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WO2025132603A1 - Nouveaux variants d'endonucléase v pour le clivage d'adn marqué - Google Patents

Nouveaux variants d'endonucléase v pour le clivage d'adn marqué Download PDF

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Publication number
WO2025132603A1
WO2025132603A1 PCT/EP2024/087133 EP2024087133W WO2025132603A1 WO 2025132603 A1 WO2025132603 A1 WO 2025132603A1 EP 2024087133 W EP2024087133 W EP 2024087133W WO 2025132603 A1 WO2025132603 A1 WO 2025132603A1
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polypeptide
nucleic acid
amino acid
seq
endo
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Manon MOLINA
Wesley LOFTIE-EATON
Jose MARTOS MARQUES
Mikhael SOSKINE
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DNA Script SAS
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DNA Script SAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/21—Endodeoxyribonucleases producing 5'-phosphomonoesters (3.1.21)
    • C12Y301/21007—Deoxyribonuclease V (3.1.21.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/27—Endoribonucleases producing 3'-phosphomonoesters (3.1.27)
    • C12Y301/27008—Ribonuclease V (3.1.27.8)

Definitions

  • the present invention relates to novel polypeptides having endonuclease V activity.
  • the present invention relates a novel synthetic polypeptide and variants thereof which exhibit improved properties such as improved stability and improved cleavage activity, in particular improved cleavage of labelled DNA.
  • Endonuclease V (herein after referred as Endo V), also called deoxyinosine 3’ endonuclease, is a DNA repair enzyme that recognizes DNA containing deoxyinosine (a deamination product of a deoxyadenosine, also referred as inosine and hypoxanthine) residues. Endo V primarily cleaves the second phosphodiester bond 3’ to an inosine residue in the same strand, leaving a nick with a 3 ’-hydroxyl and a 5 ’-phosphate.
  • Endo V also called deoxyinosine 3’ endonuclease
  • Endo V enzymes are useful in a wide variety of biochemical fields, including analysis, detection, degradation, synthesis and modification of nucleic acid molecules.
  • Endo V has been used in Enzymatic DNA Synthesis (EDS) to cleave and release single stranded DNA that has otherwise been conjugated to solid support (e.g. Creton, International patent publication W02020/165137).
  • EDS Enzymatic DNA Synthesis
  • Endo V The most widely used Endo V is the wild-type Endonuclease V from E. coli. (hereinafter referred to as the “reference Endo V”), such as the E coli Endo V commercialized by NEB under catalog number M0305S.
  • This reference Endo V presents several drawbacks: it suffers from high precipitation, low stability and variable cleavage performance on substrates with modified nucleotides.
  • Endo V is currently used in processes under conditions (e.g. high temperature, high salt, prolonged periods of time at non-reducing conditions, etc.) outside those for optimal enzymatic activity, efforts have been made in order to obtain variants of the reference Endo V that exhibit greater stability, in particular greater thermostability.
  • Such variants of E. coli Endo V have been described in a patent application (e.g. Loftie-Eaton et al. International patent publication W02022/090057).
  • a reporter dye or fluorophore
  • FRET fluorescence resonance energy transfer
  • Dual-labelled probes are traditionally produced by chemical synthesis.
  • Enzymatic DNA Synthesis such as that described in WO2020/165137, can be used for the production of dual-labelled probes.
  • such synthesis may be performed by the following steps: a) providing an initiator having a deoxyinosine penultimate to a 3’-terminal nucleotide having a free 3 ’-hydroxyl; b) repeating cycles of (i) contacting under elongation conditions the initiator or elongated fragments having free 3’-O-hydroxyls with a 3’-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3’-O-blocked nucleoside triphosphate to form 3’-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3 ’-hydroxyls, until the polynucleotide is formed; and c) treating the polynucleotide with an endonuclease V to cleave the polynucleo
  • a modified (labelled) nucleotide For the production of dual-labelled probes, it is necessary to include a modified (labelled) nucleotide during the first elongation step of EDS and during the last elongation step of the EDS. After cleavage by Endo V from the solid support, the modified nucleotides in the resulting oligonucleotide will be located respectively at the 5’ end and 3’ end of the released polynucleotide/probe, thereby producing a dual-labelled probe comprising a fluorophore and a quencher at the respective ends (5’- and 3’- or 3’- and 5’-, respectively).
  • the present invention relates to a polypeptide which (i) comprises an amino acid sequence having at least 85% identity to the full-length amino acid sequence set forth in SEQ ID NO:1 and (ii) has a deoxyinosine-specific nucleic acid cleavage activity.
  • the present invention also relates to nucleic acids encoding said polypeptides, expression cassettes or vectors comprising said nucleic acids and host cell comprising said nucleic acids, expression cassettes or vectors.
  • the present invention also relates to methods for producing the polypeptides of the invention.
  • the present invention also relates to the use of the polypeptides of the invention in methods of synthesis of a polynucleotide, such as dual-labelled probes and to kits for carrying out such methods.
  • peptide refers to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming said chain.
