Stacking geometry for non‐canonical G:U wobble base pair containing dinucleotide sequences in RNA: dispersion‐corrected DFT‐D study

Emergence of thousands of crystal structures of noncoding RNA molecules indicates its structural and functional diversity. RNA function is based upon a large variety of structural elements which are specifically assembled in the folded molecules. Along with the canonical Watson‐Crick base pairs, different orientations of the bases to form hydrogen‐bonded non‐canonical base pairs have also been observed in the available RNA structures. Frequencies of occurrences of different non‐canonical base pairs in RNA indicate their important role to maintain overall structure and functions of RNA. There are several reports on geometry and energetic stabilities of these non‐canonical base pairs. However, their stacking geometry and stacking stability with the neighboring base pairs are not well studied. Among the different non‐canonical base pairs, the G:U wobble base pair (G:U W:WC) is most frequently observed in the RNA double helices. Using quantum chemical method and available experimental data set we have studied the stacking geometry of G:U W:WC base pair containing dinucleotide sequences in roll‐slide parameters hyperspace for different values of twist. This study indicates that the G:U W:WC base pair can stack well with the canonical base pairs giving rise to large interaction energy. The overall preferred stacking geometry in terms of roll, twist and slide for the eleven possible dinucleotide sequences is seen to be quite dependent on their sequences. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 328–338, 2015.     

Hybrid simulation approach incorporating microscopic interaction along with rigid body degrees of freedom for stacking between base pairs

Stacking interaction between the aromatic heterocyclic bases plays an important role in the double helical structures of nucleic acids. Considering the base as rigid body, there are total of 18 degrees of freedom of a dinucleotide step. Some of these parameters show sequence preferences, indicating that the detailed atomic interactions are important in the stacking. Large variants of non‐canonical base pairs have been seen in the crystallographic structures of RNA. However, their stacking preferences are not thoroughly deciphered yet from experimental results. The current theoretical approaches use either the rigid body degrees of freedom where the atomic information are lost or computationally expensive all atom simulations. We have used a hybrid simulation approach incorporating Monte‐Carlo Metropolis sampling in the hyperspace of 18 stacking parameters where the interaction energies using AMBER‐parm99bsc0 and CHARMM‐36 force‐fields were calculated from atomic positions. We have also performed stacking energy calculations for structures from Monte‐Carlo ensemble by Dispersion corrected density functional theory. The available experimental data with Watson–Crick base pairs are compared to establish the validity of the method. Stacking interaction involving A:U and G:C base pairs with non‐canonical G:U base pairs also were calculated and showed that these structures were also sequence dependent. This approach could be useful to generate multiscale modeling of nucleic acids in terms of coarse‐grained parameters where the atomic interactions are preserved. This method would also be useful to predict structure and dynamics of different base pair steps containing non Watson–Crick base pairs, as found often in the non‐coding RNA structures. © 2015 Wiley Periodicals, Inc. Biopolymers 105: 212–226, 2016.     

Eukaryotic tRNA sequences present conserved and amino acid-specific structural signatures

Metazoan organisms have many tRNA genes responsible for decoding amino acids. The set of all tRNA genes can be grouped in sets of common amino acids and isoacceptor tRNAs that are aminoacylated by corresponding aminoacyl-tRNA synthetases. Analysis of tRNA alignments shows that, despite the high number of tRNA genes, specific tRNA sequence motifs are highly conserved across multicellular eukaryotes. The conservation often extends throughout the isoacceptors and isodecoders with, in some cases, two sets of conserved isodecoders. This study is focused on non-Watson–Crick base pairs in the helical stems, especially GoU pairs. Each of the four helical stems may contain one or more conserved GoU pairs. Some are amino acid specific and could represent identity elements for the cognate aminoacyl tRNA synthetases. Other GoU pairs are found in more than a single amino acid and could be critical for native folding of the tRNAs. Interestingly, some GoU pairs are anticodon-specific, and others are found in phylogenetically-specific clades. Although the distribution of conservation likely reflects a balance between accommodating isotype-specific functions as well as those shared by all tRNAs essential for ribosomal translation, such conservations may indicate the existence of specialized tRNAs for specific translation targets, cellular conditions, or alternative functions.     

