我们要感谢我们的资金来源，包括来自洁具基金会（以JPV）一份厚礼，HealthGrant T32-CA079448（到PJS）和鲍尔州立大学的启动资金（以PJS）全国学院。该资助者在研究设计，数据收集和分析，决定发表或准备手稿没有作用。

<ol><li>Qin, Y., Hurley, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structures%2C+folding+patterns%2C+and+functions+of+intramolecular+DNA+G-quadruplexes+found+in+eukaryotic+promoter+regions%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">Structures, folding patterns, and functions of intramolecular DNA G-quadruplexes found in eukaryotic promoter regions.</a> Biochimie. 90 (8), 1149-1171 (2008).</li>
<li>Stegle, O., Payet, L., Mergny, J. L., MacKay, D. J., Leon, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predicting+and+understanding+the+stability+of+G-quadruplexes%5BTitle%5D)+AND+%22Bioinformatics%22%5BJournal%5D)">Predicting and understanding the stability of G-quadruplexes.</a> Bioinformatics. 25 (12), i374-i382 (2009).</li>
<li>Todd, A. K., Johnston, M., Neidle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Highly+prevalent+putative+quadruplex+sequence+motifs+in+human+DNA%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Highly prevalent putative quadruplex sequence motifs in human DNA.</a> Nucleic Acids Res. 33 (9), 2901-2907 (2005).</li>
<li>Huppert, J. L., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Prevalence+of+quadruplexes+in+the+human+genome%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Prevalence of quadruplexes in the human genome.</a> Nucleic Acids Res. 33 (9), 2908-2916 (2005).</li>
<li>Bedrat, A., Lacroix, L., Mergny, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Re-evaluation+of+G-quadruplex+propensity+with+G4Hunter%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Re-evaluation of G-quadruplex propensity with G4Hunter.</a> Nucleic Acids Res. 44 (4), 1746-1759 (2016).</li>
<li>Mendoza, O., Bourdoncle, A., Boule, J. B., Brosh, R. M. Jr, Mergny, J. L. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/26883636">G-quadruplexes and helicases.</a> Nucleic Acids Res. 44 (5), 1989-2006 (2016).</li>
<li>Huppert, J. L., Balasubramanian, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G-quadruplexes+in+promoters+throughout+the+human+genome%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">G-quadruplexes in promoters throughout the human genome.</a> Nucleic Acids Res. 35 (2), 406-413 (2007).</li>
<li>Eddy, J., Maizels, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+function+correlates+with+potential+for+G4+DNA+formation+in+the+human+genome%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Gene function correlates with potential for G4 DNA formation in the human genome.</a> Nucleic Acids Res. 34 (14), 3887-3896 (2006).</li>
<li>Eddy, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G4+motifs+correlate+with+promoter-proximal+transcriptional+pausing+in+human+genes%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">G4 motifs correlate with promoter-proximal transcriptional pausing in human genes.</a> Nucleic Acids Res. 39 (12), 4975-4983 (2011).</li>
<li>Vaughn, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DEXH+protein+product+of+the+DHX36+gene+is+the+major+source+of+tetramolecular+quadruplex+G4-DNA+resolving+activity+in+HeLa+cell+lysates%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">The DEXH protein product of the DHX36 gene is the major source of tetramolecular quadruplex G4-DNA resolving activity in HeLa cell lysates.</a> J Biol Chem. 280 (46), 38117-38120 (2005).</li>
<li>Booy, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA+helicase+RHAU+%28DHX36%29+unwinds+a+G4-quadruplex+in+human+telomerase+RNA+and+promotes+the+formation+of+the+P1+helix+template+boundary%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary.</a> Nucleic Acids Res. 40 (9), 4110-4124 (2012).</li>
<li>Creacy, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G4+resolvase+1+binds+both+DNA+and+RNA+tetramolecular+quadruplex+with+high+affinity+and+is+the+major+source+of+tetramolecular+quadruplex+G4-DNA+and+G4-RNA+resolving+activity+in+HeLa+cell+lysates%5BTitle%5D)+AND+%22J+Biol+Chem%22%5BJournal%5D)">G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates.</a> J Biol Chem. 283 (50), 34626-34634 (2008).</li>
<li>Giri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((G4+resolvase+1+tightly+binds+and+unwinds+unimolecular+G4-DNA%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">G4 resolvase 1 tightly binds and unwinds unimolecular G4-DNA.</a> Nucleic Acids Res. 39 (16), 7161-7178 (2011).</li>
<li>Lattmann, S., Stadler, M. B., Vaughn, J. P., Akman, S. A., Nagamine, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DEAH-box+RNA+helicase+RHAU+binds+an+intramolecular+RNA+G-quadruplex+in+TERC+and+associates+with+telomerase+holoenzyme%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">The DEAH-box RNA helicase RHAU binds an intramolecular RNA G-quadruplex in TERC and associates with telomerase holoenzyme.</a> Nucleic Acids Res. 39 (21), 9390-9404 (2011).</li>