  • the amino acids are herein represented by their one-letter or three-letters code according to the following nomenclature: A: alanine (Ala); C: cysteine (Cys); D: aspartic acid (Asp); E: glutamic acid (Glu); F: phenylalanine (Phe); G: glycine (Gly); H: histidine (His); I: isoleucine (lie); K: lysine (Lys); L: leucine (Leu); M: methionine (Met); N: asparagine (Asn); P: proline (Pro); Q: glutamine (Gin); R: arginine (Arg); S: serine (Ser); T: threonine (Thr); V: valine (Vai); W: tryptophan
  • reference enzyme WT enzyme or “reference Endo V” are used interchangeably and refer to the non-mutated version of the Endo V isolated from E. coli having the amino acid sequence as set forth in SEQ ID NO: 8.
  • synthetic polypeptide refers to a polypeptide that does not occur in nature. More specifically, in the present case, the term “synthetic polypeptide” or “synthetic Endo V polypeptide” refers to the polypeptide having the sequence as set forth in SEQ ID NO:1, that was artificially designed by the inventors.
  • mutant and variant may be used interchangeably to refer to polypeptides derived from SEQ ID NO: 1 and comprising a modification or an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions and having an Endo V activity.
  • the variants may be obtained by various techniques well known in the art.
  • examples of techniques for altering the DNA sequence encoding the wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis and synthetic oligonucleotide construction.
  • substitution means that an amino acid residue is replaced by another amino acid residue.
  • substitution refers to the replacement of an amino acid residue by another selected from the naturally-occurring standard 20 amino acid residues, rare naturally occurring amino acid residues (e.g.
  • hydroxyproline hydroxylysine, allohydroxylysine, 6-N- methylysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N- methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid, ornithine, norleucine, norvaline), and non-naturally occurring amino acid residue, often made synthetically, (e.g. cyclohexyl-alanine).
  • substitution refers to the replacement of an amino acid residue by another selected from the naturally occurring standard 20 amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T).
  • G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T The sign “+” indicates a combination of substitutions.
  • Y167R denotes that amino acid residue Tyrosine (Y) at position 167 of the parent sequence is changed to an Arginine (R).
  • Y167V/I/M denotes that amino acid residue Tyrosine (Y) at position 167 of the parent sequence is substituted by one of the following amino acids: Valine (V), Isoleucine (I), or Methionine (M).
  • V Valine
  • I Isoleucine
  • M Methionine
  • conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine, asparagine and threonine), hydrophobic amino acids (methionine, leucine, isoleucine, cysteine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine and serine).
  • basic amino acids arginine, lysine and histidine
  • acidic amino acids glutmic acid and aspartic acid
  • polar amino acids glutamine, asparagine and threonine
  • hydrophobic amino acids methionine, leucine, isoleucine, cysteine and valine
  • aromatic amino acids phenylalanine, tryptophan and tyrosine
  • small amino acids glycine, alanine and serine
  • sequence identity refers to the number (or fraction expressed as a percentage %) of matches (identical amino acid residues) between two polypeptide sequences.
  • sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence gaps.
  • sequence identity may be determined using any of a number of mathematical global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g. Needleman and Wunsch algorithm; Needleman and Wunsch, 1970) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g.
  • Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software available on internet web sites such as http://blast.ncbi.nlm.nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • recombinant refers to a nucleic acid construct, a vector, a polypeptide or a cell produced by genetic engineering.
  • expression refers to any step involved in the production of a polypeptide including, but not being limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • expression cassette denotes a nucleic acid construct comprising a coding region, i.e. a nucleic acid of the invention, and a regulatory region, i.e. comprising one or more control sequences, operably linked.
  • expression vector means a DNA or RNA molecule that comprises an expression cassette of the invention.
  • the expression vector is a linear or circular double stranded DNA molecule.
  • nucleic acid As used herein, the term "nucleic acid”, “nucleic sequence,” “polynucleotide”, “oligonucleotide” and “nucleotide sequence” are used interchangeably and refer to a sequence of deoxyribonucleotides and/or ribonucleotides.
  • the nucleic acids can be DNA (cDNA or gDNA), RNA, or a mixture of the two. It can be in single stranded form or in duplex form or a mixture of the two. It can be of recombinant, artificial and/or synthetic origin and it can comprise modified nucleotides, comprising for example a modified bond, a modified purine or pyrimidine base, or a modified sugar.
  • the inventors have designed a synthetic polypeptide, having the sequence set forth in SEQ ID NO:1 , that exhibits improved properties over the Endo V polypeptides of the prior art:
  • the present invention relates to a polypeptide which (i) comprises an amino acid sequence having at least 85%, preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% identity to the full-length amino acid sequence set forth in SEQ ID NO:1 and (ii) has a deoxyinosine-specific nucleic acid cleavage activity.
  • the “deoxyinosine-specific nucleic acid cleavage activity” refers to the ability of the polypeptide to recognize a deoxyinosine (dl) residue contained in nucleic acid molecules and to cleave the second phosphodiester bond 3’ of said deoxyinosine residue.
  • the activity of an enzyme may be evaluated by the one skilled in the art, according to methods known per se in the art. For instance, the activity can be assessed by the measurement of the specific Endo V activity rate, the measurement of the specific cleavage activity rate, or the like. For instance, a standard DNA sample may be dissolved in an adequate buffer, and the Endo V enzyme is added thereto at an adequate temperature (e.g., 37°C). Whether or not the nucleic acid is cleaved into smaller subunits may then be examined by visualizing or quantifying the smaller subunits by electrophoretic techniques (e.g. agarose gel electrophoresis), changes in fluorescence resonance energy transfer (e.g.
