Classification and Identification of Non-canonical Base Pairs and Structural Motifs.

The 3D structures of many ribonucleic acid (RNA) loops are characterized by highly organized networks of non-canonical interactions. Multiple computational methods have been developed to annotate structures with those interactions or automatically identify recurrent interaction networks. By contrast, the reverse problem that aims to retrieve the geometry of a look from its sequence or ensemble of interactions remains much less explored. In this chapter, we will describe how to retrieve and build families of conserved structural motifs using their underlying network of non-canonical interactions. Then, we will show how to assign sequence alignments to those families and use the software BayesPairing to build statistical models of structural motifs with their associated sequence alignments. From this model, we will apply BayesPairing to identify in new sequences regions where those loop geometries can occur.

Modified Nucleotides and RNA Structure Prediction.

Nucleotide modifications are occurrent in all types of RNA and play an important role in RNA structure formation and stability. Modified bases not only possess the ability to shift the RNA structure ensemble towards desired functional confirmations. By changes in the base pairing partner preference, they may even enlarge or reduce the conformational space, i.e., the number and types of structures the RNA molecule can adopt. However, most methods to predict RNA secondary structure do not provide the means to include the effect of modifications on the result. With the help of a heavily modified transfer RNA (tRNA) molecule, this chapter demonstrates how to include the effect of different base modifications into secondary structure prediction using the ViennaRNA Package. The constructive approach demonstrated here allows for the calculation of minimum free energy structure and suboptimal structures at different levels of modified base support. In particular we, show how to incorporate the isomerization of uridine to pseudouridine ( Ψ ) and the reduction of uridine to dihydrouridine (D).

Ionization Patterns and Chemical Reactivity of Cytosine-Guanine Watson-Crick Pairs.

The front cover artwork is provided by Prof. Papadantonakis' group. The image shows a Watson-Crick Guanine-Cytosine pair, and the difference between vertical and adiabatic ionization potentials. Read the full text of the Research Article at 10.1002/cphc.202300946.

Binding energies and hydrogen bonds effects on DNA-cisplatin interactions: a DFT-xTB study.

CONTEXT: A systematic study of hydrogen bonds in base pairs and the interaction of cisplatin with DNA fragments was carried out. Structure, binding energies, and electron density were analyzed. xTB has proven to be an accurate method for obtaining structures and binding energies in DNA structures. Our xTB values for DNA base binding energy were in the same order and in some cases better than CAM-B3LYP values compared to experimental values. Double-stranded DNA-cisplatin structures have been calculated and the hydrogen bonds of water molecules are a decisive factor contributing to the preference for the cisplatin-Guanine interaction. Higher values of the water hydrogen bonding energies were obtained in cisplatin-Guanine structures. Furthermore, the electrostatic potential was used to investigate and improve the analysis of DNA-cisplatin structures. METHODS: We applied the xTB method and the CAM-B3LYP functional combined with def2-SVP basis set to perform and analyze of the bonding energies of the cisplatin interaction and the effects of the hydrogen bonds. Results were calculated employing the xTB and the ORCA software.

An interaction network in the polymerase active site is a prerequisite for Watson-Crick base pairing in Pol γ.

The replication accuracy of DNA polymerase gamma (Pol γ) is essential for mitochondrial genome integrity. Mutation of human Pol γ arginine-853 has been linked to neurological diseases. Although not a catalytic residue, Pol γ arginine-853 mutants are void of polymerase activity. To identify the structural basis for the disease, we determined a crystal structure of the Pol γ mutant ternary complex with correct incoming nucleotide 2'-deoxycytidine 5'-triphosphate (dCTP). Opposite to the wild type that undergoes open-to-closed conformational changes when bound to a correct nucleotide that is essential for forming a catalytically competent active site, the mutant complex failed to undergo the conformational change, and the dCTP did not base pair with its Watson-Crick complementary templating residue. Our studies revealed that arginine-853 coordinates an interaction network that aligns the 3'-end of primer and dCTP with the catalytic residues. Disruption of the network precludes the formation of Watson-Crick base pairing and closing of the active site, resulting in an inactive polymerase.

