Directed evolution and selection of biostable L-DNA aptamers with a mirror-image DNA polymerase.
basic_science · Level V
Where this comes from
- Record sourced from PubMed, PMID 35668324.
- Also identified by DOI 10.1038/s41587-022-01337-8 and PMC identifier 9646512.
- Licence recorded as CC BY.
- The licence permits redistribution, so the abstract is shown in full and the full text is available from the publisher.
Abstract
Mirror-image aptamers made from chirally inverted nucleic acids are nuclease-resistant and exceptionally biostable, opening up opportunities for unique applications. However, the directed evolution and selection of mirror-image aptamers directly from large randomized L-DNA libraries has, to our knowledge, not been demonstrated previously. Here, we developed a 'mirror-image selection' scheme for the directed evolution and selection of biostable L-DNA aptamers with a mirror-image DNA polymerase. We performed iterative rounds of enrichment and mirror-image polymerase chain reaction (PCR) amplification of L-DNA sequences that bind native human thrombin, in conjunction with denaturing gradient gel electrophoresis (DGGE) to isolate individual aptamers and L-DNA sequencing-by-synthesis to determine their sequences. Based on the selected L-DNA aptamers, we designed biostable thrombin sensors and inhibitors, which remained functional in physiologically relevant nuclease-rich environments, even in the presence of human serum that rapidly degraded D-DNA aptamers. Mirror-image selection of biostable L-DNA aptamers directly from large randomized L-DNA libraries greatly expands the range of biomolecules that can be targeted, broadening their applications as biostable sensors, therapeutics and basic research tools.
Medical subject headings
- Aptamers, Nucleotide