title: Improved nucleotide selectivity and termination of 3'-OH unblocked reversible terminators by molecular tuning of 2-nitrobenzyl alkylated HOMedU triphosphates.
abstract: We describe a novel 3'-OH unblocked reversible terminator with the potential to improve accuracy and read-lengths in next-generation sequencing (NGS) technologies. This terminator is based on 5-hydroxymethyl-2'-deoxyuridine triphosphate (HOMedUTP), a hypermodified nucleotide found naturally in the genomes of numerous bacteriophages and lower eukaryotes. A series of 5-(2-nitrobenzyloxy)methyl-dUTP analogs (dU.I-dU.V) were synthesized based on our previous work with photochemically cleavable terminators. These 2-nitrobenzyl alkylated HOMedUTP analogs were characterized with respect to incorporation, single-base termination, nucleotide selectivity and photochemical cleavage properties. Substitution at the α-methylene carbon of 2-nitrobenzyl with alkyl groups of increasing size was discovered as a key structural feature that provided for the molecular tuning of enzymatic properties such as single-base termination and improved nucleotide selectivity over that of natural nucleotides. 5-[(S)-α-tert-Butyl-2-nitrobenzyloxy]methyl-dUTP (dU.V) was identified as an efficient reversible terminator, whereby, sequencing feasibility was demonstrated in a cyclic reversible termination (CRT) experiment using a homopolymer repeat of ten complementary template bases without detectable UV damage during photochemical cleavage steps. These results validate our overall strategy of creating 3'-OH unblocked reversible terminator reagents that, upon photochemical cleavage, transform back into a natural state. Modified nucleotides based on 5-hydroxymethyl-pyrimidines and 7-deaza-7-hydroxymethyl-purines lay the foundation for development of a complete set of four reversible terminators for application in NGS technologies.
authors.affiliation: LaserGen, Inc, Houston, TX 77054, USA.
authors.forename: Vladislav A
authors.initials: VA
authors.lastname: Litosh
authors.forename: Weidong
authors.initials: W
authors.lastname: Wu
authors.forename: Brian P
authors.initials: BP
authors.lastname: Stupi
authors.forename: Jinchun
authors.initials: J
authors.lastname: Wang
authors.forename: Sidney E
authors.initials: SE
authors.lastname: Morris
authors.forename: Megan N
authors.initials: MN
authors.lastname: Hersh
authors.forename: Michael L
authors.initials: ML
authors.lastname: Metzker
mesh_terms: D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003856:Deoxyuracil Nucleotides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D055668:Photochemical Processes; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013698:Templates, Genetic; D014466:Ultraviolet Rays
chemical_list: D003856:Deoxyuracil Nucleotides; C025110:hydroxymethyldeoxyuridine triphosphate; D004259:DNA-Directed DNA Polymerase