Design and synthesis of a 3′-
            <i>O</i>
            -allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis

Ruparel, Hameer; Bi, Lanrong; Li, Zengmin; Bai, Xiaopeng; Kim, Dae Hyun; Turro, Nicholas J.; Ju, Jingyue

doi:10.1073/pnas.0501962102

Cited by 77 publications

(56 citation statements)

References 31 publications

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“…These include next-generation sequencing approaches (1)(2)(3), single molecule sequencing (4), labeling of DNA and PCR amplificates, e.g., for microarray analysis (5)(6)(7)(8), DNA conjugation (9), or the in vitro selection of ligands such as aptamers by SELEX (systematic enrichment of ligands by exponential amplification) (10). Furthermore, utilizing the intrinsic properties of DNA in combination with chemically introduced functionalities provides an entry to new classes of nucleic acids-based hybrid materials (9).…”

mentioning

confidence: 99%

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

Obeid¹,

Baccaro²,

Welte

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Numerous 2′-deoxynucleoside triphosphates (dNTPs) that are functionalized with spacious modifications such as dyes and affinity tags like biotin are substrates for DNA polymerases. They are widely employed in many cutting-edge technologies like advanced DNA sequencing approaches, microarrays, and single molecule techniques. Modifications attached to the nucleobase are accepted by many DNA polymerases, and thus, dNTPs bearing nucleobase modifications are predominantly employed. When pyrimidines are used the modifications are almost exclusively at the C5 position to avoid disturbing of Watson-Crick base pairing ability. However, the detailed molecular mechanism by which C5 modifications are processed by a DNA polymerase is poorly understood. Here, we present the first crystal structures of a DNA polymerase from Thermus aquaticus processing two C5 modified substrates that are accepted by the enzyme with different efficiencies. The structures were obtained as ternary complex of the enzyme bound to primer/template duplex with the respective modified dNTP in position poised for catalysis leading to incorporation. Thus, the study provides insights into the incorporation mechanism of the modified nucleotides elucidating how bulky modifications are accepted by the enzyme. The structures show a varied degree of perturbation of the enzyme substrate complexes depending on the nature of the modifications suggesting design principles for future developments of modified substrates for DNA polymerases.modified nucleotide | nucleobase modification | functional DNA | X-ray structure | PCR T he capability of DNA polymerases to accept modified 2′-deoxynucleoside triphosphates (dNTPs) is exploited in many important biotechnological applications. These include next-generation sequencing approaches (1-3), single molecule sequencing (4), labeling of DNA and PCR amplificates, e.g., for microarray analysis (5-8), DNA conjugation (9), or the in vitro selection of ligands such as aptamers by SELEX (systematic enrichment of ligands by exponential amplification) (10). Furthermore, utilizing the intrinsic properties of DNA in combination with chemically introduced functionalities provides an entry to new classes of nucleic acids-based hybrid materials (9). In most cases the dNTP modifications were introduced to the nucleobase. In cases where pyrimidines are employed the modifications are almost exclusively at the C5 position to avoid disturbing of Watson-Crick base pairing ability (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). In addition, the C5 modification is well accommodated into the major groove of DNA without perturbing the DNA structure. Mainly C5 alkyne modified dNTPs are used because they are accessible via metal mediated cross coupling reactions (21-23) in a straightforward manner. While many C5 modified dNTP analogs are already applied, because they are processed by DNA polymerases at least to some extent, it is still unpredictable which modification will be accepted (8, 11-23) because the detailed molecular mechanism by which C5 modif...

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confidence: 99%

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

Obeid¹,

Baccaro²,

Welte

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, Metzker et al (1994) first described the synthesis and incorporation of a 3Ј-O-allyl-dATP by DNA polymerase, with the O-allyl group being removed using the well-known palladium (Pd) catalyst chemistry (Hayakawa et al 1986(Hayakawa et al , 1993Honda et al 1997). Recently, Ruparel et al (2005) reported the synthesis of the first fluorescently labeled 3Ј-O-allyldNTPs. These unique reversible terminators require dual deprotection steps using UV light to cleave the fluorophore from the nucleotide (Fig.…”

Section: Crtmentioning

confidence: 99%

Emerging technologies in DNA sequencing

Metzker¹

2005

Genome Res.

