Kimberly de La Harpe

DNA Excited-State Dynamics: From Single Bases to the Double Helix

Ultraviolet light is strongly absorbed by DNA, producing excited electronic states that sometimes initiate damaging photochemical reactions. Fully mapping the reactive and nonreactive decay pathways available to excited electronic states in DNA is a decades-old quest. Progress toward this goal has accelerated rapidly in recent years, in large measure because of ultrafast laser experiments. Here we review recent discoveries and controversies concerning the nature and dynamics of excited states in DNA model systems in solution. Nonradiative decay by single, solvated nucleotides occurs primarily on the subpicosecond timescale. Surprisingly, excess electronic energy relaxes one or two orders of magnitude more slowly in DNA oligo- and polynucleotides. Highly efficient nonradiative decay pathways guarantee that most excited states do not lead to deleterious reactions but instead relax back to the electronic ground state. Understanding how the spatial organization of the bases controls the relaxation of excess electronic energy in the double helix and in alternative structures is currently one of the most exciting challenges in the field.

UV excitation of single DNA and RNA strands produces high yields of exciplex states between two stacked bases

Excited electronic states created by UV excitation of the diribonucleoside monophosphates ApA, ApG, ApC, ApU, and CpG were studied by the femtosecond transient-absorption technique. Bleach recovery signals recorded at 252 nm show that long-lived excited states are formed in all five dinucleosides. The lifetimes of these states exceed those measured in equimolar mixtures of the constituent mononucleotides by one to two orders of magnitude, indicating that electronic coupling between proximal nucleobases dramatically slows the relaxation of excess electronic energy. The decay rates of the long-lived states decrease with increasing energy of the charge-transfer state produced by transferring an electron from one base to another. The charge-transfer character of the long-lived states revealed by this analysis supports their assignment to excimer or exciplex states. Identical bleach recovery signals were seen for ApA, (A)4, and poly(A) at delay times >10 ps after photoexcitation. This indicates that excited states localized on a stack of just two bases are the common trap states independent of the number of stacked nucleotides. The fraction of initial excitations that decay to long-lived exciplex states is approximately equal to the fraction of stacked bases determined by NMR measurements. This supports a model in which excitations associated with two stacked bases decay to exciplex states, whereas excitations in unstacked bases decay via ultrafast internal conversion. These results establish the importance of charge transfer-quenching pathways for UV-irradiated RNA and DNA in room-temperature solution.

UV-Induced Proton Transfer between DNA Strands

UV radiation creates excited states in DNA that lead to mutagenic photoproducts. Photoexcitation of single-stranded DNA can transfer an electron between stacked bases, but the fate of excited states in the double helix has been intensely debated. Here, photoinduced interstrand proton transfer (PT) triggered by intrastrand electron transfer (ET) is detected for the first time by time-resolved vibrational spectroscopy and quantum mechanical calculations. Long-lived excited states are shown to be oppositely charged base pair radical ions. In two of the duplexes, the base pair radical anions are present as tautomers formed by interstrand PT. Charge recombination occurs on the picosecond time scale preventing the accumulation of damaging radicals or mutagenic tautomers.

Ground-State Recovery Following UV Excitation is Much Slower in G·C−DNA Duplexes and Hairpins Than in Mononucleotides

Excited states in double-stranded oligonucleotides containing G.C base pairs were studied by femtosecond transient absorption spectroscopy. Relaxation to the electronic ground state occurs about 10 times more slowly in the duplexes and hairpins studied on average than in the individual mononucleotides of G and C. Detection of long-lived excited states in G.C oligonucleotides complements the earlier observation of slow ground-state recovery in A.T DNA, showing that excited states with picosecond lifetimes are formed in DNAs containing either kind of base pair. The results show further that Watson-Crick G.C base pairs in these base-paired and base-stacked duplexes do not enable subpicosecond relaxation to the electronic ground state. A model is proposed in which fluorescent exciton states decay rapidly and irreversibly to dark exciplex states. This model explains the seemingly contradictory observations of femtosecond fluorescence and slower, picosecond recovery of the ground-state population.

