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Biochemistry Jul 2022Aromatic NH groups are essential as hydrogen-bond donors in secondary structures of DNA and RNA. Although rapid rotations of NH groups of adenine and guanine bases were...
Aromatic NH groups are essential as hydrogen-bond donors in secondary structures of DNA and RNA. Although rapid rotations of NH groups of adenine and guanine bases were previously characterized, there has been a lack of quantitative information about slow rotations of cytosine NH groups in Watson-Crick base pairs. In this study, using an NMR method we had recently developed, we determined the kinetic rate constants and energy barriers for cytosine NH rotations in a 15-base-pair DNA duplex. Our data show that the rotational dynamics of cytosine NH groups depend on local environments. Qualitative correlation between the ranges of N chemical shifts and rotational time scales for various NH groups of nucleic acids and proteins illuminates a relationship between the partial double-bond character of the C-N bond and the time scale for NH rotations.
Topics: Base Pairing; Cytosine; DNA; Guanine; Hydrogen Bonding; Nucleic Acid Conformation
PubMed: 35759792
DOI: 10.1021/acs.biochem.2c00299 -
Nature Communications Mar 2022RNA excited states represent a class of high-energy-level and thus low-populated conformational states of RNAs that are sequestered within the free energy landscape...
RNA excited states represent a class of high-energy-level and thus low-populated conformational states of RNAs that are sequestered within the free energy landscape until being activated by cellular cues. In recent years, there has been growing interest in structural and functional studies of these transient states, but the rational design of excited states remains unexplored. Here we developed a method to design small hairpin RNAs with predefined excited states that exchange with ground states through base pair reshuffling, and verified these transient states by combining NMR relaxation dispersion technique and imino chemical shift prediction. Using van't Hoff analysis and accelerated molecular dynamics simulations, a mechanism of multi-step sequential transition has been revealed. The efforts made in this study will expand the scope of RNA rational design, and also contribute towards improved predictions of RNA secondary structure.
Topics: Base Pairing; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; RNA
PubMed: 35314698
DOI: 10.1038/s41467-022-29194-8 -
International Journal of Molecular... Sep 2015Genome-wide association studies have identified numerous single-nucleotide polymorphisms (SNPs) associated with human diseases or phenotypes. However, causal... (Review)
Review
Genome-wide association studies have identified numerous single-nucleotide polymorphisms (SNPs) associated with human diseases or phenotypes. However, causal relationships between most SNPs and the associated disease have not been established, owing to technical challenges such as unavailability of suitable cell lines. Recently, efficient editing of a single base pair in the genome was achieved using programmable site-specific nucleases. This technique enables experimental confirmation of the causality between SNPs and disease, and is potentially valuable in clinical applications. In this review, I introduce the molecular basis and describe examples of single-base pair editing in human cells. I also discuss the challenges associated with the technique, as well as possible solutions.
Topics: Base Pairing; Endonucleases; Genetic Engineering; Genetic Predisposition to Disease; Genome, Human; Genome-Wide Association Study; Humans; Polymorphism, Single Nucleotide
PubMed: 26404258
DOI: 10.3390/ijms160921128 -
Nucleic Acids Research Mar 2012Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic...
Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ∼10(10)-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 10(28)-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnology.
Topics: Base Pairing; Imidazoles; Polymerase Chain Reaction; Pyridines; Pyrroles; Sequence Analysis, DNA
PubMed: 22121213
DOI: 10.1093/nar/gkr1068 -
Molecules (Basel, Switzerland) Oct 2020Quantum mechanical (QM) and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of a recently reported dinuclear mercury(II)-mediated...
Quantum mechanical (QM) and hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of a recently reported dinuclear mercury(II)-mediated base pair were performed aiming to analyse its intramolecular bonding pattern, its stability, and to obtain clues on the mechanism of the incorporation of mercury(II) into the DNA. The dynamic distance constraint was employed to find initial structures, control the dissociation process in an unbiased fashion and to determine the free energy required. A strong influence of the exocyclic carbonyl or amino groups of neighbouring base pairs on both the bonding pattern and the mechanism of incorporation was observed. During the dissociation simulation, an amino group of an adenine moiety of the adjacent base pair acts as a turnstile to rotate the mercury(II) ion out of the DNA core region. The calculations provide an important insight into the mechanism of formation of this dinuclear metal-mediated base pair and indicate that the exact location of a transition metal ion in a metal-mediated base pair may be more ambiguous than derived from simple model building.
Topics: Base Pairing; DNA; Mercury; Molecular Dynamics Simulation; Thermodynamics
PubMed: 33114568
DOI: 10.3390/molecules25214942 -
Chemistry (Weinheim An Der Bergstrasse,... Dec 2022Purine-2,6-diamine and 8-aza-7-deaza-7-bromopurine-2,6-diamine 2'-deoxyribonucleosides (1 and 2) were implemented in isothermal DNA strand displacement reactions....
DNA Strand Displacement with Base Pair Stabilizers: Purine-2,6-Diamine and 8-Aza-7-Bromo-7-Deazapurine-2,6-Diamine Oligonucleotides Invade Canonical DNA and New Fluorescent Pyrene Click Sensors Monitor the Reaction.
Purine-2,6-diamine and 8-aza-7-deaza-7-bromopurine-2,6-diamine 2'-deoxyribonucleosides (1 and 2) were implemented in isothermal DNA strand displacement reactions. Nucleoside 1 is a weak stabilizer of dA-dT base pairs, nucleoside 2 evokes strong stabilization. Strand displacement reactions used single-stranded invaders with single and multiple incorporations of stabilizers. Displacement is driven by negative enthalpy changes between target and displaced duplex. Toeholds are not required. Two new environmental sensitive fluorescent pyrene sensors were developed to monitor the progress of displacement reactions. Pyrene was connected to the nucleobase in the invader or to a dendritic linker in the output strand. Both new sensors were constructed by click chemistry; phosphoramidites and oligonucleotides were prepared. Sensors show monomer or excimer emission. Fluorescence intensity changes when the displacement reaction progresses. Our work demonstrates that strand displacement with base pair stabilizers is applicable to DNA, RNA and to related biopolymers with applications in chemical biology, nanotechnology and medicinal diagnostics.
