Patent Application
20250043259
The present disclosure generally relates to the fields of enzymology and molecular biology, and more particularly to endonucleases.
Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Two types of endonucleases are restriction nucleases and homing endonucleases.
Restriction endonucleases are enzymes that recognize a specific nucleotide sequence in a double-stranded nucleic acid called a restriction site. Upon binding to the restriction site, the restriction endonucleases cleave within or near the restriction site. These enzymes are routinely used for DNA modification in laboratories, such as for genetic engineering and molecular cloning. For example, they are used to assist insertion of genes into plasmid vectors, to distinguish gene alleles by specifically recognizing single base changes in DNA known as single-nucleotide polymorphisms (SNPs), to digest genomic DNA for gene analysis, and to insert nucleic acid molecules within the genome of an organism. Most of the known restriction enzymes recognize a restriction site, typically comprising from 4 to 8 nucleotides that are often palindromic, within a double-stranded DNA molecule, and produce a double-stranded cut in the DNA.
Homing endonucleases are double-stranded DNases that have large, asymmetric recognition sites (12-40 base pairs). However, unlike restriction endonucleases, homing endonucleases tolerate some sequence degeneracy within their recognition sequence, which means that single base changes do not abolish cleavage but reduce its efficiency to variable extents. As a result, their observed sequence specificity is typically in the range of 10-12 base pairs.
In contrast to restriction and homing endonucleases that cleaves double-stranded nucleic acids at specific sites, endonucleases that cleave single-stranded nucleic acid molecules are usually non-specific, i.e., they do not recognize a specific sequence (e.g., restriction site) within the single-stranded nucleic acid molecules but rather cleave at various sequences to degrade the nucleic acid molecules in several fragments.
Thus, there is a need for the identification of novel endonucleases that are able to recognize and cleave at specific sequences within single-stranded nucleic acid molecules such as single-stranded DNA molecules.
The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
The present disclosure provides the following items 1 to 73:
1. An isolated endonuclease specific for single-stranded desoxyribonucleic acid molecules, the isolated endonuclease having a length of 60 to 150 amino acids or less and comprising a single GIY-YIG domain.
2. The isolated endonuclease of item 1, having a length of 70 to 130 amino acids.
3. The isolated endonuclease of item 1, having a length of 80 to 120 amino acids.
4. The isolated endonuclease of any one of items 1 to 3, wherein the GIY-YIG domain is of the following sequence (I):
X1-X2-X3-B1-X4-X5-X6-B2-X7-B3-X8 (I)
5. The isolated endonuclease of item 4, wherein X1 is Y, W, V, A, F, I, C, H, R, T or S.
6. The isolated endonuclease of item 5, wherein X1 is Y.
7. The isolated endonuclease of any one of items 4 to 6, wherein X2 is V, I, L, T, A or F.
8. The isolated endonuclease of item 7, wherein X2 is V.
9. The isolated endonuclease of any one of items 4 to 8, wherein X3 is Y.
10. The isolated endonuclease of any one of items 4 to 9, wherein B1 is a sequence of 8 to 10 amino acids.
11. The isolated endonuclease of any one of items 4 to 10, wherein X4 is Y.
12. The isolated endonuclease of any one of items 4 to 11, wherein X5 is I, L, V, T, A, C, or K.
13. The isolated endonuclease of item 12, wherein X5 is I, T or V.
14. The isolated endonuclease of item 13, wherein X5 is I.
15. The isolated endonuclease of any one of items 4 to 14, wherein X6 is G.
16. The isolated endonuclease of any one of items 4 to 15, wherein B2 is a sequence of 6 to 10 amino acids.
17. The isolated endonuclease of item 16, wherein B2 is a sequence of 6 to 8 amino acids.
18. The isolated endonuclease of any one of items 4 to 17, wherein X7 is R.
19. The isolated endonuclease of any one of items 4 to 18, wherein B3 is a sequence of 35 to 40 amino acids.
20. The isolated endonuclease of any one of items 4 to 19, wherein X8 is E.
21. The isolated endonuclease of any one of items 4 to 20, wherein the GIY-YIG domain is of the following sequence (II):
X1-X2-X3-B1-X4-X5-X6-B2-X7-B4-X9-B5-X8 (II)
X1, X2, X3, B1, X4, X5, X6, B2, X7 and X8 are as defined in items 4 to 20;
22. The isolated endonuclease of item 21, wherein B4 is a sequence of 2 to 4 amino acids.
23. The isolated endonuclease of item 21 or 22, wherein X9 is H.
24 The isolated endonuclease of any one of items 21 to 23, wherein B5 is a sequence of 30 to 35 amino acids.
25. The isolated endonuclease of item 24, wherein B5 is a sequence of 31 to 33 amino acids.
26. The isolated endonuclease of any one of items 4 to 25, wherein the GIY-YIG domain is of the following sequence (III):
X1-X2-X3-B1-X4-X5-X6-B2-X7-B4-X9-B5-X8-B6-X10 (III)
27. The isolated endonuclease of item 26, wherein B6 is a sequence of 16 to 19 amino acids.
28. The isolated endonuclease of item 26, wherein X10 is N.
29. The isolated endonuclease of any one of items 1 to 28, comprising an amino acid sequence having at least 50% similarity or identity with any one of the sequences set forth in SEQ ID NOs: 2-2891.
