Crick Base Pairs Research Articles

Valid Plane Trees: Combinatorial Models for RNA Secondary Structures with Watson--Crick Base Pairs

The combinatorics of RNA plays a central role in biology. Mathematical biologists have several commonly used models for RNA: words in a fixed alphabet (representing the primary sequence of nucleotides) and plane trees (representing the secondary structure, or folding of the RNA sequence). This paper considers an augmented version of the standard model of plane trees, specifically one that incorporates some observed constraints on how the folding can occur. In particular we assume the alphabet consists of complementary pairs, for instance the Watson--Crick pairs A-U and C-G of RNA. Given a word in the alphabet, a valid plane tree is a tree for which, when the word is folded around the tree, each edge matches two complementary letters. Consider the graph whose vertices are valid plane trees for a fixed word and whose edges are given by Condon, Heitsch, and Hoos's local moves (see [C. E. Heitsch, Combinatorics on Plane Trees, Motivated by RNA Secondary Structure Configurations, preprint, Genome Center of Wisconsin and Department of Mathematics, University of Wisconsin--Madison]). We prove that this graph is connected. We give an explicit algorithm to construct a valid plane tree from a primary sequence, assuming that at least one valid plane tree exists. The tree produced by our algorithm has other useful characterizations, including a uniqueness condition defined by local moves. We also study enumerative properties of valid plane trees, analyzing how the number of valid plane trees depends on the choice of sequence length and alphabet size. Finally, we show that the proportion of words with at least one valid plane tree goes to zero as the word size increases. We also present some open questions.

Proton-detected solid-state NMR detects the inter-nucleotide correlations and architecture of dimeric RNA in microcrystals

We report a novel proton-detected MAS solid-state NMR strategy based on 15N-15N proton assisted recoupling to detect the inter-nucleotide NHN hydrogen bonds within the Watson-Crick base pairs of micro-crystallized dimeric RNA and to confirm the kissing-loop structure. This would contribute to advances in the structural determination of RNA using solid-state NMR.

Stacking interactions involving non-Watson-Crick basepairs: dispersion corrected density functional theory studies.

The G:A basepair, stabilized by hydrogen bonding through the sugar edge of guanine and Hoogsteen edge of adenine in trans orientation (G:A S:HT), appears very frequently in the solved RNA structures and is very stable. We have carried out stacking energy analyses of two sequences, namely C:G W:WC::G:A S:HT and G:C W:WC::G:A S:HT (':' represents basepairing and '::' represents stacking interactions), formed by this non-Watson-Crick basepair, by DFT-D. We have scanned nearly the complete phase space by modeling structures of these sequences using different values of the basepair orientation parameters to determine the most stable conformations. It is found that the most stable conformations are rather far from the most frequently found orientations. We have considered the effect of sugar-phosphate backbone connectivity as an energy penalty arising from deformation of pseudo bond lengths between C1' atoms of successive bases along the strands. Augmentation of stacking energy from DFT-D by this coarse grain energy gives predicted structures extremely similar to the experimentally determined ones. It has been observed that the best stacking with small twist values is associated with positive roll and negative slide values, which are similar to their values in A-RNA structures for most sequences. Among the two base pair steps, C:G W:WC::G:A S:HT appears to be more stable in terms of stacking energy as compared to G:C W:WC::G:A S:HT possibly due to larger stacking overlap in the former one.

Influence of two bulge loops on the stability of RNA duplexes.

Fifty-three RNA duplexes containing two single nucleotide bulge loops were optically melted in 1 M NaCl in order to determine the thermodynamic parameters ΔH°, ΔS°, ΔG°37, and TM for each duplex. Because of the large number of possible combinations and lack of sequence effects observed previously, we limited our initial investigation to adenosine bulges, the most common naturally occurring bulge. For example, the following duplexes were investigated: 5′GGCAXYAGGC/3′CCG YX CCG, 5′GGCAXY GCC/3′CCG YXACGG, and 5′GGC XYAGCC/3′CCGAYX CGG. The identity of XY (where XY are Watson–Crick base pairs) and the total number of base pairs in the terminal and central stems were varied. As observed for duplexes with a single bulge loop, the effect of the two bulge loops on duplex stability is primarily influenced by non-nearest neighbor interactions. In particular, the stability of the stems influences the destabilization of the duplex by the inserted bulge loops. The model proposed to predict the influence of multiple bulge loops on duplex stability suggests that the destabilization of each bulge is related to the stability of the adjacent stems. A database of RNA secondary structures was examined to determine the naturally occurring abundance of duplexes containing multiple bulge loops. Of the 2000 examples found in the database, over 65% of the two bulge loops occur within 3 base pairs of each other. A database of RNA three-dimensional structures was examined to determine the structure of duplexes containing two single nucleotide bulge loops. The structures of the bulge loops are described.