Topics: Oligonucleotides; Base Pairing; Nucleosides; DNA; Purines; Coloring Agents; Pyrenes
PubMed: 36178316
DOI: 10.1002/chem.202202412 -
RNA (New York, N.Y.) Dec 2009The maturation of RNAs includes site-specific post-transcriptional modifications that contribute significantly to hydrogen bond formation within RNA and between...
The maturation of RNAs includes site-specific post-transcriptional modifications that contribute significantly to hydrogen bond formation within RNA and between different RNAs, especially in formation of mismatch base pairs. Thus, an understanding of the geometry and strength of the base-pairing of modified ribonucleoside 5'-monophosphates, previously not defined, is applicable to investigations of RNA structure and function and of the design of novel RNAs. The geometry and free energies of base-pairings were calculated in aqueous solution under neutral conditions with AMBER force fields and molecular dynamics simulations (MDSs). For example, unmodified uridines were observed to bind to uridine and cytidine with significant stability, but the ribose C1'-C1' distances were far short ( approximately 8.9 A) of distances observed for canonical A-form RNA helices. In contrast, 5-oxyacetic acid uridine, known to bind adenosine, wobble to guanosine, and form mismatch base pairs with uridine and cytidine, bound adenosine and guanosine with geometries and energies comparable to an unmodified uridine. However, the 5-oxyacetic acid uridine base paired to uridine and cytidine with a C1'-C1' distance comparable to that of an A-form helix, approximately 11 A, when a H(2)O molecule migrated between and stably hydrogen bonded to both bases. Even in formation of canonical base pairs, intermediate structures with a second energy minimum consisted of transient H(2)O molecules forming hydrogen bonded bridges between the two bases. Thus, MDS is predictive of the effects of modifications, H(2)O molecule intervention in the formation of base-pair geometry, and energies that are important for native RNA structure and function.
Topics: Base Pairing; Models, Genetic; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; RNA; Thermodynamics
PubMed: 19861423
DOI: 10.1261/rna.1734309 -
Nucleic Acids Research May 2003Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of... (Review)
Review
Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G*A base pairs are separated by one or two pairs of purine*purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G*C or A.T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G.A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G*A, A*A, A*C or sheared-like G(anti)*C(syn) base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.
Topics: Base Pair Mismatch; Base Pairing; Base Sequence; DNA; Hydrogen Bonding; Models, Molecular; Nucleic Acid Conformation; Nucleic Acid Heteroduplexes
PubMed: 12736295
DOI: 10.1093/nar/gkg367 -
Molecules (Basel, Switzerland) Mar 2019We review the preparation of new compounds with good solution and cell uptake properties that can selectively recognize mixed A·T and G·C bp sequences of DNA. Our... (Review)
Review
We review the preparation of new compounds with good solution and cell uptake properties that can selectively recognize mixed A·T and G·C bp sequences of DNA. Our underlying aim is to show that these new compounds provide important new biotechnology reagents as well as a new class of therapeutic candidates with better properties and development potential than other currently available agents. In this review, entirely different ways to recognize mixed sequences of DNA by modifying AT selective heterocyclic cations are described. To selectively recognize a G·C base pair an H-bond acceptor must be incorporated with AT recognizing groups as with netropsin. We have used pyridine, azabenzimidazole and thiophene--methylbenzimidazole GC recognition units in modules crafted with both rational design and empirical optimization. These modules can selectively and strongly recognize a single G·C base pair in an AT sequence context. In some cases, a relatively simple change in substituents can convert a heterocyclic module from AT to GC recognition selectivity. Synthesis and DNA interaction results for initial example lead modules are described for single G·C base pair recognition compounds. The review concludes with a description of the initial efforts to prepare larger compounds to recognize sequences of DNA with more than one G·C base pairs. The challenges and initial successes are described along with future directions.
Topics: Base Pairing; Base Sequence; DNA; Heterocyclic Compounds; Nucleic Acid Conformation; Surface Plasmon Resonance
PubMed: 30866557
DOI: 10.3390/molecules24050946 -
ELife Jan 2019Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts...
Transcriptional pausing underlies regulation of cellular RNA biogenesis. A consensus pause sequence that acts on RNA polymerases (RNAPs) from bacteria to mammals halts RNAP in an elemental paused state from which longer-lived pauses can arise. Although the structural foundations of pauses prolonged by backtracking or nascent RNA hairpins are recognized, the fundamental mechanism of the elemental pause is less well-defined. Here we report a mechanistic dissection that establishes the elemental pause signal () is multipartite; () causes a modest conformational shift that puts γ-proteobacterial RNAP in an off-pathway state in which template base loading but not RNA translocation is inhibited; and () allows RNAP to enter pretranslocated and one-base-pair backtracked states easily even though the half-translocated state observed in paused cryo-EM structures rate-limits pause escape. Our findings provide a mechanistic basis for the elemental pause and a framework to understand how pausing is modulated by sequence, cellular conditions, and regulators.
Topics: Base Pairing; Base Sequence; Consensus Sequence; DNA; Kinetics; Mutation; Nucleotides; RNA; Templates, Genetic; Transcription Elongation, Genetic; Transcription, Genetic
PubMed: 30618376
DOI: 10.7554/eLife.40981