Peptide-mediated energy transfer between an anionic water-soluble conjugated polymer and Texas red labeled DNA for protease and nuclease activity study.
We report for the first time that peptide could serve as a medium to bring an anionic conjugated polymer and a dye-labeled DNA into close proximity for energy transfer. By taking advantage of the fluorescein (Fl)-labeled peptide-mediated energy transfer between poly(9,9-bis(4'-sulfonatobutyl)fluorene-alt-1,4-phenylene) sodium salt (PFP-SO(3)Na) and Texas red (TR)-labeled single-stranded DNA (ssDNA), we develop a homogeneous assay for detection and monitoring of protease and nuclease activity in one solution using peptide/DNA complexes as the substrate. The enzymes as a proof of concept are trypsin (protease) and S1 (nuclease), respectively. In the absence of enzyme, multistep fluorescence energy transfer occurs from PFP-SO(3)Na to Fl and TR and from Fl to TR, and the TR emission dominates the solution fluorescence. In the presence of trypsin, the peptide is cleaved into fragments; the relatively weak electrostatic interaction between the small peptide fragments and the polymer fails to bring the TR-ssDNA and the polymer into close proximity for energy transfer. There is a significant decrease in TR emission and an increase in PFP-SO(3)Na emission, and the solution fluorescence appears blue. When S1 nuclease is used to cleave TR-ssDNA, a significant decrease in TR emission and an obvious increase in Fl emission are found, and the solution fluorescence appears green. The developed assay is ideal for the detection of chemical and biological molecules with DNA or protein cleaving activities.
Department of Chemical and Biomolecular Engineering, 4 Engineering Drive 4, National University of Singapore, Singapore 117576.
Yong
Y
Zhang
Yanyan
Y
Wang
Bin
B
Liu
D000595:Amino Acid Sequence; D000838:Anions / Q000737:chemistry; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004720:Endonucleases / Q000032:analysis*; D004735:Energy Transfer; D005449:Fluorenes / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D010447:Peptide Hydrolases / Q000032:analysis*; D010455:Peptides / Q000737:chemistry*; D011108:Polymers / Q000737:chemistry; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism; D014867:Water / Q000737:chemistry; D014966:Xanthenes / Q000737:chemistry*
D000838:Anions; D004277:DNA, Single-Stranded; D005449:Fluorenes; D005456:Fluorescent Dyes; D010455:Peptides; D011108:Polymers; D014966:Xanthenes; C541741:poly(9,9-bis(4'-sulfonatobutyl)fluorene-alt-1,4-phenylene) sodium salt; D014867:Water; C034657:Texas red; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D010447:Peptide Hydrolases; D014357:Trypsin