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Minimizing amplification bias during reverse transcription for in vitro selections.
Lucas, Jordyn K; Gruenke, Paige R; Burke, Donald H.
Afiliación
  • Lucas JK; Department of Biochemistry, University of Missouri, Columbia, Missouri 65201, USA.
  • Gruenke PR; Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65201, USA.
  • Burke DH; Bond Life Sciences Center, University of Missouri, Columbia, Missouri 65201, USA.
RNA ; 29(8): 1301-1315, 2023 08.
Article en En | MEDLINE | ID: mdl-37192815
Systematic evolution of ligands through exponential enrichment (SELEX) is widely used to identify functional nucleic acids, such as aptamers and ribozymes. Ideally, selective pressure drives the enrichment of sequences that display the function of interest (binding, catalysis, etc.). However, amplification biases from reverse transcription can overwhelm this enrichment and leave some functional sequences at a disadvantage, with cumulative effects across multiple rounds of selection. Libraries that are designed to include structural scaffolds can improve selection outcomes by sampling sequence space more strategically, but they are also susceptible to such amplification biases, particularly during reverse transcription. Therefore, we tested five reverse transcriptases (RTs)-ImProm-II, Marathon RT (MaRT), TGIRT-III, SuperScript IV (SSIV), and BST 3.0 DNA polymerase (BST)-to determine which enzymes introduced the least bias. We directly compared cDNA yield and processivity for these enzymes on RNA templates with varying degrees of structure under various reaction conditions. In these analyses, BST exhibited excellent processivity, generated large quantities of the full-length cDNA product, displayed little bias among templates with varying structure and sequence, and performed well on long, highly structured viral RNAs. Additionally, six RNA libraries containing either strong, moderate, or no incorporated structural elements were pooled and competed head-to-head in six rounds of an amplification-only selection without external selective pressure using either SSIV, ImProm-II, or BST during reverse transcription. High-throughput sequencing established that BST maintained the most neutral enrichment values, indicating low interlibrary bias over the course of six rounds, relative to SSIV and ImProm-II, and it introduced minimal mutational bias.
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Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Transcripción Reversa / Aptámeros de Nucleótidos Idioma: En Revista: RNA Asunto de la revista: BIOLOGIA MOLECULAR Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos

Texto completo: 1 Colección: 01-internacional Banco de datos: MEDLINE Asunto principal: Transcripción Reversa / Aptámeros de Nucleótidos Idioma: En Revista: RNA Asunto de la revista: BIOLOGIA MOLECULAR Año: 2023 Tipo del documento: Article País de afiliación: Estados Unidos