title: Comparison of femtosecond laser and continuous wave UV sources for protein-nucleic acid crosslinking.
abstract: Crosslinking proteins to the nucleic acids they bind affords stable access to otherwise transient regulatory interactions. Photochemical crosslinking provides an attractive alternative to formaldehyde-based protocols, but irradiation with conventional UV sources typically yields inadequate product amounts. Crosslinking with pulsed UV lasers has been heralded as a revolutionary technique to increase photochemical yield, but this method had only been tested on a few protein-nucleic acid complexes. To test the generality of the yield enhancement, we have investigated the benefits of using approximately 150 fs UV pulses to crosslink TATA-binding protein, glucocorticoid receptor and heat shock factor to oligonucleotides in vitro. For these proteins, we find that the quantum yields (and saturating yields) for forming crosslinks using the high-peak intensity femtosecond laser do not improve on those obtained with low-intensity continuous wave (CW) UV sources. The photodamage to the oligonucleotides and proteins also has comparable quantum yields. Measurements of the photochemical reaction yields of several small molecules selected to model the crosslinking reactions also exhibit nearly linear dependences on UV intensity instead of the previously predicted quadratic dependence. Unfortunately, these results disprove earlier assertions that femtosecond pulsed laser sources provide significant advantages over CW radiation for protein-nucleic acid crosslinking.
authors.affiliation: School of Applied and Engineering Physics, Cornell University, Ithaca, NY, USA.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Fecko
authors.forename: Katherine M
authors.initials: KM
authors.lastname: Munson
authors.forename: Abbie
authors.initials: A
authors.lastname: Saunders
authors.forename: Guangxing
authors.initials: G
authors.lastname: Sun
authors.forename: Tadhg P
authors.initials: TP
authors.lastname: Begley
authors.forename: John T
authors.initials: JT
authors.lastname: Lis
authors.forename: Watt W
authors.initials: WW
authors.lastname: Webb
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D004249:DNA Damage; D007834:Lasers; D015394:Molecular Structure; D010777:Photochemistry; D017319:Photosensitizing Agents / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D035181:TATA-Box Binding Protein / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D014466:Ultraviolet Rays
chemical_list: D003432:Cross-Linking Reagents; D017319:Photosensitizing Agents; D011506:Proteins; D035181:TATA-Box Binding Protein