<li>Sexton, A. N., Collins, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+5%27+guanosine+tracts+of+human+telomerase+RNA+are+recognized+by+the+G-quadruplex+binding+domain+of+the+RNA+helicase+DHX36+and+function+to+increase+RNA+accumulation%5BTitle%5D)+AND+%22Mol+Cell+Biol%22%5BJournal%5D)">The 5' guanosine tracts of human telomerase RNA are recognized by the G-quadruplex binding domain of the RNA helicase DHX36 and function to increase RNA accumulation.</a> Mol Cell Biol. 31 (4), 736-743 (2011).</li>
<li>Smaldino, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mutational+Dissection+of+Telomeric+DNA+Binding+Requirements+of+G4+Resolvase+1+Shows+that+G4-Structure+and+Certain+3%27-Tail+Sequences+Are+Sufficient+for+Tight+and+Complete+Binding%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Mutational Dissection of Telomeric DNA Binding Requirements of G4 Resolvase 1 Shows that G4-Structure and Certain 3'-Tail Sequences Are Sufficient for Tight and Complete Binding.</a> PLoS One. 10 (7), e0132668(2015).</li>
<li>Booy, E. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+RNA+helicase+RHAU+%28DHX36%29+suppresses+expression+of+the+transcription+factor+PITX1%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">The RNA helicase RHAU (DHX36) suppresses expression of the transcription factor PITX1.</a> Nucleic Acids Res. 42 (5), 3346-3361 (2014).</li>
<li>Huang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Yin+Yang+1+contains+G-quadruplex+structures+in+its+promoter+and+5%27-UTR+and+its+expression+is+modulated+by+G4+resolvase+1%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Yin Yang 1 contains G-quadruplex structures in its promoter and 5'-UTR and its expression is modulated by G4 resolvase 1.</a> Nucleic Acids Res. 40 (3), 1033-1049 (2012).</li>
<li>Tran, H., Schilling, M., Wirbelauer, C., Hess, D., Nagamine, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Facilitation+of+mRNA+deadenylation+and+decay+by+the+exosome-bound%2C+DExH+protein+RHAU%5BTitle%5D)+AND+%22Mol+Cell%22%5BJournal%5D)">Facilitation of mRNA deadenylation and decay by the exosome-bound, DExH protein RHAU.</a> Mol Cell. 13 (1), 101-111 (2004).</li>
<li>Yoo, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DHX36+enhances+RIG-I+signaling+by+facilitating+PKR-mediated+antiviral+stress+granule+formation%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">DHX36 enhances RIG-I signaling by facilitating PKR-mediated antiviral stress granule formation.</a> PLoS Pathog. 10 (3), e1004012(2014).</li>
<li>Lai, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DEAH-box+helicase+RHAU+is+an+essential+gene+and+critical+for+mouse+hematopoiesis%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">The DEAH-box helicase RHAU is an essential gene and critical for mouse hematopoiesis.</a> Blood. 119 (18), 4291-4300 (2012).</li>
<li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DDX1%2C+DDX21%2C+and+DHX36+helicases+form+a+complex+with+the+adaptor+molecule+TRIF+to+sense+dsRNA+in+dendritic+cells%5BTitle%5D)+AND+%22Immunity%22%5BJournal%5D)">DDX1, DDX21, and DHX36 helicases form a complex with the adaptor molecule TRIF to sense dsRNA in dendritic cells.</a> Immunity. 34 (6), 866-878 (2011).</li>
<li>Kim, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Aspartate-glutamate-alanine-histidine+box+motif+%28DEAH%29%2FRNA+helicase+A+helicases+sense+microbial+DNA+in+human+plasmacytoid+dendritic+cells%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells.</a> Proc Natl Acad Sci U S A. 107 (34), 15181-15186 (2010).</li>
<li>BD Biosciences. , Available from: 
 <a target="_blank" href="http://www.bd.com/ds/technicalCenter/misc/difcobblmanual_2nded_lowres.pdf">http://www.bd.com/ds/technicalCenter/misc/difcobblmanual_2nded_lowres.pdf</a> (2016).</li>
<li>Booy, E. P., McRae, E. K., McKenna, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biochemical+characterization+of+G4+quadruplex+telomerase+RNA+unwinding+by+the+RNA+helicase+RHAU%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Biochemical characterization of G4 quadruplex telomerase RNA unwinding by the RNA helicase RHAU.</a> Methods Mol Biol. 1259, 125-135 (2015).</li>
<li>Lattmann, S., Giri, B., Vaughn, J. P., Akman, S. A., Nagamine, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+the+amino+terminal+RHAU-specific+motif+in+the+recognition+and+resolution+of+guanine+quadruplex-RNA+by+the+DEAH-box+RNA+helicase+RHAU%5BTitle%5D)+AND+%22Nucleic+Acids+Res%22%5BJournal%5D)">Role of the amino terminal RHAU-specific motif in the recognition and resolution of guanine quadruplex-RNA by the DEAH-box RNA helicase RHAU.</a> Nucleic Acids Res. 38 (18), 6219-6233 (2010).</li>
</ol>

