Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA

Zhang, Penghua; Too, Priscilla Hiu-Mei; Samuelson, James C.; Chan, Siu-Hong; Vincze, Tamas; Doucette, Stephanie; Bäckström, Stefan; Potamousis, Konstantinos; Schramm, Timothy M.; Forrest, Dan; Schwartz, David C.; Xu, Shuang-yong

doi:10.1016/j.pep.2009.09.003

Cited by 30 publications

(32 citation statements)

References 50 publications

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“…26 Enzymes were removed by phenol:chloroform extraction and the template was recovered by ethanol precipitation; its concentration was then determined by measuring UV absorbance at 260 nm.…”

Section: Experimental Methodsmentioning

confidence: 99%

Guiding the folding pathway of DNA origami

Dunn

Dannenberg

Ouldridge

et al. 2015

Nature

157

188

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*authors contributed equally to this work 2 DNA origami is a robust molecular assembly technique by which a single-stranded DNA template is folded by annealing it with hundreds of short 'staple' strands. 1-4 The guiding design principle of nanofabrication by DNA self-assembly is that the target structure is the single most stable configuration; 5 however, the pathway and kinetics of origami assembly are poorly understood. The folding transition is cooperative 4,6,7 , and there is a strong analogy with protein folding: both are governed by information encoded in polymer sequence. [8][9][10][11] Misfolded structures are kinetic traps. The yield of well-folded DNA origami can be low: 2 yield is improved by titration of cations 2,12 or by following empirical design rules, 12,13 but it is frequently necessary to separate wellfolded origami from misfolded objects. 2, 3, 14-16 Here, we present an origami structure that is designed to reveal the assembly process. Our system has the unusual property of having a small set of distinguishable, well-folded shapes that represent discrete and approximately degenerate energy minima in a vast folding landscape. We obtain a high yield of well-folded origami structures, demonstrating the existence of efficient folding pathways. The distribution of shapes provides information about individual trajectories through the folding landscape. We show that the assembly pathway can be steered by rational design and identify similarities to protein folding: assembly is highly cooperative; reversible bond-formation is important in recovering from transient misfoldings; and the early formation of long-range connections can be very effective in forcing particular folds. Expanding the rational design process to include the assembly pathway is the key to reproducible synthesis, which is essential if nucleic acid selfassembly is to continue its rapid development 1-3,17-19 and become a reliable manufacturing technology. 20 3This study is based on a simplified version of the archetypal origami tile 1 and, in particular, on the distribution of observed folds of a 'dimer' variant which contains two copies of the template sequence in head-to-tail repeat. The 'monomer' tile ( Fig. . 1) (Fig. 1c); approximately 80% of tiles appear to be well folded.The 'dimer' template is also circular. It contains two identical copies of the monomer joined head-to-tail and can therefore bind two copies of each staple (Fig. 2). Each pair of body and seam staples can bind in one of two configurations (Fig. 2a) to form either an internal link within each copy of the monomer sequence or a pair of cross-links between the two copies.The total number of possible domain pairings is 2 76 ≈ 10 23 . Although many of these configurations are sterically inaccessible it is clear that the result of reducing the specificity of staple binding is that, as in the case of protein folding, the number of possible states of the system is overwhelmingly greater than the number of well-folded structures. However, in contrast to proteins (and t...

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Section: Experimental Methodsmentioning

confidence: 99%

Guiding the folding pathway of DNA origami

Dunn

Dannenberg

Ouldridge

et al. 2015

Nature

157

188

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“…1] the N-terminal 22 aa could be deleted without significantly affecting the nicking activity in Mg 2+ and Mn 2+ buffers; [2] the first 51-aa residues were not absolutely required for the nicking activity in Mn 2+ buffer, but the truncation mutant (76-aa peptide) displayed impaired nicking activity in Mg 2+ buffer (the nicking activity can be restored by fusion with a strong DNA binding element such as a zinc finger protein); [3] larger N-terminal deletion, i.e. deletion of 54-or 66-aa residues from the N-terminus completely inactivated the enzyme activity in Mg 2+ or Mn 2+ buffer.…”

Section: Nbis30 Nφgamma and Other Phage Or Prophage-encoded Hnhementioning

confidence: 99%

“…Nt.BspQI was applied to nicking site profiling in a proofof-concept experiment on a phage DNA (2). DNA optical mapping was applied to bacterial artificial chromosome (BAC) clones containing large human DNA inserts for de novo sequence assembly and for detection of structural variations in complex regions (95).…”