Unusual Base-Pairing Interactions in Monomer-Template Complexes.

Many high-resolution crystal structures have contributed to our understanding of the reaction pathway for catalysis by DNA and RNA polymerases, but the structural basis of nonenzymatic template-directed RNA replication has not been studied in comparable detail. Here we present crystallographic studies of the binding of ribonucleotide monomers to RNA primer–template complexes, with the goal of improving our understanding of the mechanism of nonenzymatic RNA copying, and of catalysis by polymerases. To explore how activated ribonucleotides recognize and bind to RNA templates, we synthesized an unreactive phosphonate-linked pyrazole analogue of guanosine 5′-phosphoro-2-methylimidazolide (2-MeImpG), a highly activated nucleotide that has been used extensively to study nonenzymatic primer extension. We cocrystallized this analogue with structurally rigidified RNA primer–template complexes carrying single or multiple monomer binding sites, and obtained high-resolution X-ray structures of these complexes. In addition to Watson–Crick base pairing, we repeatedly observed noncanonical guanine:cytidine base pairs in our crystal structures. In most structures, the phosphate and leaving group moieties of the monomers were highly disordered, while in others the distance from O3′ of the primer to the phosphorus of the incoming monomer was too great to allow for reaction. We suggest that these effects significantly influence the rate and fidelity of nonenzymatic RNA replication, and that even primitive ribozyme polymerases could enhance RNA replication by enforcing Watson–Crick base pairing between monomers and primer–template complexes, and by bringing the reactive functional groups into closer proximity.

DNA functionalization by dynamic chemistry.

Dynamic combinatorial chemistry (DCC) is an attractive method to efficiently generate libraries of molecules from simpler building blocks by reversible reactions under thermodynamic control. Here we focus on the chemical modification of DNA oligonucleotides with acyclic diol linkers and demonstrate their potential for the deoxyribonucleic acid functionalization and generation of libraries of reversibly interconverting building blocks. The syntheses of phosphoramidite building blocks derived from D-threoninol are presented in two variants with protected amino or thiol groups. The threoninol building blocks were successfully incorporated via automated solid-phase synthesis into 13mer oligonucleotides. The amino group containing phosphoramidite was used together with complementary single-strand DNA templates that influenced the Watson–Crick base-pairing equilibrium in the mixture with a set of aldehyde modified nucleobases. A significant fraction of all possible base-pair mismatches was obtained, whereas, the highest selectivity (over 80%) was found for the guanine aldehyde templated by the complementary cytosine containing DNA. The elevated occurrence of mismatches can be explained by increased backbone plasticity derived from the linear threoninol building block as a cyclic deoxyribose analogue.

Reciprocal Self‐Assembly of Peptide–DNA Conjugates into a Programmable Sub‐10‐nm Supramolecular Deoxyribonucleoprotein

AbstractTo overcome the limitations of molecular assemblies, the development of novel supramolecular building blocks and self‐assembly modes is essential to create more sophisticated, complex, and controllable aggregates. The self‐assembly of peptide–DNA conjugates (PDCs), in which two orthogonal self‐assembly modes, that is, β‐sheet formation and Watson–Crick base pairing, are covalently combined in one supramolecular system, is reported. Despite extensive research, most self‐assembly studies have focused on using only one type of building block, which restricts structural and functional diversity compared to multicomponent systems. Multicomponent systems, however, suffer from poor control of self‐assembly behaviors. Covalently conjugated PDC building blocks are shown to assemble into well‐defined and controllable nanostructures. This controllability likely results from the decrease in entropy associated with the restriction of the molecular degrees of freedom by the covalent constraints. Using this strategy, the possibility to thermodynamically program nano‐assemblies to exert gene regulation activity with low cytotoxicity is demonstrated.

