This work was supported by grants from the Pittsburgh Foundation and the National Institutes of Health (NIH) [GM087798; CA148629; ES019498] to RWS. Support for the UPCI Lentiviral Facility was provided to RWS by the Cancer Center Support Grant from the National Institutes of Health [P30 CA047904]. Support was also provided by the University of Pittsburgh Department of Pharmacology &amp; Chemical Biology to DS. Support was also provided by ESTRO to CV.

1. Molecular Beacon Design
1.1 Designing and ordering your molecular beacon
The design of your molecular beacon must consider the following:
<ol class="lalpha">
 <li>We have good results with 43-mer DNA oligonucleotides. The GC content should be &gt;32%. Exclude a G base at the 5' end.</li>
 <li>Request 6-FAM (6-Carboxyfluorescein) conjugation on the 5' end and Dabcyl as a 3' modification.</li>
 <li>The position of the lesion should be on base #6 from the 5' end.</li...

<ol>
	<li>Mutamba, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XRCC1+and+base+excision+repair+balance+in+response+to+nitric+oxide.">XRCC1 and base excision repair balance in response to nitric oxide.</a> DNA Repair (Amst). 10, 1282-1293 (2011).</li><li>Tang, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-methylpurine+DNA+glycosylase+and+DNA+polymerase+beta+modulate+BER+inhibitor+potentiation+of+glioma+cells+to+temozolomide.">N-methylpurine DNA glycosylase and DNA polymerase beta modulate BER inhibitor potentiation of glioma cells to temozolomide.</a> Neuro-oncology. 13, 471-486 (2011).</li><li>Clegg, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+resonance+energy+transfer+and+nucleic+acids.">Fluorescence resonance energy transfer and nucleic acids.</a> Methods Enzymol. 211, 353-388 (1992).</li><li>Yaron, A., Carmel, A., Katchalski-Katzir, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramolecularly+quenched+fluorogenic+substrates+for+hydrolytic+enzymes.">Intramolecularly quenched fluorogenic substrates for hydrolytic enzymes.</a> Anal. Biochem. 95, 228-235 (1979).</li><li>Kreklau, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+fluorometric+oligonucleotide+assay+to+measure+O(+6)-methylguanine+DNA+methyltransferase,+methylpurine+DNA+glycosylase,+8-oxoguanine+DNA+glycosylase+and+abasic+endonuclease+activities:+DNA+repair+status+in+human+breast+carcinoma+cells+overexpressing+methylpurine+DNA+glycosylase.">A novel fluorometric oligonucleotide assay to measure O( 6)-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase.</a> Nucleic Acids Res. 29, 2558-2566 (2001).</li><li>Belon, C. A., Frick, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+helicase+activity+with+molecular+beacons.">Monitoring helicase activity with molecular beacons.</a> BioTechniques. 45, 433-442 (2008).</li><li>Ma, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+DNA+polymerase+activity+using+molecular+beacon.">Real-time monitoring of DNA polymerase activity using molecular beacon.</a> Anal. Biochem. 353, 141-143 (2006).</li><li>Summerer, D., Marx, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+beacon+for+quantitative+monitoring+of+the+DNA+polymerase+reaction+in+real-time.">A molecular beacon for quantitative monitoring of the DNA polymerase reaction in real-time.</a> Angew. Chem. Int. Ed. Engl. 41, 3620-3622 (2002).</li><li>Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+molecular+beacon+to+monitor+activity+of+E.+coli+DNA+ligase.">Using molecular beacon to monitor activity of E. coli DNA ligase.</a> The Analyst. 130, 350-357 (2005).</li><li>Xiao, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+beacons+for+the+detection+of+DNA+and+telomerase+activity.">Catalytic beacons for the detection of DNA and telomerase activity.</a> J. Am. Chem. Soc. 126, 7430-7431 (2004).</li><li>Kundu, L. M., Burgdorf, L. T., Kleiner, O., Batschauer, A., Carell, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavable+substrate+containing+molecular+beacons+for+the+quantification+of+DNA-photolyase+activity.">Cleavable substrate containing molecular beacons for the quantification of DNA-photolyase activity.</a> Chembiochem : a European journal of chemical biology. 3, 1053-1060 (2002).</li><li>Sokol, D. L., Zhang, X., Lu, P., Gewirtz, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+detection+of+DNA.RNA+hybridization+in+living+cells.">Real time detection of DNA.RNA hybridization in living cells.</a> Proc. Natl. Acad. Sci. U.S.A. 95, 11538-11543 (1998).</li><li>Yang, X., Tong, C., Long, Y., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+fluorescence+assay+for+hSMUG1+activity+based+on+modified+molecular+beacon.">A rapid fluorescence assay for hSMUG1 activity based on modified molecular beacon.</a> Molecular and cellular probes. 25, 219-221 (2011).</li><li>Mirbahai, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+molecular+beacon+to+track+the+activity+of+base+excision+repair+protein+OGG1+in+live+cells.">Use of a molecular beacon to track the activity of base excision repair protein OGG1 in live cells.</a> DNA Repair (Amst). , (2009).</li><li>Maksimenko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+molecular+beacon+assay+for+measuring+base+excision+repair+activities.">A molecular beacon assay for measuring base excision repair activities.</a> Biochem. Biophys. Res. Commun. 319, 240-246 (2004).</li></ol>