From Base Pairs to City Squares: Comprehensive Precision Oncology for the Future.

SUMMARY: An increased understanding of the role of the social determinants of health in cancer prevention, cancer care, and outcomes can lead to their integration into genetics and genomics as well as informing interventions and clinical trials, creating a comprehensive precision oncology framework.

Advancing Genetic Alphabet Expansion: Synthesis of 7-(2-Thienyl)-Imidazo[4,5-b]pyridine (Ds) and 4-(4-Pentyne-1,2-diol)-1-Propynyl-2-Nitropyrrole (Diol-Px) for Use in Replicable Unnatural Base Pairs for PCR Applications.

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer's chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

Insights into elastic properties of coarse-grained DNA models: q-stiffness of cgDNA vs cgDNA.

Coarse-grained models have emerged as valuable tools to simulate long DNA molecules while maintaining computational efficiency. These models aim at preserving interactions among coarse-grained variables in a manner that mirrors the underlying atomistic description. We explore here a method for testing coarse-grained vs all-atom models using stiffness matrices in Fourier space (q-stiffnesses), which are particularly suited to probe DNA elasticity at different length scales. We focus on a class of coarse-grained rigid base DNA models known as cgDNA and its most recent version, cgDNA+. Our analysis shows that while cgDNA+ closely follows the q-stiffnesses of the all-atom model, the original cgDNA shows some deviations for twist and bending variables, which are rather strong in the q → 0 (long length scale) limit. The consequence is that while both cgDNA and cgDNA+ give a suitable description of local elastic behavior, the former misses some effects that manifest themselves at longer length scales. In particular, cgDNA performs poorly on twist stiffness, with a value much lower than expected for long DNA molecules. Conversely, the all-atom and cgDNA+ twist are strongly length scale dependent: DNA is torsionally soft at a few base pair distances but becomes more rigid at distances of a few dozen base pairs. Our analysis shows that the bending persistence length in all-atom and cgDNA+ is somewhat overestimated.

High frequency of transition to transversion ratio in the stem region of RNA secondary structure of untranslated region of SARS-CoV-2.

Introduction: The propensity of nucleotide bases to form pairs, causes folding and the formation of secondary structure in the RNA. Therefore, purine (R): pyrimidine (Y) base-pairing is vital to maintain uniform lateral dimension in RNA secondary structure. Transversions or base substitutions between R and Y bases, are more detrimental to the stability of RNA secondary structure, than transitions derived from substitutions between A and G or C and T. The study of transversion and transition base substitutions is important to understand evolutionary mechanisms of RNA secondary structure in the 5'  and 3'  untranslated (UTR) regions of SARS-CoV-2. In this work, we carried out comparative analysis of transition and transversion base substitutions in the stem and loop regions of RNA secondary structure of SARS-CoV-2. Methods: We have considered the experimentally determined and well documented stem and loop regions of 5' and 3' UTR regions of SARS-CoV-2 for base substitution analysis. The secondary structure comprising of stem and loop regions were visualized using the RNAfold web server. The GISAID repository was used to extract base sequence alignment of the UTR regions. Python scripts were developed for comparative analysis of transversion and transition frequencies in the stem and the loop regions. Results: The results of base substitution analysis revealed a higher transition (ti) to transversion (tv) ratio (ti/tv) in the stem region of UTR of RNA secondary structure of SARS-CoV-2 reported during the early stage of the pandemic. The higher ti/tv ratio in the stem region suggested the influence of secondary structure in selecting the pattern of base substitutions. This differential pattern of ti/tv values between stem and loop regions was not observed among the Delta and Omicron variants that dominated the later stage of the pandemic. It is noteworthy that the ti/tv values in the stem and loop regions were similar among the later dominant Delta and Omicron variant strains which is to be investigated to understand the rapid evolution and global adaptation of SARS-CoV-2. Conclusion: Our findings implicate the lower frequency of transversions than the transitions in the stem regions of UTRs of SARS-CoV-2. The RNA secondary structures are associated with replication, translation, and packaging, further investigations are needed to understand these base substitutions across different variants of SARS-CoV-2.

