Principles of RNA base pairing: structures and energies of the trans Watson-Crick/sugar edge base pairs

Due to the presence of the 2'-OH hydroxyl group of ribose, RNA molecules utilize an astonishing variability of base pairing patterns to build up their structures and perform the biological functions. Many of the key RNA base pairing families have no counterparts in DNA. In this study, the trans Watson-Crick/sugar edge (trans WC/SE) RNA base pair family has been characterized using quantum chemical and molecular mechanics calculations. Gas-phase optimized geometries from density functional theory (DFT) calculations and RIMP2 interaction energies are reported for the 10 crystallographically identified trans WC/SE base pairing patterns. Further, stable structures are predicted for all of the remaining six possible members of this family not seen in RNAs so far. Among these novel six base pairs, the computations substantially refine two structures suggested earlier based on simple isosteric considerations. For two additional trans WC/SE base pairs predicted in this study, no arrangement was suggested before. Thus, our study brings a complete set of trans WC/SE base pairing patterns. The present results are also contrasted with calculations reported recently for the cis WC/SE base pair family. The computed base pair sizes are in sound correlation with the X-ray data for all WC/SE pairing patterns including both their cis and trans isomers. This confirms that the isostericity of RNA base pairs, which is one of the key factors determining the RNA sequence conservation patterns, originates in the properties of the isolated base pairs. In contrast to the cis structures, however, the isosteric subgroups of the trans WC/SE family differ not only in their H-bonding patterns and steric dimensions but also in the intrinsic strength of the intermolecular interactions. The distribution of the total interaction energy over the sugar-base and base-base contributions is controlled by the cis-trans isomerism.     

Non-Watson-Crick base pairing in RNA. quantum chemical analysis of the cis Watson-Crick/sugar edge base pair family

Large RNA molecules exhibit an astonishing variability of base-pairing patterns, while many of the RNA base-pairing families have no counterparts in DNA. The cis Watson-Crick/sugar edge (cis WC/SE) RNA base pairing is investigated by ab initio quantum chemical calculations. A detailed structural and energetic characterization of all 13 crystallographically detected members of this family is provided by means of B3LYP/6-31G and RIMP2/aug-cc-pVDZ calculations. Further, a prediction is made for the remaining 3 cis WC/SE base pairs which are yet to be seen in the experiments. The interaction energy calculations point at the key role of the 2'-OH group in stabilizing the sugar-base contact and predict all 16 cis WC/SE base-pairing patterns to be nearly isoenergetic. The perfect correlation of the main geometrical parameters in the gas-phase optimized and X-ray structures shows that the principle of isosteric substitutions in RNA is rooted from the intrinsic structural similarity of the isolated base pairs. The present quantum chemical calculations for the first time analyze base pairs involving the ribose 2'-OH group and unambiguously correlate the structural information known from experiments with the energetics of interactions. The calculations further show that the relative importance and absolute value of the dispersion energy in the cis WC/SE base pairs are enhanced compared to the standard base pairs. This may by an important factor contributing to the strength of such interactions when RNA folds in its polar environment. The calculations further demonstrate that the Cornell et al. force field commonly used in molecular modeling and simulations provides satisfactory performance for this type of RNA interactions.     

Vibrational dynamics of DNA. I. Vibrational basis modes and couplings

Carrying out density functional theory calculations of four DNA bases, base derivatives, Watson-Crick (WC) base pairs, and multiple-layer base pair stacks, we studied vibrational dynamics of delocalized modes with frequency ranging from 1400 to 1800 cm(-1). These modes have been found to be highly sensitive to structure fluctuation and base pair conformation of DNA. By identifying eight fundamental basis modes, it is shown that the normal modes of base pairs and multilayer base pair stacks can be described by linear combinations of these vibrational basis modes. By using the Hessian matrix reconstruction method, vibrational coupling constants between the basis modes are determined for WC base pairs and multilayer systems and are found to be most strongly affected by the hydrogen bonding interaction between bases. It is also found that the propeller twist and buckle motions do not strongly affect vibrational couplings and basis mode frequencies. Numerically simulated IR spectra of guanine-cytosine and adenine-thymine bases pairs as well as of multilayer base pair stacks are presented and described in terms of coupled basis modes. It turns out that, due to the small interlayer base-base vibrational interactions, the IR absorption spectrum of multilayer base pair system does not strongly depend on the number of base pairs.     