Section: Dna Optical Mappingmentioning

confidence: 99%

“…An initial test of hemi-phosphorothioate-modified restriction sites indicated that [1] the modification needs to be incorporated in the exact cleavage sites; [2] the first group of REases predominantly nicks the unmodified strand, resulting in nicked DNA (i.e. hemi-phosphorothioatemodified strand inhibits cleavage of that strand); [3] the second group of REases slowed down the nicking of the modified strand (nicked circular DNA can still be obtained in limited digestion); [4] the third group of REases cleaves both strands (nicked intermediate was converted to linear DNA before all supercoiled DNA was nicked) (97).…”

Section: Rease-directed Nicking Of Phosphorothioatemodified Dnamentioning

confidence: 99%

“…Nt.CviPII (↓CCD, the down arrow indicates the nicked strand as shown) originally found in chlorella virus (1), engineered NEases such as Nt.BspQI (GCTCTTCN↓), and Nt.BbvCI (CC↓TCAGC) engineered from BspQI and BbvCI restriction endonucleases (REases) (2,3). The other group of DNA NEases contains natural or engineered enzymes with more than 8-bp target sites, which includes group I intron-encoded homing endonucleases (HEs) (4,5), engineered nicking variants from LAGLIDAG HEs (6)(7)(8), engineered TALE nucleases (TALENs) by fusion of transcription activator-like effector (TALE) repeat domain with FokI nuclease domain or a MutH nicking variant (9)(10)(11), ZF nickase (ZFN) (12)(13)(14)(15), RNA-guided Cas9 nicking variants (16,17), and other chimeric NEases consisting of a DNA binding module fused to a DNA nicking domain (9,18).…”

Section: Introductionmentioning

confidence: 99%

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Sequence-specific DNA nicking endonucleases

2015

Biomolecular Concepts

Self Cite

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A group of small HNH nicking endonucleases (NEases) was discovered recently from phage or prophage genomes that nick double-stranded DNA sites ranging from 3 to 5 bp in the presence of Mg2+ or Mn2+. The cosN site of phage HK97 contains a gp74 nicking site AC↑CGC, which is similar to AC↑CGR (R=A/G) of N.ϕGamma encoded by Bacillus phage Gamma. A minimal nicking domain of 76 amino acid residues from N.ϕGamma could be fused to other DNA binding partners to generate chimeric NEases with new specificities. The biological roles of a few small HNH endonucleases (HNHE, gp74 of HK97, gp37 of ϕSLT, ϕ12 HNHE) have been demonstrated in phage and pathogenicity island DNA packaging. Another group of NEases with 3- to 7-bp specificities are either natural components of restriction systems or engineered from type IIS restriction endonucleases. A phage group I intron-encoded HNH homing endonucleases, I-PfoP3I was found to nick DNA sites of 14-16 bp. I-TslI encoded by T7-like ΦI appeared to nick DNA sites with a 9-bp core sequence. DNA nicking and labeling have been applied to optical mapping to aid genome sequence assembly and detection of large insertion/deletion mutations in genomic DNA of cancer cells. Nicking enzyme-mediated amplification reaction has been applied to rapid diagnostic testing of influenza A and B in clinical setting and for construction of DNA-based Boolean logic gates. The clustered regularly interspaced short palindromic repeats-ribonucleoprotein complex consisting of engineered Cas9 nickases in conjunction with tracerRNA:crRNA or a single-guide RNA have been successfully used in genome modifications.

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Visualization of large elongated DNA molecules

et al. 2015

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Long and linear DNA molecules are the mainstream single-molecule analytes for a variety of biochemical analysis within microfluidic devices, including functionalized surfaces and nanostructures. However, for biochemical analysis, large DNA molecules have to be unraveled, elongated, and visualized to obtain biochemical and genomic information. To date, elongated DNA molecules have been exploited in the development of a number of genome analysis systems as well as for the study of polymer physics due to the advantage of direct visualization of single DNA molecule. Moreover, each single DNA molecule provides individual information, which makes it useful for stochastic event analysis. Therefore, numerous studies of enzymatic random motions have been performed on a large elongated DNA molecule. In this review, we introduce mechanisms to elongate DNA molecules using microfluidics and nanostructures in the beginning. Secondly, we discuss how elongated DNA molecules have been utilized to obtain biochemical and genomic information by direct visualization of DNA molecules. Finally, we reviewed the approaches used to study the interaction of proteins and large DNA molecules. Although DNA-protein interactions have been investigated for many decades, it is noticeable that there have been significant achievements for the last five years. Therefore, we focus mainly on recent developments for monitoring enzymatic activity on large elongated DNA molecules.

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Engineering BspQI nicking enzymes and application of N.BspQI in DNA labeling and production of single-strand DNA

Cited by 30 publications

References 50 publications

Guiding the folding pathway of DNA origami

Guiding the folding pathway of DNA origami

Sequence-specific DNA nicking endonucleases

Visualization of large elongated DNA molecules

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