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.

an-engineered-split-tet2-enzyme-for-chemical-inducible-dna

Research

JoVE Journal

Chemistry

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Preparation of Mica and Silicon Substrates for DNA Origami Analysis and Experimentation

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of these applications require interaction with and adhesion of DNA nanostructures to a substrate. Due to its atomically flat and easily cleaned nature, mica has been the substrate of choice for DNA origami experiments. However, the practical applications of mica are relatively limited compared to those of semiconductor substrates. For this reason, a straightforward, stable, and repeatable process for DNA origami adhesion on derivatized silicon oxide is presented here. To promote the adhesion of DNA nanostructures to silicon oxide surface, a self-assembled monolayer of 3-aminopropyltriethoxysilane (APTES) is deposited from an aqueous solution that is compatible with many photoresists. The substrate must be cleaned of all organic and metal contaminants using Radio Corporation of America (RCA) cleaning processes and the native oxide layer must be etched to ensure a flat, functionalizable surface. Cleanrooms are equipped with facilities for silicon cleaning, however many components of DNA origami buffers and solutions are often not allowed in them due to contamination concerns. This manuscript describes the set-up and protocol for in-lab, small-scale silicon cleaning for researchers who do not have access to a cleanroom or would like to incorporate processes that could cause contamination of a cleanroom CMOS clean bench. Additionally, variables for regulating coverage are discussed and how to recognize and avoid common sample preparation problems is described.

Reproducible cleaning processes for substrates used in DNA origami research are described, including bench-top RCA cleaning and derivatization of silicon oxide. Protocols for surface preparation, DNA origami deposition, drying parameters, and simple experimental set-ups are illustrated.

The designed nature and controlled, one-pot synthesis of DNA origami provides exciting opportunities in many fields, particularly nanoelectronics. Many of ...

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.

A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Stable DNA Motifs, 1D and 2D Nanostructures Constructed from Small Circular DNA Molecules

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D DNA nanostructures. Over decades, the rapid development of DNA nanotechnology is attributed to the use of linear DNAs as the source materials. For example, the DAO (double crossover, antiparallel, odd half-turns) tile is well-known as a building block for construction of 2D DNA lattices; the core structure of DAO is made from two linear single-stranded (ss) oligonucleotides, like two ropes making a right hand granny&#160;knot. Herein, a new type of DNA tiles called cDAO (coupled DAO) are built using a small circular ss-DNA of c64nt or c84nt (circular 64 or 84 nucleotides) as the scaffold strand and several linear ss-DNAs as the staple strands. Perfect 1D and 2D nanostructures are assembled from cDAO tiles: infinite nanowires, nanospirals, nanotubes, nanoribbons; and finite nano-rectangles. Detailed protocols are described: 1) preparation by T4 ligase and purification by denaturing PAGE (polyacrylamide gel electrophoresis) of small circular oligonucleotides, 2) annealing of stable circular tiles, followed by native PAGE analysis, 3) assembling of infinite 1D nanowires, nanorings, nanospirals, infinite 2D lattices of nanotubes and nanoribbons, and finite 2D nano-rectangles, followed by AFM (Atomic Force Microscopy) imaging. The method is simple, robust, and affordable for most labs.

This article presents a detailed protocol for T4 ligation and denaturing PAGE purification of small circular DNA molecules, annealing and native PAGE analysis of circular tiles, assembling and AFM imaging of 1D and 2D DNA nanostructures, as well as agarose gel electrophoresis and centrifugation purification of finite DNA nanostructures.

This article presents a detailed protocol for synthesis of small circular DNA molecules, annealing of circular DNA motifs, and construction of 1D and 2D ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

An Intestine/Liver Microphysiological System for Drug Pharmacokinetic and Toxicological Assessment

