5 research outputs found

    Stability and Mismatch Discrimination of Locked Nucleic Acid–DNA Duplexes

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    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

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    <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

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    <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

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    <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

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    <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
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