title: Microenvironmental Effect of 2'-O-(1-Pyrenylmethyl)uridine Modified Fluorescent Oligonucleotide Probes on Sensitive and Selective Detection of Target RNA.
abstract: 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 π-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.
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University , Beijing, 100191, China.
authors.forename: Gülnur
authors.initials: G
authors.lastname: Imincan
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University , Beijing, 100191, China.
authors.forename: Fen
authors.initials: F
authors.lastname: Pei
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University , Beijing, 100191, China.
authors.forename: Lijia
authors.initials: L
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University , Beijing, 100191, China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Jin
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University , Beijing, 100191, China.
authors.forename: Liangren
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University , Beijing, 100191, China.
authors.forename: Xiaoda
authors.initials: X
authors.lastname: Yang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University , Beijing, 100191, China.
authors.forename: Lihe
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University , Beijing, 100191, China.
authors.forename: XinJing
authors.initials: X
authors.lastname: Tang
mesh_terms: D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D056004:Molecular Dynamics Simulation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry*; D012313:RNA / Q000032:analysis* / Q000096:biosynthesis / Q000235:genetics*; D020871:RNA Stability; D013379:Substrate Specificity; D013696:Temperature; D014529:Uridine / Q000031:analogs & derivatives* / Q000737:chemistry
chemical_list: C527209:2'-O-pyrenylmethyluridine; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012313:RNA; D014529:Uridine