Eyal Nir

Pairing of isolated nucleic-acid bases in the absence of the DNA backbone

The two intertwined strands of DNA are held together through base pairing—the formation of hydrogen bonds between bases located opposite each other on the two strands. DNA replication and transcription involve the breaking and re-forming of these hydrogen bonds, but it is difficult to probe these processes directly. For example, conventional DNA spectroscopy is dominated by solvent interactions, crystal modes and collective modes of the DNA backbone; gas-phase studies, in contrast, can in principle measure interactions between individual molecules in the absence of external effects, but require the vaporization of the interacting species without thermal degradation. Here we report the generation of gas-phase complexes comprising paired bases, and the spectroscopic characterization of the hydrogen bonding in isolated guanine–cytosine (G–C) and guanine–guanine (G–G) base pairs. We find that the gas-phase G–C base pair adopts a single configuration, which may be Watson–Crick, whereas G–G exists in two different configurations, and we see evidence for proton transfer in the G–C pair, an important step in radiation-induced DNA damage pathways. Interactions between different bases and between bases and water molecules can also be characterized by our approach, providing stringent tests for high-level ab initio computations that aim to elucidate the fundamental aspects of nucleotide interactions.

Guanine tautomerism revealed by UV–UV and IR–UV hole burning spectroscopy

The vibronic spectrum of laser desorbed and jet cooled guanine consists of bands from three different tautomers of guanine as revealed by UV–UV and IR–UV double resonance spectroscopy. 1-methylguanine, in which the Keto–Enol tautomerism is blocked, shows hole burning spectra from the 9H-and 7H-Keto form. A comparison of the vibronic pattern of the different tautomers demonstrates that the vibronic spectrum built on the redmost guanine band at 32 870 cm−1 (electronic origin 0) can be traced back to the 9H-Enol tautomer, while the spectra built on the origins at 0+404 cm−1 and 0+1044 cm−1 stem from the two Keto tautomers. The IR–UV double resonance spectra of the OH-and NH-stretch vibrations of the different tautomers support this assignment. The UV and IR spectra can be partly assigned by comparison with ab initio calculated vibrational frequencies and with the help of deuteration experiments.

Single-molecule FRET reveals sugar-induced conformational dynamics in LacY.

The N- and C-terminal six-helix bundles of lactose permease (LacY) form a large internal cavity open on the cytoplasmic side and closed on the periplasmic side with a single sugar-binding site at the apex of the cavity near the middle of the molecule. During sugar/H+ symport, an outward-facing cavity is thought to open with closing of the inward-facing cavity so that the sugar-binding site is alternately accessible to either face of the membrane. In this communication, single-molecule fluorescence (Förster) resonance energy transfer is used to test this model with wild-type LacY and a conformationally restricted mutant. Pairs of Cys residues at the ends of two helices on the cytoplasmic or periplasmic sides of wild-type LacY and the mutant were labeled with appropriate donor and acceptor fluorophores, single-molecule fluorescence resonance energy transfer was determined in the absence and presence of sugar, and distance changes were calculated. With wild-type LacY, binding of a galactopyranoside, but not a glucopyranoside, results in a decrease in distance on the cytoplasmic side and an increase in distance on the periplasmic side. In contrast, with the mutant, a more pronounced decrease in distance and in distance distribution is observed on the cytoplasmic side, but there is no change on the periplasmic side. The results are consistent with the alternating access model and indicate that the defect in the mutant is due to impaired ligand-induced flexibility on the periplasmic side.

REMPI spectroscopy of cytosine

We report resonant two-photon ionization spectra of laser desorbed, jet cooled, cytosine, 1-methyl cytosine, 5-methyl cytosine, and dimers of these. Unlike other pyrimidine bases, cytosine exhibits vibronic spectra with sharp features in two spectral regions, separated by about 5000 cm 1 . We interpret these as being due to two tautomeric forms, one keto and one enol. The dimers absorb at wavelengths that are intermediate between those of the two monomer forms. By UV–UV hole burning we determined the numbers of isomers contributing to each spectrum and by delayed two color ionization we determined triplet lifetimes. We observed hydrogen transfer between bases both in collisions between monomers and after photo-excitation in clusters. 2002 Published by Elsevier Science B.V.

Pairing of the nucleobases guanine and cytosine in the gas phase studied by IR–UV double-resonance spectroscopy and ab initio calculations

We present R2PI, IR–UV and UV–UV double resonance measurements of the guanine–cytosine (G–C) dimer formed in a supersonic jet. We show that there is only one isomer of G–C in the investigated wavelength range from 33200 to 34100 cm−1. We assigned the observed G–C isomer to a specific structure, based on comparisons of the IR spectra of the G and C monomers with the G–C dimer in the range of the OH and NH stretching vibrations and ab initio-calculated vibrational frequencies and dimer stabilities. The cluster exhibits an HNH⋯O/NH⋯N/CO⋯HNH bonding similar to the Watson–Crick G–C base pair bonding but with C as the enol tautomer. We did not observe any keto–keto or enol–enol G–C dimers in the investigated wavelength region.