作者什么都没有透露。

该协议代表一高效表达，纯化，并定量为DHX36基因产物，G4-Resolvase1（G4R1，也称为RHAU和DHX36）（ 图1）的隔离方案。这个协议利用两个纯化步骤:关于钴亲和珠His标签的亲和纯化和酶纯化上G4-DNA缀合珠。后者步骤是在，它需要在严密的亲和力，特异性高，以及催化退绕的活动，rG4R1具有G4结构优点是唯一的。对G4结构紧亲和性（在低PM范围ķ 四 ）允许高盐洗涤（邻近的NaCl的溶解度?...

G4结构是DNA和RNA的富鸟嘌呤区域内形成高度稳定的核酸的二级结构。 G4结构通过Hoogsteen碱基接合相互作用来稳定，并与一价阳离子（ 即 K +和Na +），该显著有助于G4结构1,2的显着热稳定性的中心空腔内坐标粘接。早期的生物信息学研究表明，人类基因组包含&gt; 375000"潜在G4形成的图案"3,4。最近的研究估计表明，G4基序的数目是由2-5 5倍以上，而另一项研究预测在人类基因组6 716310不同电位G4形成的序列。 G4形成序列进化上保守的，而不是随机地分散在基因组中。 G4图案富集基因编码区，以及所有基因启动子的40％以上含有G4图案7。有趣的是，在一个基因的G4基序的富集的程度已被证明表明该基因的功能。例如，参与发展原癌基因和基因比抑癌基因8,9 G4结构显著更大的富集。 具有高的热稳定性，在整个基因组中的几乎无处不在，并且显著影响主要的细胞过程的潜力，这是令人吃惊地发现，细胞已经发展酶来管理这些结构。一种这样的酶是G4 Resolvase1（G4R1;也称作RHAU和DHX36），我们表征为在人类（HeLa细胞）细胞10多数tetramolecular G4-DNA解析活性的来源。自那时以来，它已被证明塔吨G4R1紧密结合并催化开卷tetramolecular和单分子G4-DNA和G4-RNA与用于G4结合蛋白11，12，13中的紧密的报告的KDS。此外，G4R1的G4分辨活性已经牵涉在广泛的生物化学和细胞过程，包括端粒/端粒酶生物学11，14，15，16，转录和剪接17，18，19，20，发展21，造血21，及免疫调节22,23。用G4序列占优势的特别位于整个基因组和不同的对细胞processes该G4R1最近已经牵连用，表达和有效地净化活性高rG4R1将是极为重要的为阐明该蛋白的生化机制和行为的能力有关。 在这里，我们证明了一种新的表达而这需要rG4R1的ATP依赖性，G4-解决活动的优势，以有效地分离活性酶的纯化方案（ 图1）。该方案可以适用于纯化其它ATP依赖性核酸酶的量，酶反应的产物是不再结合的基板，如为G4R1的情况。

