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

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

2012-02-21

arxiv.org/abs/1202.4794v1

Biology

Quantitative Biology

Biomolecules

Scientific paper

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.

Affiliated with

Also associated with

No associations

Rating

Ultraviolet Shadowing of RNA Causes Substantial Non-Poissonian Chemical Damage in Seconds does not yet have a rating. At this time, there are no reviews or comments for this scientific paper.

If you have personal experience with Ultraviolet Shadowing of RNA Causes Substantial Non-Poissonian Chemical Damage in Seconds, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Ultraviolet Shadowing of RNA Causes Substantial Non-Poissonian Chemical Damage in Seconds will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFWR-SCP-O-413654