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WO2025155239A1 - Adn polymérases de la famille x et leurs utilisations - Google Patents

Adn polymérases de la famille x et leurs utilisations

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Publication number
WO2025155239A1
WO2025155239A1 PCT/SG2024/050772 SG2024050772W WO2025155239A1 WO 2025155239 A1 WO2025155239 A1 WO 2025155239A1 SG 2024050772 W SG2024050772 W SG 2024050772W WO 2025155239 A1 WO2025155239 A1 WO 2025155239A1
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Prior art keywords
polypeptide
amino acid
rvpolx
substitution
nucleic acid
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PCT/SG2024/050772
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English (en)
Inventor
Ee Lui Ang
Yifeng WEI
Yee Song LAW
Nazreen Binti V M Abdul MUTHALIFF
Huimin Zhao
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Agency for Science Technology and Research Singapore
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Agency for Science Technology and Research Singapore
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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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Definitions

  • a polynucleotide comprising a nucleic acid sequence encoding a polypeptide as defined herein.
  • a host cell comprising the polynucleotide as defined herein or the expression construct as defined herein.
  • a method of synthesizing a nucleic acid molecule comprising the step of contacting a nucleic acid primer with at least one nucleotide and a polypeptide as defined herein, under conditions for the addition of the at least one nucleotide to the nucleic acid primer by the polypeptide.
  • a polypeptide as defined herein for synthesizing a nucleic acid molecule with a nucleic acid primer and at least one nucleotide.
  • kits for synthesizing a nucleic acid molecule comprising a polypeptide as defined herein and at least one nucleotide.
  • ssDNA14 Denaturing —PAGE assay of RvPolX-catalyzcd template-independent extension of a 14-mer single- stranded DNA (ssDNA14) under varying conditions, including various (B) pH buffers, (C) salt concentrations, (D) divalent metal ions, and (E) ssDNA substrates (the substrates ssDNA14A, ssDNA14T, ssDNA14G, and ssDNA14C consist of ssDNA extended by A, T, G or C, respectively).
  • B pH buffers
  • C salt concentrations
  • D divalent metal ions
  • E ssDNA substrates (the substratessDNA14A, ssDNA14T, ssDNA14G, and ssDNA14C consist of ssDNA extended by A, T, G or C, respectively).
  • Figure 2 Domain structure and 3D structures of PolX family members.
  • A BRCA1 C- terminal (BRCT) domain in blue, and the low-complexity (LC) region in red.
  • the catalytic domain consists of the 8 kDa domain (yellow), fingers domain (orange), palm domain (pink), and thumb domain (green).
  • the loopl region within the palm domain is colored in black.
  • B A red arrow is used to indicate the loopl region on the 3D structures.
  • Alphafold structure of Pol mu is used in this figure as the loopl region in its crystal structure is not resolved.
  • Error bars reflect the standard deviations, and the significance was determined by one-way analysis of variance (ANOVA) with Dunnett’s posttest comparing mutants to WT (vehicle control). *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ns, not significant.
  • FIG. 9 Denaturing urea-PAGE analysis of the G513A+R522I and HsTdT templateindependent DNA polymerase assays. Denaturing urea-PAGE analysis shows the effect of G513A+R522I on template-independent DNA polymerase activity at (A) 500 mM KC1 and (B) 1000 mM KC1, with four different dNTPs. Each assay was conducted in triplicate, and the band intensities of the extension products were quantified and plotted on the graph. Error bars reflect the standard deviations. Statistical differences were based on Student’s t test. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001; ns, not significant.
  • FIG. 10 Denaturing urea-PAGE analysis of template-independent DNA polymerase assays using S’-ONEE modified nucleotides with G513A+R522I and commercial calf thymus TdT.
  • A Denaturing urea-PAGE analysis demonstrates the effect of 3’-ONH2 modified nucleotides on template-independent DNA polymerase activity between G513A+R522I and commercial calf thymus TdT. The assay was conducted in triplicate, and percentage of band intensity corresponding to the one -base extended product (+1) was quantified and plotted on the graph.