30. The isolated endonuclease of any one of items 1 to 28, wherein the isolated endonuclease comprises an amino acid sequence having at least 60% similarity or identity with any one of the sequences set forth in SEQ ID NOs: 2-2891.
31. The isolated endonuclease of any one of items 1 to 28, wherein the isolated endonuclease comprises an amino acid sequence having at least 70% similarity or identity with any one of the sequences set forth in SEQ ID NOs: 2-2891.
32. The isolated endonuclease of any one of items 1 to 28, wherein the isolated endonuclease comprises an amino acid sequence having at least 80% similarity or identity with any one of the sequences set forth in SEQ ID NOs: 2-2891.
33. The isolated endonuclease of any one of items 1 to 28, wherein the isolated endonuclease comprises an amino acid sequence having at least 90% similarity or identity with any one of the sequences set forth in SEQ ID NOs: 2-2891.
34. The isolated endonuclease of any one of items 1 to 28, wherein the isolated endonuclease comprises the amino acid sequence of any one of the sequences set forth in SEQ ID NOs: 2-2891.
35. A composition comprising (i) the isolated endonuclease of any one of items 1 to 34, and (ii) an aqueous saline solution or buffer.
36. The composition of item 35, wherein the aqueous saline solution or buffer comprises a metal.
37. The composition of item 36, wherein the metal is in the form of a metal salt.
38. The composition of item 36 or 37, wherein the metal is magnesium, manganese or nickel.
39. The composition of item 38, wherein the metal is magnesium.
40. The composition of item 39, wherein the composition comprises magnesium chloride (MgCl2).
41. The composition of any one of items 35 to 40, wherein the single-stranded nucleic acid molecule is a single-stranded DNA molecule.
42. The composition of any one of items 35 to 41, wherein the single-stranded nucleic acid molecule comprises a nucleotide sequence having at least 50% sequence identity with the sequence:
43. The composition of any one of items 35 to 42, wherein the single-stranded nucleic acid molecule comprises the following nucleotide sequence: GTCANNCCNGNNNANNONGGNNNC.
44. The composition of item 43, wherein the single-stranded nucleic acid molecule comprises the following nucleotide sequence:
45. The composition of item 43 or 44, wherein the single-stranded nucleic acid molecule comprises any one of the nucleotide sequences depicted in
46. The composition of any one of items 35 to 44, further comprising the single-stranded nucleic acid molecule defined in any one of items 41 to 45.
47. A method for cleaving a single-stranded nucleic acid molecule, the method comprising contacting the single-stranded nucleic acid molecule with the isolated endonuclease of any one of items 1 to 34 or the composition of any one of items 35 to 45 under conditions suitable for cleavage of the single-stranded nucleic acid molecule by the isolated endonuclease, wherein the single-stranded nucleic acid molecule comprises a recognition sequence for the isolated endonuclease.
48. The method of item 47, wherein said conditions comprises a temperature of about 20 to about 55° C.
49. The method of item 48, wherein said conditions comprises a temperature of about 35 to about 40° C.
50. The method of item 49, wherein said conditions comprises a temperature of about 37° C.
51. The method of any one of items 47 to 50, wherein said conditions comprises the presence of a metal.
52. The method of item 51, wherein said metal is magnesium, manganese or nickel.
53. The method of item 52, wherein the metal is magnesium.
54. The method of item 53, wherein the magnesium is in the for magnesium chloride (MgCl2).
55. The method of any one of item 51 to 54, wherein said metal is at a concentration of at least 5 mM.
56. The method of any one of items 51 to 54, wherein said metal is at a concentration of at least 10 mM.
57. The method of any one of items 47 to 56, wherein said contacting is for a period of at least 2 minutes.
58. The method of any one of items 47 to 56, wherein said contacting is for a period of at least 15 minutes.
59. The method of any one of items 47 to 58, wherein said conditions comprises a pH of about 6 to 8.
60. The method of any one of items 47 to 58, wherein the [concentration of endonuclease]/[single-stranded nucleic acid molecule] ratio is at least 0.00001.
61. The method of item 60, wherein the [concentration of endonuclease]/[single-stranded nucleic acid molecule] ratio is at least 0.01.
62. The method of item 61, wherein the [concentration of endonuclease]/[single-stranded nucleic acid molecule] ratio is at least 0.5.
63. A method for rendering a single-stranded nucleic acid susceptible to cleavage by the endonuclease defined in any one of items 1 to 34, the method comprising incorporating a nucleotide sequence comprising a recognition sequence for the isolated endonuclease into the single-stranded nucleic acid.
64. The method of item 63, wherein the nucleotide sequence comprises one of the sequence defined in any one of items 42 to 45
65. The method of item 64, wherein the method comprises adding a nucleic acid fragment comprising the nucleotide sequence defined in any one of items 42 to 45 at the 5′-end, 3′-end or within the single-stranded nucleic acid.
66. The method of item 64, wherein the method comprises introducing one or more mutations within the sequence of the single-stranded nucleic acid to obtain the nucleotide sequence defined in any one of items 42 to 45.
67. A cell comprising the endonuclease defined in any one of items 1 to 34, wherein the endonuclease is heterologous to the cell.
68. A method for expressing the endonuclease defined in any one of items 1 to 34 in a cell, the method comprising introducing a nucleic acid encoding the endonuclease into the cell.
69. The cell of item 67 or the method of item 68, wherein the cell is a prokaryotic or eukaryotic cell.
70. The cell or method of any one of items 67 to 69, wherein the cell comprises a single-stranded nucleic acid that is cleaved by the endonuclease.
