BACKGROUND OF THE INVENTION

In the field of therapeutic biotechnologies, nucleic acids have proved to be a useful tool for the regulation of gene expression, medical diagnostics, biological route modulation, molecular recognition strategies or drug design. Key strategies have been developed and have proved their efficiency for several years such as antisense nucleic acids1,2, ribozymes and riboswitches3,4and aptamers5,6. However, some innovative breakthrough in the generation of aptamer-based molecules remains needed to compete with medicinal chemistry and/or biological antibodies.

Nucleic acid aptamers consist of single-stranded DNA (ssDNA) or RNA that present defined 3D structures due to their propensity to form complementary base pairs. Their ability to fold into various secondary and tertiary structures' opens the possibility to design different conformations that are capable of specific molecular recognition of their cognate targets. RNA aptamers are prone to generate more complex 3D structures than DNA aptamers and usually display a higher binding affinity and specificity6. Triple base-pairs, hydrophobic and electrostatic interactions, van der Waals forces, shape complementarity and base stacking all combine to generate folded structures and shielded active sites that determine their binding affinity and specificity. For all of these purposes, aptamers are listed among the most important classes of drug molecules and their development is facilitated by Systematic Evolution of Ligands by EXponential enrichment (SELEX) strategies8,9. This efficient method of producing high-affinity of aptamers relies on the generation of a combinatorial library of oligonucleotides (around 1015). These libraries must contain a huge pool of random-sequence oligonucleotides to maximize the chances to select good candidates. As an alternative to the chemical synthesis of random oligonucleotides at the first step of aptamer selection, the engineering of DNA polymerases designed to synthesize nucleic acids (RNA and DNA) in a random fashion may become the cutting edge of SELEX.

In vivo, DNA polymerases are crucial to the DNA replication and the maintenance of the genome and therefore their role is critical for the propagation of the genetic information. All DNA polymerases found in eukaryotes, prokaryotes, archaea and viruses have been classified into subfamilies according to their structure and primary sequence similarity10,11. By their inherent role, replicative DNA polymerases copy the template DNA with high fidelity and consequently their native activity is of limited use for applications in modern synthetic biology, which seeks to build novel and versatile nucleic acid polymers. Indeed, DNA polymerases have an active site that is configured to incorporate the four canonical deoxyribonucleotides and to exclude ‘altered’ or modified nucleotides during cellular metabolism.

Representative members of family A with a known crystal structure areE. coliDNA Pol I11andT. aquaticusDNA pol I12in prokaryotes, and phage T713DNA pol in viruses; in eukaryotes, Pol theta (Pol θ) takes part in the repair of DNA double-strand-breaks (DSB) in the so-called Non-Homologous End Joining (NHEJ) process)14-16, pol nu (pol v) in the repair of DNA crosslinks occurring during homologous recombination (HR) and Pol gamma (Pol γ) is involved in mitochondrial DNA replication17. Very recently, human Pol theta has been described to display a robust terminal transferase activity that is apparent when it switches between three different mechanisms during alternative end-joining (alt-EJ)18,19. Indeed, human Pol theta is able to perform non-templated DNA extension, as well as instructed replication that is templated in cis or in trans of the DSB. The same study revealed that the non-templated transferase activity is enhanced in the presence of manganese divalent ions and can be randomly combined with templated extension on the 3′-end of a nucleic acid primer.

The other family of DNA polymerases which has some significant nucleotidyltransferase activity is the pol X family, especially in eukaryotes (Pol beta (Pol β), Pol lambda (Pol γ), Pol mu (Pol μ), and TdT)20,21. This activity is usually enhanced in the presence of transition metal ions. Among them, TdT (Terminal deoxynucleotidyl Transferase) is described to catalyze non-templated, random nucleotide addition at the V(D)J junctions to increase the antigen receptor diversity18,22,23. On top of this property, previous studies revealed that TdT indiscriminately incorporates ribonucleotides (NTPs) and deoxyribonucleotides (dNTPs)24,25but then fails to extend it beyond 4-5 nucleotides because the primer is no longer a DNA but an RNA. This observation is compatible with the known crystal structure of murine TdT where none of the protein residues seemed to act as steric barrier close to the 2′ or the 3′ position of the ddATP sugar25. However, engineering TdT to make it accept an RNA primer seems quite a challenge, given the conformation of the primer in the structure of the tertiary complex, where the conformation of the primer strand is a B-DNA, contrary to what would be expected for an RNA primer (A-DNA).

