Towards development of quencher-free oligonucleotide probe with single base-discriminating fluorophore enhanced by bridged nucleic acid (BNA) for genotyping SNP (single nucleotide polymorphism) linked to cancer and other diseases

Single nucleotide polymorphisms (SNP) refers to nucleotide variations affecting a specific base position in the genome.  SNPs occurring in the coding region could either be ‘synonymous’ (does not alter amino acid sequence) or ‘nonsynonymous’ (changes polypeptide sequence) type.  SNPs located in the noncoding region can also affect gene expression by altering transcription, splicing or degradation of mRNA.  SNPs associated with the risk susceptibility for various disorders including Alzheimer’s disease, cystic fibrosis, sickle cell anemia or alcoholism have been identified.  For instance, a SNP in intron 32 of DMD gene deactivates splice donor site (Thi Tran et al., 2005).

The genetic susceptibility to cancer is associated with various SNPs linked to the genes regulating cell cycle, DNA repair, metabolism, immunity, etc (Deng et al., 2016).  SNPs in promoters may affect TATA box function (EDH17B2 gene), transcription factor binding (MDM2, MMP1, survivin  genes), epigenetic regulation (BRCA1 gene) or histone modification.  Nonsynonymous SNPs in exons may promote tumorigenesis (oncogenic mutation of p53) or decrease therapeutic efficacy by eliminating targets (EGF receptor) by altering their conformation.  Synonymous SNPs in exons can affect the stability, splicing or structure of RNA (stem-loop formation of COMT mRNA; MDR1 gene).  SNPs in introns were shown to affect ‘transcriptional silence element’ (elevates FGFR2 expression in breast cancer) or alter splicing (histocompatibility antigen in renal cell carcinoma).  While SNPs in 5’-UTR (untranslated region) may affect mRNA processing, translation, etc. (CDKN2A gene in melanoma), 3’-UTR SNPs may affect degradation, polyadenylation, etc. (affect micro-RNA regulation of estrogen receptor in breast cancer).   

The development of SNP diagnostics has been guided by the need for high-throughput data acquisition and simplicity, i.e. easy to perform, low cost, portable, accuracy.  To accommodate these concerns, current methods utilize high-throughput sequencing as well as PCR.  In the case of C/T detection, the SNP genotyping methods used in high-throughput assays include ‘flap endonuclease discrimination’ that involves cleavage of fluorescent probe from the triplex structure, ‘allele specific hybridization’ wherein quencher (for the probe) is removed during amplification, ‘primer extension’ which involves single base extension of fluorescent dideoxynucleotide at the polymorphic site.  Other SNP diagnostic methods are ‘allele specific digestion’ requiring cleavage of PCR extended product incorporating dUTP by uracil-DNA glycosylase, and ‘oligonucleotide ligation’ where ligation of two oligonucleotides occurs only if one is complementary to SNP (Jenkins et al., 2002).  

                    

Several innovations are being introduced to further increase the sensitivity of SNP detection.  Molecular beacons have found great utility in detecting SNPs through PCR.  It consists of stem and loop structure with a quencher probe juxtaposed to a fluorophore to dampen its signal, which becomes restored through dissociation upon hybridization to target complementary strand.  Nevertheless, it requires dual labels that could contribute to the cost.  To improve, quencher-free probes that could distinguish the type of base in the opposite strand are being developed, i.e. probes whose fluorescence intensity changes upon hybridization to target strand (Hwang, 2018).  For instance, the quenched fluorescence of a fluorophore (ex. BODIPY, fluorescein) in a probe (when located adjacent to guanine residue) can be dequenched upon hybridizing to cytosine residue of target DNA.

One such advance involves incorporating pyrene labeled deoxyadenosine at the 5’-end of a hairpin (Panel A in Figure).  Its fluorescence was quenched when placed next to C, T or G base though was enhanced significantly upon interaction with target DNA (35 fold) or RNA (44 fold) (Seo et al., 2005).  Further increase in the ability to discriminate SNPs occurred when locked nucleic acid (LNA) was placed adjacent to pyrene-labeled uridine, i.e. emission increased with matched duplex than mismatched duplex (Kaura et al., 2015). 

