Name: single molecule analysis of laser localized psoralen adducts
Uploaded: 2017-04-20T12:30:00
Description: Watch this Scientific Journal Video about Single Molecule Analysis of Laser Localized Psoralen Adducts at JoVE.com

This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (Z01 AG000746-08) and the Fanconi Anemia Research Fund.

1. Preparation of Dig-TMP
<ol>
	<li>Mix 50 mg (0.18 mmoles) of 4&#39;-chloromethyl-4,5&#39;,8-trimethylpsoralen and 590 mg (2.7 mmoles) of 4,7,10-trioxa-1,13-tridecanedi-amine in a dry 25 mL round-bottom flask under nitrogen. Add 10 mL toluene and reflux for 12 h. Remove solvent in a rotary evaporator under reduced pressure.
	<ol>
		<li>Purify the residue by flash column chromatography over silica gel. Elute the column with chloroform, methanol, and 28% ammonia solution (9:1:0.5). Evaporate the solvent...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+the+large-scale+synthesis+of+psoralen+furan-side+monoadducts+and+diadducts.">Methods for the large-scale synthesis of psoralen furan-side monoadducts and diadducts.</a> Proc. Natl. Acad. Sci. U.S.A. 89 (10), 4514-4518 (1992).</li><li>Huang, J., 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=The+DNA+translocase+FANCM/MHF+promotes+replication+traverse+of+DNA+interstrand+crosslinks.">The DNA translocase FANCM/MHF promotes replication traverse of DNA interstrand crosslinks.</a> Mol. 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The laser localization technology requires the use of adherent cells with nuclei that are visible in bright field microscopy. We have tried to attach nonadherent cells, such as primary lymphocytes, or loosely adherent cultured cells such as AD293, to the glass surface with cell adhesive preparations such as polylysine or collagen, or more complex mixtures. Although these treatments may bind the cells to the surface, we find that they generally stay rounded making it very difficult to focus into the nuclei. Furthermore, t...