71. The cell or method of any one of items 68 to 70, wherein the nucleic acid encoding the endonuclease is present in a vector.
72. A kit comprising the endonuclease defined in any one of items 1 to 34 or the composition of any one of items 35 to 45, and instructions for cleaving single-stranded nucleic acid molecules using the endonuclease.
73. The kit of item 72, wherein said instructions comprise the method of any one of items 47 to 62.
Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
In the appended drawings:
The use of the terms “a” and “an” and “the” and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
The terms “comprising”, “having”, “including”, and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted.
All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.
The use of any and all examples, or exemplary language (“e.g.”, “such as”) provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.
No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.
Herein, the term “about” has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.
Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).
The present inventors have identified a family of endonucleases that preferentially bind to and cleave single-stranded DNA. These endonucleases recognize and cleave specific nucleotide sequences in single-stranded nucleic acids (single-stranded DNA). These endonucleases may be useful, for example, in various genetic engineering and molecular biology applications. These endonucleases are short proteins (typically less than 150 amino acids, and preferably less than 140 or 130 amino acids) and comprises a conserved GIY-YIG domain. The GIY-YIG domain comprises two short semi-conserved motifs “GIY” and “YIG” in the N-terminal part, followed by an Arg residue in the center and a Glu residue in the C-terminal part. The GIY-YIG domain has an α/β-sandwich architecture with a central three-stranded antiparallel β-sheet flanked by three-helices. The three-stranded antiparallel β-sheet contains the GIY-YIG sequence elements.
The present disclosure provides an isolated endonuclease specific for single-stranded nucleic acid molecules (e.g., binds to and cleaves a single-stranded nucleic acid such as single-stranded DNA), the isolated endonuclease having a length of 150 amino acids or less and comprising a GIY-YIG domain.
The present disclosure also provides a cell comprising an endonuclease specific for single-stranded nucleic acid molecules as described herein. In an embodiment, the endonuclease is heterogenous to the cell. “Heterogenous” as used herein means that the endonuclease is the product of a gene that is not naturally present in the cell. For example, if the endonuclease is the endonuclease of SEQ ID NO:2, the cell is not a Neisseria elongata subsp. glycolytica cell. The cell may be a cell from another bacterial species or subspecies or an eucaryotic cell (mammalian cell, human cell, yeast cell, etc.), for example.
In an embodiment, the endonuclease of the present disclosure has a length of 80 to 130 amino acids. In another embodiment, the endonuclease of the present disclosure has a length of 85 to 120 amino acids. In an embodiment, the endonuclease of the present disclosure does not comprise any additional domain, it only consists of a single GIY-YIG domain.
In an embodiment, the endonuclease of the present disclosure comprises a GIY-YIG domain of the formula I:
X1-X2-X3-B1-X4-X5-X6-B2-X7-B3-X8 (I)
In another embodiment, the endonuclease of the present disclosure comprises a GIY-YIG domain of the formula II:
X1-X2-X3-B1-X4-X5-X6-B2-X7-B4-X9-B5-X8 (II)
In another embodiment, the endonuclease of the present disclosure comprises a GIY-YIG domain of the formula III:
X1-X2-X3-B1-X4-X5-X6-B2-X7-B4-X9-B5-X8-B6-X10 (III)
In an embodiment, the isolated endonuclease of the present disclosure comprises or consists of an amino acid sequence having at least 50% similarity or identity with the any one of the sequences set forth in SEQ ID NOs: 2-5,
The term “endonuclease” as used herein refers to an enzyme having the ability to cleave a single-stranded nucleic acid molecule, such as single-stranded DNA, at or near a specific nucleotide sequence (recognition or restriction site).
The term “isolated” as used herein refers to a molecule (endonuclease) that is in a milieu or environment that is different from the natural milieu or environment where it is found in nature (i.e., that has been subjected to human manipulation), for example a endonuclease that has isolated from the natural bacteria that normally expressed it. As such, “isolated” does not necessarily reflect the extent to which the endonuclease has been purified, but indicates that the molecule has been separated in some way from the natural environment where it is normally found. An isolated endonuclease may also be produced recombinantly by cloning a nucleic acid encoding the endonuclease in a host cell capable of expressing the endonuclease, and collecting the endonuclease produced.
“Identity” refers to sequence identity between two polypeptides. Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as Clustal Omega, BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
Similarity refers to sequence similarity between two polypeptides. Percent (%) sequence similarity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are similar (identical or conserved) with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence similarity, and considering conservative substitutions as part of the sequence similarity. The similarity between amino acids can be defined either by their chemical properties (e.g., hydrophobic, hydrophilic, charged, polar, etc.) or based on a PAM matrix.
Variations in the endonucleases described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Pat. No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the endonuclease that results in a change in the amino acid sequence as compared with the native sequence of the endonuclease. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the endonuclease. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the endonuclease with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence (e.g., ability to cleave single-stranded nucleic acid molecules).
