Unstacked Conformation Research Articles

Molecular Dynamics Simulations of 2-Aminopurine-Labeled Dinucleoside Monophosphates Reveal Multiscale Stacking Kinetics.

Molecular dynamics (MD) simulations of 2-aminopurine (2Ap)-labeled DNA dinucleoside monophosphates (DNMPs) were performed to investigate the hypothesis that base stacking dynamics occur on timescales sufficiently rapid to influence the emission signals measured in time-resolved fluorescence experiments. Analysis of multiple microsecond-length trajectories shows that the DNMPs sample all four coplanar stacking motifs. In addition, three metastable unstacked conformations are detected. A hidden Markov-state model (HMSM) was applied to the simulations to estimate transition rates between the stacked and unstacked states. Transitions between different stacked states generally occur at higher rates when the number of nucleobase faces requiring desolvation is minimized. Time constants for structural relaxation range between 1.6 and 25 ns, suggesting that emission from photoexcited 2Ap, which has an excited-state lifetime of 10 ns, is sensitive to base stacking kinetics. A master equation model for the excited-state population of 2Ap predicts multiexponential emission decays that reproduce the sub-10 ns emission decay lifetimes and amplitudes seen in experiments. Combining MD simulations with HMSM analysis is a powerful way to understand the dynamics that influence 2Ap excited-state relaxation and represents an important step toward using observed emission signals to validate MD simulations.

Increasing the length of poly-pyrimidine bulges broadens RNA conformational ensembles with minimal impact on stacking energetics.

Helical elements separated by bulges frequently undergo transitions between unstacked and coaxially stacked conformations during the folding and function of noncoding RNAs. Here, we examine the dynamic properties of poly-pyrimidine bulges of varying length (n = 1–4, 7) across a range of Mg2+ concentrations using HIV-1 TAR RNA as a model system and solution NMR spectroscopy. In the absence of Mg2+, helices linked by bulges with n ≥ 3 residues adopt predominantly unstacked conformations (stacked population <15%), whereas one-bulge and two-bulge motifs adopt predominantly stacked conformations (stacked population >74%). In the presence of 3 mM Mg2+, the helices predominantly coaxially stack (stacked population >84%), regardless of bulge length, and the midpoint for the Mg2+-dependent stacking transition is within threefold regardless of bulge length. In the absence of Mg2+, the difference between free energy of interhelical coaxial stacking across the bulge variants is estimated to be ∼2.9 kcal/mol, based on an NMR chemical shift mapping with stacking being more energetically disfavored for the longer bulges. This difference decreases to ∼0.4 kcal/mol in the presence of Mg2+. NMR RDCs and resonance intensity data show increased dynamics in the stacked state with increasing bulge length in the presence of Mg2+. We propose that Mg2+ helps to neutralize the growing electrostatic repulsion in the stacked state with increasing bulge length thereby increasing the number of coaxial conformations that are sampled. Energetically compensated interhelical stacking dynamics may help to maximize the conformational adaptability of RNA and allow a wide range of conformations to be optimally stabilized by proteins and ligands.

Synthesis of Naphthyridine Dimers with Conformational Restriction and Binding to DNA and RNA.

One of the important determinants in the efficiency of a molecular interaction is the necessity for conformational changes in host and/or guest molecules upon binding. In small-molecule interactions with nucleic acids, conformational changes on both molecules are often involved, especially in intercalating binding. Mismatch binding ligands (MBLs) we described here consist of two heterocycles that predominantly exist in one conformation, so it is of interest to determine if such molecules can bind to any DNA and RNA structures. One molecule, 1-NHR, which predominantly exists as the unstacked conformation in aqueous solvent, has been successfully synthesized and characterized. Compound 1-NHR did not efficiently bind to GX/Y DNA and RNA sequences, but the binding pattern is different from that of authentic MBL naphthyridine carbamate dimer. In vitro selection of RNA that specifically binds to 1-NHR was performed from pre-miR-29a loop library RNA, and one RNA, to which 1-NHR bound with high affinity, has been successfully identified. Although it was anticipated that 1-NHR, with a predominantly unstacked conformation, would show entropy-driven binding, isothermal titration calorimetry analysis suggested that the binding of 1-NHR to RNA was enthalpy driven with an apparent Kd of about 100 nm.

