C5-Functionalized DNA, LNA, and α-L-LNA: Positional Control of Polarity-Sensitive Fluorophores Leads to Improved SNP-Typing

Single nucleotide polymorphisms (SNPs) are important markers in disease genetics and pharmacogenomic studies. Oligodeoxyribonucleotides (ONs) modified with 5‐[3‐(1‐pyrenecarboxamido)propynyl]‐2′‐deoxyuridine monomer X enable detection of SNPs at non‐stringent conditions due to differential fluorescence emission of matched versus mismatched nucleic acid duplexes. Herein, the thermal denaturation and optical spectroscopic characteristics of monomer X are compared to the corresponding locked nucleic acid (LNA) and α‐L‐LNA monomers Y and Z. ONs modified with monomers Y or Z result in a) larger increases in fluorescence intensity upon hybridization to complementary DNA, b) formation of more brightly fluorescent duplexes due to markedly larger fluorescence emission quantum yields (ΦF=0.44–0.80) and pyrene extinction coefficients, and c) improved optical discrimination of SNPs in DNA targets. Optical spectroscopy studies suggest that the nucleobase moieties of monomers X–Z adopt anti and syn conformations upon hybridization with matched and mismatched targets, respectively. The polarity‐sensitive 1‐pyrenecarboxamido fluorophore is, thereby, either positioned in the polar major groove or in the hydrophobic duplex core close to quenching nucleobases. Calculations suggest that the bicyclic skeletons of LNA and α‐L‐LNA monomers Y and Z influence the glycosidic torsional angle profile leading to altered positional control and photophysical properties of the C5‐fluorophore. SNP discrimination with LNA: Single‐nucleotide polymorphisms (SNPs) are discriminated by using DNA monomer X or the corresponding C5‐functionalized locked nucleic acid (LNA) and α‐L‐LNA monomers Y and Z. Improved positional control of the polarity‐sensitive pyrene label results in larger hybridization‐induced increases in fluorescence intensity, formation of brightly fluorescent duplexes upon hybridization to complementary nucleic acids, and efficient SNP discrimination.