5 research outputs found
Stability and Mismatch Discrimination of Locked Nucleic Acid–DNA Duplexes
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
Effects of sodium ions on melting temperatures of DNA–DNA and LNA–DNA duplexes
<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Duplexes were investigated that had a T•A base pair in the X•Y mismatch site. All duplexes had the same base sequence, 5′-ggtcctttcttggtg-3′/3′-ccaggaaagaaccac-5′, where LNA modifications were introduced at various positions (). Solid lines were calculated using a published salt correction ()
Dependence of melting temperature and mismatch discrimination on oligomer length
<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Average melting temperatures were calculated for 50% g•c DNA duplex oligomers that did not contain any mismatched base pairs as well as oligomers with single g•t or a•c mismatches. Predictions assumed total single strand concentration of 400 nM in 1 M Na buffer
Fluorescence emission spectra of target oligomer, 5′-gcgaggpggctt-3′, with single 2-aminopurine (p) reveal the magnitude of stacking interactions
<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Duplexes containing a H-bonded t•p base pair are compared with mismatched g•p base pair duplexes. Both unmodified DNA probes (blue lines) and probes with a LNA triplet at the mismatch site (red lines) were studied. UV melting experiments in 1 M Na buffer showed that LNA triplets increased mismatch discrimination for these sequences
Difference of Δ values between LNA and DNA probes for various mismatches
<p><b>Copyright information:</b></p><p>Taken from "Design of LNA probes that improve mismatch discrimination"</p><p>Nucleic Acids Research 2006;34(8):e60-e60.</p><p>Published online 2 May 2006</p><p>PMCID:PMC1456327.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Sequence Set 1 (panel A and B), and Sets 2 and 3 (panel C) are plotted. Positions of LNA residues and set names are shown in Figure 1. A positive difference indicates improved mismatch discrimination for the LNA probe relative to the DNA probe. Dashed lines denote the range (±0.8°C), which is within the experimental error of the measurements