  • the terms “increased cleavage activity” or “improved cleavage activity” or “greater cleavage activity” indicate an increased ability of the enzyme to cleave and release a nucleic acid molecule as compared to the reference WT Endo V from E. coli of SEQ ID NO: 8, submitted to identical conditions of reaction (e.g. temperature, concentration, pH, addition of stabilizing agents, etc.). Such an increase is typically of about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more.
  • the Endo V polypeptide of the invention has a cleavage activity at least 5% greater than the cleavage activity of the reference Endo V of SEQ ID N°8, preferably at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or greater.
  • the cleavage activity can be assessed according to the “DLP cleavage test” described in the Examples below, using the sequence as set forth in SEQ ID NO: 19 as a dual-labelled probe.
  • said probe can comprise different pairs of fluorophores and quenchers.
  • the polypeptide of the invention is deemed to exhibit improved cleavage activity a compared to the reference WT Endo V from E coli in the conditions described with SEQ ID NO: 19 comprising “ATTO647N” as a fluorophore at the 5’ end and “BHQ3” as the quencher at the 3’ end.
  • the polypeptides of the present invention exhibit an improved cleavage activity when cleaving substrates which comprise a label in close proximity to the cleavage site (i.e. a modified base located at position -2, -1 , + 1 or +2 with respect to the cleavable phosphodiester bond), preferably when the base located just after the cleavable phosphodiester bond is a labelled base (+1 position).
  • cleavage site i.e. a modified base located at position -2, -1 , + 1 or +2 with respect to the cleavable phosphodiester bond
  • the polypeptide may comprise one or more amino acid substitutions as compared to SEQ ID NO:1 at positions selected from Q36, L37, K38, G49, N52, K141 , A164, 1178 and M195 wherein the positions are numbered by reference to the amino acid sequence set forth in SEQ ID NO:1.
  • the polypeptide may comprise one or more amino acid substitutions selected from Q36A, L37P, K38R, G49T, N52T, K141Q, A164R, 1178V and M195T
  • the polypeptide comprises an amino acid substitution at position M195 of SEQ ID NO:1.
  • the polypeptide comprises the substitution M195T.
  • the polypeptide comprises an amino acid sequence having at least 85% 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identity with SEQ ID NO: 2.
  • the polypeptide of the invention comprises the sequence SEQ ID NO: 2.
  • the polypeptide comprises an amino acid substitution at position M195 and further comprises amino acid substitutions at position A164 and/or 1178 of SEQ ID NO:1.
  • the polypeptide further comprises the substitutions A164R and/or 1178V, even more preferably A164R and 1178V.
  • the polypeptide comprises an amino acid sequence having at least 85% 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identity with SEQ ID NO: 3.
  • the polypeptide of the invention comprises the sequence SEQ ID NO: 3.
  • the polypeptide comprises amino acid substitutions at positions A164, 1178 and M195 and further comprises amino acid substitutions at positions G49 and/or N52 of SEQ ID NO:1 .
  • the polypeptide further comprises the substitutions G49T and/or N52T, even more preferably G49T and N52T.
  • the polypeptide comprises an amino acid sequence having at least 85% 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identity with SEQ ID NO: 5.
  • the polypeptide of the invention comprises the sequence SEQ ID NO:5.
  • the polypeptide comprises amino acid substitutions at positions A164, 1178 and M195 and further comprises amino acid substitutions at positions Q36, L37 and/or K38 of SEQ ID NO:1 .
  • the polypeptide further comprises the substitutions Q36A, L37P and/or K38R, even more preferably Q36A, L37P and K38R.
  • the polypeptide comprises an amino acid sequence having at least 85% 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identity with SEQ ID NO: 6.
  • the polypeptide of the invention comprises the sequence SEQ ID NO: 6.
  • the polypeptide comprises amino acid substitutions at positions Q36, L37, K38, A164, 1178 and M195 and further comprises an amino acid substitution at positions K141 of SEQ ID NO:1.
  • the polypeptide further comprises the substitution K141Q.
  • the polypeptide comprises an amino acid sequence having at least 85% 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identity with SEQ ID NO: 4.
  • the polypeptide of the invention comprises the sequence SEQ ID NO: 4.
  • the polypeptide comprises amino acid substitutions at positions Q36, L37, K38, K141 , A164, 1178 and M195 and further comprises amino acid substitutions at positions G49 and N52 of SEQ ID NO:1.
  • the polypeptide further comprises the substitutions G49T and/or N52T, even more preferably G49T and N52T.
  • the polypeptide comprises an amino acid sequence having at least 85% 90%, 95%, 96%, 97%, 98%, 99%, 99.5% identity with SEQ ID NO: 7.
  • the polypeptide of the invention comprises the sequence SEQ ID NO: 7.
  • polypeptide of the invention does not comprise the substitution D215H. This residue corresponds to the residue D206 of SEQ ID NO:8.
  • polypeptide of the invention may further comprise a tag, such a His-tag, which can be useful for polypeptide purification and/or detection.
  • the tag can be located at any given position of the polypeptide, provided that it does not hinder the cleavage activity. Typically, the tag can be present at the N-terminus or the C- terminus of the polypeptide.
  • the tag is present at the N-terminus of the polypeptide (just after the initiation Methionine) and consists in the sequence ASSHHHHHHHHSSGSENLYFQSGSS or ASSHHHHHHHHSSGSS (SEQ ID NO:9 and SEQ ID NO: 10 respectively).
