Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries Irina Randrianjatovo-Gbalou, Sandrine Rosario, Odile Sismeiro, Hugo Varet, Rachel Legendre, Jean-Yves Coppée, Valerie Huteau, Sylvie Pochet, Marc Delarue To cite this version: Irina Randrianjatovo-Gbalou, Sandrine Rosario, Odile Sismeiro, Hugo Varet, Rachel Legendre, et al.. Enzymatic synthesis of random sequences of RNA and RNA analogues by DNA polymerase theta mutants for the generation of aptamer libraries. Nucleic Acids Research, Oxford University Press, 2018, 46 (12), pp.6271-6284. 10.1093/nar/gky413. pasteur-02170331 HAL Id: pasteur-02170331 https://hal-pasteur.archives-ouvertes.fr/pasteur-02170331 Submitted on 1 Jul 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Distributed under a Creative Commons Attribution| 4.0 International License Published online 21 May 2018 Nucleic Acids Research, 2018, Vol. 46, No. 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 Irina Randrianjatovo-Gbalou1, Sandrine Rosario1, Odile Sismeiro2, Hugo Varet2,3, 2,3 2 4 4 Rachel Legendre , Jean-Yves Coppee´ ,Valerie´ Huteau , Sylvie Pochet and Downloaded from https://academic.oup.com/nar/article-abstract/46/12/6271/5000021 by Institut Pasteur user on 03 July 2019 Marc Delarue1,* 1Unit of Structural Dynamics of Biological Macromolecules, CNRS UMR 3528, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France, 2Transcriptome and EpiGenome platform, BioMics, Center of Innovation and Technological Research, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France, 3Hub informatique et Biostatistique, Centre de Bioinformatique, Biostatistique et Biologie Integrative´ (C3BI, USR 3756 IP-CNRS), Institut Pasteur, 28 Rue du Docteur Roux, 75724 Paris Cedex 15, France and 4Unite´ de Chimie et Biocatalyse, CNRS UMR 3523, Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France Received February 08, 2018; Revised April 12, 2018; Editorial Decision May 02, 2018; Accepted May 04, 2018 ABSTRACT INTRODUCTION Nucleic acid aptamers, especially RNA, exhibit valu- In the field of therapeutic biotechnologies, nucleic acids able advantages compared to protein therapeutics have proved to be a useful tool for the regulation of gene in terms of size, affinity and specificity. However, expression, medical diagnostics, biological route modula- the synthesis of libraries of large random RNAs tion, molecular recognition strategies or drug design. Key is still difficult and expensive. The engineering of strategies have been developed and have proved their effi- ciency for several years such as antisense nucleic acids (1,2), polymerases able to directly generate these libraries ribozymes and riboswitches (3,4) and aptamers (5,6). How- has the potential to replace the chemical synthe- ever, some innovative breakthrough in the generation of sis approach. Here, we start with a DNA polymerase aptamer-based molecules remains needed to compete with that already displays a significant template-free nu- medicinal chemistry and/or biological antibodies. cleotidyltransferase activity, human DNA polymerase Nucleic acid aptamers consist of single-stranded DNA theta, and we mutate it based on the knowledge of its (ssDNA) or RNA that present defined 3D structures due to three-dimensional structure as well as previous mu- their propensity to form complementary base pairs, Hoog- tational studies on members of the same polA fam- steen base pairs, base triples or G-quartets. Their ability ily. One mutant exhibited a high tolerance towards to fold into various secondary and tertiary structures (7) ribonucleotides (NTPs) and displayed an efficient ri- opens the possibility to design molecules that are capable bonucleotidyltransferase activity that resulted in the of specific molecular recognition of their cognate targets. RNA aptamers are prone to generate more complex 3D assembly of long RNA polymers. HPLC analysis and structures than DNA aptamers and usually display a higher RNA sequencing of the products were used to quan- binding affinity and specificity (6). Van der Waals forces, tify the incorporation of the four NTPs as a function hydrophobic and electrostatic interactions, triple base-pairs of initial NTP concentrations and established the ran- and base stacking all combine to generate folded struc- domness of each generated nucleic acid sequence. tures and shielded active sites that determine their bind- The same mutant revealed a propensity to accept ing affinity and specificity. For all of these purposes, ap- other modified nucleotides and to extend them in tamers