Microenvironmental Effect of 2′-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA
Microenvironmental Effect of 2′-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA
复制标题
DOI：
10.1021/acs.analchem.6b00227
复制
发表时间：
2016-04-19
影响因子：
7.4
通讯作者：
Tang, XinJing
中科院分区：
文献类型：
--
作者：
Imincan, Gulnur;Pei, Fen;Tang, XinJing
2'-O-(1-芘基甲基)尿苷修饰寡核糖核苷酸提供高度灵敏的芘荧光探针，用于检测 RNA 靶标的特定核苷酸突变。为了开发更稳定和更具成本效益的寡核苷酸探针，我们研究了附近核碱基对 RNA 和 2'-O-(1-芘基甲基)尿苷修饰寡核苷酸双链体中芘荧光的局部微环境影响。通过在寡脱氧核苷酸探针的2'-O-(1-芘基甲基)尿苷(U-P)两侧掺入脱氧核糖核苷酸、核糖核苷酸、2'-MeO-核苷酸和2'-F-核苷酸，合成了一系列芘修饰的寡核苷酸探针。它们的芘荧光发射光谱表明，只有两个近端核苷酸对与靶RNA杂交的这些寡核苷酸探针的芘荧光特性有显着影响，荧光灵敏度顺序为2'-F-核苷酸> 2'-MeO-核苷酸>核糖核苷酸>>脱氧核糖核苷酸。虽然基于圆二色光谱，双链体的整体螺旋构象（A 型或 B 型）对探针的灵敏度具有边际影响。相反，局部取代反映了核苷酸糖环采用North型构象的倾向，因此，在局部微环境中将其螺旋几何结构转向更像A型的构象。因此，芘荧光发射的更高增强有利于探针与目标RNA的双链体小沟的局部A型螺旋结构和更具极性和疏水性的环境（2'取代处的F>MeO>OH）。进一步的动态模拟表明，2'-F-核苷酸或核糖核苷酸的局部微环境效应足以使芘部分从核碱基移出双链体的小沟；此外，2'-F-核苷酸对芘修饰的尿苷与上游和下游核碱基的p-stack影响较小。本寡核苷酸探针在 RNA 合成的体外测定过程中成功区分了目标 RNA 和单突变 RNA 分析物。
2'-O-(1-Pyrenylmethyl)uridine modified oligoribonucleotides provide highly sensitive pyrene fluorescent probes for detecting specific nucleotide mutation of RNA targets. To develop more stable and cost-effective oligonucleotide probes, we investigated the local microenvironmental effects of nearby nucleobases on pyrene fluorescence in duplexes of RNAs and 2'-O-(1-pyrenylmethyl)uridine modified oligonucleotides. By incorporation of deoxyribonucleotides, ribonucleotides, 2'-MeO-nucleotides and 2'-F-nucleotides at both sides of 2'-O-(1-pyrenylmethyl)uridine (U-P) in oligodeoxynucleotide probes, we synthesized a series of pyrene modified oligonucleotide probes. Their pyrene fluorescence emission spectra indicated that only two proximal nucleotides have a substantial effect on the pyrene fluorescence properties of these oligonucleotide probes hybridized with target RNA with an order of fluorescence sensitivity of 2'-F-nucleotides > 2'-MeO-nucleotides > ribonucleotides >> deoxyribonucleotides. While based on circular dichroism spectra, overall helix conformations (either A- or B-form) of the duplexes have marginal effects on the sensitivity of the probes. Instead, the local substitution reflected the propensity of the nucleotide sugar ring to adopt North type conformation and, accordingly, shifted their helix geometry toward a more A-type like conformation in local microenvironments. Thus, higher enhancement of pyrene fluorescence emission favored local A-type helix structures and more polar and hydrophobic environments (F > MeO > OH at 2' substitution) of duplex minor grooves of probes with the target RNA. Further dynamic simulation revealed that local microenvironmental effect of 2'-F-nucleotides or ribonucleotides was enough for pyrene moiety to move out of nucleobases to the minor groove of duplexes; in addition, 2'-F-nucleotide had less effect on p-stack of pyrene-modified uridine with upstream and downstream nucleobases. The present oligonucleotide probes successfully distinguished target RNA from single-mutated RNA analyte during an in vitro assay of RNA synthesis.