DNA is under constant assault from exogenous agents such as radiation, ultraviolet light, environmental toxins, combustion products, etc. Additionally, it is also attacked by endogenous radical species produced by oxidative metabolism. All of these have the potential to chemically or physically disrupt the integrity of DNA 1. Perturbations in the genome can activate the DNA Damage Response (DDR), a recruitment and post translational modification cascade with hundreds, if not thousands, of proteins and microRNAs involved in lesion repair, regulation of the cell cycle, apoptosis, senescence, and inflammatory pathways 2.
Most of our information about the DDR comes from studies with DSBs. This is in large part due to the availability of technologies for introducing breaks, including sequence specific breaks, in genomic DNA in living cells 3. In addition, the propensity of breaks to induce foci of DDR proteins, which can be displayed by immunofluorescence, has been very helpful for identifying the kinetics and requirements of responding proteins. One of the key technologies for studying the DDR was introduced by Bonner and colleagues, who used a laser beam to direct a stripe of DSBs in a &#34;Region of Interest&#34; (ROI) in the nuclei of living cells 4. In effect, they created a lengthy focus in which proteins of the DDR could be identified by immunofluorescence. This was illustrated by their demonstration of the strong stripe of phosphorylated histone H2AX (&#947;-H2AX) in the laser exposed cells. Since then, the laser approach has been employed in numerous studies of the DDR induced by DSBs. Although powerful and popular, and the source of dramatic immunofluorescence images, it should be noted that in most experiments the laser intensity is adjusted so as to produce observable results, without concern for lesion identity, density, or spacing. Indeed, it can be difficult to make these estimates. Thus they are largely ignored, despite the multiplicity of lesions introduced into DNA by lasers 5. This contributes to the many contradictions in the literature 6.
In contrast to DSBs, most chemical modifications of DNA do not stimulate the formation of discrete foci of DDR proteins. This is important in the light of our current understanding of lesion frequencies. It has been estimated that human cells in culture incur as many as 50 DSBs per cell cycle, formed largely during S phase 7,8,9. Fewer are formed in non-proliferating cells. This contrasts with the number of nucleobase losses or modification events, which are in the tens of thousands per cell/day 1,10. Thus, we know most about the DDR induced by events that are relatively rare, and much less about those induced by helix distorting lesions, which in aggregate are far more common.
In order to address questions about the cellular response to covalent modifications of genomic DNA, we wanted to work with a helix distorting DNA adduct that had inherent DDR induction activity. Furthermore, to facilitate experimental design and interpretation we were interested in a structure whose introduction could be controlled with respect to time and was amenable to visualization. Accordingly, we developed a strategy based on psoralen. Psoralens are well characterized photoactive DNA intercalators favoring 5&#39; TA:AT sites. Unlike other crosslinking agents such as nitrogen mustards and mitomycin C (MMC) they are not DNA reactive unless exposed to long wave UV (UVA) light. The intercalated molecules react with thymine bases on opposite strands to produce helix distorting interstrand crosslinks (ICLs) 11. With the trimethyl psoralen used in our experiments most products are ICLs, relatively few monoadducts are generated (less than 10%) 12, and intrastrand crosslinks between adjacent bases on one strand are not formed. Because they are powerful blocks to replication and transcription, psoralen and other crosslinking agents, like cis-platinum and MMC, are commonly used in chemotherapy. Thus psoralen enabled studies that followed the activation of the DDR by a helix distorting structure, and also provided insight into the cellular response to a compound with clinical importance.