IR-UV double-resonance spectroscopy of the nucleobase adenine

We present R2PI and IR–UV double resonance spectra of the nucleobase adenine seeded in a supersonic jet. We show that there is only one tautomer of adenine which absorbs in the wavelength range 36 050 to 36 700 cm−1. The IR spectra, measured in the range 3200 to 3700 cm−1, show bands at 3452, 3508 and 3569 cm−1, which we assign to the symmetric NH2 , N–H and antisymmetric NH2 stretching vibrations of a single tautomer of adenine. We compare the experimental IR–UV double resonance spectra with ab initio based normal mode calculations. The observed tautomer is most probably the 9H amino-form of adenine.

The nucleobase cytosine and the cytosine dimer investigated by double resonance laser spectroscopy and ab initio calculations

The vibronic spectrum of laser desorbed and jet cooled cytosine consists of bands from two major tautomers (keto and enol) as revealed by UV-UV and IR-UV double resonance spectroscopy and methyl blocking experiments. Only one isomer each was observed for the cytosine dimer and for the cytosine - 1-methylcytosine mixed dimer. These isomers form CO⋯HNH/NH⋯N hydrogen bonds. Cytosine - 5-methylcytosine exhibits three isomers: one again with CO⋯HNH/NH⋯N connectivity, the second with CO⋯HNH/NH⋯N interaction but one cytosine in the enol form and the third with symmetrical CO⋯NH/NH⋯OC bonds. These are also the most stable clusters according to molecular dynamics/quenching and ab initio quantum chemical calculations. The experimental IR spectra of these isomers agree well with the calculated normal mode vibrational spectra. The vibronic spectra of the clusters are blue shifted relative to the monomer spectra by more than 1000 cm−1 indicating a considerable reduction of dimer stability upon electronic excitation.

Introducing improved structural properties and salt dependence into a coarse-grained model of DNA

We introduce an extended version of oxDNA, a coarse-grained model of deoxyribonucleic acid (DNA) designed to capture the thermodynamic, structural, and mechanical properties of single- and double-stranded DNA. By including explicit major and minor grooves and by slightly modifying the coaxial stacking and backbone-backbone interactions, we improve the ability of the model to treat large (kilobase-pair) structures, such as DNA origami, which are sensitive to these geometric features. Further, we extend the model, which was previously parameterised to just one salt concentration ([Na(+)] = 0.5M), so that it can be used for a range of salt concentrations including those corresponding to physiological conditions. Finally, we use new experimental data to parameterise the oxDNA potential so that consecutive adenine bases stack with a different strength to consecutive thymine bases, a feature which allows a more accurate treatment of systems where the flexibility of single-stranded regions is important. We illustrate the new possibilities opened up by the updated model, oxDNA2, by presenting results from simulations of the structure of large DNA objects and by using the model to investigate some salt-dependent properties of DNA.

