Multiquantum photoreactions of nucleic acid components in aqueous solution by powerful ultraviolet picosecond radiation

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.

Section: Introductionsupporting

confidence: 68%

Comparison of Femtosecond Laser and Continuous Wave UV Sources for Protein–Nucleic Acid Crosslinking

Fecko¹,

Munson²,

Saunders³

et al. 2007

“…The decrease of the pulse duration from nanoseconds to picoseconds and simultaneous increase in the intensity from 10" to 10l2 W/m2 does not evoke any profound changes in the relative efficiency of ASLs at deoxyguanosine residues in comparison with deoxycytidine ones. This may be due to the alteration of the way in which higher excited level is populated because the second photon is absorbed in the S, state in the case of picosecond pulses (Kryukov et al, 1979).…”

Section: Single-and Two-quantum Rnodijcation Of Dna Residues By Lasermentioning

confidence: 99%

“…It has been shown previously that two-quantum reactions, as well as ordinary, single-quantum reactions, take place in nucleic bases, nucleosides and oligonucleotides when liquid aqueous solutions of these are exposed to UV laser radiation of an intensity over 10' W/m2 (nanosecond pulses) and over 10l2 W/m2 (picosecond pulses). These two-quantum reactions result in the base modification-mainly the opening and saturation of hetero-cycles (Kryukov et al, 1979;Budowsky et al, 1981;Yakovlev et al, 1984), the cleavage of the N-glycosidic (Menshonkova et al, 1980) and phosphoester bond (Budowsky et al, 1987). Earlier observations have shown that the action of UV laser radiation on DNA and polynucleotides leads to a considerable (as compared to the low-intensity irradiation) increase in the efficiency of direct single-strand breaks via the two-quantum mechanism (Zavilgelsky et al, 1984;Optiz and Schulte-Frohlinde, 1987;Croke et al, 1988).…”

Section: Introductionmentioning

confidence: 99%

Sequence‐specificity of the Alkali‐sensitive Lesions Induced in Dna by High‐intensity Ultraviolet Laser Radiation

Kovalsky

Panyutin

Budowsky

1990

The action of high-intensity (10(9)-10(12) W/m2) UV (266 nm) laser radiation pulses (duration ca 10 ns or ca 40 ps) on liquid aqueous solutions of DNA is known to cause not only single- but also two-quantum modification of nucleic bases. The action of hot piperidine on the laser-irradiated DNA results in non-random splitting of polynucleotide chain. Hence, at least some of the modified nucleoside residues are alkali-sensitive lesions (ASLs). The distribution of ASLs along the DNA chain shows that the position of these lesions corresponds with pyrimidines in the PyPy sequences (similar to those formed via single-quantum conversions) as well as with deoxyguanosine residues. The last ASLs result from two-quantum reactions and occur much more efficiently than the direct photo-induced cleavage of the internucleotide (phosphodiester) bond. It has been shown with fragments of plasmids pUC18, pUC19 and pBR322 (total length over 600 base pairs) that the relative efficiency of ASLs at deoxyguanosine sites depends on the primary structure context and can differ by an order of magnitude. The highest efficiency of modification is observed when a purine is 3' neighbour to the 2'-deoxyguanosine, i.e. at 5'-GPu-3' sites. However, considerable variations in the modification efficiency were also found in these sequences.

“…(19) is valid for irradiation intensities lower than the one at which saturation comes on in the population of the intermediate level S1 or T 1 (Oraevsky eta/., 1981;Nikogosyan et al, 1982). When the intermediate level is saturated and, as a consequence, saturation is reached in the efficiency of the photochemical reaction proceeding from the upper excited state, the amount of the product of this reaction forming during the pulse becomes proportional (at a standard pulse duration) to the radiation intensity (Kryukov et a/., 1979;. In this case, as well as for single-quantum reactions, the quadratic intensity-dependence of the two quantum photoreaction efficiency is not observed (cf.…”

Section: Relative Population Of Excited Statesmentioning

confidence: 99%

“…Electronic structure is the major determinant of the directions and rates of the chemical reactions in molecules. Therefore, the twoquantum photochemistry (via the states H) is characterized by final products and rates which are extraordinary from the point of view of the classical, single-quantum photochemistry (via the states sl and T 1) (Kryukov et al, 1979;Simukova et al, 1980;Menshonkova et al, 1980;Rubin eta/., 1981;Gurzadyan eta/., 1981;Nikogosyan and Letokhov, 1983;Yakovlev eta/., 1984;Budowsky eta/., 1985;Cadet et al, 1987;Croke eta/., 1988). In view of this fact and the short duration of the pulse (from nanoto femtoseconds), the use of UV pulse laser sources appears most promising for studying the structure and function of biological macromolecules and their complexes.…”

Section: Introductionmentioning

confidence: 99%

Laser (Two‐quantum) Photolysis of Polynucleotides and Nucleoproteins: Quantitative Processing of Results

Ei²

1990