Thermodynamics of DNA-RNA heteroduplex formation: Effects of locked nucleic acid nucleotides incorporated into the DNA strand
Thermodynamics of DNA-RNA heteroduplex formation: Effects of locked nucleic acid nucleotides incorporated into the DNA strand
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DOI：
10.1021/bi700996z
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发表时间：
2008-01-29
期刊：
影响因子：
2.9
通讯作者：
Maiti, Souvik
中科院分区：
文献类型：
--
作者：
Kaur, Harleen;Wengel, Jesper;Maiti, Souvik
锁定的核酸（LNA）单体是构象受限的核苷酸类似物，对互补链具有增强的杂交效率。基于na的寡核苷酸的潜力已被寻求提高用于检测和特异性靶向核酸的探针组的选择性和特异性。我们利用光谱和量热技术评价了“锁定核酸”残基对DNA中心点RNA异双工的杂化热力学、反离子和水化的影响。在DNA链的腺嘌呤（5'-AGCACCAG）或胸腺嘧啶（5'-TGCTCCTG）残基上引入了一到三个rna取代。对杂双相形成的完整热力学分析表明，lnna诱导的稳定是由于杂化焓的有利增加补偿了不利的熵变化。差示扫描量热法数据分析表明，伴随异双相形成的非零热容变化δ C-p。等温滴定量热法测量表明，随着异双链中LNA含量的增加，两条链的结合亲和力增加。总的来说，我们的结果表明，与腺嘌呤残基相比，胸腺嘧啶残基的na取代作用更为明显。此外，光学熔融研究表明，与未修饰的双相相比，lna修饰双相的形成伴随着更高的反离子吸收和更低的水分子吸收。因此，我们的结果为表征基于lnna的探针-目标集的杂交热力学提供了初步尝试，这将有助于选择杂交实验的最佳条件，以及评估杂交和克隆实验所需的最小探针长度。
A locked nucleic acid (LNA) monomer is a conformationally restricted nucleotide analogue exhibiting enhanced hybridization efficiency toward complementary strand. The potential of LNA-based oligonucleotides has been sought to improve the selectivity and specificity of probe sets employed in detection and specific targeting of nucleic acids. We have evaluated the influence of "locked nucleic acid" residues on hybridization thermodynamics, counterions and hydration of DNA center dot RNA heteroduplex using spectroscopic and calorimetric techniques. One to three LNA substitutions have been introduced either at the adenine (5'-AGCACCAG) or thymine (5'-TGCTCCTG) residues of the DNA strand. A complete thermodynamic profile for heteroduplex formation suggested that LNA-induced stabilization results from a favorable increase in the enthalpy of hybridization that compensates for the unfavorable entropy change. Analysis of differential scanning calorimetry data indicated a nonzero heat capacity change, Delta C-p, accompanying the heteroduplex formation. Isothermal titration calorimetry measurements indicated an increase in binding affinity of the two strands as the LNA content of the heteroduplex is increased. Overall our result demonstrated that the effect of LNA-substitution at the thymine residue is more pronounced compared to the adenine residue. Furthermore, optical melting studies showed that, compared to an unmodified duplex, the formation of LNA-modified duplex is accompanied by a higher uptake of counterions and a lower uptake of water molecules. Our result, thus, presents a preliminary attempt toward the characterization of hybridization thermodynamics of the LNA-based probe-target sets, which will in turn aid in the selection of optimal conditions for hybridization experiments, and evaluation of the minimum probe-length required for hybridization and cloning experiments.