In an embodiment, the isolated endonuclease comprises or consists of an amino acid sequence having at least 55% similarity or identity with the any one of the sequences set forth in SEQ ID NOs: 2-5,
The isolated endonuclease may further comprise additional amino acids at its amino-(N) and/or carboxy (C)-terminal end. For example, the isolated endonuclease may be fused to a peptide or polypeptide, for example a peptide or polypeptide that may be used as an affinity tag to facilitate the detection and/or purification of the endonuclease. Examples of affinity tags include polyhistidine (His) tags, polyarginine tags, glutathione-S-transferase (GST) tags, FLAG tags, streptavidin-binding peptide or streptavidin-binding protein (SBP) tags, streptavidin-binding tag (Strep-tag), calmodulin-binding peptide (CBP) tags, chitin-binding tags, Maltose-binding protein (MBP) tags, and natural histidine affinity tags (HAT). The peptide or polypeptide may be fused directly to the N- and/or C-terminal end of the endonuclease, or indirectly via a linker. Such a linker may be a peptide/polypeptide linker comprising one or more amino acids or another type of chemical linker (e.g., a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc.) having suitable flexibility and stability to allow the endonuclease to adopt a proper conformation. The linker may comprise at least 2, 3 or 4 amino acids. The linker may comprise about 100, 90, 80, 70, 60 or 50 amino acids or less, and preferably 20, 15 or 10 amino acids or less.
The isolated endonuclease of the disclosure may be produced by expression in a host cell comprising a nucleic acid encoding the isolated endonuclease (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). Peptides and polypeptides can be readily synthesized by manual and automated solid phase procedures well known in the art. Suitable syntheses can be performed for example by utilizing “t-Boc” or “Fmoc” procedures. Techniques and procedures for solid phase synthesis are described in for example Solid Phase Peptide Synthesis: A Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the polypeptides may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 37:933-936, 1996; Baca et al., J. Am. Chem. Soc. 117:1881-1887, 1995; Tarn et al., Int. J. Peptide Protein Res. 45:209-216, 1995; Schnolzer and Kent, Science 256:221-225, 1992; Liu and Tarn, J. Am. Chem. Soc. 116:4149-4153, 1994; Liu and Tarn, Proc. Natl. Acad. Sci. USA 91:6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31:322-334, 1988). Other methods useful for synthesizing polypeptides are described in Nakagawa et al., J. Am. Chem. Soc. 107:7087-7092, 1985.
The isolated endonuclease may also be prepared using recombinant DNA technology using standard methods. Accordingly, in another aspect, the disclosure further provides a nucleic acid (e.g., mRNA, cDNA) encoding the above-mentioned endonuclease. The disclosure also provides a vector comprising the above-mentioned nucleic acid. In yet another aspect, the present disclosure provides a cell (e.g., a host cell) comprising the above-mentioned nucleic acid and/or vector. The disclosure further provides a recombinant expression system, vectors and host cells, such as those described above, for the expression/production of an endonuclease of the disclosure, using for example culture media, production, isolation and purification methods well known in the art.
The endonuclease of the disclosure can be purified by many techniques of peptide/polypeptide purification well known in the art, such as reverse phase chromatography, high performance liquid chromatography (HPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, gel electrophoresis, and the like. The actual conditions used to purify a particular peptide or polypeptide will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those of ordinary skill in the art. For affinity chromatography purification, any ligand or antibody that specifically binds the endonuclease (or to an affinity tag fused to the endonuclease) may for example be used.
The present disclosure also provides a composition comprising (i) an isolated endonuclease specific for single-stranded nucleic acid molecules as described herein, and (ii) an aqueous saline solution or buffer.
The composition according to the present disclosure an aqueous saline solution or buffer. Such aqueous saline solutions or buffers include ingredients that stabilize the endonuclease and provide suitable conditions for the enzymatic activity of the endonuclease (e.g., conditions that permit the cleavage of single-stranded nucleic acids). The aqueous saline solution or buffer present in the composition according to the present disclosure may include suitable salts, buffering agents, minerals, co-factors, stabilizing agents, anti-oxidants, redox reagent, preservatives, etc.
In an embodiment, the composition comprises a buffering agent. The buffering agent is useful to keep the composition at a desired pH. Buffering agents are well known in the art and include potassium, acetate, citrate, acetate, phosphate, carbonate, succinate, histidine, borate, maleate, tris(hydroxymethyl) aminomethane (Tris), BIS-Tris, piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), 2-(N-morpholino) ethanesulfonic acid (MES), (3-(N-morpholino) propanesulfonic acid) (MOPS), N-(2-Acetamido)-2-aminoethanesulfonic acid (ACES), (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), magnesium and hydrochloride buffers. In an embodiment, the composition comprises a Tris buffer. In a further embodiment the Tris buffer is a Tris-HCl or a Tris-acetate buffer. In an embodiment, the buffering agent is at a concentration of about 0.1 mM to 1M. In further embodiments, the buffering agent is at a concentration of about 1 mM to about 500 mM, about 1 mM to about 200 mM, about 1 mM to about 100 mM, about 5 mM to about 100 mM, about 5 mM to about 75 mM, about 5 mM to about 50 mM, or about 5 mM to about 25 or 20 mM. In an embodiment, the buffering agent is at a concentration of about 10 mM. In an embodiment, the buffering agent has a pH of about 5 to about 10. In further embodiments, the buffering agent has a pH of about 6 to about 9, of about 6.5 to about 9, of about 7 to about 9, of about 7.5 to about 8.5, or of about 7.6 to about 8.2. In an embodiment, the buffering agent has a pH of about 7.9 or 8.0. In an embodiment, the pH is the pH at a temperature of about 20 to about 40° C. In an embodiment, the pH is the pH at a temperature of about 20 or 25° C. In an embodiment, the pH is the pH at a temperature of about 37° C.