  • polypeptides comprising the amino acid sequences SEQ ID NO:1 to 7 and comprising His-tags used in the Examples below possess the amino acid sequences SEQ ID NO: 11 to 17 respectively.
  • TSA Fluorescentbased thermal shift assays
  • Exemplary references describing thermal shift assays and their application to measure protein stability are as follows: Pantoliano et al, J. Biomolecular Screening, 6(6): 429-440 (2001); Huynh et al, Curr. Protocol. Protein Science, 79: 28.9.1- 28.9.14; Ericsson et al, Anal. Biochem., 357: 289-298 (2006); Niesen et al, Nature Protocols, 2(9): 2212-2221 (2007); and Pantoliano et al, U.S. patent 6020141, the latter of which is hereby incorporated by reference.
  • folded and unfolded proteins can be distinguished through exposure to a hydrophobic fluorescent probe.
  • a hydrophobic fluorescent probe is quenched in aqueous solution but will preferentially bind to the exposed hydrophobic interior of an unfolding protein leading to a sharp decrease in quenching so that a readily detected fluorescence emission can be studied as a function of temperature.
  • Thermally induced unfolding is an irreversible unfolding process following a typical two-state model with a sharp transition between the folded and unfolded states, where melting temperature (Tm) is defined as the midpoint of temperature of the protein-unfolding transition.
  • thermofluor Melting temperatures obtained with such a so-called “thermofluor” method have been shown to correlate well for several proteins with values determined by other biophysical methods used for measuring protein stability, such as circular dichroism (CD), turbidity measurements, and differential scanning calorimetry.
  • CD circular dichroism
  • fluorescent dyes may be used and are commercially available directly or as part of an assay kit.
  • An exemplary fluorescent dye is SYPRO Orange.
  • a conventional real-time PCR instrument may be used for temperature control and fluorescent detection. Typically, a selection of buffers, salt concentrations and compositions, and other additives is made.
  • the stability of the polypeptides of the invention is measured by the TSA described in the “Examples” section below.
  • thermostability is deemed to be improved if the Tm of a given polypeptide is increased by at least 0.3°C, preferably at least 0.4°C, at least 0.5 °C, even more preferably at least 1°C, 2°C; 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C or 10°C compared to the reference Endo V.
  • initial stability (which facilitates improved production and purification) is reflected by the lower initial fluorescence measured at the start of the TSA.
  • the initial stability is deemed to be improved if the initial fluorescence is reduced by at least 50%, preferably at reduced by at least 55%, 60% or even 65% compared to that of the reference Endo V.
  • the polypeptides of the invention exhibit improved thermal stability and/or improved initial stability compared to the reference Endo V (WT Endo V from E coli, having SEQ ID NO:8).
  • Endo V polypeptides of the present invention display both improved stability and improved cleavage activity, especially when used for cleaving labelled substrates.
  • the invention also encompasses nucleic acids which hybridize, under stringent conditions, to a nucleic acid encoding an Endo V polypeptide as defined above.
  • stringent conditions include incubations of hybridization filters at about 42° C for about 2.5 hours in 2 X SSC/0.1%SDS, followed by washing of the filters four times of 15 minutes in 1 X SSC/0.1% SDS at 65° C. Protocols used are described in such reference as Sambrook et al. (Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor N.Y. (1988)) and Ausubel (Current Protocols in Molecular Biology (1989)).
  • the invention also encompasses nucleic acids encoding Endo V polypeptides of the invention, wherein the sequence of said nucleic acids, or a portion of said sequence at least, has been engineered using optimized codon usage.
  • the nucleic acids according to the invention may be deduced from the sequence of the Endo V polypeptide according to the invention and codon usage may be adapted according to the host cell in which the nucleic acids shall be transcribed. These steps may be carried out according to methods well known to one skilled in the art and some of which are described in the reference manual Sambrook et al. (Sambrook et al., 2001).
  • Nucleic acids of the invention may further comprise additional nucleotide sequences, such as regulatory regions, i.e., promoters, enhancers, silencers, terminators, signal peptides and the like that can be used to cause or regulate expression of the polypeptide in a selected host cell or system.
  • the present invention further relates to an expression cassette comprising a nucleic acid according to the invention operably linked to one or more control sequences that direct the expression of said nucleic acid in a suitable host cell.
  • the expression cassette comprises, or consists of, a nucleic acid according to the invention operably linked to a control sequence such as transcriptional promoter and/or transcription terminator.
  • the control sequence may include a promoter that is recognized by a host cell or an in vitro expression system for expression of a nucleic acid encoding an endonuclease V of the present invention.
  • the promoter contains transcriptional control sequences that mediate the expression of the enzyme.
  • the promoter may be any polynucleotide that shows transcriptional activity in the host cell including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • the control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the nucleic acid encoding the Endo V polypeptide. Any terminator that is functional in the host cell may be used in the present invention.
  • the expression cassette comprises, or consists of, a nucleic acid according to the invention operably linked to a transcriptional promoter and a transcription terminator
  • the invention also relates to a vector comprising a nucleic acid or an expression cassette as defined above.
  • vector refers to DNA molecule used as a vehicle to transfer recombinant genetic material into a host cell.
  • the major types of vectors are plasmids, bacteriophages, viruses, cosmids, and artificial chromosomes.