We synthesized a reagent in which trimethyl psoralen was linked to digoxigenin (Dig), a plant sterol not found in mammalian cells and frequently used as an immunotag. The requirement for photoactivation permits localization by laser light (365 nm) of psoralen ICLs in defined ROI in nuclei in living cells. These can be displayed by immunofluorescence against the Dig tag. DNA repair and DDR proteins appeared in the stripes of laser localized ICLs 13,14.
The DDR activated by the high laser intensities used to produce DSBs could be due to isolated or clustered damage 15,16. Consequently, the relevance of results from these experiments to naturally occurring lesions, present at much lower concentration, is uncertain. To address similar questions about psoralen adduct frequency and spacing in DNA, we took advantage of DNA fiber technology 17 and immunoquantum dots. Quantum dots are much brighter than fluorescent dyes and are not bleached by exposure to light. Thus they are frequently used for single molecule imaging 18, an application for which fluorescent dyes are insufficiently bright. Individual DNA fibers can be stretched on glass slides and can be displayed by immunofluorescence against nucleoside analogues incorporated during incubations prior to cell harvest. We treated cells with Dig-psoralen and exposed the ROI to laser micro irradiation. Fibers were prepared from the cells and individual Dig-psoralen adducts could be visualized with the immunoquantum dots. Exposing the cells to nucleoside analogues for relatively short times (20-60 min) permits the display of replication tracts in the vicinity of the laser localized ICLs.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Digoxigenin NHS ester</td>
			<td>Sigma-Aldrich</td>
			<td>11333054001</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloro-psoralen</td>
			<td>Berry and Associates</td>
			<td>PS 5000</td>
			<td></td>
		</tr>
		<tr>
			<td>diaminoglycol</td>
			<td>Sigma-Aldrich</td>
			<td>369519</td>
			<td>4,7,10-Trioxa-1,13-tridecanediamine</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Acros Organics</td>
			<td>423550040</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher Scientific</td>
			<td>A4524</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium solution</td>
			<td>Sigma-Aldrich</td>
			<td>5002</td>
			<td></td>
		</tr>
		<tr>
			<td>TLC plates</td>
			<td>Analtech, Inc.</td>
			<td>P02511</td>
			<td></td>
		</tr>
		<tr>
			<td>Flass glass column 24/40, 100ml</td>
			<td>Chemglass Life Sciences</td>
			<td>CG-1196-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon T2000_E2 spinning disk confocal microscope, equipped with automated stage and environmental control chamber and plate holder</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>With Volocity Software</td>
		</tr>
		<tr>
			<td>Micropoint Galvo&#160;</td>
			<td>Andor Technologies</td>
			<td></td>
			<td>with a Nitrogen pulsed laser&#160;</td>
		</tr>
		<tr>
			<td>dye cell</td>
			<td>Andor Technologies</td>
			<td>MP-2250-2-365</td>
			<td></td>
		</tr>
		<tr>
			<td>365 dye</td>
			<td>Andor Technologies</td>
			<td>MP-27-365-DYE</td>
			<td></td>
		</tr>
		<tr>
			<td>IdU&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>17125</td>
			<td></td>
		</tr>
		<tr>
			<td>35mm glass botomm plates 1.5 coverslip, 10mm glass diameter, uncoated</td>
			<td>Matek</td>
			<td>P35G-1.5-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>New Comer Supply</td>
			<td>Part # 5070</td>
			<td>New Silane Slides</td>
		</tr>
		<tr>
			<td>Mouse anti BrdU antibody (IdU)</td>
			<td>BD Biosciences</td>
			<td>347580</td>
			<td>1 in 40</td>
		</tr>
		<tr>
			<td>Rat anti BrdU Antibody (CldU)</td>
			<td>Abcam</td>
			<td>ab6326</td>
			<td>1 in 200&#160;</td>
		</tr>
		<tr>
			<td>Rabbit anti Dig antibody</td>
			<td>ThermoFisher Scientific</td>
			<td>710019</td>
			<td>1 in 200</td>
		</tr>
		<tr>
			<td>Q-dot 655 goat anti Rabbit IgG</td>
			<td>ThermoFisher Scientific</td>
			<td>Q-11421MP</td>
			<td>1 in 5000</td>
		</tr>
		<tr>
			<td>AF647- goat anti Rat IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>112-605-167</td>
			<td>1 in 100</td>
		</tr>
		<tr>
			<td>AF488-goat anti mouse IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>115-545-166</td>
			<td>1 in 100</td>
		</tr>
		<tr>
			<td>Zeiss epifluorescent microscope A200</td>
			<td>Zeiss</td>
			<td></td>
			<td>&#160;with Axiovision software</td>
		</tr>
		<tr>
			<td>Q-dot 655 filter</td>
			<td>Chroma</td>
			<td>39107</td>
			<td></td>
		</tr>
	</tbody>
</table>

