Stability and Mismatch
Discrimination of Locked Nucleic Acid–DNA Duplexes
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Abstract
Locked nucleic acids (LNA; symbols of bases, +A, +C,
+G, and +T) are introduced into chemically synthesized oligonucleotides
to increase duplex stability and specificity. To understand these
effects, we have determined thermodynamic parameters of consecutive
LNA nucleotides. We present guidelines for the design of LNA oligonucleotides
and introduce free online software that predicts the stability of
any LNA duplex oligomer. Thermodynamic analysis shows that the single
strand–duplex transition is characterized by a favorable enthalpic
change and by an unfavorable loss of entropy. A single LNA modification
confines the local conformation of nucleotides, causing a smaller,
less unfavorable entropic loss when the single strand is restricted
to the rigid duplex structure. Additional LNAs adjacent to the initial
modification appear to enhance stacking and H-bonding interactions
because they increase the enthalpic contributions to duplex stabilization.
New nearest-neighbor parameters correctly forecast the positive and
negative effects of LNAs on mismatch discrimination. Specificity is
enhanced in a majority of sequences and is dependent on mismatch type
and adjacent base pairs; the largest discriminatory boost occurs for
the central +C·C mismatch within the +T+C+C sequence and the
+A·G mismatch within the +T+A+G sequence. LNAs do not affect
specificity in some sequences and even impair it for many +G·T
and +C·A mismatches. The level of mismatch discrimination decreases
the most for the central +G·T mismatch within the +G+G+C sequence
and the +C·A mismatch within the +G+C+G sequence. We hypothesize
that these discrimination changes are not unique features of LNAs
but originate from the shift of the duplex conformation from B-form
to A-form