Parallel DNA double helices incorporating isoG or m5isoC bases studied by FTIR, CD and molecular modeling

FTIR spectroscopy has been used to follow the formation of parallel stranded DNA duplexes incorporating isoG or m5isoC bases and determine their base pairing scheme. The results are discussed in comparison with data concerning anti-parallel duplexes with comparable base composition and sequence. In duplexes containing A-T and isoG-C or m5isoC-G base pairs shifts of the thymine C2=O2 and C4=O4 carbonyl stretching vibrations (to lower and higher wavenumbers, respectively, when compared to their positions in classical cis Watson-Crick (WC) base pairs) reflect the formation of trans Watson-Crick A-T base pairs. All carbonyl groups of cytosines, m5isocytosines, guanines and isoguanines are found to be involved in hydrogen bonds, indicative of the formation of isoG-C and m5isoC-G base pairs with three hydrogen bonds. Molecular modeling shows that both structures form regular right handed helices with C2'endo sugar puckers. The role of the water content on the helical conformation of the parallel duplexes has been studied by FTIR and CD. It is found that a conformational transition similar to the B --> A transition observed for anti-parallel duplexes induced by a decrease of the water content of the samples can occur for these parallel duplexes. Their helical flexibility has been evidenced by FTIR studies on hydrated films by the emergence of absorption bands characteristic of A type geometry, in particular by an S-type --> N-type repuckering of the deoxyribose. All sugars in the parallel duplex with alternating d(isoG-A)/d(C-T) sequence can adopt an N-type geometry in low water content conditions. The conformational transition of the parallel hairpin duplex with alternating d(isoG-A)/d(C-T) sequence was followed by circular dichroism in water/trifluoroethanol solutions and its free energy at 0 degrees C was estimated to be 6.6 +/- 0.3 kcal mol(-1).     

Strong enhancement of vibrational relaxation by Watson-Crick base pairing

We have studied the ultrafast dynamics of NH-stretch vibrational excitations in Watson-Crick base pairs consisting of adenine and uracil derivatives. To estimate the influence of the A:U hydrogen bonding on the vibrational dynamics, we have also studied the uracil derivative in monomeric form. The vibrational relaxation of the NH-stretching mode is found to occur much faster in the Watson-Crick base pair than in monomeric uracil. From the delay dependence of the transient vibrational spectra, it can be concluded that both in base-paired and monomeric uracil, the energy relaxation takes place in two steps, the first step being a rapid transfer of energy from the NH-stretching mode to an accepting mode, the second step the relaxation of this accepting mode. The transient spectra show evidence that in the base pair the hydrogen bond between the nucleobases acts as the accepting mode, and that the hydrogen bonding between the bases is responsible for the extremely fast vibrational relaxation in this system.     

Expanded-size bases in naturally sized DNA: evaluation of steric effects in Watson-Crick pairing

We describe physicochemical properties in DNA of altered-size nucleobases that retain Watson-Crick analogous hydrogen-bonding ability. Size-expanded analogues of adenine and thymine (xA and xT, respectively, which are expanded by benzo-fusion) were incorporated into natural DNA oligonucleotides, and their effects on helix stability were measured. Base stacking studies revealed that the two stretched analogues stack much more strongly than do their naturally sized counterparts. In contrast to this, pairing studies showed that single substitutions of the new bases are destabilizing to the natural helix as compared to A or T in standard A-T pairs in the same context, unless multiple adjacent substitutions are used. Interestingly, the size-expanded bases displayed selective recognition of the hydrogen-bonding complementary partners, suggesting that Watson-Crick analogous pairs were still formed despite local backbone strain. In an attempt to compensate for the added size of the expanded adenine, we tested a formamide deoxynucleoside, which Leonard proposed as a shortened thymine analogue (F(o)). Data showed, however, that this compound adopts a conformation unfavorable for pairing. On the basis of the combined thermodynamic data, we estimate the energetic cost of the 2.4 A stretching of an isolated base pair in DNA at ca. +1 to 2 kcal/mol. Notably, during the pairing studies, the two size-expanded nucleobases were found to display significant changes in fluorescence on formation of stacked versus unstacked structures, suggesting possible applications in probing nucleic acid structures and biochemical mechanisms.     

