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Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening.

ABSTRACT: In vitro evolution strategies have been used for >30 years to generate nucleic acid aptamers against therapeutic targets of interest, including disease-associated proteins. However, this process requires many iterative cycles of selection and amplification, which severely restricts the number of target and library design combinations that can be explored in parallel. Here, we describe a single-round screening approach to aptamer discovery that relies on function-enhancing chemotypes to increase the distribution of high-affinity sequences in a random-sequence library. We demonstrate the success of de novo discovery by affinity selection of threomers against the receptor binding domain of the S1 protein from SARS-CoV-2. Detailed biochemical characterization of the enriched population identified threomers with binding affinity values that are comparable to aptamers produced by conventional SELEX. This work establishes a highly parallelizable path for querying diverse chemical repertoires and may offer a viable route for accelerating the discovery of therapeutic aptamers.

SUBMITTER: Lozoya-Colinas A

PROVIDER: S-EPMC10690791 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening.

Lozoya-Colinas Adriana A Yu Yutong Y Chaput John C JC

Journal of the American Chemical Society 20231114 47

In vitro evolution strategies have been used for >30 years to generate nucleic acid aptamers against therapeutic targets of interest, including disease-associated proteins. However, this process requires many iterative cycles of selection and amplification, which severely restricts the number of target and library design combinations that can be explored in parallel. Here, we describe a single-round screening approach to aptamer discovery that relies on function-enhancing chemotypes to increase ...[more]

PMID: 37962593

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Project description:Here, we report on the construction of a metagenomic library from a chitin-amended disease-suppressive agricultural soil and its screening for genes that encode novel chitinolytic enzymes. The library, constructed in fosmids in an Escherichia coli host, comprised 145,000 clones containing inserts of sizes of 21 to 40 kb, yielding a total of approximately 5.8 GB of cloned soil DNA. Using genetic screenings by repeated PCR cycles aimed to detect gene sequences of the bacterial chitinase A-class (hereby named chi A genes), we identified and characterized five fosmids carrying candidate genes for chitinolytic enzymes. The analysis thus allowed access to the genomic (fosmid-borne) context of these genes. Using the chiA-targeted PCR, which is based on degenerate primers, the five fosmids all produced amplicons, of which the sequences were related to predicted chitinolytic enzyme-encoding genes of four different host organisms, including Stenotrophomonas maltophilia. Sequencing and de novo annotation of the fosmid inserts confirmed that each one of these carried one or more open reading frames that were predicted to encode enzymes active on chitin, including one for a chitin deacetylase. Moreover, the genetic contexts in which the putative chitinolytic enzyme-encoding genes were located were unique per fosmid. Specifically, inserts from organisms related to Burkholderia sp., Acidobacterium sp., Aeromonas veronii, and the chloroflexi Nitrolancetus hollandicus and/or Ktedonobacter racemifer were obtained. Remarkably, the S. maltophilia chiA-like gene was found to occur in two different genetic contexts (related to N. hollandicus/K. racemifer), indicating the historical occurrence of genetic reshufflings in this part of the soil microbiota. One fosmid containing the insert composed of DNA from the N. hollandicus-like organism (denoted 53D1) was selected for further work. Using subcloning procedures, its putative gene for a chitinolytic enzyme was successfully brought to expression in an E. coli host. On the basis of purified protein preparations, the produced protein was characterized as a chitobiosidase of 43.6 kDa, with a pI of 4.83. Given its activity spectrum, it can be typified as a halotolerant chitobiosidase.

Dataset Information

Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening.

Publications

Functionally Enhanced XNA Aptamers Discovered by Parallelized Library Screening.

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