Name: an engineered split-tet2 enzyme for chemical-inducible dna hydroxymethylation and epigenetic remodeling
Uploaded: 2017-12-18T14:00:00
Description: Watch this Scientific Journal Video about An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling at JoVE.com

We thank the financial supports from the National Institutes of Health grant (R01GM112003 to YZ), the Welch Foundation (BE-1913 to YZ), the American Cancer Society (RSG-16-215-01 TBE to YZ), the Cancer Prevention and Research Institute of Texas (RR140053 to YH, RP170660 to YZ), the American Heart Association (16IRG27250155 to YH), and the John S. Dunn Foundation Collaborative Research Award Program.

1. Cell Culture, Plasmid Transfection and Chemical Induction
<ol>
	<li>Culture an adherent cell line (e.g., the human embryonic kidney HEK293T cell) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin and streptomycin at 37 &#176;C with 5% CO2.</li>
	<li>Transfect cells with the CiDER plasmid.
	<ol>
		<li>At least 18 h before transfection, seed 0.3 x 106 cells in 2.5 mL of appropriate DMEM per well in...

<ol>
	<li>Li, E., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+in+mammals.">DNA methylation in mammals.</a> Cold Spring Harbor Perspectv Biol. 6 (5), a019133 (2014).</li><li>Tahiliani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324 (5929), 930-935 (2009).</li><li>He, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA.">Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.</a> Science. 333 (6047), 1303-1307 (2011).</li><li>Ito, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet+proteins+can+convert+5-methylcytosine+to+5-formylcytosine+and+5-carboxylcytosine.">Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.</a> Science. 333 (6047), 1300-1303 (2011).</li><li>Spruijt, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+readers+for+5-(hydroxy)methylcytosine+and+its+oxidized+derivatives.">Dynamic readers for 5-(hydroxy)methylcytosine and its oxidized derivatives.</a> Cell. 152 (5), 1146-1159 (2013).</li><li>Lio, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet2+and+Tet3+cooperate+with+B-lineage+transcription+factors+to+regulate+DNA+modification+and+chromatin+accessibility.">Tet2 and Tet3 cooperate with B-lineage transcription factors to regulate DNA modification and chromatin accessibility.</a> eLife. 5, e18290 (2016).</li><li>Kellinger, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-formylcytosine+and+5-carboxylcytosine+reduce+the+rate+and+substrate+specificity+of+RNA+polymerase+II+transcription.">5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription.</a> Nat Struct Mol Biol. 19 (8), 831-833 (2012).</li><li>Su, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Formylcytosine+Could+Be+a+Semipermanent+Base+in+Specific+Genome+Sites.">5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites.</a> Angew Chem Int Ed Engl. 55 (39), 11797-11800 (2016).</li><li>Ko, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+hydroxylation+of+5-methylcytosine+in+myeloid+cancers+with+mutant+TET2.">Impaired hydroxylation of 5-methylcytosine in myeloid cancers with mutant TET2.</a> Nature. 468 (7325), 839-843 (2010).</li><li>Huang, Y., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connections+between+TET+proteins+and+aberrant+DNA+modification+in+cancer.">Connections between TET proteins and aberrant DNA modification in cancer.</a> Trends Genet. 30 (10), 464-474 (2014).</li><li>Lu, X., Zhao, B. S., He, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TET+family+proteins:+oxidation+activity,+interacting+molecules,+and+functions+in+diseases.">TET family proteins: oxidation activity, interacting molecules, and functions in diseases.</a> Chem Rev. 115 (6), 2225-2239 (2015).</li><li>Hu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+TET2-DNA+complex:+insight+into+TET-mediated+5mC+oxidation.">Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation.</a> Cell. 155 (7), 1545-1555 (2013).</li><li>Hashimoto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+Naegleria+Tet-like+dioxygenase+in+complex+with+5-methylcytosine+DNA.">Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA.</a> Nature. 506 (7488), 391-395 (2014).</li><li>Banaszynski, L. A., Liu, C. W., Wandless, T. J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+FKBP.rapamycin.FRB+ternary+complex.">Characterization of the FKBP.rapamycin.FRB ternary complex.</a> J Am Chem Soc. 127 (13), 4715-4721 (2005).</li><li>Clackson, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redesigning+an+FKBP-ligand+interface+to+generate+chemical+dimerizers+with+novel+specificity.">Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity.</a> Proc Natl Acad Sci U S A. 95 (18), 10437-10442 (1998).</li><li>Ibrahimi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+multicistronic+lentiviral+vectors+with+peptide+2A+sequences.">Highly efficient multicistronic lentiviral vectors with peptide 2A sequences.</a> Hum Gene Ther. 20 (8), 845-860 (2009).</li><li>Kim, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+cleavage+efficiency+of+a+2A+peptide+derived+from+porcine+teschovirus-1+in+human+cell+lines,+zebrafish+and+mice.">High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice.</a> PLoS One. 6 (4), e18556 (2011).</li><li>Lee, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+Split-TET2+Enzyme+for+Inducible+Epigenetic+Remodeling.">Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling.</a> J Am Chem Soc. 139 (13), 4659-4662 (2017).</li><li>Huang, Y., Pastor, W. A., Zepeda-Mart&#237;nez, J. A., Rao, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-CMS+technique+for+genome-wide+mapping+of+5-hydroxymethylcytosine.">The anti-CMS technique for genome-wide mapping of 5-hydroxymethylcytosine.</a> Nat Protoc. 7 (10), 1897-1908 (2012).</li><li>Buenrostro, J. D., Wu, B., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATAC-seq:+A+Method+for+Assaying+Chromatin+Accessibility+Genome-Wide.">ATAC-seq: A Method for Assaying Chromatin Accessibility Genome-Wide.</a> Curr Protoc Mol Biol. 109 (21.29), 1-9 (2015).</li></ol>

