Measuring thermodynamic preferences to form non-native conformations in nucleic acids using ultraviolet melting.

Thermodynamic preferences to form non-native conformations are crucial for understanding how nucleic acids fold and function. However, they are difficult to measure experimentally because this requires accurately determining the population of minor low-abundance (<10%) conformations in a sea of other conformations. Here, we show that melting experiments enable facile measurements of thermodynamic preferences to adopt nonnative conformations in DNA and RNA. The key to this "delta-melt" approach is to use chemical modifications to render specific minor non-native conformations the major state. The validity and robustness of delta-melt is established for four different non-native conformations under various physiological conditions and sequence contexts through independent measurements of thermodynamic preferences using NMR. Delta-melt is faster relative to NMR, simple, and cost-effective and enables thermodynamic preferences to be measured for exceptionally low-populated conformations. Using delta-melt, we obtained rare insights into conformational cooperativity, obtaining evidence for significant cooperativity (1.0 to 2.5 kcal/mol) when simultaneously forming two adjacent Hoogsteen base pairs. We also measured the thermodynamic preferences to form G-C+ and A-T Hoogsteen and A-T base open states for nearly all 16 trinucleotide sequence contexts and found distinct sequence-specific variations on the order of 2 to 3 kcal/mol. This rich landscape of sequence-specific non-native minor conformations in the DNA double helix may help shape the sequence specificity of DNA biochemistry. Thus, melting experiments can now be used to access thermodynamic information regarding regions of the free energy landscape of biomolecules beyond the native folded and unfolded conformations.

Revealing A-T and G-C Hoogsteen base pairs in stressed protein-bound duplex DNA.

Watson-Crick base pairs (bps) are the fundamental unit of genetic information and the building blocks of the DNA double helix. However, A-T and G-C can also form alternative 'Hoogsteen' bps, expanding the functional complexity of DNA. We developed 'Hoog-finder', which uses structural fingerprints to rapidly screen Hoogsteen bps, which may have been mismodeled as Watson-Crick in crystal structures of protein-DNA complexes. We uncovered 17 Hoogsteen bps, 7 of which were in complex with 6 proteins never before shown to bind Hoogsteen bps. The Hoogsteen bps occur near mismatches, nicks and lesions and some appear to participate in recognition and damage repair. Our results suggest a potentially broad role for Hoogsteen bps in stressed regions of the genome and call for a community-wide effort to identify these bps in current and future crystal structures of DNA and its complexes.

Probing conformational transitions towards mutagenic Watson-Crick-like G·T mismatches using off-resonance sugar carbon R1ρ relaxation dispersion.

NMR off-resonance R1ρ relaxation dispersion measurements on base carbon and nitrogen nuclei have revealed that wobble G·T/U mismatches in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-abundance, and mutagenic Watson-Crick-like conformations. As Watson-Crick-like G·T mismatches have base pairing geometries similar to Watson-Crick base pairs, we hypothesized that they would mimic Watson-Crick base pairs with respect to the sugar-backbone conformation as well. Using off-resonance R1ρ measurements targeting the sugar C3' and C4' nuclei, a structure survey, and molecular dynamics simulations, we show that wobble G·T mismatches adopt sugar-backbone conformations that deviate from the canonical Watson-Crick conformation and that transitions toward tautomeric and anionic Watson-Crick-like G·T mismatches restore the canonical Watson-Crick sugar-backbone. These measurements also reveal kinetic isotope effects for tautomerization in D2O versus H2O, which provide experimental evidence in support of a transition state involving proton transfer. The results provide additional evidence in support of mutagenic Watson-Crick-like G·T mismatches, help rule out alternative inverted wobble conformations in the case of anionic G·T-, and also establish sugar carbons as new non-exchangeable probes of this exchange process.

Characterizing micro-to-millisecond chemical exchange in nucleic acids using off-resonance R1ρ relaxation dispersion.

This review describes off-resonance R1ρ relaxation dispersion NMR methods for characterizing microsecond-to-millisecond chemical exchange in uniformly 13C/15N labeled nucleic acids in solution. The review opens with a historical account of key developments that formed the basis for modern R1ρ techniques used to study chemical exchange in biomolecules. A vector model is then used to describe the R1ρ relaxation dispersion experiment, and how the exchange contribution to relaxation varies with the amplitude and frequency offset of an applied spin-locking field, as well as the population, exchange rate, and differences in chemical shifts of two exchanging species. Mathematical treatment of chemical exchange based on the Bloch-McConnell equations is then presented and used to examine relaxation dispersion profiles for more complex exchange scenarios including three-state exchange. Pulse sequences that employ selective Hartmann-Hahn cross-polarization transfers to excite individual 13C or 15N spins are then described for measuring off-resonance R1ρ(13C) and R1ρ(15N) in uniformly 13C/15N labeled DNA and RNA samples prepared using commercially available 13C/15N labeled nucleotide triphosphates. Approaches for analyzing R1ρ data measured at a single static magnetic field to extract a full set of exchange parameters are then presented that rely on numerical integration of the Bloch-McConnell equations or the use of algebraic expressions. Methods for determining structures of nucleic acid excited states are then reviewed that rely on mutations and chemical modifications to bias conformational equilibria, as well as structure-based approaches to calculate chemical shifts. Applications of the methodology to the study of DNA and RNA conformational dynamics are reviewed and the biological significance of the exchange processes is briefly discussed.

