REEAD hTOP1 Activity Kit Fluorescent Readout

The VPCIR™ REEAD hTOP1 Activity Kit is designed to provide an easy, sensitive, specific, and quantitative enzymatic activity detection of the human type 1B topoisomerase. REEAD stands for Rolling circle Enhanced Enzyme Detection, and the assay is based on the ability of human topoisomerase 1 to cleave and ligate DNA, converting a DNA substrate into a closed circle (1 and 2). The generated circles are hybridized to a functionalized glass slide and subsequently amplified by rolling circle amplification (3) mediated by the phi29 polymerase. The amplification gives rise to fluorescent rolling circle products (4), allowing for the visualization in a fluorescence scanner or fluorescence microscope. This allows for the quantitative measurements of 20+ samples faster than the state of the gel-based relaxation assay, giving reproducible one-day results.

APPLICATION
Type 1B eukaryotic topoisomerases are among the DNA binding enzymes with biological relevance, because they are targets of small-molecule drugs with potential activity against human cancers or pathogens causing diseases in humans. This kit provides a fast, reliable, and quantitative detection of human topoisomerase 1 and can be used as a drug screening tool. It allows to screen the efficiency of libraries of natural or synthetic small-molecule compounds as potential inhibitors of human topoisomerase 1. Moreover, since the conversion of the DNA substrate to a closed circle relies specifically on the activity of human topoisomerase 1, the assay can be used to measure enzyme activity in small crude biological samples such as cell-, or tissue extracts in the context of biological or cancer research.

KIT CONTENT
Slide mailer containing
• Functionalized slide(s)
A box containing
• DNA substrate and primer
• Phi29 enzyme
• human topoisomerase 1 enzyme for positive control
• Phi29 reaction buffer
• human topoisomerase 1 reaction buffer
• dNTPs
• Buffers

N/A

The kit components require different storage conditions:
• The functionalized slide(s) must be stored in the provided sealed foil-bag at 15-
30°C until use. Upon opening of the bag, the slide(s) need to be used immediately.
• The enzyme and reagents must be stored at -20°C upon arrival.
• Buffer 1 and Buffer 2 must be stored at 50°C.
• Buffer 3 and Buffer 4 must be stored at 15-30°C.

N/A

Safety Data Sheet for REEAD topoisomerase 1 activity kits

1. Stougaard, M.; Lohmann, J.S.; Mancino, A.; Andersen, F.F.; Koch, J.; Knudsen, B.R. Single-Molecule Detection of Human Topoisomerase I cleavge-Ligation Activity. 2009, 3, 223–233.

2. Andersen, F.F.; Stougaard, M.; Jørgensen, H.L.; Bendsen, S.; Juul, S.; Hald, K.; Andersen, A.H.; Koch, J.; Knudsen, B.R. Multiplexed detection of site specific recombinase and DNA topoisomerase activities at the single molecule level. ACS Nano 2009, 3, 4043–54, doi:10.1021/nn9012912.

3. Tesauro, C.; Keller, J.G.; Gromova, I.; Gromov, P.; Frøhlich, R.; Erlandsen, J.U.; Andersen, A.H.; Stougaard, M.; Knudsen, B.R. Different Camptothecin Sensitivities in Subpopulations of Colon Cancer Cells Correlate with Expression of Different Phospho-Isoforms of Topoisomerase I with Different Activities. Cancers (Basel). 2020, 1240, doi:10.3390/cancers12051240.

4. Tesauro, C.; Simonsen, A.K.; Andersen, M.B.; Petersen, K.W.; Kristoffersen, E.L.; Algreen, L.; Hansen, N.Y.; Andersen, A.B.; Jakobsen, A.K.; Stougaard, M.; et al. Topoisomerase I activity and sensitivity to camptothecin in breast cancer-derived cells : a comparative study. BMC Cancer 2019, 1–15.

5. Alonso, C.; Fuertes, M.; Martín-Encinas, E.; Selas, A.; Rubiales, G.; Tesauro, C.; Knudssen, B.K.; Palacios, F. Novel topoisomerase I inhibitors. Syntheses and biological evaluation of phosphorus substituted quinoline derivates with antiproliferative activity. Eur. J. Med. Chem. 2018, 149, doi:10.1016/j.ejmech.2018.02.058.

6. Pommier Editor, Y. DNA Topoisomerases and Cancer; Press, H., Ed.; 2011;

7. Keller, J.G.; Tesauro, C.; Coletta, A.; Graversen, A.D.; Ho, Y.-P.; Kristensen, P.; Stougaard, M.; Knudsen, B.R. On-slide detection of enzymatic activities in selected single cells. Nanoscale 2017, 9, doi:10.1039/c7nr05125e.

8. Keller JG, Mizielinski K, Petersen KV, Stougaard M, Knudsen BR, Tesauro C. Simple and Fast Rolling Circle Amplification-Based Detection of Topoisomerase 1 Activity in Crude Biological Samples. J Vis Exp. 2022. doi: 10.3791/64484.

9. Keller, JG, Petersen KV, Knudsen BR, Tesauro C. Simple and Fast DNA-Based Tool to Investigate Topoisomerase 1 Activity, a Biomarker for Drug Susceptibility in Colorectal Cancer. Recent Understanding of Colorectal Cancer Treatment, IntechOpen. 2022. doi:10.5772/intechopen.105758.

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