Molecular electrostatic potentials of DNA base–base pairing and mispairing

An understanding of why adenine (A) pairs with thymine (T) and cytosine (C) with guanine (G) in DNA is very useful in the design of sensors and other related devices. We report the use of dissociation energies, geometries and molecular electrostatic potentials (MEPs) to justify the canonical (AT and CG) Watson-Crick pairs. We also analyze all mismatches in both configurations—cis and trans—with respect to their glycoside bonds. As expected, we found that the most stable pair configuration corresponds to CG, providing an energy criterion for that preferred configuration. The reason why A gets together with T is much more difficult to explain as the energy of this pair is smaller than the energy of some other mismatched pairs. We tested MEPs to see if they could shed light on this problem. Interestingly, MEPs yield a unique pattern (shape) for the two canonical cases but different shapes for the mismatches. A tunnel of positive potential surrounded by a negative one is found interconnecting the three H-bonds of CG and the two of AT. This MEP tunnel, assisted partially by energetics and geometrical criteria, unambiguously determine a distinctive feature of the affinity between A and T as well as that between G and C.     

Dynamic conformational states of DNA containing T·T or BrdU·T mispaired bases: wobble H-bond pairing versus cross-strand inter-atomic contacts

The dynamic structure of 11-mer DNA duplexes of different sequences with or without homopyrimidine (T·T, or BrdU·T) mismatches was studied by molecular dynamics (MD) simulations on a time scale from 200 ps to 1 ns. The conformational analysis suggests that in mismatched duplexes the formation of classical T·T wobble H-bonding pairing is nearest-neighbor sequence-dependent and, in most cases, three-centered H-bonds and numerous alternative close cross-strand interatomic contacts exist. Thus, in duplex W1, where the central triplet is 5d(CTA)·d(TTG), two wobble conformations W () and W () are formed and exchange rapidly at 300 K. In contrast, when the central triplet is 5d(TTT)·d(ATA) (W2 duplex) wobble conformations are rarely observed at 300 K, and the T·T mispair most often adopts a twisted conformation with one largely persistent normal H-bond, plus a stable cross-strand contact involving a T flanking base. However, at elevated temperature (400 K) the same W2 duplex shows frequent exchange between the two classical wobble conformations (), as is in the case when the central triplet is 5d(TBrdUT)·d(ATA) (W3 duplex at 300 K). It is suggested that in the W2 sequence, restrictions due to thymine-methyl/ interactions prevent the formation of wobble pairing and thermal activation energy, and/or the chemical replacement of T by BrdU are required in order for the T(BrdU)·T mismatch to adopt and exchange between wobble conformations. The specific short and/or long-lived (double/triple) cross-strand dynamic interactions in W1, W2 and W3 duplexes are throughout characterized. These frequent atomic encounters exemplify possible inter-strand charge transfer pathways in the studied DNA molecules.Figure 3D structure snapshots of wobble and frequent overlapping conformers formed within the W3 central triplet during 200 ps MD: + . H-bonds (magenta) and close cross-strand contacts, Å (orange).     

The nature of the transition mismatches with Watson–Crick architecture: the G*·T or G·T* DNA base mispair or both? A QM/QTAIM perspective for the biological problem