  • the vector itself is generally a DNA sequence that consists of an insert (a heterologous nucleic acid sequence, transgene) and a larger sequence that serves as the “backbone” of the vector.
  • the purpose of a vector which transfers genetic information to the host is typically to isolate, multiply, or express the insert in the target cell.
  • Vectors called expression vectors are specifically adapted for the expression of the heterologous sequences in the target cell, and generally have a promoter sequence that drives expression of the heterologous sequences encoding a polypeptide.
  • the regulatory elements that are present in an expression vector include a transcriptional promoter, a ribosome binding site, a terminator, and optionally present operator.
  • an expression vector also contains an origin of replication for autonomous replication in a host cell, a selectable marker, a limited number of useful restriction enzyme sites, and a potential for high copy number.
  • Examples of expression vectors are cloning vectors, modified cloning vectors, specifically designed plasmids and viruses. Expression vectors providing suitable levels of polypeptide expression in different hosts are well known in the art. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the present invention thus relates to the use of a nucleic acid, expression cassette or vector according to the invention to transform, transfect or transduce a host cell.
  • the choice of the vector will typically depend on the compatibility of the vector with the host cell into which it must be introduced.
  • the host cell may be transformed, transfected or transduced in a transient or stable manner.
  • the expression cassette or vector of the invention is introduced into a host cell so that the cassette or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector.
  • the term "host cell” also encompasses any progeny of a parent host cell that is not identical to the parent host cell due to mutations that occur during replication.
  • the host cell may be any cell useful in the production of a variant of the present invention, e.g., a prokaryote or a eukaryote.
  • the prokaryotic host cell may be any Gram -positive or Gram-negative bacterium.
  • the host cell may also be a eukaryotic cell, such as a yeast, fungal, mammalian, insect or plant cell.
  • the nucleic acid, expression cassette or expression vector according to the invention may be introduced into the host cell by any method known by the skilled person, such as electroporation, conjugation, transduction, competent cell transformation, protoplast transformation, protoplast fusion, biolistic "gene gun” transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemically mediated transfection, lithium acetate-mediated transformation, liposome-mediated transformation.
  • more than one copy of a nucleic acid, cassette or vector of the present invention may be inserted into a host cell to increase production of the polypeptide.
  • Endo V polypeptides of the invention It is a further object of the invention to provide methods using an Endo V variant of the invention that can be stored in the preferred formulation buffer for 2, 4, 6 or more days at 4 °C without the presence of stabilizing or reducing agents such as BSA and TCEP and without accruing more than 20, 40 or 60% loss in enzymatic activity due to their improved stability.
  • the improved cleavage activity of the Endo V polypeptides of the invention provides a method to specifically cleave single-stranded nucleic acid at the second phosphodiester bond 3’ to a deoxyinosine residue and release a deoxyinosine-free singlestranded polynucleotide from a solid support, such as agarose beads, into solution in a 15, 30, 45, 60 or 75% shorter period of time compared to the reference WT Endo V from E coli.
  • the nucleic acid may or may not contain a modified or labelled base bearing a fluorophore, quencher, biotin or other modifications in close proximity to cleavable phosphodiester bond.
  • close proximity to the cleavable phosphodiester bond it is meant a base that is located at position -2, -1, +1 or +2 with respect to the phosphodiester bond that is cleaved by the Endo V polypeptide.
  • the modified or labelled base is immediately 3’ to the cleavable phosphodiester bond (i.e. at position +1).
  • the modified base already comprises a label such as a dye, a fluorophore, a quencher, biotin, other small molecules such as metal complexes, biosensors or molecules to facilitate cellular uptake, DNA or RNA to create linear or branched nucleic acids and peptides such as antibodies or other proteins to create DNA-protein conjugates.
  • a label such as a dye, a fluorophore, a quencher, biotin, other small molecules such as metal complexes, biosensors or molecules to facilitate cellular uptake, DNA or RNA to create linear or branched nucleic acids and peptides such as antibodies or other proteins to create DNA-protein conjugates.
  • the modified base is a base that comprises a multi-atom linker that can later be functionalized in order to comprise a label.
  • the modified or labelled base is a dNTP that is modified at a specific position of the base with a multi-atom linker terminating with an alkyne or azido group.
  • Such linkers are then compatible with either classical click chemistry (“Copper-mediated azide-alkyne “click” cycloadditions” (CuAAC)) or copper-free click reactions such as strain-promoted and fluorine-activated cycloaddition of cyclooctynes and organic azides.
  • CuAAC Copper-mediated azide-alkyne “click” cycloadditions
  • the modification can be present at specific positions. Typically, the modification can be present on position 5 of a pyrimidine or position 5 or 7 of a purine base.
  • linkers include, but are not limited to: octadiynyl, aminoallyl, aminopropargyl, ethynyl, O-propargyl-PEG, O-undeynyl and amino-modifier C6.
  • Suitable modified bases are described for instance in the following publications: Kuwahara et al. Nucleic Acids Res. 2006;34(19):5383-94; Jager et al. J. Am. Chem. Soc. 2005, 127, 43, 15071-15082 and Panattoni et al. Org. Lett. 2018, 20, 13, 3962-3965.
  • the modified base is 5-octadiynyl dllTP or 5-PEG-dllTP.