Large-Scale Analysis of 48 DNA and 48 RNA Tetranucleotides Studied by 1 μs Explicit-Solvent Molecular Dynamics Simulations.

An understanding of how the conformational behavior of single-stranded DNAs and RNAs depend on sequence is likely to be important for attempts to rationalize the thermodynamics of nucleic acid folding. In an attempt to further our understanding of such sequence dependences, we report here the results of 192 (1 μs) explicit-solvent molecular dynamics (MD) simulations of 48 DNA and 48 RNA tetranucleotide sequences performed using recently reported modifications to the AMBER force field. Each tetranucleotide was simulated starting from two different conformations, a fully natively stacked and a completely unstacked conformation, and populations of the various possible base stacking arrangements were analyzed. The simulations indicate that, for both DNA and RNA, the populations of fully natively stacked conformations increase linearly with increasing numbers of purines in the sequence, while the conformational entropies, computed by two complementary methods, decrease. Despite the comparatively short simulation times, the computed free energies of stacking of the 16 possible combinations of bases in the middle of the sequences are found to be in good correspondence with values reported recently from simulations of dinucleoside monophosphates using the same force field. Finally, consistent with recent reports from other groups, non-native stacking interactions, i.e., between bases that are not adjacent in sequence, are shown to be a recurring feature of the simulations; in particular, stacking interactions of bases in a i:i+2 relationship are shown to occur significantly more frequently when the intervening base is a pyrimidine. Given that the high prevalence of non-native stacking interactions is thought to be unrealistic, it appears that further parametrization work will be required before accurate conformational descriptions of single-stranded nucleic acids can be obtained with current force fields.

Chemically and electrochemically induced expansion and contraction of a ferrocene rotor

A 2,2'-bipyridine-appended ferrocene rotor, 1,1'-di(5-yl-ethynyl-2,2'-bipyridine)ferrocene, can be switched from a folded/stacked (syn) conformation to an extended/unstacked (anti) conformation by the addition of [Cu(CH3CN)4](PF6) and 6,6'-dimesityl-2,2'-bipyridine. This extension and contraction process was completely reversible and could be triggered either chemically or electrochemically.

Cations Form Sequence Selective Motifs within DNA Grooves via a Combination of Cation-Pi and Ion-Dipole/Hydrogen Bond Interactions

The fine conformational subtleties of DNA structure modulate many fundamental cellular processes including gene activation/repression, cellular division, and DNA repair. Most of these cellular processes rely on the conformational heterogeneity of specific DNA sequences. Factors including those structural characteristics inherent in the particular base sequence as well as those induced through interaction with solvent components combine to produce fine DNA structural variation including helical flexibility and conformation. Cation-pi interactions between solvent cations or their first hydration shell waters and the faces of DNA bases form sequence selectively and contribute to DNA structural heterogeneity. In this paper, we detect and characterize the binding patterns found in cation-pi interactions between solvent cations and DNA bases in a set of high resolution x-ray crystal structures. Specifically, we found that monovalent cations (Tl+) and the polarized first hydration shell waters of divalent cations (Mg2+, Ca2+) form cation-pi interactions with DNA bases stabilizing unstacked conformations. When these cation-pi interactions are combined with electrostatic interactions a pattern of specific binding motifs is formed within the grooves.

Base-Stacking Disorder and Excited-State Dynamics in Single-Stranded Adenine Homo-oligonucleotides

