecule, often using a 20 to 30 base pair long oligonucleotide between the fluorophore and the quencher molecule, enabling quantitative PCR. qPCR or TaqManTM probes often contain a 6-FAM reporter dye and a TAMRA quencher; however, other reporter-quencher pairs can be used as well for probe design and synthesis.

Example of how to design and construct a qPCR primer/probe set

•    Select the probe and primer to hybridize to a region unique to the targeted DNA with the probe having a Tm that is 10°C higher than the primers.

•    An online primer design tool can be used and found at https://molbiol-tools.ca/PCR.htm.

•    Design a probe sequence specific for the selected bacterial strain for detection.

•    Aim for a sequence in length of 29 base pairs with a target Tm around 70°C.

•    Select the reporter dye and quencher, for example 6-carboxyfluorescein (6-FAM)  at the 5’-end as the reporter dye and carboxyltetramethylrhodamine (TAMRA) as the quencher at the 3’-end.

•    Select the primer sequences such that they flank the probe.

•    Select the forward primer be around 25 base pairs in length and to have a Tm of approximately 60°C.

•    Select the reverse primer to be approximately 23 base pairs in length and to have a Tm of approximately 59°C.

•    Select the primer/probe set to span approximately 90 to 100 base pairs.

•    Include a normalizing standard in the PCR assay to correct for any inhibitors present in the sample DNA.

•    Use a synthetic oligonucleotide sequence or a cloned oligonucleotide as the internal standard.

•    In principle, any sequence not found in the sample is useful as an internal standard.

•    Construct a control primer/probe set for the internal standard as well with an alternative fluorochrome as the reporter dye.

•    Primers from the normalization sample are selected such that they do not amplify any sequences present in the samples tested.

•    The results will be consistent with the composition of the samples tested when sequences in the test samples as well as the internal standard are amplified with both the target primer/probe set and the normalization primer/probe set.

In summary, the design of a qPCR assay involves the selection and construction of internal standardizing sequences and the design of primer pairs plus two fluorescent probes.

Reference

Brunk CF, Li J, Avaniss-Aghajani E.;  Analysis of specific bacteria from environmental samples using a quantitative polymerase chain reaction. Curr Issues Mol Biol. 2002 Jan;4(1):13-8. [pubmed].

Ikenaga M, Sakai M. Application of Locked Nucleic Acid (LNA) oligonucleotide-PCR clamping technique to selectively PCR amplify the SSU rRNA genes of bacteria in investigating the plant-associated community structures. Microbes Environ. 2014;29(3):286-95. [pmc].

Si Ming Man, Nadeem O. Kaakoush, Sophie Octavia, Hazel Mitchell; The Internal Transcribed Spacer Region, a New Tool for Use in Species Differentiation and Delineation of Systematic Relationships within the Campylobacter Genus. Appl. Environ. Microbiol. May 2010, 76 (10) 3071-3081; DOI: 10.1128/AEM.02551-09. https://aem.asm.org/content/76/10/3071.

Priya NG, Pandey N, Rajagopal R. LNA probes substantially improve the detection of bacterial endosymbionts in whole mount of insects by fluorescent in-situ hybridization. BMC Microbiol. 2012;12:81. Published 2012 May 24. doi:10.1186/1471-2180-12-81. [pmc].

Friedrich V. Wintzingerode, Ulf B. Göbel, Erko Stackebrandt; Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis, FEMS Microbiology Reviews, Volume 21, Issue 3, 1 November 1997, Pages 213–229, https://doi.org/10.1111/j.1574-6976.1997.tb00351.x, [femsre]

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What are splice-switching oligonucleotides (SSOs)?

Splice-switching oligonucleotides are short, synthetic, oligonucleotides that act as antisense oligonucleotides. Oligonucleotides containing modified nucleic acids, for example bridged nucleic acids (BNAs), base-pair with pre-mRNA with high affinity and disrupt normal splicing of transcripts by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and pre-mRNAs. Splice-switching antisense oligonucleotides containing a backbone modified with 2’-O-methyl-phosphorothioate groups and approximately 60% BNAs, as well as two BNA-modified nucleotides at the 3’-end and one BNA-modified nucleotide at the 5’-end appear to work well for controlling and modulating the expression of specific exons hence they can act as antisense oligonucleotides enabling modulation and regulation of splicing events.

Exon skipping oligonucleotides can be designed rationally. Design approaches involve the selection of an antisense sequence targeting the exon that is supposed to be skipped.  Design parameters to be taken into account include the activity of the splice-switching oligonucleotide, the melting temperature, the guanine-cytosine content, the length of the oligonucleotide, as well as the secondary structure or sequence motif corresponding to a splicing signal of the target RNA that influence the activity of the splice-switching oligonucleotide. Since the synthesis of modified oligonucleotides is now routinely done in an automated fashion, splice-switching oligonucleotides, containing modified or unmodified nucleic acid, are now commercially available.

What is splicing? 

According to the NCI Dictionary of Genetics Terms “splicing” is defined as the “process by which introns, the noncoding regions of genes, are excised out of the primary messenger RNA transcript, and the exons (i.e., coding regions) are joined together to generate mature messenger RNA. Mature mRNA then serves as the template for synthesis of a specific protein.” Proper expression of most protein-coding genes requires the splicing of pre-mRNA. In other words, splicing refers to the editing of newly transcribed pre-messenger RNA (pre-mRNA), the removal of introns and the joining or ligation of exons. Genes encoded in the nucleus are spliced within the nucleus either during or after transcription.

Illustration explaining the process of “Alternative, or Differential Splicing.” As a result of splicing a single gene can code for multiple proteins. During alternative splicing a specific exons of a gene may be included within or excluded from the final messenger RNA (mRNA) produced from that gene.

Reference

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