Minimizing amplification bias during reverse transcription for in vitro selections

Area of Science:

• Molecular Biology
• Biotechnology
• Genomics

Background:

• Systematic Evolution of Ligands through Exponential Enrichment (SELEX) identifies functional nucleic acids like aptamers and ribozymes.
• Reverse transcription amplification biases can negatively impact SELEX efficiency, disadvantaging functional sequences.
• Structural scaffolds in libraries can improve SELEX but are also prone to amplification biases.

Purpose of the Study:

• To evaluate five reverse transcriptases (RTs) for their bias during nucleic acid amplification in SELEX.
• To identify the RT enzyme that introduces the least bias and maximizes the accuracy of SELEX outcomes.
• To compare the performance of BST 3.0 DNA polymerase (BST), SuperScript IV (SSIV), ImProm-II, Marathon RT (MaRT), and TGIRT-III.

Main Methods:

• Direct comparison of cDNA yield and processivity for five RTs on RNA templates with varying structures.
• Head-to-head competition of six RNA libraries with different structural elements in amplification-only selection.
• High-throughput sequencing to analyze interlibrary bias and mutational bias across six rounds of selection.

Main Results:

• BST 3.0 DNA polymerase (BST) demonstrated excellent processivity and high full-length cDNA yield.
• BST exhibited minimal bias across templates with diverse structures and sequences, performing well on long, structured viral RNAs.
• BST maintained the most neutral enrichment values in head-to-head library competitions, indicating low interlibrary and minimal mutational bias compared to SSIV and ImProm-II.

Conclusions:

• BST 3.0 DNA polymerase (BST) is a superior enzyme for minimizing reverse transcription bias in SELEX.
• Using BST can significantly improve the accuracy and efficiency of identifying functional nucleic acids in SELEX experiments.
• BST's performance makes it ideal for SELEX applications involving complex or highly structured RNA libraries.