410

262

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Demand for DNA sequence information has never been greater, yet current Sanger technology is too costly, time consuming, and labor intensive to meet this ongoing demand. Applications span numerous research interests, including sequence variation studies, comparative genomics and evolution, forensics, and diagnostic and applied therapeutics. Several emerging technologies show promise of delivering next-generation solutions for fast and affordable genome sequencing. In this review article, the DNA polymerase-dependent strategies of Sanger sequencing, single nucleotide addition, and cyclic reversible termination are discussed to highlight recent advances and potential challenges these technologies face in their development for ultrafast DNA sequencing.

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“…This product was then deallylated using a Pd-catalyzed deallylation mixture [1ϫ Thermopol I reaction buffer/Na 2 PdCl 4 /P(PhSO 3 Na) 3 ]. The active Pd catalyst is generated from Na 2 PdCl 4 and a ligand P(PhSO 3 Na) 3 (TPPTS) to mediate the deallylation reaction in DNA compatible aqueous condition to simultaneously cleave both the fluorophore and the 3Ј-O-allyl group (28). The deallylated product (2) was also analyzed by using MALDI-TOF MS (Fig.…”

Section: Design and Synthesis Of Chemically Cleavable Fluorescent Nucmentioning

confidence: 99%

“…We have previously established the feasibility of performing SBS on a chip using four photocleavable fluorescent nucleotide analogues (26) and discovered that an allyl group can be used as a cleavable linker to bridge a fluorophore to a nucleotide (27). We have also reported the design and synthesis of two photocleavable fluorescent nucleotides as reversible terminators for polymerase reaction (28,29).…”

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confidence: 99%

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

Kim

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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183

160

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DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleotides (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3-O-allyl-modified nucleotides, forming chemically cleavable fluorescent nucleotide reversible terminators, 3-O-allyldNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3-O-allyl group on a DNA extension product, which is generated by incorporating 3-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pdcatalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner. DNA chip DNA sequencing is driving genomics research and discovery. The completion of the Human Genome Project has set the stage for screening genetic mutations to identify disease genes on a genome-wide scale (1). Accurate high-throughput DNA sequencing methods are needed to explore the complete human genome sequence for applications in clinical medicine and health care. To overcome the limitations of the current electrophoresis-based sequencing technology (2-5), a variety of new DNA-sequencing methods have been investigated. Such approaches include sequencing by hybridization (6), mass spectrometry-based sequencing (7-9), sequence-specific detection of single-stranded DNA using engineered nanopores (10), and sequencing by ligation (11). More recently, DNA sequencing by synthesis (SBS) approaches such as pyrosequencing (12), sequencing of single DNA molecules (13), and polymerase colonies (14) have been widely explored.The concept of DNA SBS was revealed in 1988 with an attempt to sequence DNA by detecting the pyrophosphate group that is generated when a nucleotide is incorporated in a DNA polymerase reaction (15). Pyrosequencing, which was developed based on this concept and an enzymatic cascade, has been explored for genome sequencing (16). However, there are inherent difficulties in this method for determining the number of incorporated nucleotides in homopolymeric regions of the template. Additionally, each of the four nucleotides needs to be added and detected separately, which increases the overall detection time. The accumulation of undegraded nucleotides and other components could also lower the accuracy of the method when sequencing a long DNA template. It is thus desirable to have a simple method t...

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Design and synthesis of a 3′- O -allyl photocleavable fluorescent nucleotide as a reversible terminator for DNA sequencing by synthesis

Cited by 77 publications

References 31 publications

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase

Emerging technologies in DNA sequencing

Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators

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