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WO2001009363A1 - Enzymes tn5 transposases mutantes et procede d'utilisation de ces enzymes - Google Patents

Enzymes tn5 transposases mutantes et procede d'utilisation de ces enzymes Download PDF

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WO2001009363A1
WO2001009363A1 PCT/US2000/021052 US0021052W WO0109363A1 WO 2001009363 A1 WO2001009363 A1 WO 2001009363A1 US 0021052 W US0021052 W US 0021052W WO 0109363 A1 WO0109363 A1 WO 0109363A1
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transposase
mutant
amino acid
tnp
wild
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PCT/US2000/021052
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William S. Reznikoff
Todd A. Naumann
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Wisconsin Alumni Research Foundation
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Priority to CA002380850A priority Critical patent/CA2380850A1/fr
Priority to PL00353241A priority patent/PL353241A1/xx
Priority to JP2001513619A priority patent/JP2003505104A/ja
Priority to AU63967/00A priority patent/AU775043B2/en
Priority to EP00950940A priority patent/EP1198583A1/fr
Publication of WO2001009363A1 publication Critical patent/WO2001009363A1/fr

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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • 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]

Definitions

  • Tn5 Bacterial transposons such as Tn5 evolved within the cell by maintaining a low mobility level. While necessary for the transposon to survive, the low mobility level has inhibited the ability of researchers to detail the molecular transposition process and to exploit the transposition process for use, e.g., in the development of new diagnostic and therapeutic resources.
  • Tn5 is a conservative "cut and paste" transposon of the IS4 family (Rezsohazy,R., HalletB., Delcour,J., and Mahillon,J, "The IS4 family of insertion sequences: evidence for a conserved transposase motif," Mol Microbiol.
  • Tnp 53kD transposase protein
  • the Tnp protein facilitates movement of the entire element by binding initially to each of two 19bp specific binding sequences termed outside end (OE; SEQ ID NO:3), followed by formation of a nucleoprotein structure termed a synapse, blunt ended cleavage of each end, association with a target DNA, and then strand transfer (Reznikoff,W.S., Bhasin,A., Davies,D.R., Goryshin,I.Y., Mahnke,L.A., Naumann,T., Rayment,!., Stier-White,M., and Twining,S.S., "Tn5: A molecular window on transposition.” Biochem. Biophvs. Res. Commun.
  • Tn5 transposase can also promote movement of a single insertion sequence by using a combination of OE and inside end (IE; SEQ ID NO:4) sequences.
  • the IE is also 19bp long and is identical to OE at 12 of 19 positions (Fig. 1).
  • Tn5 transposase exhibits a marked preference for OE in E. coli.
  • Transposase recognition and binding to IE is inhibited in E.
  • Tnp EK/LP double mutant hyperactive form of transposase
  • the Tnp EK/LP protein differs from wild-type Tn5 Tnp at position 54 (Glu to Lys mutation) and at position 372 (Leu to Pro mutation), in addition to a non-essential but advantageous change at position 56 that prevents production of a so-called inhibitor protein.
  • the modified hyperactive Tnp protein retains the dramatic preference for OE (or OE-like) termini of wild-type Tn5 transposase.
  • Tnp EK/LP has clarified many aspects of Tn5 transposition that were not previously adequately addressable in vivo.
  • In vitro polynucleotide transposition is a powerful tool for introducing random or targeted mutations into a genome.
  • Useful in vitro transposition systems based upon the Tn5 transposon are disclosed in US Patent No. 5,925,545 and International Publication No. WO 00/17343, both of which are incorporated herein by reference in their entirety as if set forth herein.
  • a Tnp protein having an ability to discriminate between IE and OE and having a preference for binding IE is desired to permit directed nucleic acid transposition and to facilitate more complex transposition and genetic engineering strategies of the type disclosed in the above-mentioned patent and application than are available using a Tnp having a single specificity for OE.
  • a Tnp having an enhanced preference for IE 1 ⁇ is also desired because methylation of DNA in common dam+ bacterial hosts inhibits binding of existing Tn5 transposases and reduces the ability of existing transposases to facilitate movement of IE-defined transposons.
  • transposase protein modified relative to wild-type Tn5 Tnp as disclosed herein preferentially promotes transposition of a target sequence flanked with wild-type Tn5 transposon inside ends (IE) rather than outside ends (OE) without regard to whether the IE sequences are methylated.
  • a transposase modified relative to wild-type Tn5 Tnp as disclosed herein has a preference for IE over OE and is hyperactive with regard to transposition frequency.
  • the present invention is also summarized in that a transposase modified relative to wild-type Tn5 Tnp as disclosed herein has a preference for IE over OE and catalyzes transposition at a high level even when the IE sequences are methylated.
  • wild-type Tn5 transposase does not efficiently recognize methylated IE sequences.
  • a transposase according to the invention includes a mutation relative to wild type Tn5 transposase that either (1) alters binding of the transposase to the DNA termini or (2) enhances transposition or (3) both.