Dynamic basis for dG•dT misincorporation via tautomerization and ionization.

Tautomeric and anionic Watson-Crick-like mismatches have important roles in replication and translation errors through mechanisms that are not fully understood. Here, using NMR relaxation dispersion, we resolve a sequence-dependent kinetic network connecting G•T/U wobbles with three distinct Watson-Crick mismatches: two rapidly exchanging tautomeric species (Genol•T/UG•Tenol/Uenol; population less than 0.4%) and one anionic species (G•T-/U-; population around 0.001% at neutral pH). The sequence-dependent tautomerization or ionization step was inserted into a minimal kinetic mechanism for correct incorporation during replication after the initial binding of the nucleotide, leading to accurate predictions of the probability of dG•dT misincorporation across different polymerases and pH conditions and for a chemically modified nucleotide, and providing mechanisms for sequence-dependent misincorporation. Our results indicate that the energetic penalty for tautomerization and/or ionization accounts for an approximately 10-2 to 10-3-fold discrimination against misincorporation, which proceeds primarily via tautomeric dGenol•dT and dG•dTenol, with contributions from anionic dG•dT- dominant at pH 8.4 and above or for some mutagenic nucleotides.

Direct NMR Evidence that Transient Tautomeric and Anionic States in dG·dT Form Watson-Crick-like Base Pairs.

The replicative and translational machinery utilizes the unique geometry of canonical G·C and A·T/U Watson-Crick base pairs to discriminate against DNA and RNA mismatches in order to ensure high fidelity replication, transcription, and translation. There is growing evidence that spontaneous errors occur when mismatches adopt a Watson-Crick-like geometry through tautomerization and/or ionization of the bases. Studies employing NMR relaxation dispersion recently showed that wobble dG·dT and rG·rU mismatches in DNA and RNA duplexes transiently form tautomeric and anionic species with probabilities (≈0.01-0.40%) that are in concordance with replicative and translational errors. Although computational studies indicate that these exceptionally short-lived and low-abundance species form Watson-Crick-like base pairs, their conformation could not be directly deduced from the experimental data, and alternative pairing geometries could not be ruled out. Here, we report direct NMR evidence that the transient tautomeric and anionic species form hydrogen-bonded Watson-Crick-like base pairs. A guanine-to-inosine substitution, which selectively knocks out a Watson-Crick-type (G)N2H2···O2(T) hydrogen bond, significantly destabilized the transient tautomeric and anionic species, as assessed by lack of any detectable chemical exchange by imino nitrogen rotating frame spin relaxation (R1ρ) experiments. An 15N R1ρ NMR experiment targeting the amino nitrogen of guanine (dG-N2) provides direct evidence for Watson-Crick (G)N2H2···O2(T) hydrogen bonding in the transient tautomeric state. The strategy presented in this work can be generally applied to examine hydrogen-bonding patterns in nucleic acid transient states including in other tautomeric and anionic species that are postulated to play roles in replication and translational errors.

m(1)A and m(1)G disrupt A-RNA structure through the intrinsic instability of Hoogsteen base pairs.

The B-DNA double helix can dynamically accommodate G-C and A-T base pairs in either Watson-Crick or Hoogsteen configurations. Here, we show that G-C(+) (in which + indicates protonation) and A-U Hoogsteen base pairs are strongly disfavored in A-RNA. As a result,N(1)-methyladenosine and N(1)-methylguanosine, which occur in DNA as a form of alkylation damage and in RNA as post-transcriptional modifications, have dramatically different consequences. Whereas they create G-C(+) and A-T Hoogsteen base pairs in duplex DNA, thereby maintaining the structural integrity of the double helix, they block base-pairing and induce local duplex melting in RNA. These observations provide a mechanism for disrupting RNA structure through post-transcriptional modifications. The different propensities to form Hoogsteen base pairs in B-DNA and A-RNA may help cells meet the opposing requirements of maintaining genome stability, on the one hand, and of dynamically modulating the structure of the epitranscriptome, on the other.

Shortening the HIV-1 TAR RNA Bulge by a Single Nucleotide Preserves Motional Modes over a Broad Range of Time Scales.

