Analysis of the RNase T1 mediated cleavage of an immobilized gapped heteroduplex via fluorescence correlation spectroscopy

We report a new method for studying the activity of hydrolytic enzymes. Flu orescence correlation spectroscopy was used to observe online the hydrolyza tion of a rhodamine B-labeled substrate by ribonuclease T1. A gapped hetero duplex substrate - a hybrid of a ribooligonucleotide and two smaller comple mentary deoxyribooligonucleotides - was immobilized via biotin to a strepta vidin-coated surface of a coverslip. The reported method opens the possibil ity to study the cleavage of small substrates differing only slightly in mo lecular weight from the enzyme reaction product. The use of fluorescence co rrelation spectroscopy allows the detection of very low enzyme concentratio ns (down to 10(-21) mol 0.05 fM of RNase T1, corresponding to about 600 RNa se T1 molecules in 0.02 ml).