In an embodiment, the composition comprises a salt, such as a metal salt. Common salt-forming cations include ammonium (NH4+), manganese, nickel, calcium, iron, magnesium, potassium, sodium and copper. In an embodiment, the metal salt is a magnesium salt, manganese salt or zinc salt. Common salt-forming anions include acetate, carbonate, chloride, citrate, fluoride, nitrate, nitrite, oxide, phosphate and sulfate. Examples of salts include magnesium chloride (MgCl2), magnesium acetate, potassium acetate (KCH3CO2), potassium chloride (KCl), sodium acetate (CH3COONa), sodium chloride (NaCl), calcium chloride, zinc chloride, manganese sulfate, manganese chloride, nickel chloride, nickel acetate, and sodium sulfate (Na2SO4). In an embodiment, the salt comprises a magnesium, manganese, nickel and/or sodium cation. In an embodiment, the salt comprises a chloride anion. In an embodiment, the composition comprises KCl. In an embodiment, the composition comprises KCl and NaCl. In an embodiment, the concentration of salt in the composition is about 1 mM to about 500 mM. In further embodiments, the concentration of salt in the composition is about 10 mM to about 300 mM, about 20 mM to about 200 mM, about 20 mM to about 150 mM, about 30 mM to about 150 mM. In an embodiment, the composition comprises a salt comprising a magnesium cation (e.g., MgCl2) at a concentration of about 1 mM to about 100 mM, for example about 1 mM to about 50 mM, about 5 mM to about 20 mM, or about 5 to about 15 mM. In an embodiment, the composition comprises a salt comprising a magnesium cation (e.g., MgCl2) at a concentration of about 10 mM. In an embodiment, the composition comprises a salt comprising a sodium cation (e.g., NaCl) at a concentration of about 1 mM to about 200 mM, about 10 mM to about 150 mM, about 10 mM to about 100 mM, about 25 mM to about 75 mM. In an embodiment, the composition comprises a salt comprising a sodium cation (e.g., NaCl) at a concentration of about 50 mM.
In an embodiment, the composition comprises a stabilizing agent, such as a protein. In an embodiment, the protein is albumin, such as bovine serum albumin (BSA). In an embodiment, the stabilizing agent is at a concentration of about 1 μg/ml to about 1 mg/ml. In an embodiment, the stabilizing agent is at a concentration of about 10 μg/ml to about 500 μg/ml. In an embodiment, the stabilizing agent is at a concentration of about 50 μg/ml to about 200 μg/ml. In an embodiment, the stabilizing agent is at a concentration of about 50 μg/ml to about 150 μg/ml. In an embodiment, the stabilizing agent is at a concentration of about 80 to about 120 μg/ml, for example about 100 μg/ml.
In embodiments, the composition further comprises additional ingredients, such as detergents (e.g., non-ionic detergents like Triton® X-100 or Tween® 20) and/or redox reagents (DTT, beta-mercaptoethanol). For storage, the composition may further comprise a metal-chelating agent, such as ethylenediaminetetraacetic acid (EDTA).
Aqueous solutions/buffers for endonucleases are available commercially from several providers such as New England Biolabs Inc., Pomega and Thermo Scientific. Examples from New England Biolabs Inc. include NEBuffer 1 (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.0@25° C.); NEBuffer 1.1 (10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 100 μg/ml BSA. pH 7.0@25° C.); NEBuffer 2.1 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml BSA, pH 7.9@25° C.); NEBuffer 3.1 (100 mM NaCl, 50 mM Tris-HCl. 10 mM MgCl2, 100 μg/ml BSA, pH 7.9@25° C.); NEBuffer 4 (50 mM Potassium acetate, 20 mM Tris-acetate, 10 mM Magnesium Acetate, 1 mM DTT, pH 7.9@25° C.); and CutSmart Buffer (50 mM Potassium Acetate, 20 mM Tris-acetate. 10 mM Magnesium Acetate, 100 μg/ml BSA, pH 7.9@25° C.). Examples from Thermo Scientific include Buffer B (10 mM Tris-HCl (pH 7.5 at 37° C.), 10 mM MgCl2, 0.1 mg/ml BSA), Buffer G (10 mM Tris-HCl (pH 7.5 at 37° C.), 10 mM MgCl2, 50 mM NaCl, 0.1 mg/ml BSA), Buffer O (50 mM Tris-HCl (pH 7.5 at 37° C.), 10 mM MgCl2, 100 mM NaCl, 0.1 mg/ml BSA), Buffer R (10 mM Tris-HCl (pH 8.5 at 37° C.), 10 mM MgCl2, 100 mM KCl, 0.1 mg/ml BSA), and Buffer Tango™ (33 mM Tris-acetate (pH 7.9 at 37° C.), 10 mM magnesium acetate, 66 mM potassium acetate, 0.1 mg/ml BSA). Examples from Promega include Buffer A (6 mM Tris-HCl (pH 7.5 at 37° C.), 6 mM MgCl2, 6 mM NaCl, 1 mM DTT), Buffer B (6 mM Tris-HCl (pH 7.5 at 37° C.), 6 mM MgCl2, 50 mM NaCl, 1 mM DTT), Buffer C (10 mM Tris-HCl (pH 7.9 at 37° C.), 10 mM MgCl2, 50 mM NaCl, 1 mM DTT), Buffer D (6 mM Tris-HCl (pH 7.9 at 37° C.), 6 mM MgCl2, 150 mM NaCl, 1 mM DTT), Buffer E (6 mM Tris-HCl (pH 7.5 at 37° C.), 6 mM MgCl2, 100 mM NaCl, 1 mM DTT), Buffer F (10 mM Tris-HCl (pH 8.5 at 37° C.), 10 mM MgCl2, 100 mM NaCl, 1 mM DTT), Buffer G (50 mM Tris-HCl (pH 8.2 at 37° C.), 5 mM MgCl2), Buffer H (90 mM Tris-HCl (pH 7.5 at 37° C.), 10 mM MgCl2, 50 mM NaCl), Buffer J (10 mM Tris-HCl (pH 7.5 at 37° C.), 7 mM MgCl2, 50 mM KCl, 1 mM DTT), Buffer K (10 mM Tris-HCl (pH 7.4 at 37° C.), 10 mM MgCl2, 150 mM KCl), Buffer L (10 mM Tris-HCl (pH 9.0 at 37° C.), 3 mM MgCl2, 100 mM NaCl), and MULTI-CORE™ Buffer (25 mM Tris-Acetate, pH 7.5 (at 37° C.), 100 mM potassium acetate, 10 mM magnesium acetate, 1 mM DTT).
