Send to

Choose Destination
Nucleic Acids Res. 2018 Jul 6;46(12):6271-6284. doi: 10.1093/nar/gky413.

Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries.

Author information

Unit of Structural Dynamics of Biological Macromolecules, CNRS UMR 3528, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Transcriptome and EpiGenome platform, BioMics, Center of Innovation and Technological Research, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.
Hub informatique et Biostatistique, Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI, USR 3756 IP-CNRS), Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France.
Unité de Chimie et Biocatalyse, CNRS UMR 3523, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France.


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.

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center