On the wobble GoU and related pairs

The wobble GoU pairs have been implicated in several biological processes where RNA molecules play a key role. We review the geometrical and conformational properties of wobble GoU pairs on the basis of available crystal structures of RNAs at high resolution. The similarities with the wobble A+oC pairs and UoU pairs are illustrated, while the differences with the recently discovered bifurcated G x U pairs are contrasted.     

Probing the role of sequence in the assembly of three‐dimensional DNA crystals

DNA is a widely used biopolymer for the construction of nanometer‐scale objects due to its programmability and structural predictability. One long‐standing goal of the DNA nanotechnology field has been the construction of three‐dimensional DNA crystals. We previously determined the X‐ray crystal structure of a DNA 13‐mer that forms a continuously hydrogen bonded three‐dimensional lattice through Watson‐Crick and non‐canonical base pairs. Our current study sets out to understand how the sequence of the Watson‐Crick duplex region influences crystallization of this 13‐mer. We screened all possible self‐complementary sequences in the hexameric duplex region and found 21 oligonucleotides that crystallized. Sequence analysis showed that one specific Watson‐Crick pair influenced the crystallization propensity and the speed of crystal self‐assembly. We determined X‐ray crystal structures for 13 of these oligonucleotides and found sequence‐specific structural changes that suggests that this base pair may serve as a structural anchor during crystal assembly. Finally, we explored the crystal self‐assembly and nucleation process. Solution studies indicated that these oligonucleotides do not form base pairs in the absence of cations, but that the addition of divalent cations leads to rapid self‐assembly to higher molecular weight complexes. We further demonstrate that crystals grown from mixtures of two different oligonucleotide sequences contain both oligonucleotides. These results suggest that crystal self‐assembly is nucleated by the formation of the Watson‐Crick duplexes initiated by a simple chemical trigger. This study provides new insight into the role of sequence for the assembly of periodic DNA structures. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 618–626, 2015.     

Ion-induced folding of a kink turn that departs from the conventional sequence

Kink turns (k-turns) are important structural motifs that create a sharp axial bend in RNA. Most conform to a consensus in which a three-nucleotide bulge is followed by consecutive G•A and A•G base pairs, and when these G•A pairs are modified in vitro this generally leads to a failure to adopt the k-turn conformation. Kt-23 in the 30S ribosomal subunit of Thermus thermophilus is a rare exception in which the bulge-distal A•G pair is replaced by a non-Watson–Crick A•U pair. In the context of the ribosome, Kt-23 adopts a completely conventional k-turn geometry. We show here that this sequence is induced to fold into a k-turn structure in an isolated RNA duplex by Mg²⁺ or Na⁺ ions. Therefore, the Kt-23 is intrinsically stable despite lacking the key A•G pair; its formation requires neither tertiary interactions nor protein binding. Moreover, the Kt-23 k-turn is stabilized by the same critical hydrogen-bonding interactions within the core of the structure that are found in more conventional sequences such as the near-consensus Kt-7. T. thermophilus Kt-23 has two further non-Watson–Crick base pairs within the non-canonical helix, three and four nucleotides from the bulge, and we find that the nature of these pairs influences the ability of the RNA to adopt k-turn conformation, although the base pair adjacent to the A•U pair is more important than the other.     

Mispairs with Watson‐Crick base‐pair geometry observed in ternary complexes of an RB69 DNA polymerase variant

Recent structures of DNA polymerase complexes with dGMPCPP/dT and dCTP/dA mispairs at the insertion site have shown that they adopt Watson‐Crick geometry in the presence of Mn²⁺ indicating that the tautomeric or ionization state of the base has changed. To see whether the tautomeric or ionization state of base‐pair could be affected by its microenvironment, we determined 10 structures of an RB69 DNA polymerase quadruple mutant with dG/dT or dT/dG mispairs at position n‐1 to n‐5 of the Primer/Template duplex. Different shapes of the mispairs, including Watson‐Crick geometry, have been observed, strongly suggesting that the local environment of base‐pairs plays an important role in their tautomeric or ionization states.     