There are over 600 citations using the term &#x2018;molecular beacon' for detecting and quantifying numerous molecules and enzymatic activities, including helicase activity6, DNA polymerase activity7,8, DNA ligase activity9, telomerase activity10, DNA photolyase activity11 and DNA/RNA hybrids12, among many others. In addition to the use of molecular beacons for the measurement of the DNA repair activities listed above, we and others have demonstrated the ...

<table border="1">
<tbody>
 <tr>
 <td>Name of 			the reagent</td>
 <td>Company</td>
 <td>Catalog 			number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Potassium 			Chloride</td>
 <td>Fisher</td>
 <td>P217-500</td>
 <td>Molecular 			grade</td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP120-500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Glycerol</td>
 <td>Fisher</td>
 <td>BP229-1</td>
 <td>Molecular 			grade</td>
 </tr>
 <tr>
 <td>DTT</td>
 <td>Fisher</td>
 <td>BP172-5</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES- 			Solution</td>
 <td>Invitrogen</td>
 <td>15630</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>HEPES-Salt</td>
 <td>Sigma</td>
 <td>H4034-500G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Potassium 			Hydroxide</td>
 <td>Sigma</td>
 <td>17-8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.22&mu;M 			filter</td>
 <td>Nalgene</td>
 <td>167-0020</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Slide-A-Lyzer 			Dialysis Products</td>
 <td>Pierce</td>
 <td>66373</td>
 <td>MW 7000</td>
 </tr>
 <tr>
 <td>Syringe</td>
 <td>Fisher</td>
 <td>309659</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Needle</td>
 <td>Fisher</td>
 <td>305196</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1.5 mL 			Microcentrifuge Tubes</td>
 <td>Fisher</td>
 <td>05-408-129</td>
 <td>Black </td>
 </tr>
 <tr>
 <td>15 mL Falcon 			Tubes</td>
 <td>Fisher</td>
 <td>352097</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NucBuster 			Protein Extraction Kit</td>
 <td>Calbiochem</td>
 <td>71183</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Invitrogen</td>
 <td>14190</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Molecular 			Beacons</td>
 <td>IDT</td>
 <td>&nbsp; </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>BioRad 			Protein assay dye reagent concentrate</td>
 <td>BioRad</td>
 <td>Cat# 500-0006</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

quantitative, real-time analysis of base excision repair activity in cell lysates utilizing lesion-specific molecular beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

Watch this Scientific Journal Video about Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons at JoVE.com

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

quantitative-real-time-analysis-base-excision-repair-activity-cell

Research

JoVE Journal

Biology

13.5K visualizações.  University of Pittsburgh School of Medicine. Nós descrevemos um método para a quantitativa, a medição em tempo real de DNA glicosilase e as actividades de endonucleases de AP em células lisados ​​nucleares. O ensaio produz taxas de actividade de reparação de ADN passíveis de análise cinética e é adaptável para a quantificação da actividade de reparação de ADN em tecido e lisados ​​tumorais ou com proteínas purificadas.