ArticleNucleic acids research2018
Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries.
Article in Nucleic acids research, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 25 citations in OpenAlex.
- A toolbox for enzymatic modification of nucleic acids with photosensitizers for photodynamic therapy.RSC chemical biology · 2024Article
- Dynamic stem-loop extension by Pol θ and templated insertion during DNA repair.The Journal of biological chemistry · 2024Article
- Controlled enzymatic synthesis of oligonucleotides.Communications chemistry · 2024Review
- Aptamer Technologies in Neuroscience, Neuro-Diagnostics and Neuro-Medicine Development.Molecules (Basel, Switzerland) · 2024Review
- Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants.RNA (New York, N.Y.) · 2023Article
- Genome Protection by DNA Polymerase θ.Annual review of genetics · 2022Review
- DNA aptamer-based rolling circle amplification product as a novel immunological adjuvant.Scientific reports · 2020Article
- Review
- A polar filter in DNA polymerases prevents ribonucleotide incorporation.Nucleic acids research · 2019Article
- Engineering Polymerases for New Functions.Trends in biotechnology · 2019Review
- One-step enzymatic modification of RNA 3' termini using polymerase θ.Nucleic acids research · 2019Article
- Recent Advances in Aptamer Discovery and Applications.Molecules (Basel, Switzerland) · 2019Review
Corrections and comments
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Authors and funding
9 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Nucleic acid aptamers, especially RNA, exhibit valuable advantages compared to protein therapeutics in terms of size, affinity and specificity. However, the synthesis of libraries of large random RNAs is still difficult and expensive. The engineering of polymerases able to directly generate these libraries has the potential to replace the chemical synthesis approach. Here, we start with a DNA polymerase that already displays a significant template-free nucleotidyltransferase activity, human DNA polymerase theta, and we mutate it based on the knowledge of its three-dimensional structure as well as previous mutational studies on members of the same polA family. One mutant exhibited a high tolerance towards ribonucleotides (NTPs) and displayed an efficient ribonucleotidyltransferase activity that resulted in the assembly of long RNA polymers. HPLC analysis and RNA sequencing of the products were used to quantify the incorporation of the four NTPs as a function of initial NTP concentrations and established the randomness of each generated nucleic acid sequence. The same mutant revealed a propensity to accept other modified nucleotides and to extend them in long fragments. Hence, this mutant can deliver random natural and modified RNA polymers libraries ready to use for SELEX, with custom lengths and balanced or unbalanced ratios.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.