Search

Trinucleotide repeat expansion disorders are associated with the overexpansion of (CNG) repeats on the genome. Messenger RNA transcripts of sequences with greater than 60-100 (CNG) tandem units have been implicated in trinucleotide repeat expansion disorder pathogenesis. In this work, we develop a diagrammatic theory to study the structural diversity of these (CNG)n RNA sequences. Representing structural elements on the chain's conformation by a set of graphs and employing elementary diagrammatic methods, we have formulated a renormalization procedure to re-sum these graphs and arrive at a closed-form expression for the ensemble partition function. With a simple approximation for the renormalization and applied to extended (CNG)n sequences, this theory can comprehensively capture an infinite set of conformations with any number and any combination of duplexes, hairpins, multiway junctions, and quadruplexes. To quantify the diversity of different (CNG)n ensembles, the analytical equations derived from the diagrammatic theory were solved numerically to derive equilibrium estimates for the secondary structural contents of the chains. The results suggest that the structural ensembles of (CNG)n repeat sequence with n ∼60 are surprisingly diverse, and the distribution is sensitive to the ability of the N nucleotide to make noncanonical pairs and whether the (CNG)n sequence can sustain stable quadruplexes. The results show how perturbations in the form of biases on the stabilities of the various structural motifs, duplexes, junctions, helices, and quadruplexes could affect the secondary structures of the chains and how these structures may switch when they are perturbed.

While major contributors to the free energy of RNA tertiary structures such as basepairing, base-stacking, and charge and counterion interactions have been studied extensively, little is known about the intrinsic free energy of the backbone. To assess the magnitude of the entropic strains along the phosphate backbone and their impact on the folding free energy, we have formulated a mathematical treatment for computing the volume of the main-chain torsion-angle conformation space between every pair of nucleobases along any sequence to compute the corresponding backbone entropy. To validate this method, we have compared the computed conformational entropies against a statistical free energy analysis of structures in the crystallographic database from several-thousand backbone conformations between nearest-neighbor nucleobases as well as against extensive computer simulations. Using this calculation, we analyzed the backbone entropy of several ribozymes and riboswitches and found that their entropic strains are highly localized along their sequences. The total entropic penalty due to steric congestions in the backbone for the native fold can be as high as 2.4 cal/K/mol per nucleotide for these medium and large RNAs, producing a contribution to the overall free energy of up to 0.72 kcal/mol per nucleotide. For these RNAs, we found that low-entropy high-strain residues are predominantly located at loops with tight turns and at tertiary interaction platforms with unusual structural motifs.

We formulated a master equation-based mathematical model to analyze random scanning and catalysis for enzymes that act on single-stranded DNA (ssDNA) substrates. Catalytic efficiencies and intrinsic scanning distances are deduced from the distribution of positions and gap lengths between a series of catalytic events occurring over time, which are detected as point mutations in a lacZα-based reporter sequence containing enzyme target motifs. Mathematical analysis of the model shows how scanning motions become separable from the catalysis when the proper statistical properties of the mutation pattern are used to interpret the readouts. Two-point correlations between all catalytic events determine intrinsic scanning distances, whereas gap statistics between mutations determine their catalytic efficiencies. Applying this model to activation-induced deoxycytidine deaminase (AID), which catalyzes C→U deaminations processively on ssDNA, we have established that deaminations of AGC hot motifs occur at a low rate, ∼0.03 s−1, and low efficiency, ∼3%. AID performs random bidirectional movements for an average distance of 6.2 motifs, at a rate of about 15 nucleotides per second, and “dwells” at a motif site for 2.7 s while bound >4 min to the same DNA molecule. These results provide new and important insights on how AID may be optimized for generating mutational diversity in Ig genes, and we discuss how the properties of AID acting freely on a “naked” ssDNA relate to the constrained action of AID during transcription-dependent somatic hypermutation and class-switch recombination.

