title: 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.
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 (G8AE-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 G8AE-clamp does not substantially disrupt helix geometry. Primers containing G8AE-clamp demonstrated superior sensitivity in qPCR detection of dsRNA of Kemerovo virus in comparison to native oligonucleotides.
authors.affiliation: 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.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Varizhuk
authors.affiliation: 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.
authors.forename: Timofei S
authors.initials: TS
authors.lastname: Zatsepin
authors.affiliation: Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Golovin
authors.affiliation: Institute for Energy Problems of Chemical Physics of RAS, Leninskij pr. 38/2, Moscow 119334, Russia.
authors.forename: Evgeny S
authors.initials: ES
authors.lastname: Belyaev
authors.affiliation: Skolkovo Institute of Science and Technology, 3 Nobel Street, Skolkovo, Moscow Region 143026, Russia.
authors.forename: Yury I
authors.initials: YI
authors.lastname: Kostyukevich
authors.affiliation: Central Research Institute of Epidemiology, Novogireevskaya 3a, Moscow 111123, Russia.
authors.forename: Vladimir G
authors.initials: VG
authors.lastname: Dedkov
authors.affiliation: Central Research Institute of Epidemiology, Novogireevskaya 3a, Moscow 111123, Russia.
authors.forename: German A
authors.initials: GA
authors.lastname: Shipulin
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str. 16/10, Moscow 117997, Russia.
authors.forename: George V
authors.initials: GV
authors.lastname: Shpakovski
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str. 16/10, Moscow 117997, Russia. Electronic address: Baruh238@mail.ru.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Aralov
mesh_terms: D002942:Circular Dichroism; D005092:Exonucleases / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides / Q000138:chemical synthesis* / Q000737:chemistry; D018163:Orbivirus / Q000235:genetics*; D010078:Oxazines / Q000737:chemistry*; D012330:RNA, Double-Stranded / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D009841:Oligonucleotides; D010078:Oxazines; D012330:RNA, Double-Stranded; D012367:RNA, Viral; D006151:Guanosine; C039203:phenoxazine; D005092:Exonucleases; C022650:spleen exonuclease
keywords: DNA mimics; G-clamp; Modification; Oligonucleotides; qPCR