  • substitution refers to the presence of an amino acid residue at a certain position of the derivative sequence which is different from the amino acid residue which is present or absent at the corresponding position in the reference sequence.
  • 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).
  • the sign “+” herein indicates a combination of substitutions.
  • Amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.
  • a “position corresponding to” or recitation that amino acid positions “correspond to” amino acid positions in a reference sequence refers to amino acid positions identified upon alignment with the disclosed sequence to maximise identity using a standard alignment algorithm or software (such as the BLAST, ClustalW, ClustalOmega, MUSCLE, TCoffee or ProbCons software). By aligning the sequences, one skilled in the art can identify corresponding residues.
  • sequence identity refers to the extent that sequences arc identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G and 1) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Tip, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C
  • the term “at least 70%” as used herein includes at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
  • An “expression construct” generally includes at least a control sequence operably linked to a nucleotide sequence of interest.
  • promoters in operable connection with the nucleotide sequences to be expressed are provided in expression constructs for expression in an organism or part thereof including a host cell.
  • conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art, see for example, Molecular Cloning: A Laboratory Manual, 3rd edition Volumes 1, 2, and 3. J. F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000.
  • expression vector or “vector” is meant a nucleic acid molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, or plant virus, into which a nucleic acid sequence may be inserted or cloned.
  • a vector preferably contains one or more unique restriction sites and may be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible.
  • a vector system may comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon.
  • 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 vector may also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants. Examples of such resistance genes are well known to those of skill in the art.
  • polypeptide comprising an amino acid sequence having at least 70% sequence identity (such as at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity) to an amino acid sequence of SEQ ID NO: 1, or a fragment thereof.
  • the polypeptide comprises three amino acid substitutions at positions corresponding to positions 281, 513 and 522 as set forth in SEQ ID NO: 1.
  • the amino acid substitution at the position corresponding to position 281 may be a substitution to A, F, I, L, V or Y. In one embodiment, the amino acid substitution corresponding to position 281 is a substitution to L. The amino acid substitution at the position corresponding to position 513 may be a substitution to A or C.
  • the amino acid substitution at the position corresponding to position 522 may be a substitution to I, M or V.
  • the polypeptide is distinguished from the wild-type RvPolX by at least two amino acid substitutions in the wild-type RvPolX amino acid sequence.
  • the polypeptide may comprise an amino acid substitution of i) G513A or G513C, and ii) R522I, R522M or R522V.
  • Polypeptides herein may further comprise a fusion partner (such as a fusion peptide or domain) at its N-terminus, C-terminus or both.
  • a fusion partner such as a fusion peptide or domain
  • the fusion partner is recombinantly attached to the polypeptide and is expressed together with the polypeptide.
  • the fusion partner may be used for purification, identification, increasing expression, sccrctability or increasing catalytic activity.
  • a hcxahistidinc tag may be added to enable affinity chromatography during protein purification, or a solubility tag may be added to enhance protein solubility during expression.
  • Other such fusion partners are extensively described in the literature and thus all fusion partners known to a skilled person are contemplated in the present disclosure.
  • RvPolX variant polypeptides herein exhibit measurable polymerase activity at least in a range of pH from about pH 6 to about pH 10, such as at a pH of about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, or about 10.
  • RvPolX variant polypeptides herein are capable of incorporating nucleotides (including ribonucleotides and deoxyribonucleotides) and modified nucleotides, including 3’-O-modified nucleotides such as 3’-O-NH2 nucleotides.
  • a “3’-O-modified nucleotide” refers to a ribonucleotide (rNTP) or deoxyribonucleotide (dNTP) containing a blocking moiety at the 3’ position of the sugar that prevents the formation of a phosphodiester bond.
  • the blocking moiety may be a chemical group which can be removed through a specific cleaving reaction to allow further nucleotide addition, and such a nucleotide is also referred to as a “reversible terminator nucleotide”.