  • the mutation can be end-sequence-specific (as in the exemplified embodiments that alter DNA binding) or non-specific (as in the exemplified embodiment that enhances transposition.
  • a transposase according to the invention has (1) a greater preference for IE than OE and (2) differs from wild-type Tnp in at least one an amino acid selected from the group consisting of amino acid 58, amino acid 344 and amino acid 372.
  • a transposase according to the invention differs from wild-type Tnp in that it contains at least one of a mutation from glutamic acid to valine at amino acid 58, a mutation from glutamic acid to lysine at amino acid 344, and a mutation from leucine to glutamic acid at amino acid 372.
  • the transposase of the invention can also exhibit a greater preference for IE by reducing the preference for OE.
  • a mutation at amino acid position 8 relative to wild-type transposase can reduce the preference of a transposase for OE, and thereby increase the apparent preference for IE.
  • a mutation from arginine to cysteine can accomplish this modification.
  • a transposase protein of the invention can promote more transposition of an IE- flanked target sequence in vivo or in vitro than wild-type Tn5 transposase does.
  • a suitable method for determining transposase enzyme activity in vitro is disclosed herein and in US Patent No. 5,925,545, incorporated herein by reference in its entirety.
  • a suitable method for determining transposase activity in vivo is disclosed herein.
  • the modified Tn5 Tnp of the present invention differs from wild-type Tn5 Tnp by virtue of at least one a change to an amino acid position, where the change is selected from the group consisting of (1) a change at amino acid position 58 that reduces or eliminates a negative interaction between the Tnp and a methylated DNA residue and (2) a change at amino acid position 344 that alters DNA binding.
  • the modified Tnp's of the invention can also include a change at position 56 (such as a Met to Ala change) that prevents production of the so-called inhibitor protein that interferes with transposition.
  • the mutant Tn5 transposase proteins can contain mutations in addition to those noted above.
  • the invention is further summarized in that the enzymes disclosed herein facilitate a simple, in vitro system and method for introducing any transposable element from a donor DNA into a target DNA when the transposable element DNA is flanked on either side by IE termini inverted relative to one another. Few other requirements on either the donor DNA or the target DNA are envisioned. It is thought that Tn5 has few, if any, preferences for insertion sites, so it is possible to use the system to introduce desired sequences at random into target DNA. Therefore, it is believed that this system and method, employing the modified transposase described herein and a simple donor DNA, is broadly applicable to introduce changes into any target DNA, without regard to its nucleotide sequence.
  • Fig. 1 depicts the structure of Tn5 transposase and the nucleotide sequences of Tn5 outside ends (OE), inside ends (IE), methylated inside ends (IE 1 ⁇ ), and modified IE (IE12A).
  • Fig. 2 is a schematic depiction of the molecular basis for a papillation assay for observing transposition in vivo.
  • Fig. 3 A depicts the locations of mutations observed in four successive rounds (A, B, C, D) of mutagenesis/recombination.
  • Fig. 3B depicts the in vivo transposition profile of each of the mutants of Fig. 3 A.
  • Fig. 4 depicts the relative preference of mutant transposases obtained in successive rounds of mutagenesis/recombination for OE and IE in a dam- strain of E. coli.
  • Fig. 5 A depicts a plasmid suitable for use in an in vitro transposition method.
  • Fig. 5B depicts the transposition products obtained using a mutant transposase of the invention to catalyze transposition in vitro.
  • Fig. 5C further characterizes the products of lane 2 of Fig. 5B.
  • mutant transposase proteins that differ from wild type Tn5 transposase in that the mutant proteins show a preference in a transposition system for inside ends (IE), and in some cases, methylated inside ends (IE 1 ⁇ ), rather than for outside ends (OE), which are unmethylated because they lack a methylation site.
  • IE inside ends
  • OE outside ends
  • the end preference of a mutant transposase can characterized either (1) by the in vivo transposition frequency observed when it is used in a system in which the target polynucleotide is flanked with either OE and IE 1 ⁇ termini, or (2) by the ratio of the in vivo transposition frequencies observed when it is used in a pair of systems in which the target polynucleotide is flanked with OE and IE 1 ⁇ termini, respectively.
  • applicants have exemplied a number of transposase proteins differing from wild-type Tn5 Tnp at one, four, five, and seven mutations, one can reasonably predict from their analysis the effects of particular individual mutations.
  • EXAMPLE Overview A number of related methods were used to obtain the family of mutants disclosed herein. In a first method, the applicants obtained mutants that restored in vivo transposition activity to a mutated end binding sequence that is not recognized as a substrate by wild type transposase. In a second method, the applicants introduced directed mutations into certain products of the first method to determine the preferred structure of a mutant transposase according to the invention.
  • the mutant IE end binding sequence contains an adenine in place of thymine at position 12 ("IE12A"; SEQ ID NO:5).
  • IE12A adenine in place of thymine at position 12
  • the thymine-to-adenine change in IE12A destroys one of the two methylation sites of wild-type IE.