Here we have illustrated the use of an engineered split-TET2 enzyme to achieve temporal control of DNA hydroxymethylation. Following the discovery of TET family of 5-methycytosine dioxygenase, many studies have been performed to decipher the biological functions of TET proteins and their major catalytic product 5hmC1,2,3,4,5,6</sup...

DNA methylation, mostly refers to the addition of a methyl group to the carbon 5 position of cytosine to form 5-methylcytosine (5mC), is catalyzed by DNA methyl-transferases (DNMTs). 5mC acts as a major epigenetic mark in the mammalian genome that often signals for transcriptional repression, X-chromosome inactivation and transposon silencing1. The reversal of DNA methylation is mediated by the Ten-elven translocation (TET) protein family. TET enzymes belong to the iron (II) and 2-oxoglutarate dependent dioxygenases which catalyze the successive oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). TET-mediated 5mC oxidation imposes an additional layer of epigenetic control over the mammalian genome. The discovery of TET has sparked intense interest in the epigenetic field to unveil the biological functions of TET proteins and their major catalytic product 5hmC. 5hmC is not only an intermediate during TET-mediated active DNA demethylation2,3,4, but also acts as a stable epigenetic mark5,6,7,8. Although DNA hydroxymethylation is highly correlated with gene expression and aberrant changes in DNA hydroxymethylation are associated with some human disorders9,10,11, the causal relations between epigenetic modifications on DNA and the phenotypes often remain challenging to be established, which can be partially ascribed to the lack of reliable tool to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution.
Here we report the use of a chemical-inducible epigenome remodeling tool (CiDER) to overcome the hurdle facing studies of causal relationships between DNA hydroxymethylation and gene transcription. The design is based on the assumption that the catalytic domain of TET2 (TET2CD) can be split into two inactive fragments when expressed in mammalian cells and its enzymatic function can be restored by taking a chemically inducible dimerization approach (Figure 1A). To establish a split-TET2CD system, we selected six sites in TET2CD, composed of a Cys-rich region and a double-stranded &#946;-helix (DSBH) fold, on the basis of a reported crystal structure of TET2-DNA complex that lacks a low complexity region12. A synthetic gene encoding rapamycin-inducible dimerization module FK506 binding protein 12 (FKBP12) and FKBP rapamycin binding domain (FRB) of the mammalian target of rapamycin14,15, along with a self-cleaving peptide T2A polypeptide sequence16,17, was individually inserted into the selected split sites within TET2CD. The selection of TET2 as our engineering target is based on the following considerations. First, somatic mutations in TET2 with concomitant reduction in DNA hydroxymethylation are frequently observed in human disorders including myeloid disorders and cancer10, which provides useful information on sensitive sites to be avoided for insertion. Second, a large fraction of the TET2 catalytic domain, particularly the low complexity region, is dispensable for the enzymatic function12, thus enabling us to craft a minimized split-TET2 for inducible epigenetic modifications. After screening over 15 constructs, a construct that exhibited highest rapamycin-inducible restoration of its enzymatic activity in the mammalian system was chosen and designated as CiDER18. We describe herein the use of mCherry-tagged CiDER to achieve inducible DNA hydroxymethylation and epigenetic remodeling with rapamycin, and present three methods for validating CiDER-mediated 5hmC production in a model cellular system HEK293T.

