Stochastic detection of motor protein-RNA complexes by single-channel current recording

A label- and immobilization-free approach to detecting the reversible formation of complexes between nucleic acids and proteins at the single-molecule level is described. The voltage-driven translocation of individual oligoribonucleotides through a nonoscole protein pore is observed by single-channel current recordings. The oligoribonucleotide 5'-C(25)A(25)-3' gives rise to current blockades with an average duration of approximately 0.5 ms. In the presence of the RNA-binding ATPase P4, a viral packaging motor from bacteriophoge phi 8, longer events of tens to hundreds of milliseconds are observed. Upon addition of ATP the long events disappear, indicating the dissociation of the P4.RNA complex. The frequency of events also depends on the concentration of P4 and the length of the oligoribonucleotide, thereby confirming the specificity of the P4.RNA events. This study shows that single-chonnel current recordings can be used to monitor RNA-protein complex formation, thus opening up a new means to examine the motor activity of RNA- or DNA-processing enzymes.