Watson-Crick Pairing Research Articles

Molecular self-assembly of stable and small branched DNA nanostructures: Higher than a helical turn is enough for hybridization

The Watson-Crick base pairing property of DNA is widely used for fabricating DNA nanostructures with well-defined geometry. Moreover, DNA nanostructures can be easily modified in terms of shape, size and function at the nanoscale level. Therefore, investigation on smaller and stable branched DNA (bDNA) is of critical significance for biomedical applications. In the present communication, we report smaller and stable branched DNA (bDNA) which is of critical significance for biomedical applications. In the present study, a novel strategy has been used in identifying stable bDNA nanostructures with a minimum number of Watson-Crick base pairings. The importance of hybridizing regions and helical twists between multiple oligonucleotides has been explored using various biophysical techniques. The electrophoretic analysis demonstrated that hybridizing regions with ≥12 nt nucleotides can form stable bDNA structures. Substantial negative enthalpic contributions determine the significance of base stacking and the length of oligonucleotides in the hybridization process. Finally, thermal melting investigations confirmed the creation of bDNA nanostructures with ≥12 nt long hybridizing regions. In general, our findings indicate that bDNA oligonucleotides do not undergo hybridization if the number of base pairs is lesser for a single helical turn. Furthermore, the yield and stability of smaller bDNA nanostructures in physiological conditions are comparable with the earlier reported higher-order structures. Hence, smaller bDNAs are more stable which may be preferred over conventional bDNA nanostructures for advanced biomedical applications.

Cross-chiral exponential amplification of an RNA enzyme.

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D- and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other's synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D- nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D- and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson-Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids "across the mirror." There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification.

In silico study of DNA mononucleotide self-assembly.

Recent experiments have demonstrated the self-assembly and long-range ordering of concentrated aqueous solutions of DNA and RNA mononucleotides. These are found to form Watson-Crick pairs that stack into columns that become spatially organized into a columnar liquid-crystalline phase. In this work, we numerically investigate this phase behavior by adopting an extremely coarse-grained model in which nucleotides are represented as semi-disk-like polyhedra decorated with attractive (patchy) sites that mimic the stacking and pairing interactions. We carry out Monte Carlo simulations of these patchy polyhedra by adapting algorithms borrowed from computer graphics. This model reproduces the features of the experimental phase behavior, which essentially depends on the combination of pairing and stacking interactions.

Dynamics of epitranscriptomes uncover translational reprogramming directed by ac4C in rice during pathogen infection.

Messenger RNA modifications play pivotal roles in RNA biology, but comprehensive landscape changes of epitranscriptomes remain largely unknown in plant immune response. Here we report translational reprogramming directed by ac4C mRNA modification upon pathogen challenge. We first investigate the dynamics of translatomes and epitranscriptomes and uncover that the change in ac4C at single-base resolution promotes translational reprogramming upon Magnaporthe oryzae infection. Then by characterizing the specific distributions of m1A, 2'O-Nm, ac4C, m5C, m6A and m7G, we find that ac4Cs, unlike other modifications, are enriched at the 3rd position of codons, which stabilizes the Watson-Crick base pairing. Importantly, we demonstrate that upon pathogen infection, the increased expression of the ac4C writer OsNAT10/OsACYR (N-ACETYLTRANSFERASE FOR CYTIDINE IN RNA) promotes translation to facilitate rapid activation of immune responses, including the enhancement of jasmonic acid biosynthesis. Our study provides an atlas of mRNA modifications and insights into ac4C function in plant immunity.

Theoretical Investigation of the structure and Raman scattering properties of Hoogsteen- and Watson-Crick-type Adenine-Thymine base pair

Hoogsteen (HG) and Watson-Crick (WC) adenine–thymine (A-T) base pairs are fundamental to gene expression and genomic stability. Our research applied density functional theory (DFT) calculations to analyze these base pairs, demonstrating that linking them with ribose or deoxyribose and their immersion in solvents markedly enhances the distinctions in their optical properties. DFT results reveal that N···HN hydrogen bonds in both HG and WC pairs exhibit characteristics of both ionic and covalent bonds, playing a crucial role in their stability. While WC base pairs demonstrate stronger orbital interactions, they also experience greater repulsion than HG pairs. This repulsion offsets the positive interactions to a degree, making the interaction energy of WC pairs slightly lower than that of HG pairs. Furthermore, the distinct conformations of these base pairs result in different vibrational modes, suggesting Raman spectroscopy as an effective method for distinguishing between WC and HG base pairs.