Determination of redox potentials for the Watson-Crick base pairs, DNA nucleosides, and relevant nucleoside analogues

Redox potentials for the DNA nucleobases and nucleosides, various relevant nucleoside analogues, Watson-Crick base pairs, and seven organic dyes are presented based on DFT/B3LYP/6-31++G(d,p) and B3YLP/6-311+G(2df,p)//B3LYP/6-31+G* levels of calculations. The values are determined from an experimentally calibrated set of equations that correlate the vertical ionization (electron affinity) energy of 20 organic molecules with their experimental reversible oxidation (reduction) potential. Our results are in good agreement with those estimated experimentally for the DNA nucleosides in acetonitrile solutions (Seidel et al. J. Phys. Chem. 1996, 100, 5541). We have found that nucleosides with anti conformation exhibit lower oxidation potentials than the corresponding syn conformers. The lowering in the oxidation potential is due to the formation of an intramolecular hydrogen bonding interaction between the 5'-OH group of the sugar and the N3 of the purine bases or C2=O of the pyrimidine bases in the syn conformation. Pairing of adenine or guanine with its complementary pyrimidine base decreases its oxidation potential by 0.15 or 0.28 V, respectively. The calculated energy difference between the oxidation potential for the G.C base pair and that of the guanine base is in good agreement with the experimental value estimated recently (0.34 V: Caruso, T.; et al. J. Am. Chem. Soc. 2005, 127, 15040). The complete and consistent set of reversible redox values determined in this work for the DNA constituents is expected to be of considerable value to those studying charge and electronic energy transfer in DNA.     

Hydrogen-bonded nucleic acid base pairs containing unusual base tautomers: complete basis set calculations at the MP2 and CCSD(T) levels

The total interaction energies of altogether 15 hydrogen-bonded nucleic acid base pairs containing unusual base tautomers were calculated. The geometry properties of all selected adenine-thymine and guanine-cytosine hydrogen-bonded base pairs enable their incorporation into DNA. Unusual base pairing patterns were compared with Watson-Crick H-bonded structures of the adenine-thymine and guanine-cytosine pairs. The complete basis set (CBS) limit of the MP2 interaction energy and the CCSD(T) correction term, determined as the difference between the CCSD(T) and MP2 interaction energies, was evaluated. Extrapolation to the MP2 CBS limit was done using the aug-cc-pVDZ and aug-cc-pVTZ results, and the CCSD(T) correction term was determined with the 6-31G*(0.25) basis set. Final interaction energies were corrected while taking into account both tautomeric penalization determined at the CBS level and solvation/desolvation free energies. The situation for the adenine-thymine pairs is straightforward, and tautomeric pairs are significantly less stable than the Watson-Crick pair consisting of the canonical forms. In the case of the guanine-cytosine pair, the Watson-Crick structure made by canonical forms is again the most stable. The other two structures are, however, energetically rather similar (by 5 and 6 kcal/mol), which provides a very small but non-negligible chance of detecting these structures in the DNA double helix (1:5000). Due to the fact that DNA bases and base pairs incorporated into DNA are solvated less favorably than in isolated systems, this probability represents the very upper limit. The results clearly show how precisely the canonical building blocks of DNA molecules were chosen and how well their stability is maintained.     

Anharmonic Vibrational Signatures of DNA Bases and Watson-Crick Base Pairs

Changes of molecular structure and associated charge distributions, and changes of anharmonic vibrational parameters from DNA base monomers to the Watson-Crick base pairs, have been investigated at the density functional theory level. Through examination of the NH2, N H, and C=O stretching vibrational modes that are involved in the multiple H-bonds in the base pairs, sensitivity of their diagonal and off-diagonal anharmonicities, as well as anharmonic vibrational couplings, to the structure change are predicted. Our results reveal the intrinsic connection between the anharmonic vibrational potentials, H-bonding, and electrostatic interactions in DNA bases.     

How well can new-generation density functional methods describe stacking interactions in biological systems?

We compare the performance of four recently developed DFT methods (MPW1B95, MPWB1K, PW6B95, and PWB6K) and two previous, generally successful DFT methods (B3LYP and B97-1) for the calculation of stacking interactions in six nucleic acid bases complexes and five amino acid pairs and for the calculation of hydrogen bonding interactions in two Watson-Crick type base pairs. We found that the four newly developed DFT methods give reasonable results for the stacking interactions in the DNA base pairs and amino acid pairs, whereas the previous DFT methods fail to describe interactions in these stacked complexes. We conclude that the new generation of DFT methods have greatly improved performance for stacking interaction as compared to previously available methods. We recommend the PWB6K method for investigating large DNA or protein systems where stacking plays an important role.     