Another approach involves the modification of nucleic acid analogues with higher affinity to complementary strands.  Briefly, the introduction of bridged nucleic acid has greatly increased the binding affinity of an oligonucleotide to target complementary strand.  The stability of the RNA:DNA or DNA:DNA duplex can be greatly augmented through incorporation of a small number of locked nucleic acids.  This has inspired the development of 2'-O,4'-aminoethylene bridged nucleic acid (2',4'-BNA^NC), the third-generation bridged nucleic acid containing a six-member bridged structure with an N-O linkage.  BNA provides greater binding affinity, better single-mismatch discrimination, enhanced RNA specificity, stronger/selective triplex-forming properties, and considerably higher nuclease resistance (Rahman et al., 2008; Miyashita et al., 2007).  To couple heightened binding affinity with greater fluorescence, novel (phenylethynyl)pyrene–BNA constructs were developed by the investigators at University of Southern Denmark (Panel B in Figure).  Using probes incorporating these fluorescent nucleoside analogues, the drug‐resistance‐causing mutation in HIV‐1 protease cDNA and RNA gene fragments was successfully detected (Astakhova et al., 2012).

Bio-synthesis, Inc. specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA).  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as conjugate to peptides. This includes pyrene-dU fluorescent base oligonucleotide modification.  For oncogenic SNP genotyping, Bio-synthesis, Inc. provides a kit incorporating BNA for the rapid and convenient real-time PCR detection of the BRAF-V600E mutation with high sensitivity (<0.1% of mutant in wild-type background).  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology that we offer provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power was especially useful for diagnosis (ex. FISH using DNA probe).  More importantly, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides for clinical application.

https://www.biosyn.com/oligonucleotideproduct/pyrene-du-fluorescent-base-oligonucleotide-modification.aspx#!

https://www.biosyn.com/oligonucleotide-modification-services.aspx

https://www.biosyn.com/braf-mutation-analysis-kit.aspx

References

Astakhova IK, Samokhina E, Babu BR, Wengel J.  Novel (phenylethynyl)pyrene–LNA constructs for fluorescence SNP sensing in polymorphic nucleic acid targets.  (2012)  ChemBiochem  13:1509-19.  PMID: 22761036 DOI: 10.1002/cbic.201200079

Deng N, Zhou H, Fan H, Yuan Y.  Single nucleotide polymorphisms and cancer susceptibility.  (2017)  Oncotarget  8:110635-110649.   PMID: 29299175  doi: 10.18632/oncotarget.22372. eCollection

Hwang GT. Single-Labeled Oligonucleotides Showing Fluorescence Changes Upon Hybridization with Target Nucleic Acids. 2018) Molecules. 23. pii: E124. PMID: 29316733  doi: 10.3390/molecules23010124. Review.

Jenkins S, Gibson N.  High-throughput SNP.  (2002) Comp Funct Genomics.  3:57-66.   PMID: 18628885   doi: 10.1002/cfg.130.

Kaura, M.; Hrdlicka, P.J.  Locked nucleic acid (LNA) induced effect on the hybridization and fluorescence properties of oligodeoxyribonucleotides modified with nucleobase-functionalized DNA monomers. (2015)  Org. Biomol. Chem. 13, 7236–7247.  PMID: 26055658  DOI: 10.1039/c5ob00860c

Miyashita, K., Rahman, S. M., Seki, S., Obika, S., and Imanishi, T. N-Methyl substituted 2′,4′- BNANC: a highly nuclease resistant nucleic acid analogue with high-affinity RNA selective hybridization. (2007) Chem. Commun. (Cambridge, U. K.), 3765−3767.

Rahman SM, Seki S, Obika S, Yoshikawa H, Miyashita K, Imanishi T. Design, synthesis, and properties of 2',4'-BNA(NC): a bridged nucleic acid analogue.  (2008)  J Am Chem Soc. 130:4886-96. PMID: 18341342  doi: 10.1021/ja710342q.

 Seo YJ, Ryu JH, Kim BH. Quencher-free, end-stacking oligonucleotides for probing single-base mismatches in DNA.  (2005)  Org Lett. 7:4931-3.  PMID: 16235925  DOI: 10.1021/ol0518582

Thi Tran HT, Takeshima Y, Surono A, Yagi M, Wada H, Matsuo M.  A G-to-A transition at the fifth position of intron-32 of the dystrophin gene inactivates a splice-donor site both in vivo and in vitro.  (2005)  Mol Genet Metab  85:213-9.  PMID: 15979033  DOI: 10.1016/j.ymgme.2005.03.006