  • Codons in the polynucleotide or expression construct may be selected to fit the host cell in which the protein is being produced.
  • preferred codons used in bacteria may be used to express the gene in bacteria
  • preferred codons used in yeast may be used for expression in yeast
  • preferred codons used in mammals are used for expression in mammalian cells.
  • codon- optimized polynucleotides encoding the RvPolX polypeptides may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full-length coding region.
  • the vector may be any vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and can result in the expression of the RvPolX polynucleotide sequence in a suitable host cell.
  • 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 vectors may be linear or closed circular plasmids.
  • the host cell is selected from the group consisting of Grampositive bacteria, Gram-negative bacteria, filamentous fungi, yeast and algae.
  • Nonlimiting examples of host cells include Escherichia coli, Lactococcus lactis, Bacillus sp., and Pichia sp.
  • An isolated polypeptide of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., Sf21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, MD).
  • Non-limiting examples of insect cells are, Spodoptera frugiperda (ST) cells, e.g., Sf9, Sf21, Trichoplusia ni cells, e.g., High Five cells, and Drosophila S2 cells.
  • fungi including yeast host cells are S. cerevisiae, Kluyveromyces lactis (K lactis), species of Candida including C. albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces pombe (S. pombe), Komagataella pastoris (previously known as Pichia pastoris), and Yarrowia lipolytica.
  • Methods to grow cells that produce the polypeptides of the invention include, but are not limited to, batch, batch-fed, continuous and perfusion cell culture techniques.
  • cell culture is performed under sterile, controlled temperature and atmospheric conditions.
  • a bioreactor is a chamber used to culture cells in w'hich environmental conditions such as temperature, atmosphere, agitation and/or pH can be monitored.
  • the bioreactor can be a stainless steel chamber or a pre- sterilised plastic bag (e.g., Cellbag.RTM., Wave Biotech, Bridgewater, N.J.).
  • the bioreactor may be dimensioned for cultures of about 20 L to about 50,000 L.
  • the contacting is performed at a salt concentration of about 100 mM or more.
  • the salt may be a monovalent salt, such as NaCl or KC1.
  • the contacting is performed at a high salt concentration, such as a salt concentration of at least 400 mM. In some embodiments, the contacting is performed at a salt concentration of about 400 mM to about 1500 mM, such as a salt concentration of about 400 mM, about 500 mM, about 600 mM, about 700 mM, about 800 mM, about 900 mM, about 1000 mM, about 1100 mM, about 1200 mM, about 1300 mM, about 1400 mM, or about 1500 mM.
  • the constructs were transformed into Escherichia coli Rosetta 2 (DE3).
  • the bacterial culture carrying the plasmid of interest was grown in LB medium supplemented with antibiotics at 37°C with shaking at 250 rpm.
  • ODeoo reach 0.8 0.5 mM isopropyl-l-thio-p-D-galactopyranoside (1PTG) was introduced, and the culture was then incubated at 16°C with shaking at 200 rpm overnight.
  • the bacterial cultures were subsequently harvested, lysed, and the supernatant proteins were purified using TALON® Metal Affinity Resin (Takara).
  • the purified proteins were incubated with SUMO protease overnight at 4°C with an approximate molar ratio of 1 :25.
  • the purified proteins were further subjected to purification via HiTrapTM Heparin HP affinity columns (GE Healthcare Life Sciences). Finally, 4-20% mini-PROTEAN® TGXTM precast protein gel (Bio-Rad) was used to analyze the purified recombinant proteins.
  • a reaction mixture for G513A+R522I was prepared with 1 pM G513A+R522I, 50 nM Alexa-ssDNA14, 1 mM MnCh, 5 mM MgCh, 1 mM TCEP, 1% glycerol, and 50 mM Tris-HCl pH 8.8.