  • the mutated genes can be cloned into plasmids which can be selected for increased activity in vivo. Clones having desirable phenotypes are then used as substrates for subsequent rounds of mutagenesis/recombination and selection for a further improved phenotype.
  • the sPCR method can be used in conjunction with a screen (instead of a selection) in which a modest number of colonies ( ⁇ 10 4 ) are analyzed per round (Crameri, A., Whitehorn, E.A., and Stemmer, W.P., "Improved green fluorescent protein by molecular evolution using DNA shuffling," Nat. Biotechnol. 14:315-319 (1996), and Zhang, J.H., Dawes, G., and Stemmer, W.P., "Directed evolution of a fucosidase from a galactosidase by DNA shuffling and screening," Proc. Natl. Acad. Sci. U.S.A.
  • a papillation assay described in the Example that follows was used as a screen for transposase mutants that restore transposition activity to polynucleotides flanked with IE 12 A.
  • the papillation assay is a modification of the assay described by Krebs, M.P., and Reznikoff, W.S., "Use of a Tn5 derivative that creates lacZ translational fusions to obtain a transposition mutant," Gene 63:277-85 (1988), incorporated by reference in its entirety as if set forth herein.
  • mutant transposases identified using the first method to catalyze in vivo transposition of polynucleotides flanked either with wild- type OE or with wild-type IE in a dam- strain (i.e., the nucleic acid was unmethylated).
  • Tnp sC7 a mutant transposase that retained near-wild-type activity with OE-flanked polynucleotides but which had very high activity with IE-flanked polynucleotides and even higher activity with IE-flanked polynucleotides when tested in a dam+ strain (i.e., the nucleic acid was methylated, subsequently "IE 1 ⁇ ").
  • Tnp sC7 contains seven mutations relative to wild-type transposase.
  • Tnp sC7v2.0 a related mutant transposase having only four of the seven mutations of Tnp sC7 exhibited a still higher IE ⁇ OE activity ratio.
  • Both Tnp sC7 and Tnp sC7v2.0 contain a mutation that inhibits OE related activity (R8C), two mutations that specifically increase IE 1 ⁇ related activity (E58V, E344K), and a mutation that increases transposition of polynucleotides flanked by either IE 1 ⁇ or OE (L372Q).
  • a pair of inverted IE12 A termini flank a polynucleotide that contains a lacZ gene but which lacks both a promoter and a translational start site.
  • a second plasmid pRZ9904 (IE12A/IE12A)
  • Plasmids having these attributes can readily be constructed by a skilled artisan. Materials and methods are detailed below. Five random mutants that suppressed the end sequence mutation and yielded papillae after an initial cycle of mutagenesis/recombination. Equal amounts of plasmid encoding each of these mutant transposases were then used as the initial substrates for a second round of mutagenesis/recombination.
  • the mutated transposase genes were cloned into vector DNA and screened a second time for transposition activity with IE12A-defined polynucleotides via the papillation screen. From this second round, a total of 6 active mutants were isolated. A third round of mutagenesis/recombination was then performed followed by screening for activity with the mutant end sequence. This time hundreds of colonies were positive for papillation. Of these, 7 were clearly more active than the others and were isolated to serve as a template for a fourth round. In the fourth round of screening there were hundreds of transposing colonies visualized during the screening process. None of these, however, were as active as the most active mutant from the third round of mutagenesis/recombination (Tnp sC6).
  • Figs. 3 A and 3B depict the most active isolate from each round.
  • Fig. 3 A depicts the mutations relative to wild-type Tnp of Tnp sA5 (best first round papillator), sB2 (best second round papillator), sC6 (best third round papillator), and sD5 (best fourth round papillator).
  • Fig. 3B depicts the transposition activities of the four isolates in vivo in the papillation assay. Tnp WT was also tested but failed to promote a single detectable transposition event.
  • the mutation Q81H is the only mutation that distinguishes the most active mutant, sC6, isolated in the third round, from the noticeably less active mutant sB2, isolated in the second round.
  • a second isolate from the third round, Tnp sC7 is similar to the fourth round isolate sD5, except that it has two additional mutations (D217A and E344K).
  • Tnp sC7's activity with IE12A defined transposons is similar to that of the fourth round isolate sD5 (data not shown).
  • the mutant Tnp's can promote transposition of OE- and IE-defined transposons
  • the mutant Tnp's described above were of initial interest because they could restore transposition activity to transposons flanked with IE12A termini that are inactive in the presence of wild-type Tnp. Although the transposase mutants functioned increasingly well with these transposons, the transposition rate did not approach the level of activity that is required in vitro. In fact, the in vivo activity of Tnp sC6 with IE12A ends only restored activity to a level similar to that of Tnp WT with transposons defined by OE (data not shown).
  • Fig. 4 depicts the in vivo transposition activity of 25 mutant Tnps with IE and OE defined transposons in the mating-out assay in a dam- strain, normalized to the activity of wild-type Tnp (6.5X10 "5 normalized to 1).
