Stopped-flow kinetics of lacked nucleic acid (LNA)-oligonucleotide duplex formation: studies of LNA-DNA and DNA-DNA interactions

The locked nucleic acid (LNA) monomer is a conformationally restricted nucl eotide analogue with an extra 2'-O,4'-C-methylene bridge added to the ribos e ring. Oligonucleotides that contain LNA monomers have shown greatly enhan ced thermal stability when hybridized to complementary DNA and RNA and are considered most promising candidates for efficient recognition of a given m ixed sequence in a nucleic acid duplex and as an antisense molecule. Here t he kinetics and thermodynamics of a series of oligonucleotide duplex format ions of DNA-DNA and DNA-LNA octamers were studied using stopped-flow absorp tion measurements at 25 degreesC and melting curves. The reactions of the D NA octamer 5'-CAGGAGCA-3' with its complementary DNA octamer 5'-TGCTCCTG-3' : and with the LNA octamers 5'-T(L)GCTCCTG-3' (LNA-1), 5-T(L)GCT(L)CCTG-3' (LNA-2) and 5'-T(L)GCT(L)CCT(L)G-3'(LNA-3), containing respectively one, tw o or three thymidine 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotide monom ers, designated T-L, were studied. In all cases were seen fast second-order association reactions with k(obs) = 2 x 10(7) M-1 s(-1). At 25 degreesC th e dissociation constants of the duplexes obtained from melting cun es were: DNA-DNA, 10 nM; DNA-LNA-1, 20 nM; DNA-LNA-2, 2 nM; and DNA-LNA-3, 0.3 nM;t hus the greatly enhanced duplex stability induced by LNA is confirmed. Sinc e the association rates were all equal this increase in stability is due to slower rates of dissociation of the complexes.