In an embodiment, the composition comprises an aqueous solution/buffer comprising:
In a further embodiment, the composition comprises an aqueous solution/buffer comprising:
In a further embodiment, the composition comprises an aqueous solution/buffer comprising:
In a further embodiment, the composition comprises an aqueous solution/buffer comprising:
The present disclosure also provides a mixture comprising the above-described isolated endonuclease or composition and a single-stranded nucleic acid molecule (e.g., single-stranded DNA).
The present disclosure also provides a method for cleaving a single-stranded nucleic acid molecule comprising contacting the single-stranded nucleic acid molecule with the isolated endonuclease or composition defined herein under conditions suitable for cleavage of the single-stranded nucleic acid molecule by the isolated endonuclease.
The results presented in the Examples below show that the endonucleases according to the present disclosure recognize specific nucleotide sequences within the single-stranded nucleic acid molecules. The recognition motif contains a ≈30 nt repeated sequence (NTS) that is predicted to form a stem-loop secondary structure. The cleavage of the single-stranded nucleic acid molecules occurs just outside the NTS.
In an embodiment, the single-stranded nucleic acid molecule cleaved by the endonuclease comprises a nucleotide sequence having at least 50% sequence identity with the sequence:
In an embodiment, the single-stranded nucleic acid molecule cleaved by the endonuclease comprises a nucleotide sequence having at least 50% sequence identity with the sequence:
In an embodiment, the single-stranded nucleic acid molecule comprises the following nucleotide sequence:
The identification of nucleotides in the present disclosure is according to the standard 1-letter nomenclature from the International Union of Pure and Applied Chemistry (IUPAC):
In an embodiment, the single-stranded nucleic acid molecule comprises the following nucleotide sequence:
In an embodiment, the single-stranded nucleic acid molecule comprises a sequence having at least 50, 60, 70, 80, 90, 95 or 100% identity with one of the following nucleotide sequences:
As shown in the examples below, the genes encoding the endonucleases according to the present disclosure are typically surrounded by one or several repeats of their own recognition sequences in the genome of the bacteria. Thus, the skilled person would be able to easily identify the recognition sequence of any endonuclease according to the present disclosure by identifying repeating nucleotide sequences/motifs located near (i.e., just before/upstream and/or after/downstream) the gene encoding the endonuclease. Such recognition sequence is expected to have some level of sequence identity with the recognition sequences disclosed herein.
The method according to the present disclosure comprises incubating the single-stranded nucleic acid molecule with the composition defined herein for a period of time and under conditions suitable for cleavage of the single-stranded nucleic acid molecule by the endonuclease.
In an embodiment, the period of time is at least 5 minutes. In further embodiments, the period of time is at least 10 or 15 minutes. In yet further embodiments, the period of time is at least 20, 30 or 45 minutes. In embodiments, the period of time is from about 15 minutes to about 2 hours, from about 20 minutes to about 90 minutes, from about 30 minutes to about 60 minutes, or from about 45 to about 60 minutes.
In an embodiment, the conditions for incubation comprise a temperature of about 10, 15 or 20° C. to about 60, 55 or 50° C. In further embodiments, the conditions for incubation comprise a temperature of about 25 or 30° C. to about 40 or 45° C., such as a temperature of about 35 to about 40° C., e.g., about 36, 37 or 38° C.
In an embodiment, the conditions for incubation comprise the presence of a suitable amount of a metal, such as a divalent metal. Examples of divalent metals include magnesium, manganese, cadmium, calcium, cobalt, nickel, zinc, iron and copper. In a further embodiment, the divalent metal is magnesium, manganese or nickel, preferably magnesium. If the divalent metal is not present in the initial composition comprising the endonuclease, a suitable amount of the divalent metal is added to the reaction mixture prior to and/or during the incubation period. The divalent metal may be in the form of a salt, such as the salts listed above. In an embodiment, the divalent metal is magnesium and is in the form of magnesium chloride (MgCl2). In an embodiment, the concentration of metal salt (e.g., MgCl2) present during the incubation period is about 1 mM to about 100 mM, for example about 1 mM to about 50 mM, about 5 mM to about 20 mM, about 5 to about 15 mM, or about 10 mM.