How Mg2+ ion and water network affect the stability and structure of non-Watson–Crick base pairs in E. coli loop E of 5S rRNA: a molecular dynamics and reference interaction site model (RISM) study

The non-Watson–Crick (non-WC) base pairs of Escherichia coli loop E of 5S rRNA are stabilized by Mg2+ ions through water-mediated interaction. It is important to know the synergic role of Mg2+ and the water network surrounding Mg2+ in stabilizing the non-WC base pairs of RNA. For this purpose, free energy change of the system is calculated using molecular dynamics (MD) simulation as Mg2+ is pulled from RNA, which causes disturbance of the water network. It was found that Mg2+ remains hexahydrated unless it is close to or far from RNA. In the pentahydrated form, Mg2+ interacts directly with RNA. Water network has been identified by two complimentary methods; MD followed by a density-based clustering algorithm and three-dimensional-reference interaction site model. These two methods gave similar results. Identification of water network around Mg2+ and non-WC base pairs gives a clue to the strong effect of water network on the stability of this RNA. Based on sequence analysis of all Eubacteria 5s rRNA, we propose that hexahydrated Mg2+ is an integral part of this RNA and geometry of base pairs surrounding it adjust to accommodate the . Overall the findings from this work can help in understanding the basis of the complex structure and stability of RNA with non-WC base pairs.

Interfacing DNA nanodevices with biology: challenges, solutions and perspectives

The cellular machinery performs millions of complex reactions with extreme precision at nanoscale. From studying these reactions, scientists have become inspired to build artificial nanosized molecular devices with programmed functions. One of the fundamental tools in designing and creating these nanodevices is molecular self-assembly. In nature, deoxyribonucleic acid (DNA) is inarguably one of the most remarkable self-assembling molecules. Governed by the Watson–Crick base-pairing rules, DNA assembles with a structural reliability and predictability based on sequence composition unlike any other complex biological polymer. This consistency has enabled rational design of hundreds of two- and three-dimensional shapes with a molecular precision and homogeneity not preceded by any other known technology at the nanometer scale.During the last two decades, DNA nanotechnology has undergone a rapid evolution pioneered by the work of Nadrian Seeman (Kallenbach et al 1983 Nature 205 829–31). Especially the introduction of the versatile DNA Origami technique by Rothemund (2006 Nature 440 297–302) led to an efflorescence of new DNA-based self-assembled nanostructures (Andersen et al 2009 Nature 459 73–6, Douglas et al 2009 Nature 459 414–8, Dietz et al 2009 Science 325 725–30, Han et al 2011 Science 332 342–6, Iinuma et al 2014 Science 344 65–9), and variations of this technique have contributed to an increasing repertoire of DNA nanostructures (Wei et al 2012 Nature 485 623–6, Ke et al 2012 Science 338 1177–83, Benson et al 2015 Nature 523 441–4, Zhang et al 2015 Nat. Nanotechnol. 10 779–84, Scheible et al 2015 Small 11 5200–5). These advances have naturally triggered the question: What can these DNA nanostructures be used for?One of the leading proposals of use for DNA nanotechnology has been in biology and biomedicine acting as a molecular ‘nanorobot’ or smart drug interacting with the cellular machinery. In this review, we will explore and examine the perspective of DNA nanotechnology for such use. We summarize which requirements DNA nanostructures must fulfil to function in cellular environments and inside living organisms. In addition, we highlight recent advances in interfacing DNA nanostructures with biology.

Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute.

Argonaute proteins are key players in the gene silencing mechanisms mediated by small nucleic acids in all domains of life from bacteria to eukaryotes. However, little is known about the Argonaute protein that recognizes guide RNA/target DNA. Here, we determine the 2 Å crystal structure of Rhodobacter sphaeroides Argonaute (RsAgo) in a complex with 18-nucleotide guide RNA and its complementary target DNA. The heteroduplex maintains Watson–Crick base-pairing even in the 3′-region of the guide RNA between the N-terminal and PIWI domains, suggesting a recognition mode by RsAgo for stable interaction with the target strand. In addition, the MID/PIWI interface of RsAgo has a system that specifically recognizes the 5′ base-U of the guide RNA, and the duplex-recognition loop of the PAZ domain is important for the DNA silencing activity. Furthermore, we show that Argonaute discriminates the nucleic acid type (RNA/DNA) by recognition of the duplex structure of the seed region.