  • a reaction mixture was prepared with 1 pM TdT (New England Biolabs), IX Terminal Transferase Reaction Buffer (New England Biolabs), 0.25 mM CoCh (New England Biolabs), and 50 nM Alexa-ssDNA14.
  • the reaction was initiated by adding 0.25 mM of the respective 3’ ONHi modified nucleotides (Firebird Biomolccular Sciences), followed by incubation at 25°C for 40 minutes. Subsequently, the reaction was inactivated at 95°C for 2 minutes using a thermocycler. The products were then analyzed by gel electrophoresis on an 18% acrylamide gel with 8 M urea (urea-PAGE) and visualised using ChemiDoc MP Imaging System (Bio-Rad).
  • the protein sequence of RvPolX was retrieved from the UniProt database (asscssion number: A0A1D1UV65).
  • the whole modelling protocol was given as follows: (1) three initial models of RvPolX were firstly predicted by AlphaFold v2.0 program; (2) the best model with the top ranked score was selected and its N-terminus (l -234aa) containing a long-disordered loop was truncated; (3) this pruned model was subjected to the sidechain optimization in Sybyl-X package (v2.1), which afforded diverse models; (3) the model with the lowest energy was selected for the subsequent modelling; (4) two coordinated metal ions (e.g., Mn 2+ ) in the catalytic site were modelled with the following procedure: one ion was positioned in the center of two carboxyl groups of D437 and D439, while the other was placed in the center of three carboxyl groups of D437, D439, and D498; subsequently, two metal ions and the
  • the docking parameters were provided as follows: scoring function is ChemScore; population size is 400; selection pressure is 1.1; number of operations is 500,000; number of islands is 5; niche size is 2; crossover frequency is 95%; mutation frequency is 95%; migration frequency is 10%.
  • scoring function is ChemScore
  • all the sampled conformations for each incoming nucleotide were further filtered by the following criteria: (1) the base of the incoming nucleotide can form favourable TC-TC stacking with the ss DNA (5’-AGCCTG-3’); (2) the phosphate group(s) of the incoming nucleotide can form the salt bridge with the nearby residues such as lysine or arginine; (3) the phosphate group(s) of the incoming nucleotide can form the coordination interaction with a metal ion
  • the beads were separated and washed 3 times with wash buffer (25 mM HEPES pH 7.4, 500 mM NaCl, 0.01% Tween-20, 10 mM imidazole).
  • wash buffer 25 mM HEPES pH 7.4, 500 mM NaCl, 0.01% Tween-20, 10 mM imidazole.
  • the immobilized protein on the DynabeadsTM was incubated with a reaction buffer containing 25 mM HEPES pH 7.4, 1 pM ssDNA14, 1 mM dCTP, 1 mM TCEP, 1 mM MnCh, 5 mM MgCh, and 1% glycerol at 25°C and 1000 rpm for 60 minutes. After centrifugation, 30 pL of supernatant was analyzed using a UHPLC system (Shimadzu, Japan).
  • Microscale thermophore sis (MST) study was conducted to investigate the interaction between RvPolX and a mutant with four dNTPs.
  • This study utilized the MonolithTM NT.labelFree system by NanoTemper Technologies.
  • a series of samples containing nucleotides (at concentrations ranging from 0.01 to 0.15 pM) and enzyme (0.8 pM) in a reaction buffer consisting of 50 mM HEPES at pH 7.4, 5 mM MgCh, 1 mM MnCh, and 2 mM TCEP were incubated at room temperature for 10 minutes before being loaded individually into MonolithTM NT.labelFree capillaries.
  • the measurements were conducted at room temperature with 20% LED power and 20% MST power, and the resulting data were analyzed using MO Affinity Analysis to determine the dissociation constants.
  • the oligonucleotide chain growth polymerization kinetics was modelled as a Poisson distribution.
  • a specific oligonucleotide product band (k) that allowed us to observe the timedependent accumulation and subsequent depletion of the oligonucleotide product, was selected for analysis.