Advancing microRNA target site prediction with transformer and base-pairing patterns.

MicroRNAs (miRNAs) are short non-coding RNAs involved in various cellular processes, playing a crucial role in gene regulation. Identifying miRNA targets remains a central challenge and is pivotal for elucidating the complex gene regulatory networks. Traditional computational approaches have predominantly focused on identifying miRNA targets through perfect Watson-Crick base pairings within the seed region, referred to as canonical sites. However, emerging evidence suggests that perfect seed matches are not a prerequisite for miRNA-mediated regulation, underscoring the importance of also recognizing imperfect, or non-canonical, sites. To address this challenge, we propose Mimosa, a new computational approach that employs the Transformer framework to enhance the prediction of miRNA targets. Mimosa distinguishes itself by integrating contextual, positional and base-pairing information to capture in-depth attributes, thereby improving its predictive capabilities. Its unique ability to identify non-canonical base-pairing patterns makes Mimosa a standout model, reducing the reliance on pre-selecting candidate targets. Mimosa achieves superior performance in gene-level predictions and also shows impressive performance in site-level predictions across various non-human species through extensive benchmarking tests. To facilitate research efforts in miRNA targeting, we have developed an easy-to-use web server for comprehensive end-to-end predictions, which is publicly available at http://monash.bioweb.cloud.edu.au/Mimosa.

Unveiling the solution structure of a DNA duplex with continuous silver-modified Watson-Crick base pairs

The challenge of transforming organized DNA structures into their metallized counterparts persists in the scientific field. In this context, utilizing DNA molecules modified with 7-deazapurine, provides a transformative solution. In this study, we present the solution structure of a DNA duplex that can be transformed into its metallized equivalent while retaining the natural base pairing arrangement through the creation of silver-modified Watson-Crick base pairs. Unlike previously documented X-ray structures, our research demonstrates the feasibility of preserving the intrinsic DNA self-assembly while incorporating AgI into the double helix, illustrating that the binding of silver does not disrupt the canonical base-pairing organization. Moreover, in our case, the uninterrupted AgI chain deviates from forming conventional straight linear chains; instead, it adheres to a helical arrangement dictated by the underlying DNA structure. This research challenges conventional assumptions and opens the door to precisely design structures based on the organization of highly stable Ag-DNA assemblies.

Maximal Genetic Code Symmetry Is a Physicochemical Purine-Pyrimidine Symmetry Language for Transcription and Translation in the Flow of Genetic Information from DNA to Proteins.

Until now, research has not taken into consideration the physicochemical purine-pyrimidine symmetries of the genetic code in the transcription and translation processes of proteinogenesis. Our Supersymmetry Genetic Code table, developed in 2022, is common and unique for all RNA and DNA living species. Its basic structure is a purine-pyrimidine symmetry net with double mirror symmetry. Accordingly, the symmetry of the genetic code directly shows its organisation based on the principle of nucleotide Watson-Crick and codon-anticodon pairing. The maximal purine-pyrimidine symmetries of codons show that each codon has a strictly defined and unchangeable position within the genetic code. We discovered that the physicochemical symmetries of the genetic code play a fundamental role in recognising and differentiating codons from mRNA and the anticodon tRNA and aminoacyl-tRNA synthetases in the transcription and translation processes. These symmetries also support the wobble hypothesis with non-Watson-Crick pairing interactions between the translation process from mRNA to tRNA. The Supersymmetry Genetic Code table shows a specific arrangement of the second base of codons, according to which it is possible that an anticodon from tRNA recognises whether a codon from mRNA belongs to an amino acid with two or four codons, which is very important in the purposeful use of the wobble pairing process. Therefore, we show that canonical and wobble pairings essentially do not lead to misreading and errors during translation, and we point out the role of physicochemical purine-pyrimidine symmetries in decreasing disorder according to error minimisation and preserving the integrity of biological processes during proteinogenesis.