This study provides the first accurate investigation of the tautomerization of the biologically important guanine*·thymine (G*·T) DNA base mispair with Watson–Crick geometry, involving the enol mutagenic tautomer of the G and the keto tautomer of the T, into the G·T* mispair (?G?=?.99?kcal?mol^?1, population?=?15.8% obtained at the MP2 level of quantum-mechanical theory in the continuum with ε?=?4), formed by the keto tautomer of the G and the enol mutagenic tautomer of the T base, using DFT and MP2 methods in vacuum and in the weakly polar medium (ε?=?4), characteristic for the hydrophobic interfaces of specific protein–nucleic acid interactions. We were first able to show that the G*·T?G·T* tautomerization occurs through the asynchronous concerted double proton transfer along two antiparallel O6H···O4 and N1···HN3 H-bonds and is assisted by the third N2H···O2 H-bond, that exists along the entire reaction pathway. The obtained results indicate that the G·T* base mispair is stable from the thermodynamic point of view complex, while it is dynamically unstable structure in vacuum and dynamically stable structure in the continuum with ε?=?4 with lifetime of 6.4·10^?12?s, that, on the one side, makes it possible to develop all six low-frequency intermolecular vibrations, but, on the other side, it is by three orders less than the time (several ns) required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication. One of the more significant findings to emerge from this study is that the short-lived G·T* base mispair, which electronic interaction energy between the bases (?23.76?kcal?mol^?1) exceeds the analogical value for the G·C Watson–Crick nucleobase pair (?20.38?kcal?mol^?1), “escapes from the hands” of the DNA replication machinery by fast transforming into the G*·T mismatch playing an indirect role of its supplier during the DNA replication. So, exactly the G*·T mismatch was established to play the crucial role in the spontaneous point mutagenesis.     

Molecular dynamics simulations and coupled nucleotide substitution experiments indicate the nature of A·A base pairing and a putative structure of the coralyne-induced homo-adenine duplex

Coralyne is an alkaloid drug that binds homo-adenine DNA (and RNA) oligonucleotides more tightly than it does Watson–Crick DNA. Hud’s laboratory has shown that poly(dA) in the presence of coralyne forms an anti-parallel duplex, however attempts to determine the structure by NMR spectroscopy and X-ray crystallography have been unsuccessful. Assuming adenine–adenine hydrogen bonding between the two poly(dA) strands, we constructed 40 hypothetical homo-(dA) anti-parallel duplexes and docked coralyne into the six most favorable duplex structures. The two most stable structures had trans glycosidic bonds, but distinct pairing geometries, i.e. either Watson–Crick Hoogsteen (transWH) or Watson–Crick Watson–Crick (transWW) with stability of transWH > transWW. To narrow down the possibilities, 7-deaza adenine base substitutions (dA→7) were engineered into homo-(dA) sequences. These substitutions significantly reduced the thermal stability of the coralyne-induced homo-(dA) structure. These experiments strongly suggest the involvement of N7 in the coralyne-induced A·A base pairs. Moreover, due to the differential effect on melting as a function of the location of the dA→7 mutations, these results are consistent with the N1–N7 base pairing of the transWH pairs. Together, the simulation and base substitution experiments predict that the coralyne-induced homo-(dA) duplex structure adopts the transWH geometry.     

Localization and anharmonicity of the vibrational modes for GC Watson–Crick and Hoogsteen base pairs

We present an ab initio study of the vibrational properties of cytosine and guanine in the Watson–Crick and Hoogsteen base pair configurations. The results are obtained by using two different implementations of the DFT method. We assign the vibrational frequencies to cytosine or to guanine using the vibrational density of states. Next, we investigate the importance of anharmonic corrections for the vibrational modes. In particular, the unusual anharmonic effect of the H⁺ vibration in the case of the Hoogsteen base pair configuration is discussed.     

NMR spectroscopy of the ring nitrogen protons of uracil and substituted uracils; relevance to Aψ base pairing in the solution structure of transfer RNA

The chemical shifts of the well-resolved ring nitrogen protons of uracil and a series of substituted uracils, including pseudouridine (5-ribosyl-uracil) and uridine (1-ribosyl-uracil) were determined using nuclear magnetic resonance (NMR). The observed chemical shifts suggest the existence of an atypical syn conformation for pseudouridine in the Aψ base pair in regulatory tRNAs in solution.     