<table>
	<tbody>
		<tr>
			<td>Lipofectamine 2000 Transfection Reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>11668027</td>
			<td></td>
		</tr>
		<tr>
			<td>Rapamycin</td>
			<td>Sigma-Aldrich</td>
			<td>37094</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 16% Solution</td>
			<td>VWR</td>
			<td>100503-916</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Amresco</td>
			<td>0694-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric Acid</td>
			<td>Amresco</td>
			<td>0369-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin, Bovine</td>
			<td>Amresco</td>
			<td>0332-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Amresco</td>
			<td>0777-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Hydroxymethylcytosine (5-hmC) antibody (pAb)</td>
			<td>Active Motif</td>
			<td>39769</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td>
			<td>Thermo Fisher Scientific</td>
			<td>A-11008</td>
			<td></td>
		</tr>
		<tr>
			<td>4,6-Diamidino-2-phenylindolde (DAPI)</td>
			<td>Biotium</td>
			<td>40043</td>
			<td></td>
		</tr>
		<tr>
			<td>SVAC1E SHEL LAB Economy Vacuum Oven, 0.6 Cu.Ft. (17 L)</td>
			<td>SHEL LAB</td>
			<td>SVAC1E</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure SSC, 20X</td>
			<td>Thermo Fisher Scientific</td>
			<td>15557036</td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-Dot Apparatus</td>
			<td>BIO-RAD</td>
			<td>1706545</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-5-methylcytosine (5mC) Antibody, clone 33D3</td>
			<td>Millipore</td>
			<td>MABE146</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-mouse IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7076S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-rabbit IgG, HRP-linked Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>7074S</td>
			<td></td>
		</tr>
		<tr>
			<td>West-Q Pico Dura ECL Solution</td>
			<td>Gendepot</td>
			<td>W3653-100</td>
			<td></td>
		</tr>
	</tbody>
</table>

an engineered split-tet2 enzyme for chemical-inducible dna hydroxymethylation and epigenetic remodeling

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype&#8722;phenotype relations in various biological systems.

Chemical inducible 5mC-to-5hmC conversion can be validated by immunostaining at single-cell level, flow cytometry analysis on transfected (mCherry-positive) cell populations, or a more quantitative dot-blot assay as illustrated in Figure 1. The catalytic domain of TET2, or TET2CD, were used throughout the study as a positive control. See references 18-20 for further details regarding the genome-wide mapping of rapamycin-induced changes in 5hmC and chromatin a...

Watch this Scientific Journal Video about An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling at JoVE.com

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling

Detailed protocols for introducing an engineered split-TET2 enzyme (CiDER) into mammalian cells for chemical inducible DNA hydroxymethylation and epigenetic remodeling are presented.

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control ...