  • Tnp WT shows generally equivalent activity levels whether the substrate polynucleotide is flanked with IE or with OE.
  • many of the mutants exhibited higher activity with IE than with OE. This is not surprising, since the mutants were obtained in a screen using IE 12 A, which differs from IE at only 1 nucleotide. In contrast, OE differs from IE12A at 6 nucleotides.
  • Tnp sC7 displayed a very interesting phenotype. It is markedly hyperactive with IE transposons while exhibiting little change in the frequency of OE transposon movement.
  • the ability to discriminate between IE and OE is important because it facilitates multi-part transpositions that separately employ IE and OE ends, where a reaction can be directed one way or another by providing a transposase that prefers either IE or OE.
  • a preference for IE over OE of greater than about 5-fold may be suitable, though a preference of greater than about 10- fold for IE is more preferred.
  • a preference of greater than about 20-fold is still more preferred.
  • Fig. 4 demonstrates that a skilled artisan can obtain such mutant transposases using the methods disclosed herein.
  • mutants sBl , sC6, sC7, sDl and sD3 are examples of such mutants.
  • Tnp sC7 is not only not inhibited for transposition activity by methylated IE (which reduces Tnp WT levels by -102) but actually prefers IE 1 ⁇ transposons to those flanked by IE, as is shown in the mating out results of Table 1 which indicate that transposition frequency with OE is reduced in a dam+ strain for both Tnp sC7 and Tnp WT. Since binding of Tnp to OE is not affected by dam methylation, this difference merely reflects the difference in transposition activity between dam+ strains and dam- strains.
  • mutants of the second class wild-type transposase was engineered to contain exactly one of the 7 mutations from sC7.
  • the set of so-called "plus one" mutant transposases included all possible mutants having only 1 of the 7 mutations. These mutant proteins were all assayed for in vivo transposition activity with IE 1 ⁇ and OE by mating out assays. The results of the analysis are shown in Table 2.
  • a. 'Minus ones' contain all mutations present in Tnp sC7 except at indicated position, e.g. R8C 'minus one' contains the wild-type argenine at position 8.
  • b. 'Plus ones' are Tnp WT except that the indicated amino acid is mutated to the residue present in Tnp sC7. E.g. R8C 'plus one' contains a cysteine at amino acid 8.
  • the mutation E58V has the most profound effect of all the mutations on the activity of Tnp sC7.
  • This mutation in the wild-type background increases IE 1 ⁇ related transposition by 40,000 fold while removal of the mutation from Tnp sC7 (E58V 'minus one') drops the total activity by more than 1 ,000 fold.
  • the mutation has comparatively little effect on OE related activity.
  • E344K exhibits a similar, though much weaker, sequence specific effect on activity.
  • IE 1 ⁇ related activity decreases by 5 fold while OE related activity is stimulated about 5 fold.
  • This result is mirrored in the 'plus ones' data as E344K in the wild type background stimulates g ME re ⁇ a t e d activity 4 fold and decreases OE related activity about 5 fold.
  • Non-sequence-specific mutations The mutation L372Q strongly stimulates Tnp sC7 activity with IE 1 ⁇ . When removed from sC7 both IE 1 ⁇ and OE related activity are reduced to the same degree. When added to wild-type transposase, its 'plus one' phenotype stimulates activity with both substrates.
  • the mutation R8C was the most interesting of the remaining four mutations. When the mutation is added to wild-type transposase, OE related transposition was reduced nearly 10 fold. When the mutation was removed from sC7 OE related activity increased approximately 2 fold. In a methylating host, its removal from sC7 had little effect on IE 1 ⁇ related activity while by itself it decreased IE 1 ⁇ related activity to below detectable levels. None of the remaining three (A157T, T171S, D217A) mutations has much effect on either IE 1 ⁇ or OE related activity when removed from sC7.
  • Tnp sC7 is less than the sum of its individual mutations.
  • the Tnp E58V (E58V 'plus one') mutant has an activity increase of 4X10 4 over Tnp WT alone.
  • the composite mutant with E58V removed (E58V 'minus one') has an increase of 1.8X10 2 over Tnp WT. Additivity would then predict that Tnp sC7 would have an activity increase of:
  • Tnp sC7v2.0 effectively transposes IE ⁇ defined transposons in vitro
  • Tnp sC7v2.0 The ability of Tnp sC7v2.0 to promote transposition of IE 1 ⁇ defined transposons in vitro was tested under the same conditions developed for movement of OE defined transposons by Tnp EK/LP (Goryshin, I.Y., and Reznikoff, W.S., "Tn5 in vitro transposition," J. Biol. Chem. 273:7367-7374 (1998), incorporated by reference as if set forth herein in its entirety).
  • Substrate plasmid pGT4 a high-copy number pUC 19-based vector in which inverted IE 1 ⁇ end sequences flank a kanamycin resistance gene, was purified as a supercoiled monomer (see Materials and Methods).
  • Tnp sC7v2.0 was produced and purified by cloning the nucleotide sequence in an expression vector, expressing the protein in a host cell and isolating the protein from an extract from the host cell, all using standard methods known to a skilled artisan.