Helix-junction-helix (HJH) motifs are flexible building blocks of RNA architecture that help define the orientation and dynamics of helical domains. They are also frequently involved in adaptive recognition of proteins and small molecules and in the formation of tertiary contacts. Here, we use a battery of nuclear magnetic resonance techniques to examine how deleting a single bulge residue (C24) from the human immunodeficiency virus type 1 (HIV-1) transactivation response element (TAR) trinucleotide bulge (U23-C24-U25) affects dynamics over a broad range of time scales. Shortening the bulge has an effect on picosecond-to-nanosecond interhelical and local bulge dynamics similar to that casued by increasing the Mg(2+) and Na(+) concentration, whereby a preexisting two-state equilibrium in TAR is shifted away from a bent flexible conformation toward a coaxial conformation, in which all three bulge residues are flipped out and flexible. Surprisingly, the point deletion minimally affects microsecond-to-millisecond conformational exchange directed toward two low-populated and short-lived excited conformational states that form through reshuffling of bases pairs throughout TAR. The mutant does, however, adopt a slightly different excited conformational state on the millisecond time scale, in which U23 is intrahelical, mimicking the expected conformation of residue C24 in the excited conformational state of wild-type TAR. Thus, minor changes in HJH topology preserve motional modes in RNA occurring over the picosecond-to-millisecond time scales but alter the relative populations of the sampled states or cause subtle changes in their conformational features.

Characterizing RNA Excited States Using NMR Relaxation Dispersion.

Changes in RNA secondary structure play fundamental roles in the cellular functions of a growing number of noncoding RNAs. This chapter describes NMR-based approaches for characterizing microsecond-to-millisecond changes in RNA secondary structure that are directed toward short-lived and low-populated species often referred to as "excited states." Compared to larger scale changes in RNA secondary structure, transitions toward excited states do not require assistance from chaperones, are often orders of magnitude faster, and are localized to a small number of nearby base pairs in and around noncanonical motifs. Here, we describe a procedure for characterizing RNA excited states using off-resonance R1ρ NMR relaxation dispersion utilizing low-to-high spin-lock fields (25-3000 Hz). R1ρ NMR relaxation dispersion experiments are used to measure carbon and nitrogen chemical shifts in base and sugar moieties of the excited state. The chemical shift data are then interpreted with the aid of secondary structure prediction to infer potential excited states that feature alternative secondary structures. Candidate structures are then tested by using mutations, single-atom substitutions, or by changing physiochemical conditions, such as pH and temperature, to either stabilize or destabilize the candidate excited state. The resulting chemical shifts of the mutants or under different physiochemical conditions are then compared to those of the ground and excited states. Application is illustrated with a focus on the transactivation response element from the human immune deficiency virus type 1, which exists in dynamic equilibrium with at least two distinct excited states.

New insights into Hoogsteen base pairs in DNA duplexes from a structure-based survey.

Hoogsteen (HG) base pairs (bps) provide an alternative pairing geometry to Watson-Crick (WC) bps and can play unique functional roles in duplex DNA. Here, we use structural features unique to HG bps (syn purine base, HG hydrogen bonds and constricted C1'-C1' distance across the bp) to search for HG bps in X-ray structures of DNA duplexes in the Protein Data Bank. The survey identifies 106 A•T and 34 G•C HG bps in DNA duplexes, many of which are undocumented in the literature. It also uncovers HG-like bps with syn purines lacking HG hydrogen bonds or constricted C1'-C1' distances that are analogous to conformations that have been proposed to populate the WC-to-HG transition pathway. The survey reveals HG preferences similar to those observed for transient HG bps in solution by nuclear magnetic resonance, including stronger preferences for A•T versus G•C bps, TA versus GG steps, and also suggests enrichment at terminal ends with a preference for 5'-purine. HG bps induce small local perturbations in neighboring bps and, surprisingly, a small but significant degree of DNA bending (∼14°) directed toward the major groove. The survey provides insights into the preferences and structural consequences of HG bps in duplex DNA.

Visualizing transient Watson-Crick-like mispairs in DNA and RNA duplexes.

Rare tautomeric and anionic nucleobases are believed to have fundamental biological roles, but their prevalence and functional importance has remained elusive because they exist transiently, in low abundance, and involve subtle movements of protons that are difficult to visualize. Using NMR relaxation dispersion, we show here that wobble dG•dT and rG•rU mispairs in DNA and RNA duplexes exist in dynamic equilibrium with short-lived, low-populated Watson-Crick-like mispairs that are stabilized by rare enolic or anionic bases. These mispairs can evade Watson-Crick fidelity checkpoints and form with probabilities (10(-3) to 10(-5)) that strongly imply a universal role in replication and translation errors. Our results indicate that rare tautomeric and anionic bases are widespread in nucleic acids, expanding their structural and functional complexity beyond that attainable with canonical bases.

Development and application of aromatic [(13)C, (1)H] SOFAST-HMQC NMR experiment for nucleic acids.