<table><tbody><tr><td>TriEx4-DHX36 plasmid</td><td>Addgene</td><td>68368</td><td></td></tr><tr><td>Rosetta2(DE3)plysS competent cells</td><td>Novagen</td><td>71403-4</td><td></td></tr><tr><td>S.O.C medium</td><td>Thermo Fisher Scientific</td><td>15544034&amp;nbsp;</td><td></td></tr><tr><td>Difco Terrific Broth</td><td>Becton Dickinson</td><td>243820</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Chloramphenicol</td><td>Sigma-Aldrich</td><td>C1919</td><td>35 &amp;micro;g/mL in bacterial plates/large cultures</td></tr><tr><td>Carbenicillin (plant cell culture tested)</td><td>Sigma-Aldrich</td><td>C3416</td><td>50 &amp;micro;g/mL in bacterial plates/large cultures</td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Sigma-Aldrich</td><td>I6758</td><td></td></tr><tr><td>Lysozyme (from chicken egg white)</td><td>Sigma-Aldrich</td><td>L6876</td><td></td></tr><tr><td>1 M Tris-HCl, pH 8</td><td>Universal Scientific Supply Co.&amp;nbsp;</td><td>1963-B</td><td>or from standard source</td></tr><tr><td>1 M Tris-HCl, pH 7</td><td>Universal Scientific Supply Co.&amp;nbsp;</td><td>1966</td><td>or from standard source</td></tr><tr><td>1.5 M Tris-HCl, pH 8.8</td><td></td><td></td><td>For casting resolving gel (for protein quantitation gel); From standard source</td></tr><tr><td>1 M Tris-HCl, pH 6.8</td><td></td><td></td><td>For casting stacking gel (for protein quantitation gel); From standard source</td></tr><tr><td>1 M Tris-Acetate, pH 7.8</td><td>Universal Scientific Supply Co.&amp;nbsp;</td><td>1981</td><td>or from standard source</td></tr><tr><td>70% Ethanol</td><td></td><td></td><td>From standard source</td></tr><tr><td>Magnesium chloride (1&amp;nbsp;M solution)</td><td>Life Technologies</td><td>AM9530G</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>S7653</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S8750</td><td></td></tr><tr><td>20x SSC</td><td>Universal Scientific Supply Co.&amp;nbsp;</td><td>1665</td><td>or from standard source</td></tr><tr><td>&amp;beta;-mercaptoethanol (2-BME)</td><td>Sigma-Aldrich</td><td>63689</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P8849</td><td></td></tr><tr><td>Leupeptin hemisulfate</td><td>Sigma-Aldrich</td><td>L8511</td><td></td></tr><tr><td>Streptavidin paramagnetic beads</td><td>Promega</td><td>Z5482</td><td></td></tr><tr><td>0.5 M EDTA, pH 8&amp;nbsp;</td><td>Universal Scientific Supply Co.&amp;nbsp;</td><td>0718</td><td>or from standard source</td></tr><tr><td>0.2 M EDTA, pH 6</td><td>Universal Scientific Supply Co.&amp;nbsp;</td><td></td><td>From standard source; initially adjust pH with NaOH, then adjust pH back down with HCl.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>A-lactalbumin (Type 1 from bovine milk)</td><td>Sigma-Aldrich</td><td>L5385</td><td></td></tr><tr><td>Cobalt metal affinity beads</td><td>Clonetech</td><td>635502</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma-Aldrich</td><td>H8000</td><td></td></tr><tr><td>Acetic acid, glacial</td><td>Fisher Scientific</td><td>A38-500</td><td></td></tr><tr><td>Adenosine 5&#039;-Triphosphate (from bacterial source)</td><td>Sigma</td><td>A7699</td><td></td></tr><tr><td>40% acrylamide/Bis solution (37.5:1)</td><td>Biorad</td><td>161-0148</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich</td><td>50046</td><td>to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS)</td></tr><tr><td>10% Sodium dodecyl sulfate (SDS)</td><td>Universal Scientific Supply Co.&amp;nbsp;</td><td>1667</td><td>to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS); or from standard source</td></tr><tr><td>10x TBE&amp;nbsp;</td><td>Sigma-Aldrich</td><td>11666703001</td><td>or from standard source</td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-1</td><td>to make protein gel running buffer (192 mM glycine, 25 mM Tris Base, 0.1% SDS); From standard source</td></tr><tr><td>TEMED</td><td>Sigma-Aldrich</td><td>411019</td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Sigma-Aldrich</td><td>A3678</td><td></td></tr><tr><td>Broad Range Protein MW markers</td><td>Promega</td><td>V8491</td><td></td></tr><tr><td>Biotinylated Z33 oligo (&quot;Z33-Bio&quot;)</td><td>Oligos