NTPs differ from dNTPs only by the presence of an additional hydroxyl group in the 2′-position of the ribose. It was shown that human Pol theta incorporated the NTPs better than TdT18and enabled the synthesis of long polymers of RNAs, although with low yields. The joint predisposition of such DNA polymerases (Pol theta and TdT), to i) perform random nucleotides incorporation in a template-free manner and ii) the possibility to tolerate both NTPs and dNTPs and other modified nucleotides, opens the way to create a novel nucleic acid synthetic machine. This suggests that random RNA library could be enzymatically generated much easier without the need of the reverse-transcription step of DNA fragments during SELEX.

There is a need in the art for some DNA Polymerase theta mutants capable of incorporating a large diversity of nucleic analogs and generating long polymers of, nucleic acids analogs, capable of increasing the sampling as well as improving the stability, the affinity and the specificity of functional nucleic acids such as RNA aptamers.

BRIEF SUMMARY OF THE INVENTION

The invention relates to mutant DNA polymerases of the Pol theta subfamily capable of performing non-templated nucleic acid extension, or of a functional fragment of such a polymerase, methods of producing these mutant DNA polymerases, and uses and methods of using these mutant DNA polymerases.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at a position selected from the group consisting of: 2322, 2328, 2334, 2335, 2384, 2387 and 2391, the indicated positions being determined by alignment with SEQ ID NO: 1. In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution selected from the group consisting of: a Proline (P) to an aliphatic amino acid or a polar amino acid substitution at position 2322, an Alanine (A) to an aliphatic amino acid or a polar amino acid substitution at position 2328, a Leucine (L) to an aliphatic amino acid substitution at position 2334, a Glutamic acid (E) to an aliphatic amino acid or a polar amino acid substitution at position 2335, a Glutamine (Q) to an aliphatic amino acid or a polar amino acid substitution at position 2384, a Tyrosine (Y) to an aromatic amino acid or an aliphatic amino acid substitution at position 2387, and a Tyrosine (Y) to an aromatic amino acid or an aliphatic amino acid substitution at position 2391, the indicated positions being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2322, wherein the amino acid at position 2322 is substituted by an aliphatic amino acid selected from the group consisting of: Valine (V) and Alanine (A), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2322, wherein the amino acid at position 2322 is substituted by a polar amino acid selected from the group consisting of: Threonine (T) and Serine (S), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2328, wherein the amino acid at position 2328 is substituted by an aliphatic amino acid selected from the group consisting of: Valine (V) and Glycine (G), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2328, wherein the amino acid at position 2328 is substituted by a polar amino acid selected from the group consisting of: Threonine (T) and Serine (S), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2334, wherein the amino acid at position 2334 is substituted by an aliphatic amino acid selected from the group consisting of: Methionine (M), Isoleucine (I) and Alanine (A), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2335, wherein the amino acid at position 2335 is substituted by an aliphatic amino acid selected from the group consisting of: Glycine (G) and Alanine (A), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2335, wherein the amino acid at position 2335 is substituted by a polar amino acid selected from the group consisting of: Threonine (T) and Serine (S), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2384, wherein the amino acid at position 2384 is substituted by an Alanine (A), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2384, wherein the amino acid at position 2384 is substituted by a polar amino acid selected from the group consisting of: Asparagine (N), Serine (S) and Threonine (T), the indicated position being determined by alignment with SEQ ID NO: 1