Higher sensitivity of NMR spectrometers and novel isotopic labeling schemes have ushered the development of rapid data acquisition methodologies, improving the time resolution with which NMR data can be acquired. For nucleic acids, longitudinal relaxation optimization in conjunction with Ernst angle excitation (SOFAST-HMQC) for imino protons, in addition to rendering rapid pulsing, has been demonstrated to yield significant improvements in sensitivity per unit time. Extending such methodology to other spins offers a viable prospect to measure additional chemical shifts, thereby broadening their utilization for various applications. Here, we introduce the 2D [(13)C, (1)H] aromatic SOFAST-HMQC that results in overall sensitivity gain of 1.4- to 1.7-fold relative to the conventional HMQC and can also be extended to yield long-range heteronuclear chemical shifts such as the adenine imino nitrogens N1, N3, N7 and N9. The applications of these experiments range from monitoring real-time biochemical processes, drug/ligand screening, and to collecting data at very low sample concentration and/or in cases where isotopic enrichment cannot be achieved.

Increasing occurrences and functional roles for high energy purine-pyrimidine base-pairs in nucleic acids.

There are a growing number of studies reporting the observation of purine-pyrimidine base-pairs that are seldom observed in unmodified nucleic acids because they entail the loss of energetically favorable interactions or require energetically costly base ionization or tautomerization. These high energy purine-pyrimidine base-pairs include G•C(+) and A•T Hoogsteen base-pairs, which entail ∼180° rotation of the purine base in a Watson-Crick base-pair, protonation of cytosine N3, and constriction of the C1'-C1' distance by ∼2.5Å. Other high energy pure-pyrimidine base-pairs include G•T, G•U, and A•C mispairs that adopt Watson-Crick like geometry through either base ionization or tautomerization. Although difficult to detect and characterize using biophysical methods, high energy purine-pyrimidine base-pairs appear to be more common than once thought. They further expand the structural and functional diversity of canonical and non-canonical nucleic acid base-pairs.

A historical account of Hoogsteen base-pairs in duplex DNA.

In 1957, a unique pattern of hydrogen bonding between N3 and O4 on uracil and N7 and N6 on adenine was proposed to explain how poly(rU) strands can associate with poly(rA)-poly(rU) duplexes to form triplexes. Two years later, Karst Hoogsteen visualized such a noncanonical A-T base-pair through X-ray analysis of co-crystals containing 9-methyladenine and 1-methylthymine. Subsequent X-ray analyses of guanine and cytosine derivatives yielded the expected Watson-Crick base-pairing, but those of adenine and thymine (or uridine) did not yield Watson-Crick base-pairs, instead favoring "Hoogsteen" base-pairing. More than two decades ensued without experimental "proof" for A-T Watson-Crick base-pairs, while Hoogsteen base-pairs continued to surface in AT-rich sequences, closing base-pairs of apical loops, in structures of DNA bound to antibiotics and proteins, damaged and chemically modified DNA, and in polymerases that replicate DNA via Hoogsteen pairing. Recently, NMR studies have shown that base-pairs in duplex DNA exist as a dynamic equilibrium between Watson-Crick and Hoogsteen forms. There is now little doubt that Hoogsteen base-pairs exist in significant abundance in genomic DNA, where they can expand the structural and functional versatility of duplex DNA beyond that which can be achieved based only on Watson-Crick base-pairing. Here, we provide a historical account of the discovery and characterization of Hoogsteen base-pairs, hoping that this will inform future studies exploring the occurrence and functional importance of these alternative base-pairs.

Dual-function triazole-pyridine derivatives as inhibitors of metal-induced amyloid-ß aggregation.

Dysregulated metal ions are hypothesized to play a role in the aggregation of the amyloid-ß (Aß) peptide, leading to Alzheimer's disease (AD) pathology. In addition to direct effects on Aß aggregation, both Cu and Fe can catalyze the generation of reactive oxygen species (ROS), possibly contributing to significant neuronal toxicity. Therefore, disruption of metal-Aß interactions has become a viable strategy for AD therapeutic development. Herein, we report a new series of dual-function triazole-pyridine ligands [4-(2-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)ethyl)morpholine (L1), 3-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)propan-1-ol (L2), 2-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)acetic acid (L3), and 5-(4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)pentan-1-amine (L4)] that interact with the Aß peptide and modulate its aggregation in vitro. Metal chelation and Aß interaction properties of these molecules were studied by UV-vis, NMR spectroscopy and X-ray crystallography. In addition, turbidity and transmission electron microscopy (TEM) were employed to determine the anti-aggregation properties of L1-L4. All compounds demonstrated an ability to limit metal-induced Aß aggregation. Overall, our studies suggest the utility of the triazole-pyridine framework in the development of chemical reagents toward inhibitors for metal-triggered Aß aggregation.