  • the apparent rate of chain elongation (Yobs)' was estimated by fitting the changes in band intensity (I) over time (t) to a Poisson probability distribution function, with a normalization constant (A).
  • the tardigrade R. varieornatus contains three PolXs, of which two belong to the Polk- like group (UniProt IDs: A0A1D1UV65 and A0A1D1UJX5, 34 and 38% sequence identities to human Polk), and one belongs to the Pol0-like group (A0A1D1W154, 32% sequence identity to human Poip).
  • One of the R. varieornatus Polk-like enzymes (UniProt ID: A0A1D1UV65, designated as RvPolX) was recombinantly expressed in and purified from E. coli cultures for further biochemical analysis.
  • the domain structure of RvPolX consists of an N-tcrminal BRCT domain and a C- terminal catalytic domain, similar to TdT, Polp, and Polk ( Figure 1A).
  • a multiple sequence alignment of the catalytic domains of RvPolX and the mammalian PolXs suggest a conserved fold, which includes the 8 kDa, fingers, palm, and thumb subdomains.
  • the TdT crystal structure contains a specific loop (loopl) that obstructs template strand binding, and is reported to contribute to its template-independent polymerase activity.
  • a high negative surface charge density is found in many halophilic proteins, and could contribute to the stability of RvPolX under the reaction conditions. Activity was detectable up to 700 mM NaCl for incorporation of dGTP, dTTP, and dCTP, and up to 500 mM NaCl for incorporation of dATP, suggesting that RvPolX might be suitable for further development as a salt-tolerant polymerase. Tn assays with various divalent metal ions (Ca 2+ , Co 2+ , Mg 2+ , Mn 2+ , and Zn 2+ ), RvPolX activity was highest with Mn 2+ as the metal cofactor, followed by Mg 2+ and Co 2+ ( Figure 3).
  • oligonucleotide substrate sequence was examined, by employing 15-mer oligonucleotide substrates (ssDNA14A, ssDNA14T, ssDNA14G, and ssDNA14C) composed of ssDNA14 with a single base (A, T, G, or C) added at the 3' end.
  • the assays revealed that RvPolX did not exhibit a strong preference for the specific 3' base of the oligonucleotide substrate ( Figure IE).
  • RvPolX N-terminal 258-amino acid deletion mutant (Al-258aa) was constructed which contained only the catalytic domain, and its activity was compared to that of RvPolX wild-type (WT).
  • WT RvPolX wild-type
  • Figure 4C Activity of Al- 258aa is comparable to WT at 25°C and 40°C, but significantly less that WT at 45°C.
  • the activity of WT decreased between 25°C and 40°C, and was eliminated at 45°C.
  • the mammalian Poip, Poll, Poip. and TdT are the four representative enzymes that have been subjects of in-depth studies.
  • TdT has garnered significant attention for de novo DNA synthesis applications, especially in the context of DNA-bascd data storage, due to its high templateindependent polymerase activity.
  • the findings on RvPolX from the extremotolerant tardigrade R. varieornatus, expands the scope of biochemically characterized PolX enzymes to invertebrates. It is demonstrated that RvPolX possesses modest templateindependent DNA polymerase activity, despite sharing only 21% sequence identity with TdT.
  • salt tolerant versions of the template-dependent DNA polymerase from Bacillus phage phi29 have previously been employed in nanopore devices for DNA sequencing.
  • the development of salt-tolerant templateindependent polymerases could enable applications in nanopore -based devices for de novo DNA synthesis, involving electrophoretic control of the DNA synthesis process, and in situ proofreading by nanopore sequencing.
  • Targeted mutagenesis of active site residues in RvPolX was carried out to enhance its salt-tolerant template-independent DNA polymerase activity.
  • a structural model of the ternary complex of RvPolX and its substrates was constructed by molecular docking of the AlphaFold structure of RvPolX with ssDNA14, two Mn 2+ ions, and each of the four dNTPs (Figure 5A). From the structural model, 12 amino acid residues located in and around the dNTP binding pocket (R276, E281, C425, S427, R432, G513, W514, M518, Y519, R525, R522, and D537, Figure 5A) were selected for saturation mutagenesis and high throughput activity screening.
