Monitoring the direct and indirect damage of DNA bases and polynucleotides by using time-resolved infrared spectroscopy

Abstract

The nucleotide 5 -dGMP and polynucleotide poly(dGdC)⅐poly(dGdC) have been irradiated by using a 200-fs, 200-nm laser pulses and spectrally characterized by using time-resolved infrared spectroscopy. Under the experimental conditions, 200-nm excitation generates both electronic excited states and radical cations through photoionization; the former decay rapidly to vibrationally hot ground state. By using infrared signatures we have been able to follow these processes, and at time scales of >1 ns we observe an infrared marker band at 1,702 cm ؊1 within both 5 -dGMP and the polynucleotide assigned to a photoionized product of guanine. This transient has also been reproduced through indirect chemistry through the reaction with photogenerated carbonate radical with 5 -dGMP. The ability to use time-resolved infrared spectroscopy in this way paves the way for developing solution-phase studies to investigate both direct and indirect radiation chemistry of DNA.DNA damage ͉ electron transfer ͉ guanine radical cation O f the large number of ionizing events per day that are known to be a potential threat to the living cell through DNA damage, only a few are believed to lead to irreparable damage (1). This small number of irreversible events is significant because it is the fundamental step that ultimately leads to mutation and the onset of cancer (2). Oxidative stress is known to be a primary cause of such damage occurring through the formation of radical ions of nucleic acid bases, and the ease with which each individual base is ionized to produce the corresponding base radical cation is determined by its ionization potential, the order being G Ͻ A Ͻ C Ϸ T (3). Thus, within a randomly photoionized DNA strand through hole transfer from one base to the next, damage tends to favor formation of the G base radical (4-6). There is considerable research effort in trying to understand the precise mechanism of long-range electron transfer as a function of the sequence-dependent structure and how this mechanism relates to DNA damage (7-11). UV radiation to initiate DNA base ionization has proven extremely useful for studying direct DNA damage (4-6, 12) along with pulse radiolysis (13). Traditionally, reactions in DNA are monitored by either following the kinetics on the nanosecond time scale using UV͞visible absorption spectroscopy (5, 6, 12, 13) or determining the resulting base damage (single-or double-strand breaks) using hot piperidine treatment or excision enzyme analysis followed by gel electrophoresis (14, 15). Conversely, ESR spectroscopy has provided structural information on ionization products. These studies used either UV͞visible irradiation or pulse radiolysis to produce ionized DNA species in frozen glass at cryogenic temperatures (16)(17)(18)(19) and in solution (20). However, this method did not permit ultrafast time scales to be studied. It is known that the chemical processes involved in DNA damage stretch across a wide range of time domains from femtoseconds to seconds, and being able to observe the earliest event...

Triggered infrared spectroscopy for investigating metalloprotein chemistry

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Identification of charge separated states in thymine single strands

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