title: Fluoride-cleavable, fluorescently labelled reversible terminators: synthesis and use in primer extension.
abstract: Fluorescent 2'-deoxynucleotides containing a protecting group at the 3'-O-position are reversible terminators that enable array-based DNA sequencing-by-synthesis (SBS) approaches. Herein, we describe the synthesis and full characterisation of four reversible terminators bearing a 3'-blocking moiety and a linker-dye system that is removable under the same fluoride-based treatment. Each nucleotide analogue has a different fluorophore attached to the base through a fluoride-cleavable linker and a 2-cyanoethyl moiety as the 3'-blocking group, which can be removed by using a fluoride treatment as well. Furthermore, we identified a DNA polymerase, namely, RevertAid M-MuLV reverse transcriptase, which can incorporate the four modified reversible terminators. The synthesised nucleotides and the optimised DNA polymerase were used on CodeLink slides spotted with hairpin oligonucleotides to demonstrate their potential in a cyclic reversible terminating approach.
authors.affiliation: Institut für Organische Chemie und Chemische Biologie, J.W. Goethe Universität Frankfurt am Main, Max-von-Laue Strasse 7, 60438 Frankfurt am Main, Germany.
authors.forename: Diana C
authors.initials: DC
authors.lastname: Knapp
authors.forename: Saulius
authors.initials: S
authors.lastname: Serva
authors.forename: Jennifer
authors.initials: J
authors.lastname: D'Onofrio
authors.forename: Angelika
authors.initials: A
authors.lastname: Keller
authors.forename: Arvydas
authors.initials: A
authors.lastname: Lubys
authors.forename: Ants
authors.initials: A
authors.lastname: Kurg
authors.forename: Maido
authors.initials: M
authors.lastname: Remm
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Engels
mesh_terms: D017931:DNA Primers / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D005459:Fluorides / Q000737:chemistry*; D009052:Leukemia Virus, Murine / Q000201:enzymology*; D015394:Molecular Structure; D015345:Oligonucleotide Probes / Q000138:chemical synthesis* / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D012194:RNA-Directed DNA Polymerase; D005459:Fluorides