REMPI Spectroscopy of Laser Desorbed Guanosines

To observe fundamental properties of DNA building blocks it is desirable to study individual nucleosides in the gas phase without interference from solvent molecules, or macromolecular structure. As a first step, we have recently reported the first vibronic spectrum of the nucleobase guanine, obtained by a combination of laser desorption, jet cooling, and resonance enhanced multiphoton ionization (REMPI).1 Although guanine is important as a chromophore in DNA, it is more realistic for understanding the photochemistry of DNA to study the nucleosides. Those are even harder to vaporize intact because they are thermally more labile and, with their larger molecular weights, have still lower vapor pressures. Using laser desorption, we have now succeeded in forming a molecular beam of nucleosides, and we report the first REMPI spectra of a series of individual guanosines, namely guanosine (Gs), 2′deoxyguanosine (2′deoxyGs), and 3′deoxyguanosine (3′deoxyGs). We compare our results with computations at the HF 6-31G(d,p) level. The results suggest the occurrence of two different conformations, each probably stabilized by internal hydrogen bonds. One of those two conformations is absent in 2′deoxyGs implying that the 2′ hydroxyl group is required for its stabilization. Spectroscopic properties of guanosines have been studied primarily by Raman techniques in solution.2-9 A great deal of attention has been given to potential Raman markers for hydrogen bonding and for structural conformation. Observation of hydrogen bonding by Raman spectroscopy requires identification of vibrations that depend strongly on those specific atoms in guanine, that serve as either proton donor or acceptor. However, most vibrations involve the concerted motion of multiple atoms, and therefore correlation of marker frequencies with specific hydrogen bonding sites is not straightforward. Guanosine vibrations involving motion along the glycosidic bond may provide conformational markers if their frequencies are sensitive to puckering of the ribose ring or for rotation around the sugar-base bond. Interpretation of these markers requires careful analysis of complex vibrational modes. On the other hand, different conformations can be observed much more directly by vibronic spectroscopy when they produce multiple origins. As we will show below, we observe two origins in our spectra, which we can associate with the syn and the anti orientations of the base relative to the ribose moiety. We have published details of our setup for laser desorption jet cooling REMPI spectrometry elsewhere.10 Sample preparation consisted of depositing neat material in powder form on graphite substrates. We moved the substrate slowly while acquiring spectra, gradually exposing fresh material. For desorption we used pulses from a Nd:YAG laser at 1064 nm with fluences on the order of 1 mJ/cm2. Desorbed neutral molecules were entrained in a supersonic expansion with Ar drive gas, injected by a pulsed solenoid valve. Downstream, the entrained molecules were onecolor two-photon photoionized, and the ions were detected in a reflectron time-of-flight mass spectrometer. The first photon resonantly excites the molecule, while a second photon from the same laser ionizes the excited molecule. By varying the wavelength while monitoring specific mass peaks we obtained mass selected excitation spectra. The typical ionization laser fluence was on the order of 0.1 mJ/cm2. Figure 1 shows the REMPI spectra of (a) Gs, (b) 3′deoxyGs, and (c) 2′deoxyGs. We assign the lowest-energy peak in each of the spectra as a 0-0 transition to the S1 excited state. Careful scans to lower energy by 1000 cm-1 do not reveal any additional peaks. The same was true when performing two-color ionization with a second photon at 193 nm. Therefore, we do not believe that we are observing a cutoff in the spectrum related to the ionization potential of Gs. Furthermore we have measured the ionization potential of guanine as 8.1 eV by two-color ionization.11,12 That is 1 eV less than the two-photon energy at the Gs origin. † Heinrich Heine Universität. (1) Nir, E.; Grace, L.; Brauer, B.; de Vries, M. S. J. Am. Chem. Soc. 1999, 121, 4896. (2) Faurskov-Nielsen, O.; Lund, P.; Petersen, S. J. Raman Spectrosc. 1981, 11, 493. (3) Nishimura, Y.; Tsuboi, M.; Sato, T.; Aoki, K. J. Mol. Struct. 1986, 146, 123. (4) Chinsky, L.; Jolles, B.; Laigle, A.; Turpin, P. J. Raman Spectrosc. 1987, 18, 195. (5) Carmona, P.; Molina, M. J. Mol. Struct. 1990, 219, 323. (6) Toyama, A.; Takino, Y.; Takeuchi, H.; Harada, I. J. Am. Chem. Soc. 1993, 115, 11092. (7) Urabe, H.; Sugawara, Y.; Kasuya, T. Phys. ReV. B 1995, 51, 5666. (8) Toyama, A.; Hamuara, M.; Takeuchi, H. J. Mol. Struct. 1996, 15, 99. (9) Toyama, A.; Hanada, N.; Ono, J.; Yoshimitsu, E.; Takeuchi, H. J. Raman Spectrosc. 1999, 30, 623. (10) Meijer, G.; de Vries, M. S.; Hunziker, H. E.; Wendt, H. R. Appl. Phys. B 1990, 51, 395. (11) Hopkins, J. B.; Powers, D. R.; Smalley, R. E. J. Phys. Chem. 1981, 85, 3739. (12) Nir, E.; Grace, L.; de Vries, M. S. To be published. Figure 1. REMPI spectra of (a) guanosine, (b) 3′deoxyGs, and (c) 2′deoxyGs. In this energy range guanine itself does not exhibit any vibronic activity since its lowest energy peak is at 238 cm-1 above the origin. The syn and anti labels indicate origins of two possible conformers, and the numbers indicate vibrational modes and their combinations and overtones, for example 122 indicates one quantum of mode 1 and two quanta of mode 2; f indicates fundamental vibration. 8091 J. Am. Chem. Soc. 2000, 122, 8091-8092

Rational design of DNA motors: fuel optimization through single-molecule fluorescence.

While numerous DNA-based molecular machines have been developed in recent years, high operational yield and speed remain a major challenge. To understand the reasons for the limited performance, and to find rational solutions, we applied single-molecule fluorescence techniques and conducted a detailed study of the reactions involved in the operation of a model system comprised of a bipedal DNA walker that strides on a DNA origami track powered by interactions with fuel and antifuel strands. Analysis of the kinetic profiles of the leg-lifting reactions indicates a pseudo-first-order antifuel binding mechanism leading to a rapid and complete leg-lifting, indicating that the fuel-removal reaction is not responsible for the 1% operational yield observed after six steps. Analysis of the leg-placing reactions showed that although increased concentrations of fuel increase the reaction rate, they decrease the yield by consecutively binding the motor and leading to an undesirable trapped state. Recognizing this, we designed asymmetrical hairpin-fuels that by regulating the reaction hierarchy avoid consecutive binding. Motors operating with the improved fuels show 74% yield after 12 consecutive reactions, a dramatic increase over the 1% observed for motors operating with nonhairpin fuels. This work demonstrates that studying the mechanisms of the reactions involved in the operation of DNA-based molecular machines using single-molecule fluorescence can facilitate rationally designed improvements that increase yield and speed and promote the applicability of DNA-based machines.