7-Deaza-2'-deoxyisoguanosine, a Noncanonical Nucleoside for Nucleic Acid Code Expansion and New DNA Constructs: Nucleobase Functionalization of Inverse Watson-Crick and Purine-Purine Base Pairs.

7-Deaza-2'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2'-deoxyisocytidine and purine-purine base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2'-deoxyisoguanosine/5-aza-7-deaza-2'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.

Design of novel broad-spectrum antiviral nucleoside analogues using natural bases ring-opening strategy.

The global prevalence of RNA virus infections has presented significant challenges to public health in recent years, necessitating the expansion of its alternative therapeutic library. Due to its evolutional conservation, RNA-dependent RNA polymerase (RdRp) has emerged as a potential target for broad-spectrum antiviral nucleoside analogues. However, after over half a century of structural modification, exploring unclaimed chemical space using frequently-used structural substitution methods to design new nucleoside analogues is challenging. In this study, we explore the use of the "ring-opening" strategy to design new base mimics, thereby using these base mimics to design new nucleoside analogues with broad-spectrum antiviral activities. A total of 29 compounds were synthesized. Their activity against viral RdRp was initially screened using an influenza A virus RdRp high-throughput screening model. Then, the antiviral activity of 38a was verified against influenza virus strain A/PR/8/34 (H1N1), demonstrating a 50% inhibitory concentration (IC50) value of 9.95 μM, which was superior to that of ribavirin (the positive control, IC50 = 11.43 μM). Moreover, 38a also has inhibitory activity against coronavirus 229E with an IC50 of 30.82 μM. In addition, compounds 42 and 46f exhibit an 82% inhibition rate against vesicular stomatitis virus at a concentration of 20 μM and hardly induce cytotoxicity in host cells. This work demonstrates the feasibility of designing nucleoside analogues with "ring-opening" bases and suggests the "ring-opening" nucleosides may have greater polarity, and designing prodrugs is an important aspect of optimizing their antiviral activity. Future research should focus on enhancing the conformational restriction of open-loop bases to mimic Watson-Crick base pairing better and improve antiviral activity.

Towards combining backbone and sugar constraint in 3'-3' bis-phosphonate tethered 2'-4' bridged LNA oligonucleotide trimers.

Therapeutic oligonucleotides are chemically modified to enhance their drug-like properties - including binding affinity for target RNA. Many nucleic acid analogs that enhance RNA binding affinity constrain the furanose sugar in an RNA-like sugar pucker. The improvements in binding affinity result primarily from increased off-rates with minimal effects on on-rates for hybridization. To identify alternate chemical modification strategies that can modulate on- and off-rates for oligonucleotide hybridization, we hypothesized that extending conformational restraint across multiple nucleotides could modulate hybridization kinetics by restricting rotational freedom of the sugar-phosphate backbone. As part of that effort, we recently reported that using hydrocarbon tethers to bridge adjacent phosphodiester linkages as phosphonate tethered bridges can pre-organize nucleic acids in conformations conducive for Watson-Crick base-pairing and modulate hybridization kinetics. In this report, we describe the synthesis of locked nucleic acid (LNA) trimers linked through alkylphosphonate tethers which restrict conformation of the furanose sugar in addition to restricting conformational mobility of the sugar-phosphate backbone across three nucleotide units.

Rational Design of Chimeric Antisense Oligonucleotides on a Mixed PO-PS Backbone for Splice-Switching Applications.

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson-Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO's pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO-PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs.

Defined folding pattern of poly(rG) supports inherent ability to encode biological information.