Any nucleic acid structure that contains single-stranded (ss) segments is dominated by entropy, but molecular-level information about these entropic determinants are inaccessible to direct experiments. A new Monte Carlo (MC) simulation has been developed based on a recently reported backbone conformational formula (BCV/-c) for RNAs. BCV/-c analytically enumerates all conformational states on the sugar-phosphate backbone. It has been validated rigorously against experiments, and the new MC algorithm, NUCLEIC, is atomistically accurate, computationally efficient, as well as predictive. We used NUCLEIC to investigate the entropic landscape of a comprehensive set of RNA secondary structures including: hairpins, stem-loops, symmetric and asymmetric bulges, 2-way, 3-way and 4-way junctions, as well as triplexes and quadruplexes. We found strong evidence that the kinetic barriers to the formation of many of these secondary structural elements are determined by the entropic bottlenecks associated with the unpaired ss tracts on each structure, though the thermodynamic free energy factor that stabilizes them come predominantly from base stacking. Based on these entropy data, we computed the rates of formation of various secondary structures and found that the kinetics of different pathways leading to the same product structure often have significantly different rates. These results highlight the importance of a molecular-level understanding of entropy as a defining factor behind RNA structures.

Functional RNAs can fold into intricate structures using a number of different secondary and tertiary structural motifs. Many factors contribute to the overall free energy of the target fold. This study aims at quantifying the entropic costs coming from the loss of conformational freedom when the sugar-phosphate backbone is subjected to constraints imposed by secondary and tertiary contacts. Motivated by insights from topology theory, we design a diagrammatic scheme to represent different types of RNA structures so that constraints associated with a folded structure may be segregated into mutually independent subsets, enabling the total conformational entropy loss to be easily calculated as a sum of independent terms. We used high-throughput Monte Carlo simulations to simulate large ensembles of single-stranded RNA sequences in solution to validate the assumptions behind our diagrammatic scheme, examining the entropic costs for hairpin initiation and formation of many multiway junctions. Our diagrammatic scheme aids in the factorization of secondary/tertiary constraints into distinct topological classes and facilitates the discovery of interrelationships among multiple constraints on RNA folds. This perspective, which to our knowledge is novel, leads to useful insights into the inner workings of some functional RNA sequences, demonstrating how they might operate by transforming their structures among different topological classes.

A transferable tight-binding potential has been constructed for heteroatomic systems containing carbon and hydrogen. The electronic degree of freedom is treated explicitly in this potential using a small set of transferable parameters which has been fitted to small hydrocarbons and radicals. Transferability to other higher hydrocarbons was tested by comparison with ab initio calculations and experimental data. The potential can correctly reproduce changes in the electronic configuration as a function of the local bonding geometry around each carbon atom. This type of potential is well suited for computer simulations of covalently bonded systems in both gas-phase and condensed-phase systems.

Rigorous quantum dynamics simulations have been performed to test the path-integral centroid formulation proposed by Voth, Chandler and Miller for quantal rate constants in a simple class of models for electron transfer reactions in the tight-binding limit. Fully dynamical quantum Monte Carlo simulations show that inside and slightly away from the diabatic limit the electron flux is invariant with the electronic centroid density near the dividing surface instead of being sharply peaked there. Despite this, we show analytically that the rate in this regime is largely governed by the centroid density on the dividing surface. But on approaching the adiabatic limit with larger electronic coupling, complex large-amplitude coherent oscillations develop in the quantum flux, suggesting that in addition to the imaginary-time centroid density, the dynamical factors also play an important role in determining the full quantal electron transfer rate.

The decomposition of water on Si(111)7 × 7 was studied using laser-induced thermal desorption (LITD), temperature programmed desorption (TPD) and Auger electron spectroscopy (AES). Product yields versus H 2 O exposure were determined using TPD measurements. The only TPD reaction products were H 2 and SiO which desorbed at approximately 800 and 950 K, respectively. The LITD yield from H 2 O on Si(111)7 × 7 also produced H 2 and SiO desorption products. Moreover, H 2 O and SiOH were detected in the LITD yield after larger water exposures at temperatures above the H 2 O multilayer desorption at 160 K. The LITD desorption yield for H 2 O persisted until 550 K, whereas the SiOH LITD signals were observed up to 600 K. These H 2 O and SiOH LITD signals were associated with reaction intermediates on Si(111)7 × 7 because neither H 2 O nor SiOH was observed in the TPD yield above 160 K. Isothermal LITD experiments were performed to measure the disappearance of SiOH and H 2 O LITD signals from Si(111)7 × 7. The results for the isothermal decay were consistent with self-poisoning of the H 2 O decomposition caused by site-blocking by the reaction products. In addition, the effects of preadsorbed hydrogen on the H 2 O decomposition reaction were investigated. Hydrogen was observed to decrease the H 2 O decomposition reaction on Si(111)7 × 7 nearly linearly with hydrogen coverage.