Etc</td><td></td><td>5&amp;rsquo; AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT-biotin 3&amp;rsquo;</td></tr><tr><td>TAMRA-Z33 oligo (&quot;Z33-TAM&quot;)</td><td>Oligos Etc</td><td></td><td>5&amp;rsquo; TAMRA-AAA GTG ATG GTG GTG GGG GAA GGA TTC GGA CCT 3&amp;rsquo;</td></tr><tr><td>Fluor-coated TLC plate</td><td>Life Technologies</td><td>AM10110</td><td></td></tr><tr><td>Ficoll</td><td>Sigma-Aldrich</td><td>F2637</td><td>30% in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Coomassie R-250</td><td>Sigma-Aldrich</td><td>27816</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412</td><td></td></tr><tr><td>Multiband UV lamp</td><td></td><td></td><td>Capable of emitting UV light at 365 nm</td></tr><tr><td>Table-top centrifuge (with swinging bucket rotor)</td><td></td><td></td><td>Capable of being cooled to 4 &amp;deg;C</td></tr><tr><td>Microcentrifuge</td><td></td><td></td><td>Capable of being cooled to 4 &amp;deg;C</td></tr><tr><td>Digital Sonfier</td><td>Branson</td><td></td><td>Or equivalent capable of delivering sonication pulses (30% amplitude, 2 s ON/2 s OFF)</td></tr><tr><td>50 &amp;deg;C water bath</td><td></td><td></td><td>For formation of Z33 into quadruplex</td></tr><tr><td>37 &amp;deg;C incubator for bacteria</td><td></td><td></td><td>For bacterial transformations and initial overnight growth of large cultures of Rosetta2 &lt;em&gt;E. coli&lt;/em&gt; transformed with TriEx4-DHX36</td></tr><tr><td>37 &amp;deg;C/14&amp;nbsp;&amp;deg;C shaking incubator for bacteria</td><td></td><td></td><td>For growth and protein induction of large cultures of Rosetta2 &lt;em&gt;E. coli&lt;/em&gt; transformed with TriEx4-DHX36</td></tr><tr><td>Spectrophotometer</td><td></td><td></td><td>capable of reading OD600; capable of reading oligomer concentrations based on base sequence (such as Biorad SmartSpec 3000)</td></tr><tr><td>Thermometer</td><td></td><td></td><td>From standard source</td></tr><tr><td>PCR strip tubes</td><td></td><td></td><td>From standard source</td></tr><tr><td>15 and 50 mL centrifuge tubes (polypropylene)</td><td></td><td></td><td>From standard source</td></tr><tr><td>Microcentrifuge tubes (2.0 mL)</td><td></td><td></td><td>From standard source</td></tr><tr><td>500 mL centrifuge bottles (polypropylene)</td><td>Thermo Scientific</td><td>3141-0500</td><td></td></tr><tr><td>Standard array of pipet tips and serological pipettes</td><td></td><td></td><td>From standard source</td></tr><tr><td>Gel-loading tips</td><td></td><td></td><td>From standard source</td></tr><tr><td>Automatic repeating pipette</td><td></td><td></td><td>For quick aliquoting of rG4R1; From standard source</td></tr><tr><td>Thermal cycler</td><td></td><td></td><td>From standard source</td></tr><tr><td>Liquid Nitrogen</td><td></td><td></td><td>From standard source</td></tr><tr><td>Dry ice</td><td></td><td></td><td>From standard source</td></tr><tr><td>Laemlli sample buffer&amp;nbsp;</td><td>Biorad</td><td>161-0737</td><td></td></tr><tr><td>Apparatus for running large slab gels</td><td>Biorad</td><td></td><td>We have used the Protean II xi cell apparatus from Biorad</td></tr><tr><td>Magnet</td><td>Life Technologies</td><td>12301D</td><td>We use a magnet from One Lambda (Now a Thermo Fisher Scientific brand); and Life is also a subsidiary of Thermo, and thus the magnet listed here should be a suitable replacement</td></tr><tr><td>Razor blades</td><td></td><td></td><td>From standard source</td></tr><tr><td>Filter paper and funnel</td><td></td><td></td><td>From standard source</td></tr><tr><td>Glass casserole dish</td><td></td><td></td><td>From standard source</td></tr><tr><td>Orbital shaker</td><td></td><td></td><td>From standard source</td></tr><tr><td>Kimwipes</td><td></td><td></td><td>From standard source</td></tr><tr><td>Clear sheet protectors</td><td></td><td></td><td>From standard source</td></tr><tr><td>Scanner and associated TWAIN software</td><td></td><td></td><td>From standard source</td></tr><tr><td>Image analysis software</td><td></td><td></td><td>Such as Fuji Multiguage, or equivalent</td></tr><tr><td>Microsoft Excel</td><td></td><td></td><td>Or equivalent&amp;nbsp;</td></tr></tbody></table>

