Nucleic acid structural engineering using pyrene-functionalized 2'-amino-α-L-LNA monomers and abasic sites

Oligonucleotides (ONs) modified with a 2'-N-(pyren-1-yl)acetyl-2'-amino-alpha-L-LNA thymine monomer Y flanked on the 3'-side by an abasic site Phi (i.e., Y Phi)-unit) exhibit unprecedented increases in thermal affinity (Delta T-m values) toward target strands containing abasic sites (Delta T-m per Y Phi unit >+33.0 degrees C in 9-mer duplexes relative to unmodified ONs). Biophysical studies along with force field calculations suggest that the conformationally locked 2-oxo-5-azabicyclo[2.2.1]heptane skeleton of monomer Y, in concert with the short rigid acetyl linker, efficiently forces the thymine and pyrene moieties to adopt an interplanar distance of similar to 3.4 angstrom. This precisely positions the pyrene moiety in the duplex core void formed by abasic sites (Phi:Phi pair) for optimal pi-pi overlap. Duplexes with multiple Y Phi:A Phi units separated by one base pair are tolerated extraordinarily well, as exemplified by a 13-mer duplex containing four separated Y Phi: A Phi units (8 abasic sites distributed over 13 base pairs), which exhibit a thermal denaturation temperature of 60.5 degrees C. The Y Phi probes display up to 16-fold increases in fluorescence intensity at 380 nm upon hybridization with abasic target strands, whereby self-assembly of these complex architectures can be easily monitored. This study underlines the potential of N2'-functionalized 2'-amino-alpha-L-LNA as building blocks in nucleic acid based diagnostics and nanomaterial engineering.