Single-stranded adenine homo-oligonucleotides were investigated in aqueous solution by femtosecond transient absorption spectroscopy in order to study the effect of strand length on the nature and dynamics of excited states formed by UV absorption. Global fitting analysis of bleach recovery signals recorded at a probe wavelength of 250 nm and pH 7 reveals that the same lifetimes of 2.72 and 183 ps reproduce the pronounced biexponential decays observed in all (dA)n oligomers, containing between 2 and 18 residues. Although the lifetimes are invariant, the amplitudes of the short- and long-lived components depend sensitively on the number of residues. For example, the 183 ps component increases with strand length and is greater for DNA vs RNA single strands with the same number of adenines. Inhomogeneous kinetics arising from two classes of adenine bases in each oligomer best explains the observations. A subset of adenine residues produce short-lived excited states upon excitation, while absorption by the remaining adenines yields long-lived excited states that are responsible for the long-lived signal. By assuming that each short-lived excited state in the oligomer makes the same contribution to the transient absorption signal as an excited state of the adenine mononucleotide, the fraction of each type of base in the oligomer can be estimated along with the quantum yield of long-lived excited states. The fraction of oligonucleotides that yield long-lived excited states increases with oligomer length in precisely the same manner as the fraction of bases that are found in base stacks. Corroborating evidence that base stacking leads to distinct decay channels comes from experiments conducted at low pH on (dA)2. Coulombic repulsion between the two protonated bases at pH 2 results in open, unstacked conformations causing the long-lived component seen in (dA)2 at neutral pH to vanish completely. The fast component seen in oligomers with two or more bases is assigned to vibrational cooling following ultrafast internal conversion to the electronic ground state. This monomer-like decay channel is operative for the subset of adenine residues that are either poorly or not at all stacked with neighboring bases. This study shows that static base stacking disorder fully accounts for the length-dependent transient absorption signals. Although absorption likely creates delocalized excitons of unknown spatial extent, the results from this study suggest that long-lived excitations in single-stranded A tracts are already fully localized on no more than two bases no later than 1 ps after UV excitation.

Intramolecular Base Stacking of Dinucleoside Monophosphate Anions in Aqueous Solution

Time-dependent motions of 32 deoxyribodinucleoside and ribodinucleoside monophosphate anions in aqueous solution at 310 K were monitored during 40 ns using classical molecular dynamics (MD). In all studied molecules, spontaneous stacking/unstacking transitions occurred on a time-scale of 10 ns. To facilitate the structural analysis of the sampled configurations we defined a reaction coordinate for the nucleobase stacking that considers both the angle between the planes of the two nucleobases and the distance between their mass-centers. Additionally, we proposed a physically meaningful transient point on this coordinate that separates the stacked and unstacked states. We applied this definition to calculate free energies for stacking of all pairwise combinations of adenine, thymine (uracil), cytosine and guanine moieties embedded in studied dinucleosides monophosphate anions. The stacking equilibrium constants decreased in the order 5'-AG-3' > GA ~ GG ~ AA > GT ~ TG ~ AT ~ GC ~ AC > CG ~ TA > CA ~ TC ~ TT ~ CT ~ CC. The stacked conformations of AG occurred 10 times more frequently than its unstacked conformations. On the other hand, the last five base combinations showed a greater preference for the unstacked than the stacked state. The presence of an additional 2'-OH group in the RNA-based dinucleoside monophosphates increased the fraction of stacked complexes but decreased the compactness of the stacked state. The calculated MD trajectories were also used to reveal prevailing mutual orientation of the nucleobase dipoles in the stacked state.

Conformational dynamics of di-(perylene bisimide acrylate) and its footprints in steady-state, time-resolved, and fluorescence-correlation spectroscopy

Employing steady-state spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence (cross-) correlation spectroscopy we investigated di-(perylene bisimide acrylate) and compared the results with those from monomeric perylene bisimide acrylate. For the dimeric structure two emitting species were found. By comparison with the spectroscopic results from monomeric perylene bisimide one species was assigned to perylene moieties in an isolated, unstacked conformation, whereas the other species is attributed to aggregated π-stacked perylene moieties. The transition dynamics between these conformations was uncovered by fluorescence (cross) correlation spectroscopy. A consistent description of the various correlation curves is derived for a dynamic model that considers an additional non-fluorescent dark state that is associated exclusively with the aggregated species.

