title: Use of the SYBR Green dye for measuring helicase activity.
abstract: Here we describe a non-radioactive assay that exploits the fluorescent dye SYBR Green to measure the helicase enzyme activity. SYBR Green I emits fluorescence upon intercalation with double-stranded DNA or RNA. The fluorescence is lost proportionally as the nucleic acid is converted to single strands by a helicase, and this decrease in fluorescence intensity can be used to measure the activity of the helicase enzyme. The reaction was prepared by mixing a double-stranded substrate with the helicase enzyme, buffer, ATP and SYBR Green I. After completion, the reaction was terminated by EDTA and fluorescence was measured. Using this technique, a linear increase in substrate release was observed with increasing time and helicase concentrations. The assay described here is speedy, efficient and economical; it holds promise for use in large-scale screening of drugs that target helicases.
authors: Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi, Pakistan.
authors: Sammer
authors: S
authors: Siddiqui
authors: Irfan
authors: I
authors: Khan
authors: Shamshad
authors: S
authors: Zarina
authors: Syed
authors: S
authors: Ali
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000952:Antigens, Polyomavirus Transforming / Q000032:analysis* / Q000378:metabolism; D052160:Benzothiazoles; D002939:Ciprofloxacin / Q000494:pharmacology; D004265:DNA Helicases / Q000032:analysis* / Q000378:metabolism; D003959:Diamines; D005456:Fluorescent Dyes / Q000032:analysis*; D005470:Fluorometry / Q000379:methods*; D016174:Hepacivirus / Q000201:enzymology; D009695:Nucleic Acid Renaturation; D009841:Oligonucleotides / Q000378:metabolism; D009930:Organic Chemicals / Q000032:analysis*; D011804:Quinolines; D013539:Simian virus 40 / Q000201:enzymology; D017361:Viral Nonstructural Proteins / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000255:Adenosine Triphosphate; D000952:Antigens, Polyomavirus Transforming; D052160:Benzothiazoles; D002939:Ciprofloxacin; D004265:DNA Helicases; D003959:Diamines; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D009930:Organic Chemicals; D011804:Quinolines; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C098022:SYBR Green I