Ultrafast Photodynamics of Nucleic Acids

Introduction

The recently completed map of the human genome and the ever-expanding crystallographic database of nucleic acid structures are two examples that illustrate the highly detailed information currently available about static properties of nucleic acids. In contrast, much less is known about dynamics. In this project we are studying the dynamics of the excited singlet states formed in DNA by ultraviolet (UV) light. Rising skin cancer rates and concern about anthropogenic modification of the ozone layer have heightened public awareness of the health risks of the suns UV rays.Understanding the fate of electronic energy in nucleic acids is a first step toward a molecular-level understanding of photocarcinogenesis.

Ultrafast Internal Conversion in Single Bases

Our group succeeded in directly measuring the singlet excited state lifetimes (i.e. fluorescence lifetimes) of a series of DNA and RNA nucleosides for the first time. In our experiments, we have used the femtosecond pump-probe technique to monitor excited-state absorption. The results show that the bases return to their lowest electronic ground states in just hundreds of femtoseconds.

TThe signals above shows the decay of excited-state absorption for the single bases in water at a probe wavelength of 600 nm. The remarkably short lifetimes of the DNA bases measured by us has been confirmed by the femtosecond fluorescence up-conversion technique.

Current work on single base photophysics includes the study of effects due to the solvent as well as chemical substitution towards the goal of elucidating the mechanism of excited-state deactivation. The decay mechanism is also being investigated through continuing collaborations with Professors Massimo Olivucci and Michael Robb on high-level quantum studies. Solvent studies have show that the short lifetimes above are an intrinsic property of the bases. We have found that chemical modification results in significantly increased lifetimes. Both suggest that the structure of the bases were naturally selected for the ability to dissipate energy.

"Biology's Natural Sunscreens"

Internal conversion produces vibrationally highly excited ground-state molecules, which exhibit strongly red-shifted absorption at near UV wavelengths, as shown for adenosine above. This hot-band absorption decays in a highly probe-wavelength dependent manner on the time scale of a few picoseconds in aqueous solution. The intermolecular transfer of vibrational energy to the solvent is promoted by solute-solvent hydrogen bonding. The bases thus convert potentially dangerous electronic energy into heat on an ultrafast time scale. This is the same photophysical functionality of sunscreens that work by the absorption of UV light. Nonradiative decay may be as highly evolved in nature as the better-known ultrafast photoprocesses of visual signal transduction, light harvesting, and electron transfer in the photosynthetic reaction center. From this perspective the building blocks of DNA have a kinship to other photoprotective compounds found in nature like the flavonoids of plants, and cyanobacterial pigments such as scytonemin and the mycosporine-like amino acids.

The photoprotective properties of the DNA bases are likely to have played a critical role at the dawn of life. Current evidence suggests that life arose a billion or more years before the presence of a significant ozone layer. The first organisms would have therefore been subjected to a much higher UV irradiance than today. Ultrafast nonradiative decay may have provided critical photoprotection long before the advent of enzymatic repair. Rather than a photophysical curiosity, nonradiative decay by the bases appears to be an essential function of these remarkable, multifunctional molecules. The nucleobases may have served as primordial sunscreen for the fragile, self-replicating molecules on the pre-biotic earth.

Excited-State Dynamics in Di- and Polynucleotides

Base stacking, base pairing, solvent accessibility, and polynucleotide conformation are all expected to influence singlet energy relaxation significantly, but the significance of each is poorly understood.The proximity of the bases to one another in DNA/RNA influences their singlet photophysics in two ways. First, they create a solvent environment substantially different than bulk water. Hydrophobic interactions exclude water from the interior of duplex DNA. Studies of solvent effects for single bases in different solvents is helping us to understand the significance of this effect in polynucleotides. The second way that the polynucleotide environment affects singlet dynamics is through electronic coupling between stacked bases. This coupling is evident in reduced ground state absorption (hypochromism). Photophysical consequences of this coupling are energy delocalization (exciplex and excimer formation) and energy transport.

Single-stranded poly(riboadenylic) acid (poly(A), blue) and poly(2-deoxyriboadenylic) acid polymers (poly(dA), red) have shown multiexponential decays, with lifetimes of 1.33 ps, 154 ps, and a third time constant in the nanosecond regime. The long-lived decays components are assigned to excitations over segments of stacked bases, while the fastest component is assigned to excitations localized in unstacked polymer regions. Temperature dependent studies and TDDFT calculations have supported this assignment. The larger amplitudes observed in poly(dA) demonstrate that electronic energy relaxation depends sensitively on the secondary structure of the adenine homopolymers.

Studies ofpoly(ribocytidylic) acid, poly(C), as a function of pH have also shown long-lived and short components. The short component in poly(C) is similarly assigned to unstacked regions of the polymer, while the long component is due to the ability of poly(C) to form double-stranded helices at low pH.

Conclusions

Finally, the knowledge obtained in this study about singlet excited state dynamics will strongly shape mechanistic thinking about DNA photochemistry. The sheer speed of nonradiative decay imposes severe constraints on the ability of singlet states to participate in bimolecular reactions. During the hundreds of femtoseconds required for relaxation to the electronic ground state, diffusive encounter of reactants is impossible. This may indicate a greater significance of singlet excimer states for those DNA photoreactions that have non-triplet intermediates.

References

1. Daniels, M.; Hauswirth, W., "Fluorescence of the purine and pyrimidine bases of the nucleic acids in neutral aqueous solution at 300 K," Science 1971, 171, 675-677.

2. Pecourt, J.-M. L.; Peon, J.; Kohler, B., "Ultrafast internal conversion of electronically excited RNA and DNA nucleosides in water," J. Am. Chem. Soc. 2000, 122, 9348-9349 [erratum J. Am. Chem. Soc. 2001, 123, 5166].

3. Pecourt, J.-M. L.; Peon, J.; Kohler, B., "DNA Excited-State Dynamics: Ultrafast Internal Conversion and Vibrational Cooling in a Series of Nucleosides," J. Am. Chem. Soc. 2001, 123, 10370-10378.

Other Information

"Literature Recipes For Calculating Extinction Coefficients and Other Physical Properties of Short Oligonucleotides and DNA. Web Pages for Order Custom Oligonucleotides and Polynucleotides."
Compiled by Carlos E. Crespo-Hernndez

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See also the August 2, 2001 issue of Nature, p. 476.