  • Fig. 5C is a reproduction of lane 2 of Fig. 5B.
  • Band 1 is the excised transposon. It is an intermediate that has undergone double ended break from the plasmid but has not undergone strand transfer.
  • Band 2 is the donor backbone DNA that is released upon double-ended excision of the transposon. These bands have the same molecular weight as the products of the PvuII digest shown in lane 3 of Fig. 5B.
  • Band 3 represents substrate plasmid that has undergone cleavage at one transposon end.
  • Bands 4 and 5 are two different types of strand transfer products.
  • Band 4 is the result of a transposon inserting intermolecularly into an unreacted plasmid. This results in a relaxed circular DNA that is longer than the original substrate plasmid by the length of the inserted transposon.
  • the bands denoted as 5 are the result of intramolecular inversion events.
  • These transposition products are the size of the transposon but circularized. These circularized transposition products can contain differing numbers of nodes and hence migrate in different positions on the gel.
  • the hyperactive Tn5 transposase mutant Tnp sC7v2.0 which increases transposition of flanked transposons by 7.4X10 5 times, includes mutations at amino acids 8, 58, 344, and 372. Analysis of Tnp sC7 revealed that the hyperactivity conferred by both the E58V and E344K mutations depends upon the transposon termini sequence.
  • position 7 is not one of the seven that distinguishes IE from OE, it is between both position 4 and the region of nucleotides 10, 11, and 12 that differentiate the two. It is therefore plausible that the activity difference of Tnp E344K with IE and OE can be attributed to context effects of these two regions. We therefore propose that both mutations E58V and E344K are interacting with end sequence DNA and alter transposase function at the level of primary sequence recognition.
  • the mutation R8C which reduces OE related transposition almost 10-fold in the wild-type background (R8C 'plus one') and increases it approximately 2-fold when removed from the sC7 background (R8C 'minus one') is less easy to interpret.
  • the arginine residue is not in an area of DNA contact.
  • this structure only represents a still picture of the complex after cleavage has occurred and it is possible that this region contacts DNA in the initial synapse.
  • the inside end of the Tn5 transposon contains two GATC signal sequences that add four methyl groups into the major groove of each end.
  • E58V a single mutation
  • the co-crystal structure of Tnp EK/LP complexed with pre-cleaved DNA shows that glutamate 58 interacts directly with position 10 of OE in the major groove.
  • This region is in the vicinity of the methyl group that is present on the adenine of the non-transferred strand 0 jg ME -r/kg f act mat a s i n gie amino acid change can result in this extreme change in phenotype leads us to propose that this methyl group alone is responsible for the inhibition of binding of transposase to IE 1 ⁇ .
  • the inhibition of binding by Tnp WT to IE 1 ⁇ is likely caused by an interaction between this methyl group and the negatively charged side chain of glutamate 58. Replacing this residue with a valine can not only remove this unfavorable interaction but also lead to an increase in binding affinity due to hydrophobic packaging between the side chain of the valine residue and this methyl group.
  • Plasmid pGT4 was constructed as a high copy number plasmid containing a kanamycin resistance gene flanked by two inside ends. It is designed so that digestion with PvuII releases the transposon from its pUC vector backbone.
  • the fourteen pRZ9905 (sC7) derivatives and pRZ9905 (sC7 version 2.0) were constructed by swapping restriction fragments between pRZ9905 and pRZ9905 (sC7).
  • the assembly mix contained 0.2mM dNTP's; 2.0mM MgCl; 50mM KC1; lOmM Tris-HCl (pH9.0 at 25C); and 0.1% Triton X-100.
  • the DNA was reassembled by the following thermo cycling program: 94C for 30seconds; 50 cycles of 94C for 20 seconds, 65C for 1 minute, and 72C for 2 minutes; and cooling to 4C.
  • a standard PCR amplification reaction using 5 ⁇ l of the assembly reaction product as a DNA template was performed for each sample (200-600bp and 600-lOOObp) to amplify the transposase gene. This transposase-encoding fragment was digested to completion with Aflll and Bglll and ligated into purified Aflll/Bglll digested vector
  • Ligation products were transformed into electrocompetent JM109 cells that contained plasmid pRZ9904 (IE12A IE12A). After outgrowth the cells were plated on Trp ⁇ XG-PG plates with chloramphenicol and ampicillin selection. The plates were incubated at 32C for 14 days. At this time pRZ9905 plasmid DNA from all colonies that exhibited at least one papillae were isolated and re-transformed into the papillation assay to confirm their papillation plus phenotype. A total of 5 pRZ9905 derivatives (out of 20,000 original colonies screened) were confirmed to be papillation plus. An equal amount of all five plasmids was then used as the substrate for a second round of mutagenesis and screening. This process was repeated for a total of four rounds of screening ( ⁇ 20,000 colonies / round)
  • the IE12A in vivo transposition activity of Tnp WT, Tnp sA5, Tnp sB2, Tnp sC6, and Tnp sD5 were compared by a quantitative papillation assay. Competent cells of strain JM109 harboring plasmid pRZ9904 (IE12A/IE12A) were transformed with the appropriate transposase-encoding version of pRZ9905. After outgrowth, transformed cells were plated on Trp ⁇ XG-PG plates with chloramphenicol and ampicillin. The plates were grown at 32C until colonies began to appear (-18 hours). Individual colonies were then picked with sterile sticks and spotted onto a fresh plate in a 4X4 grid pattern to evenly space all colonies. One plate of 16 colonies was spotted for each protein. Plates were incubated at 32C and quantified for transposition by observing the appearance of papillae at 24-hour intervals. Data are expressed as the average number of papillae present per colony.
