3′‐Hydroxymethyl 2′‐deoxynucleoside 5′‐triphosphates are inhibitors highly specific for reverse transcriptase

Kutateladze, Tamara; Kritzyn, Anatoli M.; Florentjev, Vladimir L.; Kavsan, V. M.; Chidgeavadze, Zurab G.; Beabealashvilli, Robert Sh.

doi:10.1016/0014-5793(86)81489-2

Cited by 14 publications

(24 citation statements)

References 12 publications

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“…The results reported here may indicate that the polymerase-bound 3' end of DNA lies in the vicinity of a powerful nucleophilic group in the active site involved either in polymerization or in 3'-substituent removal, a finding in accordance with crystallographic data for several DNA polymerases (8)(9)(10)(11)(12). "Readthrough" (16), "quenching" (18), and results reported by others (23,26,27) may be explained by a quantitative removal of the fluorescent reporter upon enzymatic chain extension.…”

Section: Discussionsupporting

confidence: 77%

Catalytic editing properties of DNA polymerases.

Canard¹,

Cardona²,

Sarfati³

1995

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

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Enzymatic incorporation of 2',3'-dide-ixynucleotides into DNA results in chain termination. We report that 3'-esterified 2'-deoxynucleoside 5'-triphosphates (dNTPs) are false chain-terminator substrates since DNA polymerases, including human immunodeficiency virus reverse transcriptase, can incorporate them into DNA and, subsequently, use this new 3' end to insert the next correctly paired dNTP. Likewise, a DNA substrate with a primer chemically esterified at the 3' position can be extended efficiently upon incubation with dNTPs and T7 DNA polymerase lacking 3'-to-5' exonuclease activity. This enzyme is also able to use dTTP-bearing reporter groups in the 3' position conjugated through amide or thiourea bonds and cleave them to restore a DNA chain terminated by an amino group at the 3' end. Hence, a number of DNA polymerases exhibit wide catalytic versatility at the 3' end of the nascent DNA strand. As part of the polymerization mechanism, these capabilities extend the number of enzymatic activities associated with these enzymes and also the study of interactions between DNA polymerases and nucleotide analogues.

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Section: Discussionsupporting

confidence: 77%

Catalytic editing properties of DNA polymerases.

Canard¹,

Cardona²,

Sarfati³

1995

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

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“…The 17-member oligonucleotide d(GTAAAACCACGGCCAGT) and the 14-member d(CCCAGTCACGACGT) were labeled with [32p] at the 5'-position using polynucleotide kinase and [7-32p]ATP. dNTP(3'-NH2) and dNTP(3'-N3) [10], dNTP(3'-F) Jill dNTP(3'-OCH3) [12], araNTP(3'-N3) and ara-NTP(3'-NH2) [13] were synthesized as described.…”

Section: Methodsmentioning

confidence: 99%

“…From tracks 4-5 it can be seen that dddTTP terminates DNA chain elongation catalyzed by AMV reverse transcriptase, the effectiveness of this process being significantly greater than that in control tracks. For these control tracks, termination was achieved with dNTP(3'-OCH3) (tracks 2,3) [12]. Strong termination of DNA synthesis in the presence of dddTTP is observed in a set of bands with shorter DNA fragments (the fragments that terminated on 8T, 9T, 14T, 20T, 25T, 27T, 34T) in comparison with DNA fragments in tracks 2 and 3.…”

Section: Data Polymerase I Kf From E Coli Amvmentioning

confidence: 99%

“…track 6). In the case of DNA polymerase I KF, the effect of dddTTP was compared with that of dTTP(3'-F) [11]; dTTP(3'-F) is known to be one of the strongest termination substrates for the system with DNA polymerase I KF [12]. From tracks 12 and 14,15 it FEBS is clear that both compounds terminate DNA synthesis but the effect of dddTTP is stronger.…”

Section: Data Polymerase I Kf From E Coli Amvmentioning

confidence: 99%

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Properties of 2′,3′‐dideoxy‐2′,3′‐dehydrothymidine 5′‐triphosphate in terminating DNA synthesis catalyzed by several different DNA polymerases

Dyatkina¹,

Minassian²,

Kukhanova³

et al. 1987

FEBS Letters

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-dehydrothymidine 5'-triphosphate (dddTTP) shows termination substrate properties in the DNA synthesis catalyzed by E. coli DNA polymerase I KF, rat liver DNA polymerase r, reverse transcriptases of avian myeloblastosis virus and Raus sarcoma virus and calf thymus terminal deoxynucleotidyl transferase. This implies that the mononucleotide residue of dddTTP incorporates into 3'-termini of newly synthesized DNA chains. However, dddTTP has no influence on the DNA synthesis catalyzed by calf thymus DNA polymerase ct. In the case of some DNA polymerases dddTTP was one order of magnitude more effective in comparison with the other known termination substrates.

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“…It has been reported that nucleotides with ether linkages at the 3Ј position can be incorporated by some DNA polymerases, whereas those with ester linkages are not generally accepted by most of the polymerases tested (18). Significant efforts have been dedicated to evaluating a wide variety of 3Ј-modified nucleotides to be used as terminators for various DNA polymerases and reverse transcriptases, but none of the functional groups tested have had established methods to regenerate a free 3Ј-OH (19)(20)(21)(22).…”

mentioning

confidence: 99%

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

Ruparel

Bi²,

Li³

et al. 2005

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

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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-3␣,4␣-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. 2-nitrobenzyl linker ͉ photocleavageT he completion of the Human Genome Project (1, 2) has led to an increased demand for high-throughput and rapid DNA sequencing methods to identify genetic variants for applications in pharmacogenomics (3), disease gene discovery (4, 5), and gene function studies (6). Current state-of-the-art DNA sequencing technologies (7-11) to some extent address the accuracy and throughput requirements but suffer limitations with respect to cost and data quality. Thus, a new DNA sequencing approach is required to broaden the applications of genomic information in medical research and health care. In this regard, DNA sequencing by synthesis (SBS) offers an alternative approach to possibly address the limitations of current DNA sequencing techniques. We have previously described the design of a parallel chip-based SBS system, which uses a self-priming DNA template covalently linked to the glass surface of a chip and four modified nucleotides (12-14). The nucleotides are modified such that they have a photocleavable fluorescent moiety attached to the base (5 position of pyrimidines, 7 position of purines) and a chemically cleavable group to cap the 3Ј-OH. When the correct nucleotide is incorporated in a DNA polymerase reaction, specific to the template sequence, the reaction is temporarily terminated because of the lack of a free 3Ј-OH group. After the fluorescent signal is detected ...

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3′‐Hydroxymethyl 2′‐deoxynucleoside 5′‐triphosphates are inhibitors highly specific for reverse transcriptase

Cited by 14 publications

References 12 publications

Catalytic editing properties of DNA polymerases.

Catalytic editing properties of DNA polymerases.

Properties of 2′,3′‐dideoxy‐2′,3′‐dehydrothymidine 5′‐triphosphate in terminating DNA synthesis catalyzed by several different DNA polymerases

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

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