Ultrafast Fluorescence Decay Profiles Reveal Differential Unstacking of 2-Aminopurine Residues Positioned Within Binding Subsites of a Single-Stranded DNA Binding Protein

DNAs bound by proteins are kinetically dynamic and exist in multiple conformations, including conformations that differ in stacking interactions. We have utilized ultrafast time resolved fluorescence spectroscopy to investigate changes in base stacking of single-stranded DNA (ssDNA) upon binding of the dimeric gene 5 protein (g5p) of fd, f1, and M13 strains of E. coli bacteriophages. DNA oligomer hairpins of 44 nucleotides were designed to have two antiparallel 16-nucleotide ssDNA tails for binding of a cluster of g5p dimers. Otherwise identical oligomers each contained a single fluorescent 2-aminopurine (2AP) base. Time domain fluorescence measurements showed that the label exists in an ensemble of conformations, including stacked, partially stacked, and unstacked species that are altered upon binding of the g5p. Two oligomers had 2AP labels at different subsite locations within one of the four-nucleotide DNA-binding sites of a given g5p monomer when the oligomers were saturated with g5p. These labels showed increases of the unstacked conformation from 22-24% for the free DNAs to 47-74% for the bound DNAs, where the extent of this increase was specific for the subsite location within the DNA-binding site of the g5p monomer. Time resolved anisotropy measurements indicated that the 2AP labels were in relatively inflexible conformations within the g5p-saturated oligomers.

Cy3-DNA Stacking Interactions Strongly Depend on the Identity of the Terminal Basepair

We characterized the effect of the first basepair on the conformational dynamics of the fluorescent dye Cy3 attached to the 5′ end of double-stranded DNA using Gaussian-mixture adaptive umbrella sampling simulations. In the simulations, the sampling of all five dihedral angles along the linker was enhanced, so that both stacked and unstacked states were sampled. The affinity of Cy3 for a T·A basepair (with the dye attached to T) was found to be significantly less than for the other basepairs. This was verified experimentally by measuring the activation energies for cis-trans isomerization of the dye. The simulation and experimental results indicate the existence of partially unstacked conformations amenable to photoisomerization. The simulations also showed that stacking of Cy3 straightens the DNA while stabilizing the first basepair. Our findings indicate that fluorescence is modulated by Cy3-DNA interactions in a sequence-dependent manner.

Molecular Dynamics of Potential rRNA Binders: Single-Stranded Nucleic Acids and Some Analogues

By hindering or "silencing" protein translation in vivo, antisense nucleic acid analogues that hybridize to bacterial rRNA could serve as a promising class of antibacterial compounds. Thus, we performed a comparative analysis of the dynamical properties of modified oligonucleotides based upon a sequence (5')r(UGUUACGACU)(3') that is complementary to bacterial ribosomal A-site RNA. In particular, 25 ns explicit solvent molecular dynamics simulations were computed for the following six single-stranded decamers: (1) the above RNA in unmodified form; (2) the 2'-O-methyl-modified RNA; (3) peptide nucleic acid (PNA) analogues of the above sequence, containing either (a) T or (b) U; and (4) two serine-substituted PNAs. Our results show that 2'-O-methylation attenuates RNA backbone dynamics, thereby preventing interconversion between stacked and unstacked conformations. The PNA analogue is rendered less flexible by replacing uracil with thymine; in addition, we found that derivatizing the PNA backbone with serine leads to enhanced base-stacking interactions. Consistent with known solubility properties of these classes of molecules, both RNAs exhibited greater localization of water molecules than did PNA. In terms of counterions, the initially helical conformation of the 2'-O-methyl RNA exhibits the highest Na(+) density among all the simulated decamers, while Na(+) build-up was most negligible for the neutral PNA systems. Further studies of the conformational and physicochemical properties of such modified single-stranded oligomers may facilitate better design of nucleic acid analogues, particularly those capable of serving as specific, high-affinity ribosomal A-site binders.

DNA models of trinucleotide frameshift deletions: the formation of loops and bulges at the primer–template junction

Although mechanisms of single-nucleotide residue deletion have been investigated, processes involved in the loss of longer nucleotide sequences during DNA replication are poorly understood. Previous reports have shown that in vitro replication of a 3′-TGC TGC template sequence can result in the deletion of one 3′-TGC. We have used low-energy circular dichroism (CD) and fluorescence spectroscopy to investigate the conformations and stabilities of DNA models of the replication intermediates that may be implicated in this frameshift. Pyrrolocytosine or 2-aminopurine residues, site-specifically substituted for cytosine or adenine in the vicinity of extruded base sequences, were used as spectroscopic probes to examine local DNA conformations. An equilibrium mixture of four hybridization conformations was observed when template bases looped-out as a bulge, i.e. a structure flanked on both sides by duplex DNA. In contrast, a single-loop structure with an unusual unstacked DNA conformation at its downstream edge was observed when the extruded bases were positioned at the primer–template junction, showing that misalignments can be modified by neighboring DNA secondary structure. These results must be taken into account in considering the genetic and biochemical mechanisms of frameshift mutagenesis in polymerase-driven DNA replication.