The RNA World hypothesis posits that RNA can represent a primitive life form by reproducing itself and demonstrating catalytic activity. However, this hypothesis is incapable of addressing several major origin-of-life (OoL) questions. A recently described paradox-free alternative OoL hypothesis, the Quadruplex (G4) World, is based on the ability of poly(dG) to fold into a stable architecture with an unambiguous folding pattern using G-tetrads as building elements. Because of the folding pattern of three G-tetrads and single-G loops, dG15 is programmable and has the capability to encode biological information. Here, we address two open questions of the G4 World hypothesis: (1) Does RNA follow the same folding pattern as DNA? (2) How do stable quadruplexes evolve into the present-day system of information transfer, which is based on Watson-Crick base pair complementarity? To address these questions, we systematically studied the thermodynamic and optical properties of both DNA and RNA G15- and G3T (GGGTGGGTGGGTGGG)-derived sequences. Our study revealed that similar to DNA sequences, RNAs adopt quadruplexes with only three G-tetrads. Thus, both poly(dG) and poly(rG) possess inherent ability to fold into 3D quadruplex architecture with strictly defined folding pattern. The study also revealed that despite high stability of both DNA and RNA quadruplexes, they are vulnerable to single-nucleotide substitutions, which drop the thermal stability by ~40°C and can facilitate introduction of the complementarity principle into the G4 World.

Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

Proximity-Dependent Activation of Split DNAzyme Kinase.

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted Dk1sub and Dk1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis.

Building RNA-Mediated Artificial Signaling Pathways between Endogenous Genes.

ConspectusSophisticated genetic networks play a pivotal role in orchestrating cellular responses through intricate signaling pathways across diverse environmental conditions. Beyond the inherent complexity of natural cellular signaling networks, the construction of artificial signaling pathways (ASPs) introduces a vast array of possibilities for reshaping cellular responses, enabling programmable control of living organisms. ASPs can be integrated with existing cellular networks and redirect output responses as desired, allowing seamless communication and coordination with other cellular processes, thereby achieving designable transduction within cells. Among diversified ASPs, establishing connections between originally independent endogenous genes is of particular significance in modifying the genetic networks, so that cells can be endowed with new capabilities to sense and deal with abnormal factors related to differentiated gene expression (i.e., solve the issues of the aberrant gene expression induced by either external or internal stimuli). In a typical scenario, the two genes X and Y in the cell are originally expressed independently. After the introduction of an ASP, changes in the expression of gene X may exert a designed impact on gene Y, subsequently inducing the cellular response related to gene Y. If X represents a disease signal and Y serves as a therapeutic module, the introduction of the ASP empowers cells with a new spontaneous defense system to handle potential risks, which holds great potential for both fundamental and translational studies.In this Account, we primarily review our endeavors in the construction of RNA-mediated ASPs between endogenous genes that can respond to differentiated RNA expression. In contrast to other molecules that may be restricted to specific pathways, synthetic RNA circuits can be easily utilized and expanded as a general platform for constructing ASPs with a high degree of programmability and tunability for diversified functionalities through predictable Watson-Crick base pairing. We first provide an overview of recent advancements in RNA-based genetic circuits, encompassing but not limited to utilization of RNA toehold switches, siRNA and CRISPR systems. Despite notable progress, most reported RNA circuits have to contain at least one exogenous RNA X as input or one engineered RNA Y as a target, which is not suitable for establishing endogenous gene connections. While exogenous RNAs can be engineered and controlled as desired, constructing a general and efficient platform for manipulation of naturally occurring RNAs poses a formidable challenge, especially for the mammalian system. With a focus on this goal, we are devoted to developing efficient strategies to manipulate cell responses by establishing RNA-mediated ASPs between endogenous genes, particularly in mammalian cells. Our step-by-step progress in engineering customized cell signaling circuits, from bacterial cells to mammalian cells, from gene expression regulation to phenotype control, and from small RNA to long mRNA of low abundance and more complex secondary structures, is systematically described. Finally, future perspectives and potential applications of these RNA-mediated ASPs between endogenous genes are also discussed.

Simulating the Lyotropic Phase Behavior of a Partially Self-Complementary DNA Tetramer.