In an embodiment, the conditions for incubation comprise a pH of about 5 to about 10. In further embodiments, the conditions for incubation comprise a pH of about 6 to about 9, of about 6.5 to about 9, of about 7 to about 9, of about 7.5 to about 8.5, or of about 7.6 to about 8.2. In an embodiment, the conditions for incubation comprise a pH of about 7.9 or 8.0.
The amount of the endonuclease relative to that of the single-stranded nucleic acid molecule may be adjusted to obtain a suitable cleavage efficiency. In an embodiment, the [concentration of endonuclease]/[concentration of single-stranded nucleic acid molecule] ratio is at least 0.00005, 0.0001, 0.001, 0.01, 0.05, 0.1 or 0.5. In an embodiment, the [concentration of endonuclease]/[concentration of single-stranded nucleic acid molecule] ratio is from 0.01 to 100, from 0.05 to 50 or from 0.1 to 10.
In another aspect, the present disclosure provides a method for rendering a single-stranded nucleic acid susceptible to cleavage by the endonuclease described herein, the method comprising incorporating the nucleotide sequence defined above (recognition motif) into the single-stranded nucleic acid. The incorporation of the nucleotide sequence (recognition motif) may be achieved by adding the nucleotide sequence defined above (or a portion thereof) at the 5′-end, 3′-end or within the single-stranded nucleic acid, and/or by introducing one or more mutations (e.g., substitutions) within the sequence of the single-stranded nucleic acid to obtain the desired nucleotide sequence (recognition motif). Methods to modify nucleic acids are well known in the art and include, for example, cassette mutagenesis, PCR site-directed mutagenesis and genome-editing technologies using nucleases such as zinc finger nucleases (ZPNs) (Gommans et al., J. Mol Biol, 354 (3): 507-519 (2005)), transcription activator-like effector nucleases (TALENs) (Zhang et al., Nature Biotechnol, 29:149-153 (2011)), the CRISPR/Cas system (Cheng et al., Cell Res., 23:1163-71] (2013)), and engineered meganucleases (Riviere et al., Gene Ther., 21 (5): 529-32 (2014)).
The present disclosure also provides a method for expressing the endonuclease defined herein in a cell, the method comprising introducing a nucleic acid encoding the endonuclease into the cell. The cell may be a procaryotic or eucaryotic cell. The nucleic acid may be an mRNA or cDNA molecule, naked or incorporated into a vector or plasmid, and it may be incorporated into the cell using any suitable methods for introducing nucleic acids into a cell (e.g., transfection, transformation, etc.).
In an embodiment, the cell comprises a single-stranded nucleic acid that is cleaved by the endonuclease.
In another aspect, the present disclosure provides a kit or commercial package comprising the endonuclease or composition described herein. In an embodiment, the kit or package further comprises instructions setting forth a method for cleaving a single-stranded nucleic acid with the endonuclease, such as the method described herein. The kit or package may further comprise various components such as solutions or buffers (e.g., a reaction buffer) such as those described herein, containers, vials, etc.
The present disclosure is illustrated in further details by the following non-limiting examples.
Gene expression and purification (
GeneID: 12395323 Altname: NMV_RS00225 Accession_number: WP_002216166.1 Prot_locus: CAX49033. Embl_accession: FM999788.1. The SsnA gene is known as NMB0047 or NMB_RS00250 (CDS WP_002216166.1) in the reference strain N. meningitidis MC58, and NMA0292 or NMA_RS01540 in N. meningitidis Z2491 (CDS WP_002246543.1).
For SsnA homologs from different species, NEIELOOT_01219 (EFE49965.1) was taken from Neisseria elongata subsp. glycolytica, WP_011213498 was taken from Legionella pneumophila subsp. pneumophila str Paris and WP_011271370 was taken from Rickettria felis URRWXCal2. Expression and purification of 6×His-recombinant proteins were done by affinity chromatography using a nickel resin. Expression and purification of GST-recombinant proteins were done by affinity chromatography using a glutathione resin (
Electrophoretic mobility shift assay (EMSA). Gel shift assays, or EMSA, were performed by diluting the proteins in Diluent A (NEB) to the indicated working concentrations. 5′carboxyfluorescin-tagged oligos corresponding to genomic regions of N. meningitidis MC58 were synthesized from Sigma. When needed, complementary oligonucleotides were annealed by mixing equimolar amounts in annealing buffer (10 mM Tris-HCl PH8, 50 mM NaCl, 1 mM EDTA), incubating them 5 minutes at 95° C. and letting them slowly cool down. For the gel-shift assays, proteins were mixed with the fluorescent oligonucleotides in a reaction buffer containing 50 mM NaCl, 10 mM Tris-HCl, 100 μg/ml BSA, pH7.9. The mixes were incubated at 37° C. for 30 minutes before adding native loading dye. Samples were resolved on native 10% TBE-acrylamide gels and imaged with a Typhoon FLA9500 scanner. For branched DNA binding assays, a similar approach was used but the gel was stained with GelStain (Biotium) and imaged on a GelDoc (BioRad) since the oligonucleotides were not fluorescently labelled.