Chemical Repair of Radical Damage to the GC Base Pair by DNA-Bound Bisbenzimidazoles.

The migration of an electron-loss center (hole) in calf thymus DNA to bisbenzimidazole ligands bound in the minor groove is followed by pulse radiolysis combined with time-resolved spectrophotometry. The initially observed absorption spectrum upon oxidation of DNA by the selenite radical is consistent with spin on cytosine (C), as the GC• pair neutral radical, followed by the spectra of oxidized ligands. The rate of oxidation of bound ligands increased with an increase in the ratio (r) ligands per base pair from 0.005 to 0.04. Both the rate of ligand oxidation and the estimated range of hole transfer (up to 30 DNA base pairs) decrease with the decrease in one-electron reduction potential between the GC• pair neutral radical of ca. 1.54 V and that of the ligand radicals (E0', 0.90-0.99 V). Linear plots of log of the rate of hole transfer versus r give a common intercept at r = 0 and a free energy change of 12.2 ± 0.3 kcal mol-1, ascribed to the GC• pair neutral radical undergoing a structural change, which is in competition to the observed hole transfer along DNA. The rate of hole transfer to the ligands at distance, R, from the GC• pair radical, k2, is described by the relationship k2 = k0 exp(constant/R), where k0 includes the rate constant for surmounting a small barrier.

The effect of pseudoknot base pairing on cotranscriptional structural switching of the fluoride riboswitch.

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of Escherichia coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37°C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65°C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

Base pair dynamics, electrostatics, and thermodynamics at the LTR-III quadruplex:duplex junction.

G-quadruplexes (GQs) play key regulatory roles within the human genome and have also been identified to play similar roles in other eukaryotes, bacteria, archaea, and viruses. Human immunodeficiency virus 1, the etiological agent of acquired immunodeficiency syndrome, can form two GQs in its long terminal repeat (LTR) promoter region, each of which act to regulate viral gene expression in opposing manners. The major LTR GQ, called LTR-III, is a distinct hybrid GQ containing a 12-nucleotide duplex loop attached to the quadruplex motif. The resulting quadruplex:duplex junction (QDJ) has been hypothesized to serve as a selective drug targeting site. To better understand the dynamics of this QDJ, we performed conventional and enhanced-sampling molecular dynamics simulations using the Drude-2017 force field. We observed unbiased and reversible formation of additional base pairs in the QDJ, between Ade4:Thy14 and Gua3:Thy14. Both base pairs were electrostatically favored, but geometric constraints within the junction may drive the formation of, and preference for, the Ade4:Thy14 base pair. Finally, we demonstrated that the base pairs are separated only by small energy barriers that may enable transitions between both base-paired states. Together, these simulations provide new insights into the dynamics, electrostatics, and thermodynamics of the LTR-III QDJ.

Expanding the Horizon of the Xeno Nucleic Acid Space: Threose Nucleic Acids with Increased Information Storage.

Xeno nucleic acids (XNAs) constitute a class of synthetic nucleic acid analogues characterized by distinct, non-natural modifications within the tripartite structure of the nucleic acid polymers. While most of the described XNAs contain a modification in only one structural element of the nucleic acid scaffold, this work explores the XNA chemical space to create more divergent variants with modifications in multiple parts of the nucleosidic scaffold. Combining the enhanced nuclease resistance of α-l-threofuranosyl nucleic acid (TNA) and the almost natural-like replication efficiency and fidelity of the unnatural hydrophobic base pair (UBP) TPT3:NaM, novel modified nucleoside triphosphates with a dual modification pattern were synthesized. We investigated the enzymatic incorporation of these nucleotide building blocks by XNA-compatible polymerases and confirmed the successful enzymatic synthesis of TPT3-modified TNA, while the preparation of NaM-modified TNA presented greater challenges. This study marks the first enzymatic synthesis of TNA with an expanded genetic alphabet (exTNA), opening promising opportunities in nucleic acid therapeutics, particularly for the selection and evolution of nuclease-resistant, high-affinity aptamers with increased chemical diversity.