DNA oligomers in solution have been found to develop liquid crystal phases via a hierarchical process that involves Watson-Crick base pairing, supramolecular assembly into columns of duplexes, and long-range ordering. The multiscale nature of this phenomenon makes it difficult to quantitatively describe and assess the importance of the various contributions, particularly for very short strands. We performed molecular dynamics simulations based on the coarse-grained oxDNA model, aiming to depict all of the assembly processes involved and the phase behavior of solutions of the DNA GCCG tetramers. We find good quantitative matching to experimental data at both levels of molecular association (thermal melting) and collective ordering (phase diagram). We characterize the isotropic state and the low-density nematic and high-density columnar liquid crystal phases in terms of molecular order, size of aggregates, and structure, together with their effects on diffusivity processes. We observe a cooperative aggregation mechanism in which the formation of dimers is less thermodynamically favored than the formation of longer aggregates.

Liquid biopsy multiomics enable clinical triage for early colorectal cancer diagnosis.

e15599 Background: Colorectal cancer (CRC) is the second most common cause of cancer death in the United State. Despite advancements in screening, diagnosis, and treatment, the disease remains a formidable public health issue. In 2023, approximately 153020 individuals were diagnosed with CRC and 52550 will die from the disease. In order to meet early diagnostic, we developed this multiomics assay to enable clinical triage for liquid biopsy screening. Methods: Xenonucleic acid (XNAs) are artificial genetic polymers that retain the Watson-Crick base-pairing capability and exhibit high chemical and biological stability. They can be employed as molecular clamps in RT-qPCR due to the stronger hybridizing/binding capability seen in XNA/DNA duplexes than DNA/DNA duplexes. We are developing an XNA-based multiplex RT-qPCR assay for APC, BRAF, CTNNB1, KRAS, NRAS, PIK3CA, SMAD4 and TP53 gene mutation panel and CRC 9 genes methylation panel combined with multiplex protein panel, called ColoscapeTM Plus. We enrolled in FIT positive subjects of both genders, aged 50-74, attending the CRC screening program, we tested 152 plasma samples, 75 health, 50 AA and 27 early CRC with this multiomics assay. Results: The assay results showed that its sensitivity was about 44% (95% CI: 0.302- 0.587) and specificity about 80% (95%CI: 0.689- 0.880) for advanced adenoma (AA). For CRC stage I and II, its sensitivity is about 63% and 78% with 80% specificity separately. Conclusions: The primary data has shown that this low cost and rapid turnaround time multiomics assay has a comparable sensitivity for early colorectal cancer diagnostic.

Unveiling the Intermolecular Interactions between Drug 5-Fluorouracil and Watson-Crick/Hoogsteen Base Pairs: A Computational Analysis.

The adsorption of 5-fluorouracil (5FU) on Watson-Crick (WC) base pairs and Hoogsteen (HT) base pairs has been studied using the dispersion-corrected density functional theory (DFT). The adsorption, binding energy, and thermochemistry for the drug 5FU on the WC and HT base pairs were determined. The most stable geometries were near planar geometry, and 5FU has a higher preference for WC than HT base pairs. The adsorption energies of 5FU on nucleobase pairs are consistently higher than pristine nucleobase pairs, indicating that nucleobase pair cleavage is less likely during the adsorption of the 5FU drug. The enthalpy change for the formation of 5FU-DNA base pairs is higher than that for the formation of 5FU-nucleobases and is enthalpy-driven. The E gap of AT base pairs is higher, suggesting that their chemical reactivity toward further reaction would be less than that of GC base pairs. The electron density difference (EDD) analysis shows a significant decrease in electron density in aromatic regions on the purine bases (adenine/guanine) compared to the pyrimidine bases. The MESP diagram of the stable 5FU-nucleobase pair complexes shows a directional interaction, with the positive regions in a molecule interacting with the negative region of other molecules. The atoms in molecule analysis show that the ρ(r) values of C=O···H-N are higher than those of N···H/N-H···O. The N···H intermolecular bonds between the base pair/drug and nucleobases are weak, closed shell interactions and are electrostatic in nature. The noncovalent interaction analysis shows that several new spikes are engendered along with an increase in their strength, which indicates that the H-bonding interactions are stronger and play a dominant role in stabilizing the complexes. Energy decomposition analysis shows that the drug-nucleobase pair complex has a marginal increase in the electrostatic contributions compared to nucleobase pair complexes.

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.