Abstract
AbstractMirror-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.
Funder
National Natural Science Foundation of China
Publisher
Springer Science and Business Media LLC
Subject
Biomedical Engineering,Molecular Medicine,Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
Cited by
29 articles.
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