  • Preferred modified bases are bases that comprise linkers compatible with click chemistry, such as for example a 5-octadiynyl linker, and which can later be functionalized with a label such as a dye, a fluorophore, a quencher, biotin, other small molecules such as metal complexes, biosensors or molecules to facilitate cellular uptake, DNA or RNA to create linear or branched nucleic acids and peptides such as antibodies or other proteins to create DNA- protein conjugates.
  • linkers compatible with click chemistry such as for example a 5-octadiynyl linker
  • a label such as a dye, a fluorophore, a quencher, biotin, other small molecules such as metal complexes, biosensors or molecules to facilitate cellular uptake
  • DNA or RNA to create linear or branched nucleic acids and peptides such as antibodies or other proteins to create DNA- protein conjugates.
  • One or more embodiments of the present invention provide compositions, methods and kits for various nucleic acid assays, wherein an Endo V polypeptide that is capable of nicking an inosine-containing strand of a single or double-stranded polynucleotide at a location 3' to the inosine residue is employed.
  • nucleic acid assay employing an Endo V polypeptide of the present invention, as described above, comprising:
  • nucleic acid that is conjugated to a solid support at its 5’ terminal, which contains an inosine residue and which contains a label such as a biotin or similar on the base of the second nucleotide 3’ to the inosine residue;
  • the method may further comprise the step of
  • nucleic acid assay employing an Endo V polypeptide of the present invention, as described above, comprising:
  • nucleic acid that is conjugated to a solid support at its 5’ terminal, which contains an inosine residue and which contains a FRET fluorophore on the base of the second nucleotide 3’ to the inosine residue and a FRET quencher at the 3’ terminal of the nucleic acid or a FRET quencher on the base of the second nucleotide 3’ to the inosine residue and a FRET fluorophore at the 3’ terminal of the nucleic acid
  • This nucleic acid is also commonly called a “dual-labelled probe” (DLP) and is useful in various applications.
  • DLP dual-labelled probe
  • the method described above may further comprise the step of
  • FRET fluorophore and quencher-containing nucleic acid as a probe in fluorescence-based assays such as TaqMan-based, real-time polymerase chain reaction or fluorescent in situ hybridization.
  • nucleic acid assay employing a variant of the present invention, as described above, comprising:
  • the invention also relates to a method for producing a 5’ labelled polynucleotide comprising the steps of: a) providing an initiator DNA having a deoxyinosine penultimate to a 3’-terminal nucleotide having a free 3’-hydroxyl; b) repeating cycles of (i) contacting under elongation conditions the initiator or elongated fragments having free 3’-O-hydroxyls with a 3’-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3’-O-blocked nucleoside triphosphate to form 3’-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3’-hydroxyls, until the polynucleotide is formed; c) treating the polynucleotide with an Endo V poly
  • the above method may also include washing steps after the reaction, or extension, step, as well as after the de-blocking step.
  • the above method may also include a step of subjecting said linker to a click chemistry reaction. This step can be carried out before or after step c), preferably before step c).
  • an “initiator” refers to a short oligonucleotide sequence with a free 3’-end, which can be further elongated by a template-free polymerase, such as a terminal deoxynucleotidyl transferase (TdT).
  • a template-free polymerase such as a terminal deoxynucleotidyl transferase (TdT).
  • the initiating fragment is a DNA initiating fragment.
  • the initiating fragment is an RNA initiating fragment.
  • the initiating fragment possesses between 3 and 100 nucleotides, in particular between 3 and 20 nucleotides.
  • the initiating fragment is single-stranded. In an alternative embodiment, the initiating fragment is doublestranded. In a particular embodiment, an initiator oligonucleotide synthesized with a 5’- primary amine may be covalently linked to magnetic beads using the manufacturer’s protocol. Likewise, an initiator oligonucleotide synthesized with a 3’-primary amine may be covalently linked to magnetic beads using the manufacturer’s protocol.
  • reaction conditions for an extension or elongation step may comprising the following: 2.0-50 pM purified TdT, preferably around 25 pM purified TdT; 125- 600 pM 3’-O-blocked dNTP (e.g.
  • 3’-O-NH2-blocked dNTP about 10 to about 500 mM potassium cacodylate buffer (pH between 6.5 and 7.5) and from about 0.01 to about 10 mM of a divalent cation (e.g. C0CI2 or MnCh), where the elongation reaction may be carried out in a 50 pL reaction volume, at a temperature within the range from room temperature (RT) to 45°C, for 3 minutes.
  • a divalent cation e.g. C0CI2 or MnCh
  • reaction conditions for a deblocking step may comprise the following: 700 mM NaNCh; 1 M sodium acetate (adjusted with acetic acid to pH in the range of 4.8-6.5), where the deblocking reaction may be carried out in a 50 pL volume, at a temperature within the range of from RT to 70°C, preferably RT to 47°C, even more preferably from RT to 45°C for 30 seconds to several minutes.
  • the deblocking agent (also sometimes referred to as a de-blocking reagent or agent and sometimes referred to cleavage agent used for the de-blocking step) is a chemical deblocking agent, such as, for example, dithiothreitol (DTT).
  • DTT dithiothreitol
  • a deblocking agent may be an enzymatic agent, such as, for example, a phosphatase, which may cleave a 3’-phosphate blocking group. It will be understood by the person skilled in the art that the selection of deblocking agent depends on the type of 3’-nucleotide blocking group used, whether one or multiple blocking groups are being used, whether initiators are attached to living cells or organisms or to solid supports, and the like, that necessitate mild treatment.