Structure of myostatin·follistatin-like 3: N-terminal domains of follistatin-type molecules exhibit alternate modes of binding

TGF-β family ligands are involved in a variety of critical physiological processes. For instance, the TGF-β ligand myostatin is a staunch negative regulator of muscle growth and a therapeutic target for muscle-wasting disorders. Therefore, it is important to understand the molecular mechanisms of TGF-β family regulation. One form of regulation is through inhibition by extracellular antagonists such as the follistatin (Fst)-type proteins. Myostatin is tightly controlled by Fst-like 3 (Fstl3), which is the only Fst-type molecule that has been identified in the serum bound to myostatin. Here, we present the crystal structure of myostatin in complex with Fstl3. The structure reveals that the N-terminal domain (ND) of Fstl3 interacts uniquely with myostatin as compared with activin A, because it utilizes different surfaces on the ligand. This results in conformational differences in the ND of Fstl3 that alter its position in the type I receptor-binding site of the ligand. We also show that single point mutations in the ND of Fstl3 are detrimental to ligand binding, whereas corresponding mutations in Fst have little effect. Overall, we have shown that the NDs of Fst-type molecules exhibit distinctive modes of ligand binding, which may affect overall affinity of ligand·Fst-type protein complexes.     

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

T4 RNA ligase 2 (Rnl2) repairs 3′-OH/5′-PO₄ nicks in duplex nucleic acids in which the broken 3′-OH strand is RNA. Ligation entails three chemical steps: reaction of Rnl2 with ATP to form a covalent Rnl2–(lysyl-Nζ)–AMP intermediate (step 1); transfer of AMP to the 5′-PO₄ of the nick to form an activated AppN– intermediate (step 2); and attack by the nick 3′-OH on the AppN– strand to form a 3′–5′ phosphodiester (step 3). Here we used rapid mix-quench methods to analyze the kinetic mechanism and fidelity of single-turnover nick sealing by Rnl2–AMP. For substrates with correctly base-paired 3′-OH nick termini, k_step2 was fast (9.5 to 17.9 sec⁻¹) and similar in magnitude to k_step3 (7.9 to 32 sec⁻¹). Rnl2 fidelity was enforced mainly at the level of step 2 catalysis, whereby 3′-OH base mispairs and oxoguanine, oxoadenine, or abasic lesions opposite the nick 3′-OH elicited severe decrements in the rate of 5′-adenylylation and relatively modest slowing of the rate of phosphodiester synthesis. The exception was the noncanonical A:oxoG base pair, which Rnl2 accepted as a correctly paired end for rapid sealing. These results underscore (1) how Rnl2 requires proper positioning of the 3′-terminal ribonucleoside at the nick for optimal 5′-adenylylation and (2) the potential for nick-sealing ligases to embed mutations during the repair of oxidative damage.     

Lack of correlation between binding of Eco RII methyltransferase to DNA duplexes containing mismatches and the promotion of C to T mutations

The cytosine methyltransferases (MTases) M. HhaIand M. HpaII bind substrates in which the target cytosine is replaced by uracil or thymine, i.e. DNA containing a U:G or a T:G mismatch. We have extended this observation to the EcoRII MTase (M. EcoRII) and determined the apparent K_d for binding. Using a genetic assay we have also tested the possibility that MTase binding to U:G mismatches may interfere with repair of the mismatches and promote C:G to T:A mutations. We have compared two mutants of M. EcoRII that are defective for catalysis by the wild-type enzyme for their ability to bind DNA containing U:G or T:G mismatches and for their ability to promote C to T mutations. We find that although all three proteins are able to bind DNAs with mismatches, only the wild-type enzyme promotes C:G to T:A mutations in vivo. Therefore, the ability of M. EcoRII to bind U:G mismatched duplexes is not sufficient for their mutagenic action in cells.     