Nuclease assays. Nuclease assays were performed similarly to gel-shift assays, with the addition of 10 mM MgCl2 in the reaction buffer. Reactions were stopped by adding formamide loading dye and incubating 3 minutes at 95° C. Samples were resolved on denaturing 17.5% TBE-Urea (8M) acrylamide gels, and imaged with a Typhoon FLA9500 scanner. The sequences of the DNA constructs used in the studies described herein are depicted in the table below.
pneumophila)
The NMV0044 gene from Neisseria meningitidis 8013 2C4.3 was cloned in pET15-MHL, allowing its expression with a 6×His-tag in N-terminal. Purification was done by affinity chromatography with a nickel resin. The resulting protein was diluted in reaction buffer and used directly for in vitro assays to determine its enzymatic activity.
SsnA possesses a single functional domain, belonging to the GIY-YIG nuclease superfamily. Its nuclease activity was therefore tested on different nucleic acids (
Removing magnesium from the reaction mix or adding the metal chelator EDTA completely abolishes the nuclease activity of SsnA (
The glutamic acid residue in position 64 of SsnA is well conserved within the GIY-YIG superfamily, where it often corresponds to a metal cofactor (e.g., magnesium) binding site (
The 100 nt ssDNA that is specifically bound and cleaved by SsnA contains a ≈30 nt repeated sequence (named NTS) frequently found in the Neisseria genomes and predicted to form a stem-loop (hairpin) secondary structure. Reducing the ssDNA length to 37 nt did not significantly affect the binding activity nor the nuclease activity of SsnA (
To assess whether SsnA could bind to and cleave all types of nucleic acids, its activity on both RNA and ssDNA with uracils instead of thymines was also tested (
To confirm that the enzymatic activity of SsnA is specific to the NTS, the assays were repeated on ssDNA from different regions of the Neisseria genome which harbor the full-length NTS with different flanking sequences (
The specific cutting site of SsnA on ssDNA was determined precisely by running the cleaved product from a 75 nt ssDNA next to fluorescent oligonucleotides of increasing length (
Several proteins from the GIY-YIG superfamily interact with branched DNA to accomplish various functions. Since branched DNA have dsDNA and ssDNA regions just like the hairpin formed by the NTS repeat, it was tested whether SsnA could bind different DNA constructs. No binding activity was detected against Holliday junctions, D-loops, forks and pseudo-Y DNA in absence of the recognition sequence (
Several parameters of SsnA's nuclease activity were determined using the 100 nt ssDNA containing its recognition sequence (
Tens of thousands of hypothetical proteins throughout the bacterial domain contain a single GIY-YIG domain covering most of their sequence. Apart from the YhbQ nuclease which only shares about 22% identity with SsnA, none of these single-domain GIY-YIG proteins have been characterized to our knowledge. Since SsnA exhibits a unique enzymatic activity, a search for potential homologs was performed. Using a cut-off of 50% identity and a strict size threshold, thousands of predicted homologs were found in bacteria from all orders (
In N. meningitidis and closely related species such as N. elongata, the gene encoding SsnA is surrounded by several repeats of its own recognition sequence. Although the enzyme cannot cut genomic DNA in a double-stranded form, SsnA might also act as a mobile genetic element, similarly to transposases. It is shown here that the genes encoding SsnA homologs in unrelated species are located near highly similar sequences, which could be an indication of their nuclease specificity (
To truly define the Ssn proteins as a novel family of single-stranded endonucleases, SsnA homologs from three unrelated bacterial species, Neisseria elongata, Rickettsia felis and Legionella pneumophila (sequence alignment in
For each homolog, a DNA sequence similar to the NTS repeat found around the N. meningitidis SsnA gene was identified in close vicinity of their respective genes (
Therefore, it may be concluded that Ssn proteins can indeed be grouped as a novel family of enzymes, more specifically specific single-stranded endonucleases (Ssn) with a potentially wide array of unique specificities.
Mutants of N. meningitidis 8013 2C4.3 were generated; a knock-out strain (KO) in which the SsnA gene was deleted, and a complemented strain (Compl) in which the gene is overexpressed. The uptake and homologous recombination of DNA by natural transformation was then compared between these strains. Briefly, 200 ng of linearized plasmid DNA is incubated for 2 hours with bacterial suspensions of OD600=1. The transformations mixes are then serially diluted, and the appropriate dilutions are plated on non-selective media (GCB agar) and selective media containing either 5 μg/ml chloramphenicol (Cm) or 75 μg/ml spectinomycin (Sp). Colonies are counted after an overnight incubation at 37 C with 5% CO2, and the rate of transformation is determined, corresponding to the number of resistant CFUs divided by the number of total CFUs.
Two plasmids were assayed, each containing an antibiotic resistance gene (chloramphenicol or spectinomycin) flanked by sequences homologous to the N. meningitidis genome, which allow integration to the host genome by double recombination. One plasmid did not contain the recognition sequence of SsnA, while the other one contains several repeats of it in the homologous regions.
Neisseria species are naturally competent, meaning they can readily update DNA from their environment by transformation and integrate it into their own genome if there is sufficient homology. Of note, only one strand of DNA (ssDNA) is imported in the cytoplasm of the bacteria during natural transformation. When transforming N. meningitidis mutant strains with a plasmid that does not contain SsnA's recognition sequence, a slight decrease of transformation efficiency was observed in the SsnA KO strain (
Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”.
The present application claims the benefit of U.S. provisional patent application Ser. No. 63/263,896, filed on Nov. 11, 2021, which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CA2022/051668 | 11/11/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63263896 | Nov 2021 | US |