a g-quadruplex dna-affinity approach for purification of enzymatically active g4 resolvase1

高阶核酸结构称为G-四链（台G4，G4结构）可以在DNA和RNA的富含鸟嘌呤区形成并且是高度热稳定的。有在人类基因组&gt; 375000推定G4形成的序列，它们在启动子区域，非翻译区（UTR）富集和端粒重复内。由于对这些结构以影响细胞过程，如复制和转录的潜力，所述细胞已演变的酶来管理它们。一种这样的酶是G4解离1（G4R1），将其生化共同特征在于我们的实验室和长峰等。并发现极紧到两个G4-DNA和G4-RNA（K d。在低PM范围）结合。 G4R1是多数在HeLa细胞裂解物的G4分辨活性的来源，并一直牵连发挥端粒代谢，淋巴开发，基因转录，造血和免疫监视作用。 EF英孚的能力ficiently表达和纯化催化活性G4R1是重要的，针对有意获得进一步的深入了解G4结构和G4-解决酶的动力学相互作用实验室。在这里，我们描述了重组G4R1（rG4R1）的提纯的详细方法。所描述的过程结合的C-末端组氨酸标记的酶的传统的基于亲和纯化在具有rG4R1的能力利用人密码子优化的细菌中表达结合和放松G4-DNA的纯化高活性酶在ATP的依赖洗脱步骤。该协议还包括其中rG4R1的酶活性是通过检查纯化的酶的放松G4-DNA的能力测得的质量控制步骤。的方法还描述了允许纯化rG4R1的量化。该协议的另类改编进行了讨论。