Pulse sequences for detection of NH2···N hydrogen bonds in sheared G⋅A mismatches via remote, non-exchangeable protons

The non-detectability of NH...N hydrogen bonds in nucleic acids due to exchange broadened imino/amino protons has recently been addressed via the use of non-exchangeable protons for detecting internucleotide 2hJ(NN) couplings. In these applications, the appropriate non-exchangeable proton is separated by two bonds from the NH...N bond. In this paper, we extend the scope of this approach to protons which are separated by four bonds from the NH...N moiety. Specifically, we consider the case of the commonly occurring sheared G x A mismatch alignment, in which we use the adenine H2 proton to report on the (A)N6H6(1.2)...N3(G) hydrogen bond, in the presence of undetectable, exchange broadened N6H6(1.2) protons. Two sequences, the 'straight-through' (H6)N6N3H2 and 'out-and-back' H2N6N3 experiments, are presented for observing these correlations in H2O and D2O solution, respectively. The sequences are demonstrated on two uniformly 15N,13C labelled DNA samples: d(G1G2G3T4T5C6A7G8G9)2, containing a G3 x (C6-A7) triad involving a sheared G3 x A7 mismatch, and d(G1G2G3C4A5G6G7T8)4, containing an A5 x (G3 x G6 x G3 x G6) x A5 hexad involving a sheared G3 x A5 mismatch.     

Thermodynamic contributions of single internal rA·dA, rC·dC, rG·dG and rU·dT mismatches in RNA/DNA duplexes

The thermodynamic contributions of rA·dA, rC·dC, rG·dG and rU·dT single internal mismatches were measured for 54 RNA/DNA duplexes in a 1 M NaCl buffer using UV absorbance thermal denaturation. Thermodynamic parameters were obtained by fitting absorbance versus temperature profiles using the curve-fitting program Meltwin. The weighted average thermodynamic data were fit using singular value decomposition to determine the eight non-unique nearest-neighbor parameters for each internal mismatch. The new parameters predict the ΔG°₃₇, ΔH° and melting temperature (T_m) of duplexes containing these single mismatches within an average of 0.33 kcal/mol, 4.5 kcal/mol and 1.4°C, respectively. The general trend in decreasing stability for the single internal mismatches is rG·dG > rU·dT > rA·dA > rC·dC. The stability trend for the base pairs 5′ of the single internal mismatch is rG·dC > rC·dG > rA·dT > rU·dA. The stability trend for the base pairs 3′ of the single internal mismatch is rC·dG > rG·dC >> rA·dT > rU·dA. These nearest-neighbor values are now a part of a complete set of single internal mismatch thermodynamic parameters for RNA/DNA duplexes that are incorporated into the nucleic acid assay development software programs Visual oligonucleotide modeling platform (OMP) and ThermoBLAST.     

Preferential intracellular pH regulation represents a general pattern of pH homeostasis during acid–base disturbances in the armoured catfish,Pterygoplichthys pardalis

Preferential intracellular pH (pH_i) regulation, where pH_i is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO₂. To determine whether preferential pH_i regulation may represent a general pattern for acid–base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2–3 h following the respective treatments and extracellular blood pH, pH_i of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO₂ were measured. All treatments resulted in significant changes in extracellular pH and RBC pH_i that likely cover a large portion of the pH tolerance limits of this species (pH 7.15–7.86). In all tissues other than RBC, pH_i remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pH_i after anoxia and an increase in liver pH_i following a metabolic alkalosis. Thus preferential pH_i regulation appears to be a general pattern for acid–base homeostasis in the armoured catfish and may be a common response in Amazonian fishes.     

Insights into binding modes of tumstatin peptide T7 with the active site of αvβ3 integrin

Through interaction with the active site of α_vβ₃ integrin, tumstatin T7 peptide inhibits both the angiogenesis and the proliferation of tumour cells. In this work, docking in conjunction with molecular dynamics simulation was used to explore the binding mode of T7 peptide and α_vβ₃ integrin. The binding mode analysis revealed that the residues Ser⁹⁰, Arg⁹¹, Asp⁹³ and Tyr⁹⁴ in T7 peptide, and (α)-Asp¹⁵⁰, (β)-Arg²¹⁴, (α)-Asp¹⁴⁸ (α)-Gln²¹⁴ and (α)-Glu¹²³ in the active site of α_vβ₃ integrin were most likely the key interaction sites. The hydroxyl of Tyr⁹⁴ coordinates α_vβ₃ via a Mn²⁺ ion, revealing that Mn²⁺ is also an important factor for the interaction. The insight into these key interaction sites not only suggests that the active site of α_vβ₃ integrin can bind to molecules through multiple binding mechanisms, but also provides some useful information for structure-based drug design.     