РЕПАРАЦИЯ НЕСПАРЕННЫХ ОСНОВАНИЙ И ПЕТЕЛЬ ДЕЛЕЦИИ/ВСТАВКИ ДНК У ЭУКАРИОТ

The mismatch repair (MMR) system detects non-Watson – Crick base pairs as well as the defects, appearing in course of DNA replication, and helps to eliminate them by catalyzing the excision of the defect-containing region of daughter DNA and its error-free resynthesis. Thus, MMR remarkably improves the fidelity of replication. After separation, both strands contain non-repairable damages and the mismatches may generate DNA mutation in 50 % of cell progeny after next replication. MMR dysfunction causes surge of mutation rate, abnormal recombination, and cancer in humans and animals. Therefore, the main MMR efficiency parameter is mismatch correction before the next replication cycle. Mismatch detection is made by the MSH2 protein, which forms a heterodimer with either MSH6 or MSH3 (Mut S), depending on the damage (MSH6 is needed for the amendment of single base mispairs, whereas both MSH3 and MSH6 can correct IDLs). A heterodimer of MLH1 and PMS2 (Mut L) controls the interaction between the mismatch-detecting complex of proteins and other proteins essential for MMR, including exonuclease 1, helicase, nuclear antigen of proliferating cells, single-stranded DNA-binding protein and DNA polymerases δ and ε. MLH1 can form a heterodimer with two additional proteins – MLH3 and PMS1. PMS2 is required for the correction of single based mismatches, and PMS2 and MLH3 contribute to the correction of IDLs. The Nobel Prize in Chemistry 2015 was awarded for the studies of DNA repair, i.a. MMR.

Frontispiece: Highly Stable Double‐Stranded DNA Containing Sequential Silver(I)‐Mediated 7‐Deazaadenine/Thymine Watson–Crick Base Pairs

Frontispiece: Highly Stable Double‐Stranded DNA Containing Sequential Silver(I)‐Mediated 7‐Deazaadenine/Thymine Watson–Crick Base Pairs

Frontispiz: Highly Stable Double‐Stranded DNA Containing Sequential Silver(I)‐Mediated 7‐Deazaadenine/Thymine Watson–Crick Base Pairs

Frontispiz: Highly Stable Double‐Stranded DNA Containing Sequential Silver(I)‐Mediated 7‐Deazaadenine/Thymine Watson–Crick Base Pairs

Trinucleotide’s quadruplet symmetries and natural symmetry law of DNA creation ensuing Chargaff’s second parity rule

For almost 50 years the conclusive explanation of Chargaff’s second parity rule (CSPR), the equality of frequencies of nucleotides A=T and C=G or the equality of direct and reverse complement trinucleotides in the same DNA strand, has not been determined yet. Here, we relate CSPR to the interstrand mirror symmetry in 20 symbolic quadruplets of trinucleotides (direct, reverse complement, complement, and reverse) mapped to double-stranded genome. The symmetries of Q-box corresponding to quadruplets can be obtained as a consequence of Watson–Crick base pairing and CSPR together. Alternatively, assuming Natural symmetry law for DNA creation that each trinucleotide in one strand of DNA must simultaneously appear also in the opposite strand automatically leads to Q-box direct-reverse mirror symmetry which in conjunction with Watson–Crick base pairing generates CSPR. We demonstrate quadruplet’s symmetries in chromosomes of wide range of organisms, from Escherichia coli to Neanderthal and human genomes, introducing novel quadruplet-frequency histograms and 3D-diagrams with combined interstrand frequencies. These “landscapes” are mutually similar in all mammals, including extinct Neanderthals, and somewhat different in most of older species. In human chromosomes 1–12, and X, Y the “landscapes” are almost identical and slightly different in the remaining smaller and telocentric chromosomes. Quadruplet frequencies could provide a new robust tool for characterization and classification of genomes and their evolutionary trajectories.

A general strategy for expanding polymerase function by droplet microfluidics.

Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered polymerase. We then applied our method to evolve a manganese-independent α-L-threofuranosyl nucleic acid (TNA) polymerase that functions with >99% template-copying fidelity. Based on our findings, we suggest that DrOPS is a versatile tool that could be used to evolve any polymerase function, where optical detection can be achieved by Watson–Crick base pairing.