该协议（ 图1）经常产生几乎纯，催化活性rG4R1。作为酶活性的量度，它通常被观察到，0.2皮摩尔TAMRA标记tetramolecular G4-DNA的50％在0.2内转化成单体- rG4R1 0.013微升范围，由上述（ 图中概述的G4活性测定为测定2）。纯化rG4R1的考马斯染色在约75 kDa的，这可能代表截短rG4R1已保持其结合G4-DNA的珠和在ATP依赖洗脱能力表明在预期120 kDa的大小的单一条带，具有轻微的污?...

Watch this Scientific Journal Video about A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1 at JoVE.com

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

G4 Resolvase1结合G-四（G4）的结构与一个G4结合蛋白的紧密报道的亲和力，并表示在HeLa细胞中的大多数G4-DNA解旋活动。我们描述了线束的亲和力和G4-Resolvase1的ATP依赖的平仓活动，特别是净化催化活性的重组G4R1一个新的协议。

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

a-g-quadruplex-dna-affinity-approach-for-purification-enzymatically

Research

JoVE Journal

Biochemistry

9.6K 次观看.  Wake Forest School of Medicine.  G4 Resolvase1结合G-四（G4）的结构与一个G4结合蛋白的紧密报道的亲和力，并表示在HeLa细胞中的大多数G4-DNA解旋活动。我们描述了线束的亲和力和G4-Resolvase1的ATP依赖的平仓活动，特别是净化催化活性的重组G4R1一个新的协议。 

视频: A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

从外膜囊泡内蛋白质导演包装<em&gt;大肠杆菌</em&gt;:设计，生产和纯化

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

科研

生物化学

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 replisome is one of the simplest replication systems known, composed of only four proteins, which is an attractive feature for biochemical experiments. Special emphasis is placed on the synthesis of ribonucleotide primers by the T7 primase-helicase, which are used by DNA polymerase to initiate DNA synthesis. Because the mechanisms of DNA replication are conserved across evolution, these protocols should be applicable, or useful as a conceptual springboard, to investigators using other model systems. The protocols described here are highly sensitive and an experienced investigator can perform these experiments and obtain data for analysis in about a day. The only specialized piece of equipment required is a rapid-quench flow instrument, but this piece of equipment is relatively common and available from various commercial sources. The major drawbacks of these assays, however, include the use of radioactivity and the relative low throughput.

Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins

We describe sensitive, gel-based discontinuous assays to examine the kinetics of lagging-strand initiation using the replication proteins of bacteriophage T7.

滞后链DNA合成的动力学研究<em&gt;体外</em&gt;由T7噬菌体复制蛋白

Here we provide protocols for the kinetic examination of lagging-strand DNA synthesis in vitro by the replication proteins of bacteriophage T7. The T7 ...

遗传学

Expression, Purification, and Antimicrobial Activity of S100A12

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some S100 proteins have the capacity to bind transition metals with high affinity and effectively sequester them away from invading microbial pathogens in a process that is termed "nutritional immunity". S100A12 (EN-RAGE) binds both zinc and copper and is highly abundant in innate immune cells such as macrophages and neutrophils. We report a refined method for the expression, enrichment and purification of S100A12 in its active, metal-binding configuration. Utilization of this protein in bacterial growth and viability analyses reveals that S100A12 has antimicrobial activity against the bacterial pathogen, Helicobacter pylori. The antimicrobial activity is predicated on the zinc-binding activity of S100A12, which chelates nutrient zinc, thereby starving H. pylori which requires zinc for growth and proliferation.