Database proton NMR chemical shifts for RNA signal assignment and validation

The Biological Magnetic Resonance Data Bank contains NMR chemical shift depositions for 132 RNAs and RNA-containing complexes. We have analyzed the ¹H NMR chemical shifts reported for non-exchangeable protons of residues that reside within A-form helical regions of these RNAs. The analysis focused on the central base pair within a stretch of three adjacent base pairs (BP triplets), and included both Watson–Crick (WC; G:C, A:U) and G:U wobble pairs. Chemical shift values were included for all 4³ possible WC-BP triplets, as well as 137 additional triplets that contain one or more G:U wobbles. Sequence-dependent chemical shift correlations were identified, including correlations involving terminating base pairs within the triplets and canonical and non-canonical structures adjacent to the BP triplets (i.e. bulges, loops, WC and non-WC BPs), despite the fact that the NMR data were obtained under different conditions of pH, buffer, ionic strength, and temperature. A computer program (RNAShifts) was developed that enables convenient comparison of RNA ¹H NMR assignments with database predictions, which should facilitate future signal assignment/validation efforts and enable rapid identification of non-canonical RNA structures and RNA-ligand/protein interaction sites.     

Non-Watson Crick base pairs might stabilize RNA structural motifs in ribozymes — a comparative study of group-I intron structures

In recent decades studies on RNA structure and function have gained significance due to discoveries on diversified functions of RNA. A common element for RNA secondary structure formed by series of non-Watson/Watson Crick base pairs, internal loops and pseudoknots have been the highlighting feature of recent structural determination of RNAs. The recent crystal structure of group-I introns has demonstrated that these might constitute RNA structural motifs in ribozymes, playing a crucial role in their enzymatic activity. To understand the functional significance of these non-canonical base pairs in catalytic RNA, we analysed the sequences of group-I introns from nuclear genes. The results suggest that they might form the building blocks of folded RNA motifs which are crucial to the catalytic activity of the ribozyme. The conservation of these, as observed from divergent organisms, argues for the presence of non-canonical base pairs as an important requisite for the structure and enzymatic property of ribozymes by enabling them to carry out functions such as replication, polymerase activity etc. in primordial conditions in the absence of proteins.     

New information content in RNA base pairing deduced from quantitative analysis of high-resolution structures

Non-canonical base pairs play important roles in organizing the complex three-dimensional folding of RNA. Here, we outline methodology developed both to analyze the spatial patterns of interacting base pairs in known RNA structures and to reconstruct models from the collective experimental information. We focus attention on the structural context and deformability of the seven pairing patterns found in greatest abundance in the helical segments in a set of well-resolved crystal structures, including (i–ii) the canonical A·U and G·C Watson–Crick base pairs, (iii) the G·U wobble pair, (iv) the sheared G·A pair, (v) the A·U Hoogsteen pair, (vi) the U·U wobble pair, and (vii) the G·A Watson–Crick-like pair. The non-canonical pairs stand out from the canonical associations in terms of apparent deformability, spanning a broader range of conformational states as measured by the six rigid-body parameters used to describe the spatial arrangements of the interacting bases, the root-mean-square deviations of the base-pair atoms, and the fluctuations in hydrogen-bonding geometry. The deformabilties, the modes of base-pair deformation, and the preferred sites of occurrence depend on sequence. We also characterize the positioning and overlap of the base pairs with respect to the base pairs that stack immediately above and below them in double-helical fragments. We incorporate the observed positions of the bases, base pairs, and intervening phosphorus atoms in models to predict the effects of the non-canonical interactions on overall helical structure.     

RNA structure and dynamics: A base pairing perspective

RNA is now known to possess various structural, regulatory and enzymatic functions for survival of cellular organisms. Functional RNA structures are generally created by three-dimensional organization of small structural motifs, formed by base pairing between self-complementary sequences from different parts of the RNA chain. In addition to the canonical Watson–Crick or wobble base pairs, several non-canonical base pairs are found to be crucial to the structural organization of RNA molecules. They appear within different structural motifs and are found to stabilize the molecule through long-range intra-molecular interactions between basic structural motifs like double helices and loops. These base pairs also impart functional variation to the minor groove of A-form RNA helices, thus forming anchoring site for metabolites and ligands. Non-canonical base pairs are formed by edge-to-edge hydrogen bonding interactions between the bases. A large number of theoretical studies have been done to detect and analyze these non-canonical base pairs within crystal or NMR derived structures of different functional RNA. Theoretical studies of these isolated base pairs using ab initio quantum chemical methods as well as molecular dynamics simulations of larger fragments have also established that many of these non-canonical base pairs are as stable as the canonical Watson–Crick base pairs. This review focuses on the various structural aspects of non-canonical base pairs in the organization of RNA molecules and the possible applications of these base pairs in predicting RNA structures with more accuracy.     

