Selection of Primer–Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase
| dc.contributor.author | DeStefano, Jeffrey J. | |
| dc.contributor.author | Iseni, Frédéric | |
| dc.contributor.author | Tarbouriech, Nicolas | |
| dc.date.accessioned | 2023-10-27T18:21:17Z | |
| dc.date.available | 2023-10-27T18:21:17Z | |
| dc.date.issued | 2022-02-10 | |
| dc.description.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 (KD,app) of 93 ± 7 nM. The dissociation rate constant (koff) and binding half-life (t1/2) 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. | |
| dc.description.uri | https://doi.org/10.3390/v14020369 | |
| dc.identifier | https://doi.org/10.13016/dspace/nnep-1z38 | |
| dc.identifier.citation | DeStefano, J.J.; Iseni, F.; Tarbouriech, N. Selection of Primer–Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase. Viruses 2022, 14, 369. | |
| dc.identifier.uri | http://hdl.handle.net/1903/31159 | |
| dc.language.iso | en_US | |
| dc.publisher | MDPI | |
| dc.relation.isAvailableAt | Cell Biology & Molecular Genetics | en_us |
| dc.relation.isAvailableAt | Digital Repository at the University of Maryland | en_us |
| dc.relation.isAvailableAt | College of Computer, Mathematical & Natural Sciences | en_us |
| dc.relation.isAvailableAt | University of Maryland (College Park, MD) | en_us |
| dc.subject | vaccinia virus polymerase | |
| dc.subject | E9 | |
| dc.subject | DNA polymerase sequence preference | |
| dc.subject | SELEX | |
| dc.title | Selection of Primer–Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase | |
| dc.type | Article | |
| local.equitableAccessSubmission | No |