single molecule analysis of laser localized psoralen adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Laser localized Dig-TMP (Figure 1A) ICLs can be displayed by immunofluorescence against the Dig-tag linked to the psoralen. Although the laser can be directed to strike in an area of any contour, stripes are not &#34;natural&#34; shapes in cells, and legitimate signals can be easily distinguished from artifacts due to non-specific binding by primary or secondary antibodies. This feature is helpful when using antibodies of less than perfect specificity. An example of the w...

Watch this Scientific Journal Video about Single Molecule Analysis of Laser Localized Psoralen Adducts at JoVE.com

Single Molecule Analysis of Laser Localized Psoralen Adducts

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

The overall goal of this procedure is to perform single molecule analysis of the distribution of laser localized interstrand crosslinks in genomic DNA in order to study cellular responses to DNA interstrand crosslinks. This method can help answer key questions in the DNA repair field, regarding the distribution of DNA lesions introduced by laser technology, a widely used experimental tool. The main advantage of this technique is that it allows direct imaging of DNA lesions introduced by the laser activated compound in genomic DNA.This has not been possible before. Demonstrating the procedure will be Julia Gichimu, and Hima Gali from our lab. Every time the targeting laser is used it is important to perform a mirror slide calibration to verify that the laser is hitting at the appropriate XYZ coordinates, and to establish the area of the field captured by the camera where the laser can be set to strike.Find a field on the slide that has been previously etched. Set the exposure time and laser power. Then adjust the focus to move to a nearby non targeted field on the mirror slide.Start the calibration procedure. The software will fire the laser to etch the mirror slide along horizontal and diagonal line. These lines define the boundaries of the field in which the laser can strike a target cell.Set several regions of interest, or ROIs, inside the field and fire the laser to confirm that it is targeting the selected areas and thus striking at the appropriate XYZ coordinates. A biological calibration must also be performed when setting up the microscope targeting laser system. The first step is to define the minimum laser power required to photoactivate trimethylpsoralen, or TMP, induce interstrand crosslinks, and provoke recruitment of a GFP tagged repair factor, for which kinetics can previously be characterized.In this laboratory a U2OS cell line stably transfacted with a plasmid expressing GFP fan one is used. As shown here, when cells are exposed to six micromolar TMP and 1.2%laser an image for 10 minutes, viewing the sequence of images as a video confirms the recruitment of GFP fan one to the targeted ROI. The second step is to verify that this power setting does not trigger recruitment in the absence of the compound.As shown here, when cells are exposed to 1.2%laser alone image for 10 minutes and the image is viewed as a video there is no recruitment of GFP fan one to the targeted ROI. Thirdly, it is important to define the minimum power setting that is able to blow out a cell. The ability to blow out a cell at this power setting should be tested as a routine biological calibration every day the targeting laser is used.Set the ROI inside the nucleus while avoiding the nucleoli. Adjust the focus. Set the laser power to 12%and fire.Finally, it is essential to set the focal plane at mid cell to achieve correct targeting. To demonstrate this three cells are targeted at three focal planes. Mid cell.Too high. And too low. Three minutes after targeting a stack of images spanning three microns is generated to examine the distribution of the GFP fan one stripe throughout the imaged focal plane.Only the cell targeted at mid cell shows a GFP fan one stripe in all Z planes. Prior to performing laser localization prepare a 35 millimeter glass bottom cell culture dishes for growing cells. With a marking pen make a vertical mark on the side of each dish.Use a diamond pen to scratch a cross in the center of the glass on the culture surface, such that the black stripe on the side of the dish is at the top of one arm of the cross. The cross is marked on the growth surface of the glass so that it and the cells will be in the same focal plane. Plate cells and sterilized cross marked culture dishes and incubate for 24 hours in one micromolar five chloro deoxyuridine to uniformly label DNA.On the day of the experiment, add digoxigenin linked TMP or dig TMP in 50%ethanol to the cell culture medium to a final concentration of 20 micromolar. Bring the medium to 37 degrees Celsius. Replace the medium in the culture dishes with dig TMP containing medium and incubate at 37 degrees Celsius and 5%CO2 for 30 minutes to allow the dig TMP to equilibrate.While the cells are incubating perform mirror slide calibration and 12%blow out of a cell as demonstrated earlier. Place the plate in the environmental chamber at 37 degrees Celsius with controlled CO2 and humidity on the microscope stage. Focus with the 60X oil objective on the intersection of the cross.Select the first field to target and use the shape tool to set the ROIs in individual cells located in the immediate vicinity of the intersection of the cross. Select nucleoplasmic areas outside the nucleoli. Adjust the Z focal plane to target the laser midway through the nuclei.Next set the power setting to the minimum required to activate the psoralen in the interstrand crosslink induced DNA damage response. 1.2%is used here. Fire the laser.After all cells in a field have been targeted, move the plate to a new field, staying close to the intersection of the cross. Repeat the procedure just demonstrated in 12 to 18 fields to target a total of 80 to 100 cells. To harvest the cells, remove the culture dish from the stage.Remove the medium and wash the cells with PBS solution. Remove the PBS and place a 10 microliter drop of a commercial trypsin EDTA solution on the intersection of the cross in the middle of the glass surface. Incubate for three to four minutes at room temperature and then draw the trypsin solution with detached cells into a pipette tip.Place the drop of trypsin EDTA with the cells at one end of silanized glass microscope slide. To lyse the cells and release the DNA add 10 microliters of 5%SDS solution and incubate for three to four minutes at room temperature, mixing gently with pipette tip, allowing the periphery of the pool to dry. Tilt the slide 20 degrees and allow the liquid to run down to the end.The hydrodynamic forces of the flowing liquid will extend and stretch the DNA fibers. Let the slide air dry for 10 minutes. Immerse the slide in a solution of three to one methanol to acetic acid and fix for 10 minutes.Then remove the slide from the fix solution and air dry again. At this point the slide can be stored indefinitely in 70%ethanol at 20 degrees Celsius. To process the slide for detection of dig signals on DNA fibers by fluorescence microscopy remove the slide with the stretched DNA from the 70%ethanol solution and allow it to air dry.Subsequently denature the DNA fibers in hydrogen chloride and then transfer them to a container of tris hydrochloridic at PH eight for neutralization. Next wash the samples in PBS. And finally, stain them with the appropriate antibodies following a previously published procedure.The structure of trimethylpsoralen with a dioxigenin tag is shown. Interstrand DNA crosslinks, or ICLs, can be displayed by immuno florescence against dig TMP. This example shows cells with an accumulation of a well known marker of the DNA damage response gamma H2AX, visualized in green, in ROIs containing laser localized ICLs visualized in red.The DAPI stained nucleus is in blue. The bright field shows a cell partially on the mark made by the diamond pen. Note that ROIs are placed outside the nucleoli.To determine the frequency and spacing of adducts cells were incubated in five chloro deoxyuridine for 24 hours prior to introduction of the ICLs. After harvesting the cells and spreading the DNA fibers the dig signals were detected with an amino quantum dot visualized in red in the five chloro deoxyuridine by amino florescence visualized in green. Analysis of the dig signals on the fibers reveal that the inter ICL distances range from less than 10 KB to greater than 160 KB.There was no evidence for clustered adducts.Following these guidelines it should be possible to extend this approach to other DNA adducts, such as oxidative lesions that can be localized by lasers of appropriate wavelength.

single-molecule-analysis-of-laser-localized-psoralen-adducts

Research

JoVE Journal

Genetics

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Metagenomic Analysis of Silage

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. 
In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

Metagenomics was used to investigate the microbiome of silage cattle feed. Analysis was performed by shotgun sequencing. This approach was used to characterize the composition of the microbial community within the cattle feed.

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (&#947;-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Studying DNA damage repair kinetics requires a system to induce lesions at defined sub-nuclear regions. We describe a method to create localized double-stranded breaks using a laser-scanning confocal microscope equipped with a 405 nm laser and provide automated procedures to quantify the dynamics of repair factors at these lesions.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.