Alternative heterocycles for DNA recognition: an N-methylpyrazole/N-methylpyrrole pair specifies for A.T/T.A base pairs

Side-by-side pairs of three five-membered rings, N-methylpyrrole (Py), N-methylimidazole (Im), and N-methylhydroxy-pyrrole (Hp), have been demonstrated to distinguish each of the four Watson Crick base pairs in the minor groove of DNA. However, not all DNA sequences targeted by these pairing rules achieve affinities and specificities comparable to DNA binding proteins. We have initiated a search for new heterocycles which can expand the sequence repetoire currently available. Two heterocyclic aromatic amino acids. N-methylpyrazole (Pz) and 4-methylthiazole (Th), were incorporated into a single position of an eight-ring polyamide of sequence ImImXPy-gamma-lmPyPyPy-beta-Dp to examine the modulation of affinity and specificity for DNA binding by a Pz/Py pair and or a Th/Py pair. The X/Py pairings Pz/Py and Th/Py were evaluated by quantitative DNase I footprint titrations on a DNA fragment with the four sites 5'-TGGNCA-3' (N=T, A, G, C). The Pz/Py pair binds T.A and A.T with similar affinity to a Py/Py pair but with improved specificity. disfavoring both G.C and C.G by about 100-fold. The Th/Py pair binds poorly to all four Watson Crick base pairs. These results demonstrate that in some instances new heterocyclic aromatic amino acid pairs can be incorporated into imidazole-pyrrole polyamides to mimic the DNA specificity of Py/Py pairs which may be relevant as biological criteria in animal studies become important.     

Control of box C/D snoRNP assembly by N6‐methylation of adenine

N⁶‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N⁶‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N⁶‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N⁶mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.     

Structural basis of error‐prone replication and stalling at a thymine base by human DNA polymerase ι

Human DNA polymerase ι (polι) is a unique member of Y‐family polymerases, which preferentially misincorporates nucleotides opposite thymines (T) and halts replication at T bases. The structural basis of the high error rates remains elusive. We present three crystal structures of polι complexed with DNA containing a thymine base, paired with correct or incorrect incoming nucleotides. A narrowed active site supports a pyrimidine to pyrimidine mismatch and excludes Watson–Crick base pairing by polι. The template thymine remains in an anti conformation irrespective of incoming nucleotides. Incoming ddATP adopts a syn conformation with reduced base stacking, whereas incorrect dGTP and dTTP maintain anti conformations with normal base stacking. Further stabilization of dGTP by H‐bonding with Gln59 of the finger domain explains the preferential T to G mismatch. A template ‘U‐turn’ is stabilized by polι and the methyl group of the thymine template, revealing the structural basis of T stalling. Our structural and domain‐swapping experiments indicate that the finger domain is responsible for polι's high error rates on pyrimidines and determines the incorporation specificity.     

Effect of flanking residues on CA and AA dinucleotides: some rationale

Effect of flanking base pairs on CA and AA dinluceotide step-geometry has been studied using molecular dynamics method. Sixteen dodecameric sequences are constructed for each doublet with all possible bases at their 5' and 3' positions along with their complementary sequences. Structural parameter, formation of Extra Watson Crick bifurcated hydrogen bonds along or across the strands and effect of sodium ions are studied for these sequences. It is found that geometry of CA doublet step is perturbed by the neighboring base pairs, which might be due to inherent flexibility of the step. Flexible character of CA step is reflected in its low bifurcated hydrogen bond formation capability and lower preference of sodium ions to enter in minor or major grooves. AA step on the other hand is quite rigid according to different structural parameters and respond much less to environmental changes due to formation of strong Extra Watson-Crick hydrogen bonds.     

