Enhanced Diagnostic Tools
BNAs are structurally rigid oligo-nucleotides with increased binding affinities and stability. Oligonucleotide modifications are characterized by the presence of one or more bicyclic ribose analogs. The structural similarity to native nucleic acids and the presence of a nitrogen atom within the bicyclic ring leads to very good solubility in water and allows for easy handling of synthetic primers and probes.
In contrast to peptide nucleic acids (PNAs) and minor groove binders (MGBs), but similar to LNAs BNA monomers can be used for both primers and probes in real time quantitative polymerase chain reaction (RT-Q-PCR) assays.
Compared to locked nucleic acids (LNAs) the substitution of DNA monomers with BNA monomers in oligonucleotides adds exceptional biological stability, resistance to nucleases and a significantly increased affinity to their complementary DNA targets.
The thermal stability is depending on the number of BNA monomers present in the sequence. Furthermore, BNA modifications greatly increase the melting temperature of oligonucleotides. Differences in melting temperatures (Tm) between perfectly and imperfectly matched nucleic acid duplexes allow for the discrimination even of single base mutations.
BNA-containing oligonucleotides useful for RT-Q-PCR assays can range in length between 12 and 20 nucleotides (nt), which is significantly shorter than unmodified primers and probes displaying the same Tm.
Furthermore, primers and probes modified with BNA monomers provide greater flexibility in designing consensus primers and probes for the detection of partially homologous target sequences, such as related viral species and serotypes. Finally, BNA-modified oligonucleotides in RT-Q-PCR assays reveal a specificity and sensitivity superior to other types of primers and probes.
The proposed action mechanism for antisense oligonucleotides may involve translation arrest, mRNA degradation mediated by RNase H and splicing arrest. This is illustrated in the following figure.
N = position of BNA NC [NMe] monomer.
Probes are usually labeled with Cy3 or FITC at the 5’ or 3’ end but other dyes may be used as well.
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