  • Mating out assays Mating out assays were performed as described previously (Yin et al., 1988;
  • Bacterial cells with the transposon containing plasmids pFMA52-187 (with either two OEs or two IEs) and the F factor pox-Gen were transformed with the appropriate transposase encoding plasmid pRZ9905.
  • the donor used for the library screening was the strain JCMIOI [ ⁇ lacZX74, raps, dam-3]. All other mating out was performed in E. coli strain RZ212 [ ⁇ (lac-proA,B), ara, str, recA56, srl, thi].
  • the recipient strain used was 14R525[F-nalr]. A total of three assays were performed for each combination of transposase and end sequence. The values reported are the average of these three data points.
  • Substrate plasmid pGT4 was isolated from DH5 ⁇ cells using a qiafilter plasmid mega kit (Qiagen). Supercoiled monomer plasmid was isolated from a 1% agarose gel by use of the qiaquick gel purification kit (Qiagen). Reactions were performed at 37C under conditions determined by Reznikoff and Goryshin (1998). The concentration of pGT4 was 35.5nM. Tnp sC7v2.0 was added to a concentration of 280nM.

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Abstract

L'invention se rapporte à des protéines Tn5 transposases modifiées possédant une préférence pour les extrémités internes plutôt que pour les externes du transposon Tn5. Ces protéines peuvent être utilisées en association à des enzymes transposases qui préfèrent les extrémités externes aux extrémités internes lors de la mise en oeuvre d'un procédé de transposition directe in vitro ou in vivo, spécifique de certaines extrémités.
PCT/US2000/021052 1999-08-02 2000-08-02 Enzymes tn5 transposases mutantes et procede d'utilisation de ces enzymes WO2001009363A1 (fr)

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CA002380850A CA2380850A1 (fr) 1999-08-02 2000-08-02 Enzymes tn5 transposases mutantes et procede d'utilisation de ces enzymes
PL00353241A PL353241A1 (en) 1999-08-02 2000-08-02 Mutant tn5 transposase enzymes and method for their use
JP2001513619A JP2003505104A (ja) 1999-08-02 2000-08-02 変異体ｔｎ５トランスポザーゼ酵素及びその使用方法
AU63967/00A AU775043B2 (en) 1999-08-02 2000-08-02 Mutant Tn5 transposase enzymes and method for their use
EP00950940A EP1198583A1 (fr) 1999-08-02 2000-08-02 Enzymes tn5 transposases mutantes et procede d'utilisation de ces enzymes

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Cited By (55)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002004629A3 (fr) * 2000-07-07 2002-08-29 Maxygen Inc Filiation moléculaire d'éléments transposables
EP1115856B1 (fr) * 1998-09-23 2004-11-17 Wisconsin Alumni Research Foundation Methode permettant d'effectuer des mutations d'insertions
US7338801B2 (en) 2000-12-05 2008-03-04 Wisconsin Alumni Research Foundation Double transposition methods for manipulating nucleic acids
WO2018156519A1 (fr) 2017-02-21 2018-08-30 Illumina Inc. Tagmentation utilisant des transposomes immobilisés avec des lieurs
WO2020047002A1 (fr) 2018-08-28 2020-03-05 10X Genomics, Inc. Procédé de marquage spatial induit par transposase et d'analyse d'adn génomique dans un échantillon biologique
WO2020112604A2 (fr) 2018-11-30 2020-06-04 Illumina, Inc. Analyse d'analytes multiples à l'aide d'un seul dosage
WO2020131626A1 (fr) 2018-12-17 2020-06-25 Illumina, Inc. Procédés et moyens de préparation d'une bibliothèque pour le séquençage
WO2021009494A1 (fr) 2019-07-12 2021-01-21 Illumina Cambridge Limited Préparation de bibliothèque d'acides nucléiques par électrophorèse
US11156603B2 (en) 2010-04-05 2021-10-26 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11162132B2 (en) 2015-04-10 2021-11-02 Spatial Transcriptomics Ab Spatially distinguished, multiplex nucleic acid analysis of biological specimens
US11208684B2 (en) 2010-04-05 2021-12-28 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11286515B2 (en) 2013-06-25 2022-03-29 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US11332790B2 (en) 2019-12-23 2022-05-17 10X Genomics, Inc. Methods for spatial analysis using RNA-templated ligation
US11352659B2 (en) 2011-04-13 2022-06-07 Spatial Transcriptomics Ab Methods of detecting analytes
