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

Viruses

Jeffrey J. DeStefano¹  Nicolas Tarbouriech²  Frédéric Iseni³ 

[1] Department of Cell Biology and Molecular Genetics, Bioscience Research Building, University of Maryland, College Park, MD 20742, USA;Institut de Biologie Structurale, Université Grenoble Alpes, Centre National de la Recherche Scientifique, Commissariat à l’Energie Atomique et aux Energies Alternatives, IBS, 38000 Grenoble, France;Unité de Virologie, Institut de Recherche Biomédicale des Armées, BP73, 91223 Brétigny-sur-Orge, France;

关键词: vaccinia virus polymerase;    E9;    DNA polymerase sequence preference;    SELEX;   

DOI  :  10.3390/v14020369

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown