Stochastic Detection of Motor Protein–RNA Complexes by Single‐Channel Current Recording

Abstract

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 nanoscale protein pore is observed by single‐channel current recordings. The oligoribonucleotide 5′‐C₂₅A₂₅‐3′ 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 bacteriophage ϕ8, 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‐channel 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.