Design and synthesis of a 3'-O-allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis.
DNA sequencing by synthesis (SBS) offers an approach for potential high-throughput sequencing applications. In this method, the ability of an incoming nucleotide to act as a reversible terminator for a DNA polymerase reaction is an important requirement to unambiguously determine the identity of the incorporated nucleotide before the next nucleotide is added. A free 3'-OH group on the terminal nucleotide of the primer is necessary for the DNA polymerase to incorporate an incoming nucleotide. Therefore, if the 3'-OH group of an incoming nucleotide is capped by a chemical moiety, it will cause the polymerase reaction to terminate after the nucleotide is incorporated into the DNA strand. If the capping group is subsequently removed to generate a free 3'-OH, the polymerase reaction will reinitialize. We report here the design and synthesis of a 3'-modified photocleavable fluorescent nucleotide, 3'-O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleavable 4,4-difluoro-4-bora-3alpha,4alpha-diaza-s-indacene), as a reversible terminator for SBS. This nucleotide analogue contains an allyl moiety capping the 3'-OH group and a fluorophore Bodipy-FL-510 linked to the 5 position of the uracil through a photocleavable 2-nitrobenzyl linker. Here, we have shown that this nucleotide is a good substrate for a DNA polymerase. After the nucleotide was successfully incorporated into a growing DNA strand and the fluorophore was photocleaved, the allyl group was removed by using a Pd-catalyzed reaction to reinitiate the polymerase reaction, thereby establishing the feasibility of using such nucleotide analogues as reversible terminators for SBS.
Ruparel
Hameer
H
Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
Bi
Lanrong
L
Li
Zengmin
Z
Bai
Xiaopeng
X
Kim
Dae Hyun
DH
Turro
Nicholas J
NJ
Ju
Jingyue
J
D001483:Base Sequence; D001896:Boron Compounds; D004247:DNA / Q000138:chemical synthesis* / Q000737:chemistry*; D017931:DNA Primers / Q000138:chemical synthesis / Q000737:chemistry; D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D017931:DNA Primers; D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009838:Oligodeoxyribonucleotides; D004247:DNA