Highly Stable Double‐Stranded DNA Containing Sequential Silver(I)‐Mediated 7‐Deazaadenine/Thymine Watson–Crick Base Pairs

The oligonucleotide d(TX)9 , which consists of an octadecamer sequence with alternating non-canonical 7-deazaadenine (X) and canonical thymine (T) as the nucleobases, was synthesized and shown to hybridize into double-stranded DNA through the formation of hydrogen-bonded Watson-Crick base pairs. dsDNA with metal-mediated base pairs was then obtained by selectively replacing W-C hydrogen bonds by coordination bonds to central silver(I) ions. The oligonucleotide I adopts a duplex structure in the absence of Ag(+) ions, and its stability is significantly enhanced in the presence of Ag(+) ions while its double-helix structure is retained. Temperature-dependent UV spectroscopy, circular dichroism spectroscopy, and ESI mass spectrometry were used to confirm the selective formation of the silver(I)-mediated base pairs. This strategy could become useful for preparing stable metallo-DNA-based nanostructures.

Highly Stable Double‐Stranded DNA Containing Sequential Silver(I)‐Mediated 7‐Deazaadenine/Thymine Watson–Crick Base Pairs

AbstractThe oligonucleotide d(TX)9, which consists of an octadecamer sequence with alternating non‐canonical 7‐deazaadenine (X) and canonical thymine (T) as the nucleobases, was synthesized and shown to hybridize into double‐stranded DNA through the formation of hydrogen‐bonded Watson–Crick base pairs. dsDNA with metal‐mediated base pairs was then obtained by selectively replacing W‐C hydrogen bonds by coordination bonds to central silver(I) ions. The oligonucleotide I adopts a duplex structure in the absence of Ag+ ions, and its stability is significantly enhanced in the presence of Ag+ ions while its double‐helix structure is retained. Temperature‐dependent UV spectroscopy, circular dichroism spectroscopy, and ESI mass spectrometry were used to confirm the selective formation of the silver(I)‐mediated base pairs. This strategy could become useful for preparing stable metallo‐DNA‐based nanostructures.

Effects of single water molecule on proton transfer reaction in uracil dimer cation

Ionizing radiation to DNA induces sometimes the DNA damage. In this report, the ionization dynamics of uracil dimer (U)2 and its water complex (U)2–H2O have been investigated by means of direct ab initio molecular dynamics (AIMD) method in order to elucidate the effects of single water molecule on the reaction rate of proton transfer (PT) in DNA model base pair. The (U)2 dimer is widely used as a simplified mimetic model of Watson–Crick base pair. The static ab initio calculation showed that two conformers exist as neutral complex of (U)2–H2O. The direct AIMD calculation of ionization process of (U)2–H2O showed that the rate of PT is affected even by a single water molecule, while it was dependent on the position of H2O around (U)2. The interaction of water molecule with (U)2 affected the potential energy curve for PT. Especially, the activation barrier along the PT coordinate was significantly changed by the interaction with one H2O molecule. The effects of one H2O molecule on the PT process were discussed on the basis of theoretical results.

Density Functional Computations on 6-Aminouracil: Effect of Amino Group in the 6th Position on the Watson–Crick Base Pair Uridine–Adenosine

The predicted infrared and Raman spectra of 6-aminouracil in the solid state by density functional theory methods were analyzed and compared with the experimental spectra. The effect of amino substitution in the sixth position of uridine on the stability of the Watson–Crick (WC) base pairs with deoxyadenosine was evaluated. Different WC pairs of 5-aminouridine, 6-aminouridine, and uridine with deoxyadenosine were simulated, and the counterpoise-corrected interaction energies were determined. 6-Aminouridine produces a stronger WC pair than that involving uridine, and its high dipole moment facilitates interaction with water molecules.

Watson–Crick base pairing in 9-methyladenine and ethylene-9,9′-diadenine structures with close to 70% solvent content

In small organic molecules with adenine (A) moieties the nucleobases usually form hydrogen-bonded AAA tapes with trans Watson–Crick/Hoogsteen base pairs. In the crystal structure of ethylene-9,9′-diadenine tetra-1,1,1,3,3,3-hexafluoro-2-propanol solvate presented here, the hydroxyl group of the co-crystallized solvent molecules replaces the adenine –NH2 group as donor for the attractive N7 acceptor, leading instead to a highly solvated structure with less common trans Watson–Crick/Watson–Crick base pairs. The properties of hexafluoro-2-propanol as a hydrogen bond pattern modifier was subsequently demonstrated by using it as a crystallization solvent for 9-methyladenine, yielding a structure with the same hydrogen bonds and solvent content as the diadenine analogue.