Investigation of the dynamical structures of double-chain deoxyribonucleic acid model in biological sciences.

The present research investigates the double-chain deoxyribonucleic acid model, which is important for the transfer and retention of genetic material in biological domains. This model is composed of two lengthy uniformly elastic filaments, that stand in for a pair of polynucleotide chains of the deoxyribonucleic acid molecule joined by hydrogen bonds among the bottom combination, demonstrating the hydrogen bonds formed within the chain's base pairs. The modified extended Fan sub equation method effectively used to explain the exact travelling wave solutions for the double-chain deoxyribonucleic acid model. Compared to the earlier, now in use methods, the previously described modified extended Fan sub equation method provide more innovative, comprehensive solutions and are relatively straightforward to implement. This method transforms a non-linear partial differential equation into an ODE by using a travelling wave transformation. Additionally, the study yields both single and mixed non-degenerate Jacobi elliptic function type solutions. The complexiton, kink wave, dark or anti-bell, V, anti-Z and singular wave shapes soliton solutions are a few of the creative solutions that have been constructed utilizing modified extended Fan sub equation method that can offer details on the transversal and longitudinal moves inside the DNA helix by freely chosen parameters. Solitons propagate at a consistent rate and retain their original shape. They are widely used in nonlinear models and can be found everywhere in nature. To help in understanding the physical significance of the double-chain deoxyribonucleic acid model, several solutions are shown with graphics in the form of contour, 2D and 3D graphs using computer software Mathematica 13.2. All of the requisite constraint factors that are required for the completed solutions to exist appear to be met. Therefore, our method of strengthening symbolic computations offers a powerful and effective mathematical tool for resolving various moderate nonlinear wave problems. The findings demonstrate the system's potentially very rich precise wave forms with biological significance. The fundamentals of double-chain deoxyribonucleic acid model diffusion and processing are demonstrated by this work, which marks a substantial development in our knowledge of double-chain deoxyribonucleic acid model movements.

Utility of intercalator displacement assays for screening of ligands for i-motif DNA structures.

Cytosine rich sequences can form intercalated, i-motif DNA structures stabilized by hemi-protonated cytosine:cytosine base pairing. These sequences are often located in regulatory regions of genes such as promoters. Ligands targeting i-motif structures may provide potential leads for treatments for genetic disease. The focus on ligands interacting with i-motif DNA has been increasing in recent years. Here, we describe the fluorescent intercalator displacement (FID) assay using thiazole orange binding i-motif DNA and assess the binding affinity of a ligand to the i-motif DNA by displacing thiazole orange. This provides a time and cost-effective high throughput screening of ligands against secondary DNA structures for hit identification.

Potentiometric titrations to study ligand interactions with DNA i-motifs.

i-Motifs are non-canonical secondary structures of DNA formed by mutual intercalation of hemi-protonated cytosine-cytosine base pairs, most typically in slightly acidic conditions (pH<7.0). These structures are well-studied in vitro and have recently been suggested to exist in cells. Despite nearly a decade of active research, the quest for small-molecule ligands that could selectively bind to and stabilize i-motifs continues, and no reference, bona fide i-motif ligand is currently available. This is, at least in part, due to the lack of robust methods to assess the interaction of ligands with i-motifs, since many techniques well-established for studies of other secondary structures (such as CD-, UV-, and FRET-melting) may generate artifacts when applied to i-motifs. Here, we describe an implementation of automated, potentiometric (pH) titrations as a robust isothermal method to assess the impact of ligands or cosolutes on thermodynamic stability of i-motifs. This approach is validated through the use of a cosolute previously known to stabilize i-motifs (PEG2000) and three small-molecule ligands that are able to stabilize, destabilize, or have no effect on the stability of i-motifs, respectively.

