Selection of Primer-Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:14 |
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| Enthalten in: |
Viruses - 14(2022), 2 vom: 10. Feb. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
DeStefano, Jeffrey J [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 14.03.2022 Date Revised 21.09.2022 published: Electronic Citation Status MEDLINE |
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| doi: |
10.3390/v14020369 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 100 | 1 | |a DeStefano, Jeffrey J |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Selection of Primer-Template Sequences That Bind with Enhanced Affinity to Vaccinia Virus E9 DNA Polymerase |
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| 500 | |a Date Revised 21.09.2022 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Extramural | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a DNA polymerase sequence preference | |
| 650 | 4 | |a E9 | |
| 650 | 4 | |a SELEX | |
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| 650 | 7 | |a DNA-Directed DNA Polymerase |2 NLM | |
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| 700 | 1 | |a Iseni, Frédéric |e verfasserin |4 aut | |
| 700 | 1 | |a Tarbouriech, Nicolas |e verfasserin |4 aut | |
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