  • a phosphine such as tris(2-carboxyethyl)phosphine (TCEP) can be used to cleave a 3’0- azidomethyl groups
  • TCEP tris(2-carboxyethyl)phosphine
  • palladium complexes can be used to cleave a 3’O-allyl groups
  • sodium nitrite can be used to cleave a 3’0-amino group.
  • the deblocking reaction involves TCEP, a palladium complex or sodium nitrite.
  • kits of the invention comprise an Endo V polypeptide of the invention in a formulation suitable for carrying out cleavage as described herein and solid supports with initiator DNA linked to said solid support, said initiator DNA comprising a dl nucleotide at the penultimate position.
  • kits of the invention may also include a template-independent polymerase such as TdT, synthesis buffers that provide reaction conditions for optimizing the template-free addition or incorporation of a 3’-O-protected dNTP to a growing strand.
  • kits of the invention further include 3’-O- reversibly protected dNTPs.
  • the 3’- O-reversibly protected dNTPs may comprise 3’-O-amino-dNTPs or 3’-O-azidomethyl-dNTPs.
  • kits may also include modified 3’-O-reversibly protected dNTPs such as 5-octadiynyl dllTP.
  • kits may include one or more of the following items, either separately or together with the above-mentioned items: (i) deprotection reagents for carrying out a deprotecting step as described herein, (ii) wash reagents or buffers for removing unreacted 3’-O-protected dNTPs at the end of an enzymatic addition or coupling step, and (iii) post-synthesis processing reagents, such as purification columns, desalting reagents, eluting reagents, and the like.
  • kits of the invention may include arrays of reaction wells for carrying out multiple synthesis reactions in a single operation. In some embodiments, such arrays may be conventional filter plates comprising 24-, 48-, 96-, 384- or 1536-wells.
  • polypeptide comprising the sequence set forth in SEQ ID NO:1 and corresponding to a synthetic sequence having endonuclease V activity designed by the inventors;
  • Variants of the polypeptide having the sequence SEQ ID NO:1 and having substitutions at one or more positions selected from Q36, L37, K38, G49, N52, K141 , A164, 1178 and M195 with respect to SEQ ID NO:1 polypeptides comprising the sequences SEQ ID 2, 3, 4, 5, 6 and 7;
  • the reference polypeptide comprising the sequence set forth in SEQ ID NO:8 and corresponding to the wild-type Endonuclease V protein from E. coli.
  • a His-tag (having the sequence set forth in SEQ ID NO:9 or SEQ ID NO: 10) was introduced at the N-terminal of the polypeptides for purification purposes.
  • Variants of the synthetic sequence having the sequence SEQ ID NO:2 were constructed using the Megaprimer Polymerase Chain Reaction (PCR) method as described by Ke and Madison, 1997 and detailed below.
  • the first step was the construction of the megaprimer using PCR.
  • PCR mix included 20 ng of pET28a vector with the SEQ ID NO:2 gene, 0.5 pM of appropriate forward and reverse primers (Eurogentec) 0.2 mM of each dNTP, 1X Phire II reaction buffer and 1 pL of PhireTM Hot Start II polymerase (ThermoFisher Scientific).
  • the program used was 30 sec of initial denaturation at 98°C followed by 30 cycles of 5 sec denaturation at 98°C, 5 sec annealing at 70°C and 15 sec extension at 72°C. After the 30 cycles, a final extension was performed at 72°C for 1 min.
  • the PCR reaction was then purified using QIAquick PCR Purification kit (QIAGEN).
  • the second step involved a PCR mix as followed: 80 ng of pET28a vector with the SEQ ID NO:2 gene, 300 to 400 ng of the previously constructed megaprimer, 0.2 mM of each dNTP,
  • the digested product was transformed into 40 pL of home-made E. coli BL21 DE3 electro competent cells using 0.5 pL of product.
  • the cells plus DNA to transform were transferred into a 1 mm gap precooled electroporation cuvette (ThermoFisher Scientific), the cells were subjected to one pulse of 1700 V using Eporator electroporator (Eppendorf) and 1 mL of recovery media (e.g. SOC 20 g/L tryptone, 5 g/L Yeast extract, 10 mM NaCI, 2.5 mM KCI, 10 mM MgCI2, 10 mM MgSO4 and 20 mM glucose) was added.
  • Eporator electroporator Eppendorf
  • 1 mL of recovery media e.g. SOC 20 g/L tryptone, 5 g/L Yeast extract, 10 mM NaCI, 2.5 mM KCI, 10 mM MgCI2, 10 mM MgSO4 and 20 mM
  • 5 mL of 2YT media (8 g/L tryptone, 5 g/L Yeast Extract, 5 g/L NaCI) supplemented with 0.05% glucose and 50 pg/mL Kanamycin were inoculated with 10 pL of E. coli BL21 DE3 mutant glycerol stock. After 37°C overnight growth at 180 RPM, 50 mL cultures of the same media were inoculated using 500 pL of starter culture. Cultures were incubated at 37°C 180 RPM until optical density at 600 nm (GD600) reached between 1 to 1.5. Then, the cultures were cooled down in a 20°C incubator during 30 min with 180 RPM shaking.