The Dynamic Structural Basis of Differential Enhancement of Conformational Stability by 5′- and 3′-Dangling Ends in RNA

Unpaired bases at the end of an RNA duplex (dangling ends) can stabilize the core duplex in a sequence-dependent manner and are important determinants of RNA folding, recognition, and functions. Using 2-aminopurine as a dangling end purine base, we have employed femtosecond time-resolved fluorescence spectroscopy, combined with UV optical melting, to quantitatively investigate the physical and structural nature of the stacking interactions between the dangling end bases and the terminal base pairs. A 3'-dangling purine base has a large subpopulation that stacks on the guanine base of the terminal GC or UG pair, either intrastrand or cross-strand depending on the orientation of the pair, thus providing stabilization of different magnitudes. On the contrary, a 5'-dangling purine base only has a marginal subpopulation that stacks on the purine of the same strand (intrastrand) but has little cross-strand stacking. Thus a 5'-dangling purine does not provide significant stabilization. These stacking structures are not static, and a dangling end base samples a range of stacked and unstacked conformations with respect to the terminal base pair. Femtosecond time-resolved anisotropy decay reveals certain hindered base conformational dynamics that occur on the picosecond to nanosecond time scales, which allow the dangling base to sample these substates. When the dangling purine is opposite to a U and is able to form a potential base pair at the end of the duplex, there is an interplay of base stacking and hydrogen-bonding interactions that depends on the orientation of the base pair relative to the adjacent GC pair. By resolving these populations that are dynamically exchanging on fast time scales, we elucidated the correlation between dynamic conformational distributions and thermodynamic stability.

DNA base flipping by a base pair-mimic nucleoside

On the basis of non-covalent bond interactions in nucleic acids, we synthesized the deoxyadenosine derivatives tethering a phenyl group (X) and a naphthyl group (Z) by an amide linker, which mimic a Watson–Crick base pair. Circular dichroism spectra indicated that the duplexes containing X and Z formed a similar conformation regardless of the opposite nucleotide species (A, G, C, T and an abasic site analogue F), which was not observed for the natural duplexes. The values among the natural duplexes containing the A/A, A/G, A/C, A/T and A/F pairs differed by 5.2 kcal mol−1 while that among the duplexes containing X or Z in place of the adenine differed by only 1.9 or 2.8 kcal mol−1, respectively. Fluorescence quenching experiments confirmed that 2-amino purine opposite X adopted an unstacked conformation. The structural and thermodynamic analyses suggest that the aromatic hydrocarbon group of X and Z intercalates into a double helix, resulting in the opposite nucleotide base flipping into an unstacked position regardless of the nucleotide species. This observation implies that modifications at the aromatic hydrocarbon group and the amide linker may expand the application of the base pair-mimic nucleosides for molecular biology and biotechnology.

RNA–protein recognition: Single-residue ultrafast dynamical control of structural specificity and function

The transcription antiterminator N protein from bacteriophage lambda uses its arginine-rich motif to specifically bind a stem-loop RNA hairpin (boxB) as a bent alpha-helix. A single stacking interaction between a tryptophan (Trp-18) and an adenosine (A7) in the RNA loop is robust and necessary for antitermination activity in vivo. Previously, femtosecond fluorescence up-conversion experiments from this laboratory indicated that the N/boxB complex exists in a dynamical two-state equilibrium between stacked and unstacked conformations and that the extent of stacking depends on the identity of peptide residues 14 and 15. In the present work, we have combined transient absorption and fluorescence up-conversion to determine the nature of interactions responsible for this sequence-dependent behavior. Analysis of mutant complexes supports the idea that the beta-carbon of residue 14 enforces the stacked geometry by hydrophobic interaction with the ribose of A7, whereas a positive charge at this residue plays only a secondary role. A positive charge at position 15 substantially disfavors the stacked state but retains much of the binding energy. Remarkably, in vivo antitermination experiments show strong correlation with our femtosecond dynamics, demonstrating how conformational interplay can control the activity of a macromolecular machine.