Free energy landscape and transition pathways from Watson–Crick to Hoogsteen base pairing in free duplex DNA

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson–Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine–thymine (A–T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events.     

Weak base pairing in both seed and 3′ regions reduces RNAi off-targets and enhances si/shRNA designs

The use of RNA interference is becoming routine in scientific discovery and treatment of human disease. However, its applications are hampered by unwanted effects, particularly off-targeting through miRNA-like pathways. Recent studies suggest that the efficacy of such off-targeting might be dependent on binding stability. Here, by testing shRNAs and siRNAs of various GC content in different guide strand segments with reporter assays, we establish that weak base pairing in both seed and 3′ regions is required to achieve minimal off-targeting while maintaining the intended on-target activity. The reduced off-targeting was confirmed by RNA-Seq analyses from mouse liver RNAs expressing various anti-HCV shRNAs. Finally, our protocol was validated on a large scale by analyzing results of a genome-wide shRNA screen. Compared with previously established work, the new algorithm was more effective in reducing off-targeting without jeopardizing on-target potency. These studies provide new rules that should significantly improve on siRNA/shRNA design.     

Phototautomerism of the GC base pair of DNA

Electronic spectra and ground and excited state electronic structures of normal G and rare tautomeric G1z.sbnd;C1 base pairs as well as of the individual rare tautomeric bases (purines and pyrimidines) have been studied using the VE-PPP molecular orbital method. The nature and consequences of the lowest energy purine-localized and purine to pyrimidine charge transfer type π?π1 singlet excitations of the base pairs have been investigated. The results indicate that in these excited states, particularly in the charge transfer excited state, the probability for the GC base pair to change over to G1C1 would be larger than in the ground state. The likeliness of the relevance of results obtained experimentally by other workers from the study of a model system to the GC base pair is discussed.     

Fluorescence analysis of G·C versus A·T binding of quinacrine to DNA

The affinity of quinacrine for native DNA has been determined from fluorescence measurements and equilibrium dialysis in Tris-HC10.05 m, NaCl0.1 m, EDTA 10^?3m, pH 7.5. When considering M. lysodeiktikus, E. coli calf thymus and C. perfringens the affinities of DNA for quaniactive have been found to change by a factor of two and the fluorescence intensities to change by a factor of 25. The varying affinities and fluoroescence intensities of bound quinacrine are attributed to heterogeneous binding. For all DNAs we have assumed that there exist three classes of intercalation sites: I, A·T-A·T; 2, G·C-G·C; and 3, A·T-G·C, assuming that base pair ordering is less relevant than base composition of sites. By fitting the affinities of native DNAs with this model it was found that quinacrine binds to site 2 three times more strongly than it does to site 1. When flucrescence intensity is studied, triplets of A·T pairs appear to be responsible for the high quantum yield of A·T rich DNA whereas the quenching properties of a G·C base pair adjacent to an intercalated quinacrine are well known.     

Thermodynamic signature of DNA damage: characterization of DNA with a 5-hydroxy-2'-deoxycytidine·2'-deoxyguanosine base pair

Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. One of the oxidation lesions formed, 5-hydroxy-2'-deoxycytidine, has been shown to miscode by some replicative DNA polymerases but not by error prone polymerases capable of translesion synthesis. The 5-hydroxy-2'-deoxycytidine lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine base to be extrahelical so it can enter into the enzyme's active site where it is excised off the DNA backbone to afford an abasic site. The thermodynamic and nuclear magnetic resonance results presented here describe the effect of a 5-hydroxy-2'-deoxycytidine·2'-deoxyguanosine base pair on the stability of two different DNA duplexes. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2'-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that requires the lesion to be extrahelical.