  • 2YT media 8 g/L tryptone, 5 g/L Yeast Extract, 5 g/L NaCI
  • 50 mL cultures of the same media were inoculated using 500 pL of starter culture. Cultures were incubated at 37°C 180 RPM until optical density at 600 nm (GD600
  • the supernatant was transferred to a new 50 mL Falcon tube and 100 pL of HisPur Ni-NTA Resin (ThermoFisher Scientific) were added to each tube. Binding of the His-tagged enzymes was performed during 1 hour at RT under shaking. The resin from each tube was then loaded into a Bio-Spin Disposable Chromatography Column (Bio-Rad) and was washed with 50 mL of Wash Buffer (25 mM Tris-HCI pH 7.5, 10 mM imidazole, 0.5 M NaCI) using NucleoVac 24 Vacuum Manifold.
  • Wash Buffer 25 mM Tris-HCI pH 7.5, 10 mM imidazole, 0.5 M NaCI
  • Enzymes were purified as described above. Directly after elution and quantification, enzymes were standardized at the same concentration of 200 pM using Elution Buffer and kept at 4°C. After 4, 7, 11, 18 and 32 days, tubes were centrifuged 10 min at 4°C 16,000g and enzyme concentration was remeasured on Nanodrop instrument using three technical replicates. The quantity lost by precipitation was calculated as the percentage of decrease in the concentration measured and presented in Table 2. Table 2: Percentage of protein lost due to precipitation over time at 4°C
  • Thermal Shift Assay was performed using a final concentration of 7.2 pM of pure enzyme and 4X of SYPROTM Orange dye (ThermoFisher Scientific) diluted in Dulbecco’s Phosphate Buffered Saline (DPBS) buffer (Sigma). Changes in fluorescence were monitored by a C1000 Touch Thermocycler (Bio-rad) using denaturation program used ranged from 25°C, to 95°C with increments of 0.3°C every 5 sec and FRET acquisition. Three technical replicates were used for each enzyme.
  • Table 3 Melting temperature measured using TSA A second parameter of TSA was analyzed: the initial fluorescence, which is measured at the start of the assay, is representative of the enzyme quality. Indeed, high initial fluorescence means that partially unfolded protein is present in the formulated enzyme preparation.
  • polypeptides according to the invention had around three times lower initial fluorescence than the reference WT E. coli EndoV (Table 4).
  • polypeptides of the invention are more stable than the reference WT E coli EndoV, in the absence of stabilizing agents such as BSA or TCEP.
  • Enzymatic DNA Synthesis was performed as described for example in Creton (International patent publication WO2020/165137), with a few extra steps in order to make a Dual Labelled Probe (DLP) that includes a fluorophore on its 5’ and a quencher on its 3’.
  • DLP Dual Labelled Probe
  • Such DLP are typically used as probes for quantitative Taqman PCR.
  • the EDS protocol was adapted and performed with the following steps to synthesize the DLPs having the sequence SEQ ID NO: 19 (NCCACCACCCAGAGAAGCCAAAGAAN), where N represents a modified “U” nucleotide with a fluorophore or quencher attached to said “U” by a linker.
  • Pre-synthesis step providing an initiator DNA having a deoxyinosine penultimate to a 3’-terminal nucleotide having a free 3’-hydroxyl having the sequence SEQ ID NO: 20 (CCCCCCCCCCCCTTdIT);
  • Synthesis step repeating cycles of (i) contacting under elongation conditions the initiator or elongated fragments having free 3’-O-hydroxyls with a 3’-O-blocked nucleoside triphosphate and a template-independent DNA polymerase so that the initiator or elongated fragments are elongated by incorporation of a 3’-O-blocked nucleoside triphosphate to form 3’-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3’- hydroxyls, until the polynucleotide is formed.
  • the first and last nucleotides to be incorporated were a modified 3’-O-blocked deoxyuridine triphosphate containing a 5-Octadiynyl linker compatible with click chemistry.
  • a first click reaction was performed after the first cycle in order to functionalize the first added nucleotide with a fluorophore.
  • a second click reaction was performed after the last cycle in order to functionalize the last added nucleotide with a quencher.
  • Labels used were AF488, ATTO565, ATTO647N (fluorophores), BHQ1, BHQ2 or BHQ3 (quenchers).
  • Post-synthesis step treating the polynucleotide with an endonuclease V enzyme to cleave the modified polynucleotide from the initiator (liberation).
  • the enzyme was used at 4 pM and the cleavage was performed 30 min at 37°C in Liberation Buffer (NaCI 0.173 mol/L, MgCI 2 50 mM, TRIS pH 8.0 10 mM).
  • the DNA was then desalted, recovered by elution in water and quantified by measuring the absorbance at 260nm.
  • the polypeptides according to the present invention display improved cleavage activity compared to the reference Endo V (WT Endo V from E. coli).
  • the polypeptides of the invention are therefore particularly useful in methods for producing dual labelled probes.

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Abstract

La présente invention concerne de nouveaux polypeptides présentant une activité d'endonucléase V accrue et leurs utilisations, plus particulièrement pour cliver l'ADN marqué.
PCT/EP2024/087133 2023-12-18 2024-12-18 Nouveaux variants d'endonucléase v pour le clivage d'adn marqué Pending WO2025132603A1 (fr)

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US5808045A (en) 1994-09-02 1998-09-15 Andrew C. Hiatt Compositions for enzyme catalyzed template-independent creation of phosphodiester bonds using protected nucleotides
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