Single-molecule dynamics of conformational changes in flavin adenine dinucleotide

Dynamic conformational changes of the prosthetic group, flavin adenine dinucleotide (FAD), in flavoprotein NADH peroxidase (Npx), in thioredoxin reductase (TrxR), and in free solution were monitored with total internal reflection fluorescence microscopy. FAD bound loosely in the proteins changed from the stacked conformation to the unstacked conformation upon laser excitation. On the other hand, our results show that FAD in free solution not only underwent conformational changes but also reacted with each other to form a dimer.

Stacked–Unstacked Equilibrium at the Nick Site of DNA

Stability of duplex DNA with respect to separation of complementary strands is crucial for DNA executing its major functions in the cell and it also plays a central role in major biotechnology applications of DNA: DNA sequencing, polymerase chain reaction, and DNA microarrays. Two types of interaction are well known to contribute to DNA stability: stacking between adjacent base-pairs and pairing between complementary bases. However, their contribution into the duplex stability is yet to be determined. Now we fill this fundamental gap in our knowledge of the DNA double helix. We have prepared a series of 32, 300 bp-long DNA fragments with solitary nicks in the same position differing only in base-pairs flanking the nick. Electrophoretic mobility of these fragments in the gel has been studied. Assuming the equilibrium between stacked and unstacked conformations at the nick site, all 32 stacking free energy parameters have been obtained. Only ten of them are essential and they govern the stacking interactions between adjacent base-pairs in intact DNA double helix. A full set of DNA stacking parameters has been determined for the first time. From these data and from a well-known dependence of DNA melting temperature on G.C content, the contribution of base-pairing into duplex stability has been estimated. The obtained energy parameters of the DNA double helix are of paramount importance for understanding sequence-dependent DNA flexibility and for numerous biotechnology applications.

Fluorescence quenching of flavin adenine dinucleotide in aqueous solution by pH dependent isomerisation and photo-induced electron transfer

Abstract The fluorescence behaviour of flavin adenine dinucleotide (FAD) in aqueous solution is investigated over a wide hydrogen ion range from pH=1 to pH=11.3. The accessible pH range is limited by room temperature hydrolysis of FAD to flavin-mononucleotide and adenosine-monophosphate at high and low pH. Fluorescence quantum distributions, fluorescence quantum yields, and fluorescence signal decays are measured. The results are compared with the pH dependence of riboflavin in aqueous solution. In the range from pH ≈ 4 to pH ≈ 9 the fluorescence quantum yield is approximately constant at φ F ≈0.033. Below pH=4 the fluorescence rises up to φ F ≈0.13 around pH=2.5 and then reduces similar as riboflavin. The fluorescence behaviour is discussed in terms of a two conformation system. In the range from pH ≈ 3.5 to pH ≈ 11 the stacked (closed) isoalloxazine–adenine conformation dominates over the un-stacked (open) conformation in the S 0 ground-state. At higher and lower pH the ground-state isomerisation equilibrium shifts towards un-stacked conformation dominance. S 1 state photo-isomerisation converts un-stacked molecules to stacked molecules, especially in the range from pH ≈ 3 to pH ≈ 11. The stacked molecules are practically non-fluorescent due to reductive photo-induced electron transfer from the adenine moiety to the isoalloxazine moiety.

Binding modes of two novel dinucleotide inhibitors of HIV-1 integrase

Insights into the binding modes on HIV-1 integrase of our novel dinucleotide inhibitors (pisodApdC and pdCpisodU) have been obtained using molecular docking experiments. In contrast to their base-stacked unbound state, these dinucleotides in their integrase-bound state prefer unstacked conformations for a more extensive interaction with the active site. The calculated free energies of binding are in concert with the experimentally acquired anti-HIV-1 integrase data.