WO2022164615A1 (fr) 2021-01-29 2022-08-04 10X Genomics, Inc. Procédé de marquage spatial induit par transposase et d'analyse d'adn génomique dans un échantillon biologique
US11408029B2 (en) 2020-06-25 2022-08-09 10X Genomics, Inc. Spatial analysis of DNA methylation
US11407992B2 (en) 2020-06-08 2022-08-09 10X Genomics, Inc. Methods of determining a surgical margin and methods of use thereof
US11434524B2 (en) 2020-06-10 2022-09-06 10X Genomics, Inc. Methods for determining a location of an analyte in a biological sample
US11519033B2 (en) 2018-08-28 2022-12-06 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic DNA in a biological sample
US11535887B2 (en) 2020-04-22 2022-12-27 10X Genomics, Inc. Methods for spatial analysis using targeted RNA depletion
US11560592B2 (en) 2020-05-26 2023-01-24 10X Genomics, Inc. Method for resetting an array
US11608520B2 (en) 2020-05-22 2023-03-21 10X Genomics, Inc. Spatial analysis to detect sequence variants
US11618897B2 (en) 2020-12-21 2023-04-04 10X Genomics, Inc. Methods, compositions, and systems for capturing probes and/or barcodes
US11624086B2 (en) 2020-05-22 2023-04-11 10X Genomics, Inc. Simultaneous spatio-temporal measurement of gene expression and cellular activity
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US11702698B2 (en) 2019-11-08 2023-07-18 10X Genomics, Inc. Enhancing specificity of analyte binding
US11702693B2 (en) 2020-01-21 2023-07-18 10X Genomics, Inc. Methods for printing cells and generating arrays of barcoded cells
US11733238B2 (en) 2010-04-05 2023-08-22 Prognosys Biosciences, Inc. Spatially encoded biological assays
US11732300B2 (en) 2020-02-05 2023-08-22 10X Genomics, Inc. Increasing efficiency of spatial analysis in a biological sample
US11732299B2 (en) 2020-01-21 2023-08-22 10X Genomics, Inc. Spatial assays with perturbed cells
US11753673B2 (en) 2021-09-01 2023-09-12 10X Genomics, Inc. Methods, compositions, and kits for blocking a capture probe on a spatial array
US11761038B1 (en) 2020-07-06 2023-09-19 10X Genomics, Inc. Methods for identifying a location of an RNA in a biological sample
WO2023225519A1 (fr) 2022-05-17 2023-11-23 10X Genomics, Inc. Transposons modifiés, compositions et utilisations de ceux-ci
US11827935B1 (en) 2020-11-19 2023-11-28 10X Genomics, Inc. Methods for spatial analysis using rolling circle amplification and detection probes
US11891654B2 (en) 2020-02-24 2024-02-06 10X Genomics, Inc. Methods of making gene expression libraries
US11898205B2 (en) 2020-02-03 2024-02-13 10X Genomics, Inc. Increasing capture efficiency of spatial assays
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
US11926822B1 (en) 2020-09-23 2024-03-12 10X Genomics, Inc. Three-dimensional spatial analysis
US11933957B1 (en) 2018-12-10 2024-03-19 10X Genomics, Inc. Imaging system hardware
US11965213B2 (en) 2019-05-30 2024-04-23 10X Genomics, Inc. Methods of detecting spatial heterogeneity of a biological sample
US11981960B1 (en) 2020-07-06 2024-05-14 10X Genomics, Inc. Spatial analysis utilizing degradable hydrogels
US11981958B1 (en) 2020-08-20 2024-05-14 10X Genomics, Inc. Methods for spatial analysis using DNA capture
US12031177B1 (en) 2020-06-04 2024-07-09 10X Genomics, Inc. Methods of enhancing spatial resolution of transcripts
USRE50065E1 (en) 2012-10-17 2024-07-30 10X Genomics Sweden Ab Methods and product for optimising localised or spatial detection of gene expression in a tissue sample
US12071655B2 (en) 2021-06-03 2024-08-27 10X Genomics, Inc. Methods, compositions, kits, and systems for enhancing analyte capture for spatial analysis
US12076701B2 (en) 2020-01-31 2024-09-03 10X Genomics, Inc. Capturing oligonucleotides in spatial transcriptomics
US12110541B2 (en) 2020-02-03 2024-10-08 10X Genomics, Inc. Methods for preparing high-resolution spatial arrays
US12117439B2 (en) 2019-12-23 2024-10-15 10X Genomics, Inc. Compositions and methods for using fixed biological samples
US12129516B2 (en) 2020-02-07 2024-10-29 10X Genomics, Inc. Quantitative and automated permeabilization performance evaluation for spatial transcriptomics