Rational design of hetero-ring-expanded guanine analogs with enhanced properties for modified DNA building blocks

The properties and modes of recognition of physiological DNAs associated with the four natural nucleobases might be extended, in principle, by the design of non-natural nucleobase derivatives. The goal is an expansion of the genetic alphabet, with the possible outcome of producing new DNAs with improved physical or biological properties. In this work, a new series of hetero-ring-expanded guanine analogs are proposed, and their relevant structural characteristics and electronic properties are determined by density functional theory. The stabilities of the decamer DNA duplexes (dn.dC)10 (where n represents the corresponding expanded guanine analog designed here) are also examined, using molecular dynamics. The simulations show that the designed motifs can form stable DNA-like structures. We determined the pairing energies for the Watson-Crick (WC) hydrogen-bonded dimers between the expanded G-analogs and the natural C, and found that the pairing energies are close to those of the natural GC pair. The calculated adiabatic ionization potentials (IPs) of the size-expanded guanine analogs and their base pairs, and the corresponding vertical ionization potentials, show that some are distinctly smaller than the corresponding natural versions. The HOMO-LUMO energy gaps for most of the size-expanded guanine analogs and their WC base pairs are considerably lower than those of the corresponding natural base and base pairs. Thus, the expanded G bases may be considered as DNA genetic motifs, and they may serve as building blocks for potential biological applications and the development of molecular electronic devices.     

Fluorous base-pairing effects in a DNA polymerase active site

We describe selective "fluorous" effects in the active site of a DNA polymerase, by using nucleotide analogues whose pairing edges are perfluorinated. The 5'-triphosphate deoxynucleotide derivatives of DNA base analogues 2,3,4,5-tetrafluorobenzene ((F)B) and 4,5,6,7-tetrafluoroindole ((F)I), as well as hydrocarbon controls benzene (B) and indole (I), were synthesized and studied as substrates for the DNA Polymerase I Klenow fragment (KF exo-). Modified nucleotides were present in the DNA template or were supplied as nucleoside triphosphates in studies of the steady-state kinetics of single nucleotide insertion. When supplied opposite the non-natural bases in the template strand, the hydrophobic nucleoside triphosphates were incorporated by up to two orders of magnitude more efficiently than the natural deoxynucleoside triphosphates. The purine-like fluorinated indole nucleotide ((F)I) was the most efficiently inserted of the four hydrophobic analogues, with the most effective incorporation occurring opposite the pyrimidine-like tetrafluorobenzene ((F)B). In all cases, the polyfluorinated base pairs were more efficiently processed than the analogous hydrocarbon pairs. A preliminary test of polymerase extension beyond these pairs showed that only the (F)B base is appreciably extended; the inefficient extension is consistent with recently published data regarding other nonpolar base pairs. These results suggest the importance of hydrophobicity, stacking, and steric interactions in the polymerase-mediated replication of DNA base pairs that lack hydrogen bonds. These findings further suggest that the enhanced hydrophobicity of polyfluoroaromatic bases could be employed in the design of new, selective base pairs that are orthogonal to the natural Watson-Crick pairs used in replication.     

MP2 study on the hydrogen bonding interaction between 5-hydroxy-5-methylhydantoin and DNA bases: A,C, G,T

The 5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) is a nucleobase lesion induced by the action of ionizing radiation on thymine residue in DNA. In this study, we present the hydrogen bonding base pairs involving 5-OH-5-Me-dHyd bound to the four bases in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T). Full geometry optimizations have been performed for the studied complexes by MP2 method. The interaction energies were corrected for the basis-set superposition error (BSSE), using the full Boys–Bernardi counterpoise correction scheme. Hydrogen bonding patterns of these base pairs were characterized using NBO analysis and AIM analysis. According to the calculated binding energies and structural parameters, the stability of the base pairs decrease in the following order: 5-OH-5-Me-dHyd:G>5-OH-5-Me-dHyd:A>5-OH-5-Me -dHyd:C~5-OH-5-Me-dHyd:T.     

Thermodynamic stability of Hoogsteen and Watson-Crick base pairs in the presence of histone H3-mimicking peptide

We found that Hoogsteen base pairs were stabilized by molecular crowding and a histone H3-mimicking peptide, which was not observed for Watson-Crick base pairs. Our findings demonstrate that the type of DNA base pair is critical for the interaction between DNA and histones.     

