Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators: Synthesis and Use in Primer Extension

• Published in: Chemistry : a European journal Vol. 17; no. 10; pp. 2903 - 2915
• Main Authors: Knapp, Diana C., Serva, Saulius, D'Onofrio, Jennifer, Keller, Angelika, Lubys, Arvydas, Kurg, Ants, Remm, Maido, Engels, Joachim W.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.03.2011; WILEY‐VCH Verlag; Wiley; Wiley Subscription Services, Inc
• Subjects: Chemical compounds; Chemistry; Chemistry, Multidisciplinary; Deoxyribonucleic acid; DNA; DNA polymerase; DNA Primers - metabolism; Fluorescence; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; fluoride ions; Fluorides; Fluorides - chemistry; Leukemia Virus, Murine - enzymology; Molecular Structure; Nucleotides; Oligonucleotide Probes - chemical synthesis; Oligonucleotide Probes - chemistry; Oligonucleotides; Physical Sciences; reverse transcriptase; reversible terminators; RNA-Directed DNA Polymerase - metabolism; Science & Technology; Stopping; Synthesis; fluorescence; reverse transcriptase; fluoride ions; DNA; NUCLEOSIDES; reversible terminators; nucleotides
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.201001952

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. Bright new things: A new set of fluorescently labelled reversible terminators has been synthesised (see picture). A polymerase able to incorporate these compounds was found, and the complete system of terminator–polymerase was successfully used in a cyclic reversible terminating approach on CodeLink slides spotted with oligonucleotide probes.