US12195790B2 (en) 2021-12-01 2025-01-14 10X Genomics, Inc. Methods for improved in situ detection of nucleic acids and spatial analysis
US12203134B2 (en) 2021-04-14 2025-01-21 10X Genomics, Inc. Methods of measuring mislocalization of an analyte
US12209280B1 (en) 2020-07-06 2025-01-28 10X Genomics, Inc. Methods of identifying abundance and location of an analyte in a biological sample using second strand synthesis
US12275988B2 (en) 2021-11-10 2025-04-15 10X Genomics, Inc. Methods, compositions, and kits for determining the location of an analyte in a biological sample
US12281357B1 (en) 2020-02-14 2025-04-22 10X Genomics, Inc. In situ spatial barcoding
US12297486B2 (en) 2020-01-24 2025-05-13 10X Genomics, Inc. Methods for spatial analysis using proximity ligation

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101698848B (zh) * 2009-08-26 2012-10-03 上海海洋大学 金鱼自主转座子基因gfTol2、该基因编码的转座酶及该基因的用途
CN103602654B (zh) * 2013-11-20 2016-02-24 浙江农林大学 人工改造的高活性Mariner-Like转座酶
SG11201608585RA (en) * 2014-04-15 2016-11-29 Illumina Inc Modified transposases for improved insertion sequence bias and increased dna input tolerance
CN106754811B (zh) * 2016-12-21 2019-05-14 南京诺唯赞生物科技有限公司 一种突变型Tn5转座酶及其制备方法和应用
CN108265040B (zh) * 2017-09-24 2021-02-09 天津强微特生物科技有限公司 一种嗜盐古细菌中的转座酶高活性突变体

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DEVINE S E ET AL: "EFFICIENT INTEGRATION OF ARTIFICIAL TRANSPOSONS INTO PLASMID TARGETS IN VITRO: A USEFUL TOOL FOR DNA MAPPING, SEQUENCING AND GENETIC ANALYSIS", NUCLEIC ACIDS RESEARCH,GB,OXFORD UNIVERSITY PRESS, SURREY, vol. 22, no. 18, 1994, pages 3765 - 3772, XP002072909, ISSN: 0305-1048 *
GORYSHIN I Y ET AL: "Tn5 in vitro transposition", JOURNAL OF BIOLOGICAL CHEMISTRY,US,AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, vol. 273, no. 13, 27 March 1998 (1998-03-27), pages 7367 - 7374, XP002129603, ISSN: 0021-9258 *
KREBS M P ET AL: "USE OF A TRANSPOSON TN5 DERIVATIVE THAT CREATES LACZ TRANSLATIONAL FUSIONS TO OBTAIN A TRANSPOSITION MUTANT", GENE (AMSTERDAM), vol. 63, no. 2, 1988, pages 277 - 285, XP002152830, ISSN: 0378-1119 *
NAUMANN, T.A. ET AL.: "Trans catalysis in Tn5 transposition.", PROC.NAT'L.ACAD.SCI.USA, vol. 97, no. 16, 1 August 2000 (2000-08-01), pages 8944 - 9, XP002152831 *
ZHOU M ET AL: "TN5 TRANSPOSASE MUTANT THAT ALTER DNA BINDING SPECIFICITY", JOURNAL OF MOLECULAR BIOLOGY,GB,LONDON, vol. 271, no. 3, 1997, pages 362 - 373, XP002052643, ISSN: 0022-2836 *

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US7338801B2 (en) 2000-12-05 2008-03-04 Wisconsin Alumni Research Foundation Double transposition methods for manipulating nucleic acids
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WO2018156519A1 (fr) 2017-02-21 2018-08-30 Illumina Inc. Tagmentation utilisant des transposomes immobilisés avec des lieurs
US12234451B2 (en) 2017-02-21 2025-02-25 Illumina, Inc. Tagmentation using immobilized transposomes with linkers
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US11708573B2 (en) 2017-02-21 2023-07-25 Illumina, Inc. Tagmentation using immobilized transposomes with linkers
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US12104281B2 (en) 2018-11-30 2024-10-01 Illumina, Inc. Analysis of multiple analytes using a single assay
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US11840724B2 (en) 2021-09-01 2023-12-12 10X Genomics, Inc. Methods, compositions, and kits for blocking a capture probe on a spatial array
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US12275988B2 (en) 2021-11-10 2025-04-15 10X Genomics, Inc. Methods, compositions, and kits for determining the location of an analyte in a biological sample
US12195790B2 (en) 2021-12-01 2025-01-14 10X Genomics, Inc. Methods for improved in situ detection of nucleic acids and spatial analysis
WO2023225519A1 (fr) 2022-05-17 2023-11-23 10X Genomics, Inc. Transposons modifiés, compositions et utilisations de ceux-ci

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PL353241A1 (en) 2003-11-03
CN1367840A (zh) 2002-09-04
JP2003505104A (ja) 2003-02-12
AU775043B2 (en) 2004-07-15
AU6396700A (en) 2001-02-19
EP1198583A1 (fr) 2002-04-24
RU2002105508A (ru) 2004-01-27

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