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2387, wherein the amino acid at position 2387 is substituted by an aromatic amino acid selected from the group consisting of: Phenylalanine (F) and Tryptophan (W), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2387, wherein the amino acid at position 2387 is substituted by an aliphatic amino acid selected from the group consisting of: Alanine (A) and Valine (V), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2391, wherein the amino acid at position 2391 is substituted by an aromatic amino acid selected from the group consisting of: Phenylalanine (F) and Tryptophan (W), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution at position 2391, wherein the amino acid at position 2391 is substituted by an aliphatic amino acid selected from the group consisting of: Alanine (A) and Valine (V), the indicated position being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution selected from the group consisting of: P to V substitution at position 2322 (P2322V), A to V substitution at position 2328 (A2328V), L to M substitution at position 2334 (L2334M), E to G substitution at position 2335 (E2335G), Q to N at position 2384 (Q2384N), Y to F substitution at position 2387 (Y2387F); and Y to F substitution at position 2391 (Y2391 F), the indicated positions being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least one amino acid substitution selected from the group consisting of: P to V substitution at position 2322 (P2322V), A to V substitution at position 2328 (A2328V), L to M substitution at position 2334 (L2334M), E to G substitution at position 2335 (E2335G), and Y to F substitution at position 2387 (Y2387F), the indicated positions being determined by alignment with SEQ ID NO: 1.

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises at least two amino acid substitutions at positions 2334 and 2335, the indicated position being determined by alignment with SEQ ID NO: 1. In various embodiments, the amino acid at position 2334 is substituted by an amino acid selected from the group consisting of: Methionine (M), Isoleucine (I) and Alanine (A), and preferably by a Methionine (M). In some embodiments, the amino acid at position 2335 is substituted by an amino acid selected from the group consisting of: Glycine (G), Alanine (A), Threonine (T) and Serine (S), and preferably by a Glycine (G).

In various embodiments, the mutant DNA polymerase theta (Pol theta) or a functional fragment thereof comprises a double amino acid substitution L2334M and E2335G, the indicated positions being determined by alignment with SEQ ID NO: 1.

In various embodiments, the invention encompasses nucleic acids comprising a nucleotide sequence encoding the mutant DNA polymerase or a functional fragment thereof, a DNA vector comprising these nucleic acids, and a host cell comprising these vectors.

In various embodiments, the invention encompasses a method for generating a mutant DNA polymerase of the Pol theta subfamily or a functional fragment thereof, comprising substituting at least one amino acid in a DNA polymerase of the Pol theta subfamily at a position selected from the group consisting of: 2322, 2328, 2334, 2335, 2384, 2387 and 2391, the indicated positions being determined by alignment with SEQ ID NO: 1.

In various embodiments, the invention encompasses the use of the nucleic acids, vectors, and host cells of the invention for producing a mutant DNA polymerase or a functional fragment thereof.

In various embodiments, the invention encompasses a method for producing a mutant DNA polymerase culturing the host cell of the invention under culture conditions allowing expression of the polynucleotide encoding said mutant, and optionally recovering said mutant thus expressed from the medium culture or host cells.

In one embodiment, the invention encompasses a method for incorporating nucleotides in a template-free manner comprising incubating the mutant DNA polymerase of the invention or a functional fragment thereof with nucleotide triphosphates under conditions that allow nucleotide incorporation in the absence of a template.

In one embodiment, the invention encompasses the use the mutant DNA polymerase of the invention or a functional fragment thereof, for incorporating nucleotides in a template-free manner.

In various embodiments, the invention encompasses a method for producing degenerate or random nucleotide sequences comprising incubating the mutant DNA polymerase of the invention or a functional fragment thereof with nucleotide triphosphates under conditions that allow degenerate or random nucleotide incorporation to produce degenerate or random nucleotide sequences. In some embodiments, a fixed nucleotide sequence can be added to the 3′ end of the degenerate or random nucleotide sequences. In some embodiments, the degenerate or random nucleotide sequences can be amplified. In some embodiments, the amplified sequences can be cloned into a vector to generate a library of degenerate or random nucleotide sequences.

In one embodiment, the invention encompasses use the mutant DNA polymerase of the invention or a functional fragment thereof for generating an aptamer library.

In various embodiments, the invention encompasses a kit for generating an aptamer library comprising the mutant DNA polymerase of the invention or a functional fragment thereof. In some embodiments, the kit comprises reagents for degenerate or random nucleotide incorporation. In some embodiments, the kit comprises a vector for generating a library of degenerate or random nucleotide sequences.

