Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA

Anna M Varizhuk; Timofei S Zatsepin; Andrey V Golovin; Evgeny S Belyaev; Yury I Kostyukevich; Vladimir G Dedkov; German A Shipulin; George V Shpakovski; Andrey V Aralov

doi:10.1016/j.bmc.2017.03.062

Synthesis of oligonucleotides containing novel G-clamp analogue with C8-tethered group in phenoxazine ring: Implication to qPCR detection of the low-copy Kemerovo virus dsRNA

Bioorg Med Chem. 2017 Jul 15;25(14):3597-3605. doi: 10.1016/j.bmc.2017.03.062. Epub 2017 Mar 31.

Authors

Anna M Varizhuk¹, Timofei S Zatsepin², Andrey V Golovin³, Evgeny S Belyaev⁴, Yury I Kostyukevich⁵, Vladimir G Dedkov⁶, German A Shipulin⁶, George V Shpakovski⁷, Andrey V Aralov⁸

Affiliations

¹ Research and Clinical Center for Physical Chemical Medicine, 119435 Moscow, Russia; Department of Structure-Functional Analysis of Biopolymers, Engelhardt Institute of Molecular Biology, Vavilov Str. 32, Moscow 119991, Russia.
² Chemistry Department, Lomonosov Moscow State University, Leninskie Gory, Moscow 119991, Russia; Central Research Institute of Epidemiology, Novogireevskaya 3a, Moscow 111123, Russia; Skolkovo Institute of Science and Technology, 3 Nobel Street, Skolkovo, Moscow Region 143026, Russia. Electronic address: t.zatsepin@skoltech.ru.
³ Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia.
⁴ Institute for Energy Problems of Chemical Physics of RAS, Leninskij pr. 38/2, Moscow 119334, Russia.
⁵ Skolkovo Institute of Science and Technology, 3 Nobel Street, Skolkovo, Moscow Region 143026, Russia.
⁶ Central Research Institute of Epidemiology, Novogireevskaya 3a, Moscow 111123, Russia.
⁷ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str. 16/10, Moscow 117997, Russia.
⁸ Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str. 16/10, Moscow 117997, Russia. Electronic address: Baruh238@mail.ru.

PMID: 28396019
DOI: 10.1016/j.bmc.2017.03.062

Abstract

Nowadays modified oligonucleotides are widely used in diagnostics and as novel therapeutics. Introduction of modified or unnatural residues into oligonucleotides allows fine tuning of their binding properties to complementary nucleic acids and leads to improved stability both in vitro and in vivo. Previously it was demonstrated that insertion of phenoxazine nucleotides with various groups in C9-position into oligonucleotides leads to a significant increase of duplex stability with complementary DNA and RNA. Here the synthesis of a novel G-clamp nucleoside analogue (G^8AE-clamp) bearing 2-aminoethyl tether at C8-atom is presented. Introduction of such modified residues into oligonucleotides lead to enhanced specificity of duplex formation towards complementary DNA and RNA targets with increased thermal and 3'-exonuclease stability. According to CD-spectroscopy studies G^8AE-clamp does not substantially disrupt helix geometry. Primers containing G^8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides.

Keywords: DNA mimics; G-clamp; Modification; Oligonucleotides; qPCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Circular Dichroism
Exonucleases / metabolism
Guanosine / analogs & derivatives*
Guanosine / metabolism
Molecular Dynamics Simulation
Nucleic Acid Conformation
Nucleic Acid Hybridization
Oligonucleotides / chemical synthesis*
Oligonucleotides / chemistry
Orbivirus / genetics*
Oxazines / chemistry*
RNA, Double-Stranded / analysis
RNA, Double-Stranded / genetics
RNA, Double-Stranded / metabolism
RNA, Viral / genetics
RNA, Viral / metabolism*
Real-Time Polymerase Chain Reaction

Substances

Oligonucleotides
Oxazines
RNA, Double-Stranded
RNA, Viral
Guanosine
phenoxazine
Exonucleases
spleen exonuclease