DFT and MP2 investigations on the hydrogen bonding interaction between 5,6-dihydrothymine and dna bases: A, C, G, T

5,6-Dihydrothymine (DHT) is a nucleobase lesion induced by the action of ionizing radiation on thymine residue in DNA. In this work, we present the hydrogen bonding base pairs involving 5,6-dihydrothymine bound to four bases in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T). Full geometry optimizations are performed for the studied complexes by the B3LYP method. Interaction energies are corrected for the basis set superposition error, using the full Boys-Bernardi counterpoise correction scheme. Hydrogen bonding patterns of these base pairs are characterized using NBO and AIM analysis. According to the calculated binding energies and structural parameters, the stability of the base pairs decrease in the following order: DHT:G ～ DHT:A > DHT:C > DHT:T.     

Structures and energetics of base flipping of the thymine dimer depend on DNA sequence

The cis,syn-cyclobutane pyrimidine dimer (CPD) is a photoinduced DNA lesion leading to a significant distortion of the DNA structure. Its repair by DNA photolyase requires a flip of the damaged base into an extrahelical position. This base flip is expected to be sequence-dependent, but the structures and energetics as a function of the bases 3' and 5' to the CPD lesion are unknown. Eight-nanosecond MD simulations of four different hexadecamer duplexes with the CPD were performed for the flipped-in and flipped-out structures. Analysis of these results indicates clear sequence-dependent differences. Significant disruptions of the base pairs to the 3' side of the CPD are observed for the flipped-out structures with adjacent A-T pairs, whereas those with G-C pairs adjacent show no such distortions. The conformational spaces occupied by these two duplexes are significantly different. The structural differences correlate well with the free energy differences for base flipping calculated using the previously established 2D potential of mean force (PMF) method. The energy differences for base flipping in duplexes containing A, T, G, and C pairs adjacent to the CPD were found to be 6.25-6.5, 5.25-5.5, 7.25-7.5, and 6.5-6.75 kcal/mol, respectively. These energy differences of up to 2 kcal/mol should be large enough to be detected experimentally using sensitive probes.     

Direct assessment of interresidue forces in Watson-Crick base pairs using theoretical compliance constants

This communication presents quantum mechanical calculations of interresidue potential constants for the Watson-Crick base pairs adenine-thymine (AT) and guanine-cytosine (GC), permitting a unique quantification of hydrogen bond strengths. Due to the calculated compliance constants, large differences between individual H bonds were found, while a repulsive character of the C-H...O bond in AT can be excluded.     

MP2 study on the hydrogen-bonding interaction between 5-hydroxymethyl-uracil and DNA bases: A,C, G,T

The 5-hydroxymethyl-uracil (HmU) is a product of oxidative attack on the methyl group of thymine in DNA. In this work, we present the hydrogen bonding complexes formation involving HmU bound to the four bases in DNA: adenine (A), cytosine (C), guanine (G), and thymine (T). Full geometry optimizations have been performed for the studied complexes by MP2 method. The interaction energies were corrected for the basis-set superposition error (BSSE), using the full Boys-Bernardi counterpoise correction scheme. Hydrogen bonding patterns of these base pairs were characterized using NBO analysis and AIM analysis. According to the calculated binding energies and structural parameters, the stability of the base pairs decrease in the following order: HmU:A > HmU:G > HmU:C > HmU:T.     

C-F...H-C hydrogen bonds in ribonucleic acids

We report the synthesis of 1'-deoxy-1'-(benzimidazol-1-yl)-beta-D-ribofuranose 7 and 1'-deoxy-1'-phenyl-beta-D-ribofuranose 2. With these two ribonucleoside analogues we have a set of nine different RNA building blocks in hand, which are isostere to the natural bases. Now it is possible to investigate their duplex stabilizing forces. These forces are hydrogen bonds, base stacking, and solvation. The phosphoramidites of all building blocks were incorporated into a 12mer RNA, and the resulting RNA duplexes were investigated by UV- and CD-spectroscopy. We found that some of the RNA analogues are universal bases. The best universal bases with the lowest destabilization and the smallest discrimination between the natural bases are 1 (B) and 9 (E). On the basis of UV measurements we determined the melting points and the thermodynamic data. We were able to show that there are no hydrogen bonds between the natural bases and the RNA analogues. From thermodynamic data we calculated the contributions for base stacking and solvation of all modified building blocks. Comparison of calculated and measured data of double modified base pairs in 12mer RNA duplexes showed a further duplex stabilizing force in base pairs containing fluorine atoms at the Watson-Crick binding site. This stabilizing force can be defined as C-F.H-C hydrogen bond as is observed in crystal structures of 1'-deoxy-1'-(4-fluorophenyl)-beta-D-ribofuranose.