The Paradox of Dual Roles in the RNA World: Resolving the Conflict Between Stable Folding and Templating Ability

The hypothesized dual roles of RNA as both information carrier and biocatalyst during the earliest stages of life require a combination of features: good templating ability (for replication) and stable folding (for ribozymes). However, this poses the following paradox: well-folded sequences are poor templates for copying, but poorly folded sequences are unlikely to be good ribozymes. Here, we describe a strategy to overcome this dilemma through G:U wobble pairing in RNA. Unlike Watson–Crick base pairs, wobble pairs contribute highly to the energetic stability of the folded structure of their sequence, but only slightly, if at all, to the stability of the folded reverse complement. Sequences in the RNA World might thereby combine stable folding of the ribozyme with an unstructured, reverse-complementary genome, resulting in a “division of labor” between the strands. We demonstrate this strategy using computational simulations of RNA folding and an experimental model of early replication, nonenzymatic template-directed RNA primer extension. Additional study is needed to solve other problems associated with a complete replication cycle, including separation of strands after copying. Interestingly, viroid RNA sequences, which have been suggested to be relics of an RNA World (Diener, Proc Natl Acad Sci USA 86:9370–9374, 1989), also show significant asymmetry in folding energy between the infectious (+) and template (?) strands due to G:U pairing, suggesting that this strategy may even be used by replicators in the present day.     

89 Computational mapping reveals effect of Hoogsteen breathing on duplex DNA reactivity with formaldehyde

Formaldehyde has long been recognized as a hazardous environmental agent highly reactive with DNA. Recently, it has been realized that due to the activity of histone demethylation enzymes within the cell nucleus, formaldehyde is produced endogenously in direct vicinity of genomic DNA. Should it lead to extensive DNA damage? We address this question with the aid of a computational mapping method, analogous to X-ray and nuclear magnetic resonance techniques for observing weakly specific interactions of small organic compounds with a macromolecule in order to establish important functional sites. We concentrate on the leading reaction of formaldehyde with free bases: hydroxymethylation of cytosine amino groups. Our results show that in B-DNA, cytosine amino groups are totally inaccessible for the formaldehyde attack. Then, we explore the effect of recently discovered transient flipping of Watson–Crick (WC) pairs into Hoogsteen (HG) pairs (HG breathing). Our results show that the HG base pair formation dramatically affects the accessibility for formaldehyde of the cytosine-aminonitrogens within WC-base pairs adjacent to HG-base pairs. The extensive literature on DNA interaction with formaldehyde is analysed in light of the new findings. The obtained data emphasize the significance of DNA HG breathing.     

A review on architecture of the gag-pol ribosomal frameshifting RNA in human immunodeficiency virus: a variability survey of virus genotypes

Programmed ‘-1’ ribosomal frameshifting is necessary for expressing the pol gene overlapped from a gag of human immunodeficiency virus. A viral RNA structure that requires base pairing across the overlapping sequence region suggests a mechanism of regulating ribosome and helicase traffic during expression. To get precise roles of an element around the frameshift site, a review on architecture of the frameshifting RNA is performed in combination of reported information with augments of a representative set of 19 viral samples. In spite of a different length for the viral RNAs, a canonical comparison on the element sequence allocation is performed for viewing variability associations between virus genotypes. Additionally, recent and historical insights recognized in frameshifting regulation are looked back as for indel and single nucleotide polymorphism of RNA. As specially noted, structural changes at a frameshift site, the spacer sequence, and a three-helix junction element, as well as two Watson–Crick base pairs near a bulge and a C–G pair close a loop, are the most vital strategies for the virus frameshifting regulations. All of structural changes, which are dependent upon specific sequence variations, facilitate an elucidation about the RNA element conformation-dependent mechanism for frameshifting. These facts on disrupting base pair interactions also allow solving the problem of competition between ribosome and helicase on a same RNA template, common to single-stranded RNA viruses. In a broad perspective, each new insight of frameshifting regulation in the competition systems introduced by the RNA element construct changes will offer a compelling target for antiviral therapy.     

Effect of Flanking Residues on CA and AA Dinucleotides: Some Rationale

Abstract

Effect of flanking base pairs on CA and AA dinluceotide step-geometry has been studied using molecular dynamics method. Sixteen dodecameric sequences are constructed for each doublet with all possible bases at their 5′ and 3′ positions along with their complementary sequences. Structural parameter, formation of Extra Watson Crick bifurcated hydrogen bonds along or across the strands and effect of sodium ions are studied for these sequences. It is found that geometry of CA doublet step is perturbed by the neighboring base pairs, which might be due to inherent flexibility of the step. Flexible character of CA step is reflected in its low bifurcated hydrogen bond formation capability and lower preference of sodium ions to enter in minor or major grooves. AA step on the other hand is quite rigid according to different structural parameters and respond much less to environmental changes due to formation of strong Extra Watson-Crick hydrogen bonds.