Here, we present a method to express and purify S100A12 (calgranulin C). We describe a protocol to measure its antimicrobial activity against the human pathogen H. pylori.

S100A12的表达，纯化和抗菌活性

Calgranulin proteins are important mediators of innate immunity and are members of the S100 class of the EF-hand family of calcium binding proteins. Some ...

A Tandem Liquid Chromatography&#8211;Mass Spectrometry-based Approach for Metabolite Analysis of Staphylococcus aureus

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the abundances of key metabolites to which regulatory factors respond, adjusting cellular metabolism. In recent years, a number of proteins and RNA have emerged as important regulators of virulence gene expression. For example, the CodY protein responds to levels of branched-chain amino acids and GTP and is widely conserved in low G+C Gram-positive bacteria. As a global regulator in Staphylococcus aureus, CodY controls the expression of dozens of virulence and metabolic genes. We hypothesize that S. aureus uses CodY, in part, to alter its metabolic state in an effort to adapt to nutrient-limiting conditions potentially encountered in the host environment. This manuscript describes a method for extracting and analyzing metabolites from S. aureus using liquid chromatography coupled with mass spectrometry, a protocol that was developed to test this hypothesis. The method also highlights best practices that will ensure rigor and reproducibility, such as maintaining biological steady state and constant aeration without the use of continuous chemostat cultures. Relative to the USA200 methicillin-susceptible S. aureus isolate UAMS-1 parental strain, the isogenic codY mutant exhibited significant increases in amino acids derived from aspartate (e.g., threonine and isoleucine) and decreases in their precursors (e.g., aspartate and O-acetylhomoserine). These findings correlate well with transcriptional data obtained with RNA-seq analysis: genes in these pathways were up-regulated between 10- and 800-fold in the codY null mutant. Coupling global analyses of the transcriptome and the metabolome can reveal how bacteria alter their metabolism when faced with environmental or nutritional stress, providing potential insight into the physiological changes associated with nutrient depletion experienced during infection. Such discoveries may pave the way for the development of novel anti-infectives and therapeutics.

Here we describe a protocol for the extraction of metabolites from Staphylococcus aureus and their subsequent analysis via liquid chromatography and mass spectrometry.

对于代谢物分析基于法 - 串联液相色谱 - 质谱法<em&gt;金黄色葡萄球菌</em

In an effort to thwart bacterial pathogens, hosts often limit the availability of nutrients at the site of infection. This limitation can alter the ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

磁性镊子 quadruplexes 单分子操作

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

使用化学作图鉴定 DNA 模板中的 G-四链体

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. 
Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

作者聚焦：通过基于 Bis-3-氯哌啶的化学定位表征 DNA G-四链体 

Bis-3-chloropiperidines (B-CePs) are useful chemical probes to identify and characterize G-quadruplex structures in DNA templates in vitro. This protocol details the procedure to perform probing reactions with B-CePs and to resolve reaction products by high-resolution polyacrylamide gel electrophoresis.

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing ...

<meta charset="utf8"></head><body>通过 ssDNA-RPA 束生长观察到的 CMG 介导的 DNA 解旋

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

<meta charset="utf8"></head><body>作者聚焦:通过单分子可视化揭示真核 DNA 复制的动力学

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

对酶活性G4 Resolvase1净化用G-四DNA亲和方法

摘要

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从外膜囊泡内蛋白质导演包装

滞后链DNA合成的动力学研究

S100A12的表达，纯化和抗菌活性

对于代谢物分析基于法 - 串联液相色谱 - 质谱法

磁性镊子 quadruplexes 单分子操作

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

体外研究双-3-氯哌啶对G-四链体DNA结构的化学定位

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化

通过 CMG 解旋酶对 DNA 解旋进行单分子实时可视化