Ultraviolet Shadowing of RNA Causes Substantial Non-Poissonian Chemical Damage in Seconds

TL;DR

UV shadowing rapidly causes significant, non-random chemical damage to RNA molecules within seconds, potentially affecting high-precision RNA studies by introducing artifacts and heterogeneity.

Contribution

This study quantitatively characterizes UV-induced RNA damage, revealing rapid, widespread, and non-Poissonian lesions that challenge assumptions about RNA integrity during gel visualization.

Findings

Damage detectable within seconds of UV exposure

Damage distribution is more heterogeneous than Poisson

200-nt RNAs can incur 20% damage in 20 seconds

Abstract

Chemical purity of RNA samples is critical for high-precision studies of RNA folding and catalytic behavior, but such purity may be compromised by photodamage accrued during ultraviolet (UV) visualization of gel-purified samples. Here, we quantitatively assess the breadth and extent of such damage by using reverse transcription followed by single-nucleotide-resolution capillary electrophoresis. We detected UV-induced lesions across a dozen natural and artificial RNAs including riboswitch domains, other non-coding RNAs, and artificial sequences; across multiple sequence contexts, dominantly at but not limited to pyrimidine doublets; and from multiple lamps that are recommended for UV shadowing in the literature. Most strikingly, irradiation time-courses reveal detectable damage within a few seconds of exposure, and these data can be quantitatively fit to a 'skin effect' model that accounts for the increased exposure of molecules near the top of irradiated gel slices. The results indicate that 200-nucleotide RNAs subjected to 20 seconds or less of UV shadowing can incur damage to 20% of molecules, and the molecule-by-molecule distribution of these lesions is more heterogeneous than a Poisson distribution. Photodamage from UV shadowing is thus likely a widespread but unappreciated cause of artifactual heterogeneity in quantitative and single-molecule-resolution RNA biophysical measurements.