The surface diffusion coefficients for hydrogen on Ru(001) were calculated using transition state theory. The calculations were based on an empirical potential surface constructed using experimental values for the adsorption energy and vibrational frequencies of hydrogen on Ru(001). The calculated activation barrier was 3.84 kcalmole and the diffusion pre-exponential was D0 = 2.2 × 10−3cm2s. These calculated results are in excellent agreement with recent experimental results for hydrogen surface diffusion on Ru(001) obtained using laser-induced thermal desorption (LITD) techniques.

Laser-induced thermal desorption (LITD) was used to remove surface reaction intermediates from Si(111)7 × 7. Silicon-containing species were detected in the LITD yield from Si(111)7 × 7 surfaces covered with hydrogen, water or methanol. These silicon-containing LITD products include SiHn, Si(OH)n and Si(OCH3)n where n = 1 − 3. None of these species were observed in the conventional temperature programmed desorption (TPD) spectra. TPD and temperature-pro-grammed LITD studies were also performed and correlated for Si(111)7 × 7 surfaces as a function of hydrogen coverage. The SiH2 LITD products were related directly to the low-temperature β2-peak in the H2 TPD spectra. Previous infrared studies have demonstrated that this β2-peak corresponds to H2 desorption from silicon dihydride species on the silicon surface. The ability of LITD to desorb silicon-containing surface reaction intermediates should be extremely useful in investigations of silicon surface reaction kinetics.

The coverage dependence of the surface diffusion coefficient for hydrogen on Ru(001) was studied using laser-induced thermal desorption(LITD) techniques. The LITD measurements were performed for a wide range of initial hydrogen surface coverages in the temperature range 230–270 K. At a given temperature, the surface diffusion coefficient was found to be constant as a function of coverage. The absence of coverage dependence in the surface diffusion coefficient indicates that adsorbate-adsorbate interactions between adsorbed hydrogen atoms on Ru(001) are negligible. This observation suggests that the coverage-dependent features observed in work function, high resolution electron energy loss spectroscopy and possibly temperature programmed desorption studies of hydrogen on Ru(001) are associated with two inequivalent hydrogen sites rather than repulsive lateral hydrogen interactions. Monte Carlo simulations reveal that the surface diffusion coefficient should be coverage independent on a surface with two inequivalent sites in the absence of adsorbate-adsorbate interactions.

The surface diffusion of hydrogen and deuterium on Ru(001) at low coverage was studied using laser-induced thermal desorption techniques. In the temperature range from 260 to 300 K, the surface diffusion coefficients could be expressed in Arrhenius form as D H =6.9×10 −4 cm 2 /s exp(−3.6±0.5 kcal mol −1 / RT ) and D D =4.6×10 −4 cm 2 /s exp(−4.1±0.5 kcal mol −1 / RT ) for hydrogen and deuterium, respectively. The observed isotope effect was somewhat larger than predicted by simple transition state theory but was within the limits of experimental error. Quantum mechanical tunneling can be playing, at best, only a minor role in the surface migration of hydrogen on Ru(001) at these temperatures.

Laser-induced thermal desorption (LITD) was used to remove surface reaction intermediates from Si(111)7×7. Silicon-containing species were detected in the LITD yield from Si(111)7×7 surfaces covered with hydrogen, water or methanol. These silicon-containing LITD products include SiH n , Si(OH) n and Si(OCH 3 ) n where n = 1–3. None of these species were observed in the conventional temperature programmed desorption (TPD) spectra. TPD and temperature-programmed LITD studies were also performed and correlated for Si(111)7×7 surfaces as a function of hydrogen coverage. The SiH 2 LITD products were related directly to the low-temperature β 2 -peak in the H 2 TPD spectra. Previous infrared studies have demonstrated that this β 2 -peak corresponds to H 2 desorption from silicon dihydride species on the silicon surface. The ability of LITD to desorb silicon-containing surface reaction intermediates should be extremely useful in investigations of silicon surface reaction kinetics.