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Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides

Ashley, J, Schaap-Johansen, A-L, Mohammadniaei, M, Naseri, M, Marcatili, P, Prado, M and Sun, Y (2021) Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides. Nucleic Acids Research, 49 (2). pp. 1065-1074. ISSN 0305-1048

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Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.

Item Type: Article
Uncontrolled Keywords: Science & Technology; Life Sciences & Biomedicine; Biochemistry & Molecular Biology; Thrombin; DNA Nucleotidylexotransferase; Lactoferrin; DNA; DNA, Single-Stranded; Electrophoretic Mobility Shift Assay; Drug Evaluation, Preclinical; Nucleic Acid Amplification Techniques; Binding Sites; Protein Binding; Substrate Specificity; Gene Library; Aptamers, Nucleotide; High-Throughput Nucleotide Sequencing; V(D)J Recombination; Aptamers, Nucleotide; Binding Sites; DNA; DNA Nucleotidylexotransferase; DNA, Single-Stranded; Drug Evaluation, Preclinical; Electrophoretic Mobility Shift Assay; Gene Library; High-Throughput Nucleotide Sequencing; Lactoferrin; Nucleic Acid Amplification Techniques; Protein Binding; Substrate Specificity; Thrombin; V(D)J Recombination; Developmental Biology; 05 Environmental Sciences; 06 Biological Sciences; 08 Information and Computing Sciences
Subjects: R Medicine > RM Therapeutics. Pharmacology
Divisions: Pharmacy & Biomolecular Sciences
Publisher: Oxford University Press (OUP)
SWORD Depositor: A Symplectic
Date Deposited: 13 Jun 2022 10:17
Last Modified: 13 Jun 2022 10:17
DOI or ID number: 10.1093/nar/gkaa1263
URI: https://researchonline.ljmu.ac.uk/id/eprint/17051
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