A beginner's handbook to identify and characterize i-motif DNA.

Genomic DNA exhibits an innate ability to manifest diverse sequence-dependent secondary structures, serving crucial functions in gene regulation and cellular equilibrium. While extensive research has confirmed the formation of G-quadruplex structures by guanine-rich sequences in vitro and in cells, recent investigations have turned the quadruplex community's attention to the cytosine (C)-rich complementary strands that can adopt unique tetra-stranded conformation, termed as intercalated motif or i-motif. I-motifs are stabilized by hemi-protonated C:CH+ base pairs under acidic conditions. Initially, the in vivo occurrence of i-motifs was underestimated because their formation is favored at non-physiological pH. However, groundbreaking research utilizing the structure-specific iMab antibody and high-throughput sequencing have recently detected their conserved dispersion throughout the genome, challenging previous assumptions. Given the evolving nature of this research field, it becomes imperative to conduct independent in vitro experiments aimed at identifying potential i-motif formation in C-rich sequences and consolidating the findings to address the properties of i-motifs. This chapter serves as an introductory guide for the swift identification of novel i-motifs, where we present an experimental framework for investigating and characterizing i-motif sequences in vitro. In this chapter, we selected a synthetic oligonucleotide (C7T3) sequence and outlined appropriate methodologies for annealing the i-motif structure into suitable buffers. Then, we validated its formation by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopy. Finally, we provided a thorough account of the step-by-step procedures to investigate the effect of i-motif formation on the stalling or retardation of DNA replication using high resolution primer extension assays.

Comprehensive Comparison and Critical Assessment of RNA-Specific Force Fields.

Molecular dynamics simulations play a pivotal role in elucidating the dynamic behaviors of RNA structures, offering a valuable complement to traditional methods such as nuclear magnetic resonance or X-ray. Despite this, the current precision of RNA force fields lags behind that of protein force fields. In this work, we systematically compared the performance of four RNA force fields (ff99bsc0χOL3, AMBERDES, ff99OL3_CMAP1, AMBERMaxEnt) across diverse RNA structures. Our findings highlight significant challenges in maintaining stability, particularly with regard to cross-strand and cross-loop hydrogen bonds. Furthermore, we observed the limitations in accurately describing the conformations of nonhelical structural motif, terminal nucleotides, and also base pairing and base stacking interactions by the tested RNA force fields. The identified deficiencies in existing RNA force fields provide valuable insights for subsequent force field development. Concurrently, these findings offer recommendations for selecting appropriate force fields in RNA simulations.

Accurate prediction of RNA secondary structure including pseudoknots through solving minimum-cost flow with learned potentials.

Pseudoknots are key structure motifs of RNA and pseudoknotted RNAs play important roles in a variety of biological processes. Here, we present KnotFold, an accurate approach to the prediction of RNA secondary structure including pseudoknots. The key elements of KnotFold include a learned potential function and a minimum-cost flow algorithm to find the secondary structure with the lowest potential. KnotFold learns the potential from the RNAs with known structures using an attention-based neural network, thus avoiding the inaccuracy of hand-crafted energy functions. The specially designed minimum-cost flow algorithm used by KnotFold considers all possible combinations of base pairs and selects from them the optimal combination. The algorithm breaks the restriction of nested base pairs required by the widely used dynamic programming algorithms, thus enabling the identification of pseudoknots. Using 1,009 pseudoknotted RNAs as representatives, we demonstrate the successful application of KnotFold in predicting RNA secondary structures including pseudoknots with accuracy higher than the state-of-the-art approaches. We anticipate that KnotFold, with its superior accuracy, will greatly facilitate the understanding of RNA structures and functionalities.