Time-Resolved Infrared Spectroscopy of RNA Folding

We introduce time-resolved infrared spectroscopy as a powerful method to study the kinetics of RNA folding and unfolding transitions. A laser-induced temperature jump is used to initiate a perturbation in the RNA structure. A probe laser, tuned to a specific infrared absorption of the RNA, is then used to monitor the subsequent relaxation kinetics. A 10-ns pump pulse permits the investigation of fast, nanosecond events. In this work we probe two vibrational transitions, one at 1620 cm−1 and one at 1661 cm−1. The former transition reports mainly on the dynamics of A and U interactions, the latter is attributed to mainly G and C interactions. Our results reveal three distinct kinetic phases for each vibrational transition probed. We propose two models to describe the data. In one mechanism, the unfolded state partitions into two separate populations; each is conformationally biased to proceed via one of two distinct pathways. In an alternative model, folding proceeds through a series of sequentially populated intermediates. In both cases, the first step in the proposed folding mechanism is rate limiting (hundreds of microseconds) and involves a collapse into incorrectly folded intermediate populations. Two faster kinetic phases (tens of microseconds and hundreds of nanoseconds) follow in which the intermediate populations undergo localized reorganizational motions in the search for native contacts.