Deuterium Isotope Effect on Excited-State Dynamics in an Alternating GC Oligonucleotide

Isotope effects on the excited-state dynamics of single- and double-stranded GC-containing DNAs were studied by femtosecond transient absorption spectroscopy. A pronounced deuterium isotope effect was observed in alternating d(GC)(9).d(GC)(9), but none was seen in the nonalternating or single-stranded variations investigated. These findings demonstrate that an interstrand process involving proton-coupled electron transfer contributes to the excited-state dynamics in DNAs having an appropriate base sequence.

Excited-State Dynamics of DNA Duplexes with Different H-Bonding Motifs

The excited-state dynamics of three distinct forms of the d(GC)9·d(GC)9 DNA duplex were studied by combined time-resolved infrared experiments and quantum mechanical calculations. In the B- and Z-forms, bases on opposite strands form Watson-Crick (WC) base pairs but stack differently because of salt-induced changes in backbone conformation. At low pH, the two strands associate by Hoogsteen (HG) base pairing. Ultraviolet-induced intrastrand electron transfer (ET) triggers interstrand proton transfer (PT) in the B- and Z-forms, but the PT pathway is blocked in the HG duplex. Despite the different decay mechanisms, a common excited-state lifetime of ∼ 30 ps is observed in all three duplex forms. The ET-PT pathway in the WC duplexes and the solely intrastrand ET pathway in the HG duplex yield the same pair of π-stacked radicals on one strand. Back ET between these radicals is proposed to be the rate-limiting step behind excited-state deactivation in all three duplexes.

The Excited‐State Lifetimes in a G⋅C DNA Duplex are Nearly Independent of Helix Conformation and Base‐Pairing Motif

DNA photophysics: Femtosecond transient absorption experiments reveal that excited states produced by UV light in a duplex DNA oligonucleotide decay at essentially the same rate in B and Z helix conformers (see figure).

Excited-State Dynamics of a DNA Duplex in a Deep Eutectic Solvent Probed by Femtosecond Time-Resolved IR Spectroscopy

To better understand how the solvent influences excited-state deactivation in DNA strands, femtosecond time-resolved IR (fs-TRIR) pump-probe measurements were performed on a d(AT)9·d(AT)9 duplex dissolved in a deep eutectic solvent (DES) made from choline chloride and ethylene glycol in a 1:2 mol ratio. This solvent, known as ethaline, is a member of a class of ionic liquids capable of solubilizing DNA with minimal disruption to its secondary structure. UV melting analysis reveals that the duplex studied here melts at 18 °C in ethaline compared to 50 °C in aqueous solution. Ethaline has an excellent transparency window that facilitates TRIR measurements in the double-bond stretching region. Transient spectra recorded in deuterated ethaline at room temperature indicate that photoinduced intrastrand charge transfer occurs from A to T, yielding the same exciplex state previously detected in aqueous solution. This state decays via charge recombination with a lifetime of 380 ± 10 ps compared to the 300 ± 10 ps lifetime measured earlier in D2O solution. The TRIR data strongly suggest that the long-lived exciplex forms exclusively in the solvated duplex, and not in the denatured single strands, which appear to have little, if any, base stacking. The longer lifetime of the exciplex state in the DES compared to aqueous solution is suggested to arise from reduced stabilization of the charge transfer state, resulting in slower charge recombination on account of Marcus inverted behavior.

Chemical cross-linking of a variety of green fluorescent proteins as Förster resonance energy transfer donors for Yukon orange fluorescent protein: A project-based undergraduate laboratory experience: Chemical Cross-Linking of Green and Yukon Orange Fluorescent Proteins

Förster resonance energy transfer (FRET) is the basis for many techniques used in biomedical research. Due to its wide use in molecular sensing, FRET is commonly introduced in many biology, chemistry, and physics courses. While FRET is of great importance in the biophysical sciences, the complexity and difficulty of constructing FRET experiments has resulted in limited usage in undergraduate laboratory settings. Here, we present a practical undergraduate laboratory experiment for teaching FRET using a diverse set of green‐emitting fluorescent proteins (FPs) as donors for a cross‐linked Yukon orange FP. This laboratory experiment enables students to make the connection of basic lab procedures to real world applications and can be applied to molecular biology, biochemistry, physical chemistry, and biophysical laboratory courses. Published 2018. This article is a U.S. Government work and is in the public domain in the USA., 46(5):516–522, 2018.