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests phase of drug developing process because they are genetically human and recapitulate the interplay among tissues. In this study, the human intestinal barrier (emulated by a co-culture of Caco-2 and HT-29 cells) and the liver equivalent (emulated by spheroids made of differentiated HepaRG cells and human hepatic stellate cells) were integrated into a two-organ chip (2-OC) microfluidic device to assess some acetaminophen (APAP) pharmacokinetic (PK) and toxicological properties. The MPS had three assemblies: Intestine only 2-OC, Liver only 2-OC, and Intestine/Liver 2-OC with the same media perfusing both organoids. For PK assessments, we dosed the APAP in the media at preset timepoints after administering it either over the intestinal barrier (emulating the oral route) or in the media (emulating the intravenous route), at 12 &#181;M and 2 &#181;M respectively. The media samples were analyzed by reversed-phase high-pressure liquid chromatography (HPLC). Organoids were analyzed for gene expression, for TEER values, for protein expression and activity, and then collected, fixed, and submitted to a set of morphological evaluations. The MTT technique performed well in assessing the organoid viability, but the high content analyses (HCA) were able to detect very early toxic events in response to APAP treatment. We verified that the media flow does not significantly affect the APAP absorption whereas it significantly improves the liver equivalent functionality. The APAP human intestinal absorption and hepatic metabolism could be emulated in the MPS. The association between MPS data and in silico modeling has great potential to improve the predictability of the in vitro methods and provide better accuracy than animal models in pharmacokinetic and toxicological studies.

We exposed a microphysiological system (MPS) with intestine and liver organoids to acetaminophen (APAP). This article describes the methods for organoid production and APAP pharmacokinetic and toxicological property assessments in the MPS. It also describes the tissue functionality analyses necessary to validate the results.

The recently introduced microphysiological systems (MPS) cultivating human organoids are expected to perform better than animals in the preclinical tests ...

DNA Origami-Mediated Substrate Nanopatterning of Inorganic Structures for Sensing Applications

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA nanofabrication technique is called DNA origami, and it allows high-throughput synthesis of accurate and highly versatile structures with nanometer-level precision. Here, it is shown how the spatial information of DNA origami can be transferred to metallic nanostructures by combining the bottom-up DNA origami with the conventionally used top-down lithography approaches. This allows fabrication of billions of tiny nanostructures in one step onto selected substrates. The method is demonstrated using bowtie DNA origami to create metallic bowtie-shaped antenna structures on silicon nitride or sapphire substrates. The method relies on the selective growth of a silicon oxide layer on top of the origami deposition substrate, thus resulting in a patterning mask for following lithographic steps. These nanostructure-equipped surfaces can be further used as molecular sensors (e.g., surface-enhanced Raman spectroscopy (SERS)) and in various other optical applications at the visible wavelength range owing to the small feature sizes (sub-10 nm). The technique can be extended to other materials through methodological modifications; therefore, the resulting optically active surfaces may find use in development of metamaterials and metasurfaces.

Here, we describe a protocol to create discrete and accurate inorganic nanostructures on substrates using DNA origami shapes as guiding templates. The method is demonstrated by creating plasmonic gold bowtie-shaped antennas on a transparent substrate (sapphire).

Structural DNA nanotechnology provides a viable route for building from the bottom-up using DNA as construction material. The most common DNA ...

A Femtoliter Droplet Array for Massively Parallel Protein Synthesis from Single DNA Molecules

Advances in spatial resolution and detection sensitivity of scientific instrumentation make it possible to apply small reactors for biological and chemical research. To meet the demand for high-performance microreactors, we developed a femtoliter droplet array (FemDA) device and exemplified its application in massively parallel cell-free protein synthesis (CFPS) reactions. Over one million uniform droplets were readily generated within a finger-sized area using a two-step oil-sealing protocol. Every droplet was anchored in a femtoliter microchamber composed of a hydrophilic bottom and a hydrophobic sidewall. The hybrid hydrophilic-in-hydrophobic structure and the dedicated sealing oils and surfactants are crucial for stably retaining the femtoliter aqueous solution in the femtoliter space without evaporation loss. The femtoliter configuration and the simple structure of the FemDA device allowed minimal reagent consumption. The uniform dimension of the droplet reactors made large-scale quantitative and time-course measurements convincing and reliable. The FemDA technology correlated the protein yield of the CFPS reaction with the number of DNA molecules in each droplet. We streamlined the procedures about the microfabrication of the device, the formation of the femtoliter droplets, and the acquisition and analysis of the microscopic image data. The detailed protocol with the optimized low running cost makes the FemDA technology accessible to everyone who has standard cleanroom facilities and a conventional fluorescence microscope in their own place.

The overall goal of the protocol is to prepare over one million ordered, uniform, stable, and biocompatible femtoliter droplets on a 1 cm2 planar substrate that can be used for cell-free protein synthesis.