are listed among the most important classes of drug long fragments. Hence, this mutant can deliver ran- molecules and their development is facilitated by Systematic dom natural and modified RNA polymers libraries Evolution of Ligands by EXponential enrichment (SELEX) ready to use for SELEX, with custom lengths and strategies (8,9). This efficient method of producing high- balanced or unbalanced ratios. affinity aptamers relies on the generation of a combinato- rial library of oligonucleotides. These libraries must contain a huge pool of random-sequence oligonucleotides to max- *To whom correspondence should be addressed. Tel: +33 1 45 68 86 05; Email: [email protected] C The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 6272 Nucleic Acids Research, 2018, Vol. 46, No. 12 imize the chances to select good candidates. As an alterna- sition metal ions such as Mn2+. Among them TdT (Termi- tive to the chemical synthesis of random oligonucleotides at nal deoxynucleotidyl Transferase) is known to catalyze ef- the first step of aptamer selection, the engineering of DNA ficient, non-templated, random nucleotide addition at the polymerases designed to synthesize nucleic acids (RNA and V(D)J junctions to increase the diversity of the adaptive im- DNA) in a random fashion may provide a faster way to gen- mune system repertoire (24). It was also recently shown that erate the initial libraries. TdT can, in some conditions, have an in trans templated ac- In vivo, DNA polymerases are responsible for the DNA tivity on a DNA synapsis (25,26). On top of this property, replication and the maintenance of the genome and there- previous studies revealed that TdT indiscriminately incor- fore their role is critical for the propagation of the ge- porates ribonucleotides (NTPs) and deoxyribonucleotides netic information. All DNA polymerases found in eukary- (dNTPs) (27) but then fails to extend the primer beyond Downloaded from https://academic.oup.com/nar/article-abstract/46/12/6271/5000021 by Institut Pasteur user on 03 July 2019 otes, prokaryotes, archaea and viruses have been classi- 4–5 ribonucleotides when the substrate sensed by the en- fied into subfamilies according to their structure and pri- zyme is no longer a DNA but an RNA. This observation mary sequence similarity (10–12). By construction, replica- is compatible with the known crystal structure of murine tive DNA polymerases have evolved to copy the template TdT which shows that the steric barrier close to the 2 po- DNA with high fidelity and consequently their native ac- sition of the incoming dATP sugar in pol ␤ and ␭,aty- tivity is of limited use for applications in modern synthetic rosine residue, is replaced by a glycine in pol ␮ and Tdt biology, which seeks to build novel and versatile nucleic (28). However, engineering TdT to make it accept an RNA acid polymers. Indeed, DNA polymerases have an active primer seems quite a challenge, given that the conformation site that is configured to incorporate the four canonical de- of the primer is a B-DNA structure in the tertiary complex, oxyribonucleotides and to exclude ‘altered’ or modified nu- contrary to what would be favored for an RNA primer (A- cleotides during cellular metabolism. However, the design DNA). In other words, there seems to be no ‘gate’ to the ri- of mutants based on the known three-dimensional structure bonucleotides in the catalytic site of TdT but rather one or of a DNA polymerase, if available, has the potential to lead several ‘checkpoints’ in guiding the primer single-stranded to engineered enzyme(s) with the desired property in a rel- oligonucleotide. atively straightforward way. Taken together, the availability of crystal structures of Pol DNA polymerases are divided into several families, based ␪ and TdT as well as their known predisposition to perform on sequence alignments (11,12). Classically, polA, polB, random nucleotides incorporation in a template-free man- polY and reverse transcriptases (RT) all share the same fold- ner suggest the possibility to engineer them so as to make ing type, while polC and polX share a different one. Never- them tolerate both NTPs and dNTPs, as well as other mod- theless, they all have the same kind of catalytic site, based on ified nucleotides, opening the way to create novel random the two-metal ion mechanism (13). To our knowledge, only nucleic acid synthesizing machines. members of the polA and polX families display a significant Given previous studies on the engineering of the gate of nucleotidyltransferase activity.