Sequence-dependent thermodynamic parameters; for locked nucleic acid (LNA)-DNA duplex formation

The design of modified nucleic acid probes, primers, and therapeutics is improved by considering their thermodynamics. Locked nucleic acid (LNA) is one of the most useful modified backbones, with incorporation of a single LNA providing a substantial increase in duplex stability. In this work, the hybridization DeltaHdegrees, DeltaSdegrees, and melting temperature (T-M) were measured from absorbance melting curves for 100 duplex oligonucleotides with single internal LNA nucleotides on one strand, and the results provided DeltaDeltaHdegrees, DeltaDeltaSdegrees, DeltaDeltaGdegrees(37), and DeltaT(M) relative to reference DNA oligonucleotides. LNA pyrimidines contribute 37 00, more stability than purines, especially A(L), but there is substantial context dependence for each LNA base. Both the 5' and 3' neighbors must be considered in predicting the effect of an LNA incorporation, with purine neighbors providing more stability. Enthalpy-entropy compensation in DeltaDeltaHdegrees and DeltaDeltaSdegrees is observed across the set of sequences, suggesting that LNA can stabilize the duplex by either preorganization or improved stacking, but not both simultaneously. Singular value decomposition analysis provides predictive sequence-dependent rules for hybridization of singly LNA-substituted DNA oligonucleotides to their all-DNA complements. The results are provided as sets of DeltaDeltaHdegrees, DeltaDeltaSdegrees, and DeltaDeltaG(37)degrees parameters for all 32 of the possible nearest neighbors for LNA+DNA:DNA hybridization (5' MXL and 5' (XN)-N-L, where M, N, and X A, C, G, or T and X-L represents LNA). The parameters are applicable within the standard thermodynamic prediction algorithms. They provide T-M estimates accurate to within 2 degreesC for LNA-containing oligonucleotides, which is significantly better accuracy than previously available.