Wolk Steven K, Shoemaker Richard K, Mayfield Wes S, Mestdagh Andrew L, Janjic Nebojsa
SomaLogic, Inc., Boulder, CO 80301, USA
Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA.
Nucleic Acids Res. 2015 Oct 30;43(19):9107-22. doi: 10.1093/nar/gkv981. Epub 2015 Oct 4.
We have recently shown that the incorporation of modified nucleotides such as 5-N-carboxamide-deoxyuridines into random nucleic acid libraries improves success rates in SELEX experiments and facilitates the identification of ligands with slow off-rates. Here we report the impact of these modifications on the thermodynamic stability of both duplexes and intramolecular 'single-stranded' structures. Within duplexes, large, hydrophobic naphthyl groups were destabilizing relative to the all natural DNA duplex, while the hydrophilic groups exhibited somewhat improved duplex stability. All of the significant changes in stability were driven by opposing contributions from the enthalpic and entropic terms. In contrast, both benzyl and naphthyl modifications stabilized intramolecular single-stranded structures relative to their natural DNA analogs, consistent with the notion that intramolecular folding allows formation of novel, stabilizing hydrophobic interactions. Imino proton NMR data provided evidence that elements of the folded structure form at temperatures well below the Tm, with a melting transition that is distinctly less cooperative when compared to duplex DNA. Although there are no data to suggest that the unmodified DNA sequences fold into structures similar to their modified analogs, this still represents clear evidence that these modifications impart thermodynamic stability to the folded structure not achievable with unmodified DNA.
我们最近发现，将修饰核苷酸（如5-N-羧酰胺-脱氧尿苷）掺入随机核酸文库中，可提高SELEX实验的成功率，并有助于鉴定解离速率较慢的配体。在此，我们报告这些修饰对双链体和分子内“单链”结构热力学稳定性的影响。在双链体内，相对于全天然DNA双链体，大的疏水性萘基具有去稳定作用，而亲水性基团则使双链体稳定性有所提高。稳定性的所有显著变化均由焓项和熵项的相反贡献驱动。相比之下，相对于天然DNA类似物，苄基和萘基修饰均使分子内单链结构稳定，这与分子内折叠允许形成新型稳定疏水相互作用的观点一致。亚氨基质子核磁共振数据表明，折叠结构的元件在远低于熔点温度（Tm）时形成，与双链DNA相比，其熔解转变的协同性明显较低。虽然没有数据表明未修饰的DNA序列会折叠成与其修饰类似物相似的结构，但这仍然清楚地证明了这些修饰赋予折叠结构热力学稳定性，而未修饰的DNA无法实现这一点。