  • a total of 240 clones from the mutant library were individually expressed and purified using DynabeadsTM, and screened for template-independent DNA polymerase activity, with reaction products analyzed by high-performance liquid chromatography (HPLC).
  • HPLC high-performance liquid chromatography
  • substitutions to hydrophobic amino acids e.g., A, C, F, I, L, M, V, W, and Y
  • WT high-performance liquid chromatography
  • the G513A+R522T mutant exhibits an enhanced salt-tolerant polymerase activity, and increased promiscuity towards dNTP substrates, with a particularly marked increase in proficiency for incorporation of dATP.
  • the formation of a continuous hydrophobic patch in the nucleotide binding pocket is crucial for further improvement in catalytic activity.
  • a potentially optimal combination of residues in this hydrophobic patch can be A or C at 513, W at 514, M at 518, V at 521, and I, M or V at 522.
  • the dissociation constants for binding of the four dNTPs to RvPolX WT and G513A+R552I were determined using label-free microscale thermophoresis (MST, Figure 7D).
  • MST label-free microscale thermophoresis
  • the Kd for binding of dGTP to G513A+R5521 was ⁇ 5-fold lower than WT, while the Kd for binding of the other dNTPs to the G513A+R552I was comparable to WT.
  • the absence of correlation between dNTP binding affinity and enhancement of catalytic activity indicates the need for alternative explanations, particularly for the nonconservative R522I mutation.
  • the residue corresponding to R552 is conserved in Polk, Polp, and TdT, and in the crystal structure of human Polp gap filling pre- and post- catalytic complexes, this residue interacts with the phosphodiester backbone of the template DNA strand.
  • loopl of TdT adopts a lariat-like conformation, impeding the binding of a continuous template DNA strand, a feature believed to be important in its template-independent DNA polymerase activity. Deletion of loopl or substitution of amino acid residues within the loop reduces or completely abolishes template-independent DNA polymerise activity. Interestingly, loopl is absent in both RvPolX and the well-studied Polk, which has also been reported to have modest template-independent DNA polymerase activity.
  • RvPolX is the only Polk-like enzyme containing the GW motif, while the others contain YF, HY or other motifs ( Figure 11). Given its proximity to the dNTP substrate, the increased activity of the G513A mutation may result from the binding of the dNTP in a more reactive conformation, or through rigidification of the dNTP binding pocket.

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Abstract

La présente invention concerne un variant ingénierisé d'une ADN polymérase de la famille Ramazzottius varieornatus X (RvPoIX) présentant une activité d'ADN polymérase indépendante de la matrice améliorée et tolérante au sel, comportant au moins une substitution d'acide aminé correspondant aux positions 281, 513 et/ou 522, comme représenté dans SEQ ID NO : 1. La substitution d'acide aminé au niveau de la position correspondant à la position 281 peut être une substitution en A, F, I, L, V ou Y. La substitution d'acide aminé au niveau de la position correspondant à la position 513 peut être une substitution en A ou C. La substitution d'acide aminé au niveau de la position correspondant à la position 522 peut être une substitution en I, M ou V. La présente invention concerne également des procédés de synthèse d'acides nucléiques faisant appel à ladite ADN-polymérase.
PCT/SG2024/050772 2024-01-17 2024-12-04 Adn polymérases de la famille x et leurs utilisations Pending WO2025155239A1 (fr)

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Citations (2)

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US20200002690A1 (en) * 2016-06-14 2020-01-02 Dna Script Variants of a DNA Polymerase of the Polx Family
US20210164008A1 (en) * 2017-05-26 2021-06-03 Nuclera Nucleics Ltd. Use of Terminal Transferase Enzyme in Nucleic Acid Synthesis

Patent Citations (2)

* Cited by examiner, † Cited by third party
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