The overall goal of this series of experiments is to introduce an engineered split-TET2 enzyme called CiDER into mammalian cells for inducible DNA modification such as hydroxymethylation as well as epigenetic remodeling. This method can help answer key questions in the epigenetics field such as The main advantage of this technique is that the engineered split-TET2 enzyme allow temporal control of 5-methylcytosine oxidation and subsequent remodeling of epigenetic states in mammalian cells with chemical additives. To begin this procedure, culture an adherent cell line in DMEM supplemented with 10%heat-inactivated FBS as well as 100 units per milliliter of penicillin and streptomycin.Incubate the cells at 37 degrees Celsius with 5%CO2. Transfect the cells with the CiDER plasmid as described in the text protocol. Then incubate the cells overnight at 37 degrees Celsius.The following day, replace the transfection reagent-containing media with fresh complete DMEM. To chemically induce the cells, add 20 microliters of diluted Rapamycin to the transfected cells maintained in two milliliters of medium to reach a final concentration of 200 nanomolar. To perform flow cytometry, resuspend the transfected and Rapamycin-induced cells in FACS buffer.For the control group, use CiDER-expressing cells without Rapamycin treatment. Fix and permeabilize the cells as described in the text protocol. Then add two normal hydrochloric acid to the cells to denature the DNA for 10 minutes.After neutralizing and blocking the cells as described in the text protocol, add the diluted anti-5hmC antibody to the cells. Incubate the cells for one hour at room temperature or overnight at four degrees Celsius. Following incubation, wash the cells with FACS buffer three times.Remove the FACS buffer thoroughly. Add a diluted fluorophore conjugated secondary antibody for FACS analysis and incubate for one hour at room temperature. Then wash the cells with FACS buffer three times.Following the wash, the samples are ready for FACS analysis. Isolate genomic DNA from transfected and Rapamycin-induced cells using a commercial DNA extraction kit. For the control group, use CiDER transfected cells without Rapamycin treatment.Heat the vacuum oven to 80 degrees Celsius. Pre-soak the nitrocellulose membrane in double distilled water and then in 6X SSC buffer for 10 minutes. Next, load 60 microliters of equal amounts of DNA to a 96-well PCR plate.Add 30 microliters of TE buffer to the rest wells. Make two-fold serial dilutions vertically on the 96-well plate by transferring 30 microliters of solution in row one to the same column position in row two. Mix again and transfer 30 microliters of solution to the wells in the subsequent row until the boundary is reached.Then remove the last 30 microliters from the well. Next, add 20 microliters of one molar sodium hydroxide and 10 millimolar EDTA to each well. Seal the plate and heat the mixture for 10 minutes at 95 degrees Celsius to completely denature the DNA duplex.Immediately cool down the samples on ice. Add 50 microliters of ice cold two molar ammonium acetate to each well and incubate on ice for 10 minutes. Meanwhile, assemble the dot-blot apparatus with the pre-soaked membrane.Remove the residual buffer using a full vacuum. Wash the membrane by adding 200 microliters of TE buffer to each well of the dot-blot apparatus. Turn on the vacuum to remove TE buffer.Next, load the denatured DNA to each well of the dot-blot apparatus. Turn on the vacuum to remove solution. Wash the membrane by adding 200 microliters of 2X SSC and remove residual solutions completely using a vacuum.Disassemble the dot-blot apparatus. Then take out the membrane and rinse the whole membrane with 20 milliliters of 2X SSC. Air dry the membrane for 20 minutes.To further dry the membrane, bake it at 80 degrees under vacuum for two hours. Add the blocking solution to the membrane and incubate for one hour at room temperature. After discarding the blocking solution, add the diluted 5-hmC or 5-mC antibodies to the membrane and incubate overnight at four degrees Celsius.Wash the membrane with TBST three times for 10 minutes to remove residual antibodies. After removing the TBST thoroughly, add an HRP conjugated secondary antibody to the membrane. Incubate the membrane for one hour at room temperature.Wash the membrane with TBST three times for 10 minutes. Finally, add ECL western blotting substrate to the membrane for visualization of 5-hmC signals using a chemiluminescence detector. A representative quantification result of CiDER-mediated 5-hmC production by flow cytometry is shown here.Only cells expressing CiDER under Rapamycin-treated conditions show significant increase of 5-hmC levels. A representative result of the global change of 5-hmC level in the whole cell population by dot-blot assay is shown here. The loading control is visualized by ethylene blue staining of total amounts of input DNA.The results demonstrate that Rapamycin treatment induces robust production of 5-hmC in CiDER-expressing HEK293T cells with negligible background activity. Once mastered, this technique can be done in a week if it is performed properly. After its development, this technique paved the way for researchers in the field of epigenetics to explore cellular function without altering the genetic code and to probe the epigenotype and phenotype relations in various biological systems.

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