Dataset Information

Selection of Primer-Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase.

ABSTRACT: A modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) pr,otocol (referred to as PT SELEX) was used to select primer-template (P/T) sequences that bound to the vaccinia virus polymerase catalytic subunit (E9) with enhanced affinity. A single selected P/T sequence (referred to as E9-R5-12) bound in physiological salt conditions with an apparent equilibrium dissociation constant (K_D,app) of 93 ± 7 nM. The dissociation rate constant (k_off) and binding half-life (t_1/2) for E9-R5-12 were 0.083 ± 0.019 min^-1 and 8.6 ± 2.0 min, respectively. The values indicated a several-fold greater binding ability compared to controls, which bound too weakly to be accurately measured under the conditions employed. Loop-back DNA constructs with 3'-recessed termini derived from E9-R5-12 also showed enhanced binding when the hybrid region was 21 nucleotides or more. Although the sequence of E9-R5-12 matched perfectly over a 12-base-pair segment in the coding region of the virus B20 protein, there was no clear indication that this sequence plays any role in vaccinia virus biology, or a clear reason why it promotes stronger binding to E9. In addition to E9, five other polymerases (HIV-1, Moloney murine leukemia virus, and avian myeloblastosis virus reverse transcriptases (RTs), and Taq and Klenow DNA polymerases) have demonstrated strong sequence binding preferences for P/Ts and, in those cases, there was biological or potential evolutionary relevance. For the HIV-1 RT, sequence preferences were used to aid crystallization and study viral inhibitors. The results suggest that several other DNA polymerases may have P/T sequence preferences that could potentially be exploited in various protocols.

SUBMITTER: DeStefano JJ

PROVIDER: S-EPMC8880465 | biostudies-literature | 2022 Feb

REPOSITORIES: biostudies-literature

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Selection of Primer-Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase.

DeStefano Jeffrey J JJ Iseni Frédéric F Tarbouriech Nicolas N

Viruses 20220210 2

A modified SELEX (Systematic Evolution of Ligands by Exponential Enrichment) pr,otocol (referred to as PT SELEX) was used to select primer-template (P/T) sequences that bound to the vaccinia virus polymerase catalytic subunit (E9) with enhanced affinity. A single selected P/T sequence (referred to as E9-R5-12) bound in physiological salt conditions with an apparent equilibrium dissociation constant (KD,app) of 93 ± 7 nM. The dissociation rate constant (koff) and bind ...[more]

PMID: 35215961

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Dataset Information

Selection of Primer-Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase.

Publications

Selection of Primer-Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase.

Similar Datasets

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