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.

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.

A single base pair substitution in zebrafish distinguishes between innate and acute startle behavior regulation.

Behavioral thresholds define the lowest stimulus intensities sufficient to elicit a behavioral response. Establishment of baseline behavioral thresholds during development is critical for proper responses throughout the animal's life. Despite the relevance of such innate thresholds, the molecular mechanisms critical to establishing behavioral thresholds during development are not well understood. The acoustic startle response is a conserved behavior whose threshold is established during development yet is subsequently acutely regulated. We have previously identified a zebrafish mutant line (escapist) that displays a decreased baseline or innate acoustic startle threshold. Here, we identify a single base pair substitution on Chromosome 25 located within the coding sequence of the synaptotagmin 7a (syt7a) gene that is tightly linked to the escapist acoustic hypersensitivity phenotype. By generating animals in which we deleted the syt7a open reading frame, and subsequent complementation testing with the escapist line, we demonstrate that loss of syt7a function is not the cause of the escapist behavioral phenotype. Nonetheless, escapist mutants provide a powerful tool to decipher the overlap between acute and developmental regulation of behavioral thresholds. Extensive behavioral analyses reveal that in escapist mutants the establishment of the innate acoustic startle threshold is impaired, while regulation of its acute threshold remains intact. Moreover, our behavioral analyses reveal a deficit in baseline responses to visual stimuli, but not in the acute regulation of responses to visual stimuli. Together, this work eliminates loss of syt7a as causative for the escapist phenotype and suggests that mechanisms that regulate the establishment of behavioral thresholds in escapist larvae can operate independently from those regulating acute threshold regulation.

The origin of different bending stiffness between double-stranded RNA and DNA revealed by magnetic tweezers and simulations.

The subtle differences in the chemical structures of double-stranded (ds) RNA and DNA lead to significant variations in their biological roles and medical implications, largely due to their distinct biophysical properties, such as bending stiffness. Although it is well known that A-form dsRNA is stiffer than B-form dsDNA under physiological salt conditions, the underlying cause of this difference remains unclear. In this study, we employ high-precision magnetic-tweezer experiments along with molecular dynamics simulations and reveal that the relative bending stiffness between dsRNA and dsDNA is primarily determined by the structure- and salt-concentration-dependent ion distribution around their helical structures. At near-physiological salt conditions, dsRNA shows a sparser ion distribution surrounding its phosphate groups compared to dsDNA, causing its greater stiffness. However, at very high monovalent salt concentrations, phosphate groups in both dsRNA and dsDNA become fully neutralized by excess ions, resulting in a similar intrinsic bending persistence length of approximately 39 nm. This similarity in intrinsic bending stiffness of dsRNA and dsDNA is coupled to the analogous fluctuations in their total groove widths and further coupled to the similar fluctuation of base-pair inclination, despite their distinct A-form and B-form helical structures.

Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability.

Multi-segmented viruses often multimerize their genomic segments to ensure efficient and stoichiometric packaging of the correct genetic cargo. In the bipartite Nodaviridae family, genome heterodimerization is also observed and conserved among different species. However, the nucleotide composition and biological function for this heterodimer remain unclear. Using Flock House virus as a model system, we developed a next-generation sequencing approach ("XL-ClickSeq") to probe heterodimer site sequences. We identified an intermolecular base-pairing site which contributed to heterodimerization in both wild-type and defective virus particles. Mutagenic disruption of this heterodimer site exhibited significant deficiencies in genome packaging and encapsidation specificity to viral genomic RNAs. Furthermore, the disruption of this intermolecular interaction directly impacts the thermostability of the mature virions. These results demonstrate that the intermolecular RNA-RNA interactions within the encapsidated genome of an RNA virus have an important role on virus particle integrity and thus may impact its transmission to a new host.IMPORTANCEFlock House virus is a member of Nodaviridae family of viruses, which provides a well-studied model virus for non-enveloped RNA virus assembly, cell entry, and replication. The Flock House virus genome consists of two separate RNA molecules, which can form a heterodimer upon heating of virus particles. Although similar RNA dimerization is utilized by other viruses (such as retroviruses) as a packaging mechanism and is conserved among Nodaviruses, the role of heterodimerization in the Nodavirus replication cycle is unclear. In this research, we identified the RNA sequences contributing to Flock House virus genome heterodimerization and discovered that such RNA-RNA interaction plays an essential role in virus packaging efficiency and particle integrity. This provides significant insight into how the interaction of packaged viral RNA may have a broader impact on the structural and functional properties of virus particles.

A 76-base pair duplication within the enhancer region of the HMX1 gene causes sheep microtia.

Sheep congenital microtia is characterized by underdeveloped ears and provides an ideal basis for studying human microtia. This study identified the causal mutation and regulatory mechanisms underlying this disorder. Whole-genome association analysis was conducted using 23 ear tissue samples from sheep with microtia and 28 samples from normal-eared sheep. A significant correlation was found between microtia and a 76-base pair duplication in the enhancer region of the HMX1 gene. Further analysis of offspring phenotypes confirmed an autosomal dominant inheritance pattern. Genotypic analysis showed that individuals that are homozygous for this duplication were earless, heterozygous individuals exhibited shortened ears, and wild-type individuals had normal ears. Moreover, luciferase assays confirmed that this duplication increased HMX1 gene expression, and duplication knock-in mice also exhibited shorter and narrower external ears compared to wild-type mice. Transcriptomic analysis further demonstrated that this duplication enhanced HMX1 gene expression in animal models. This study characterized the causal regulatory mutation underlying sheep microtia.

Melting and Bubble Formation in a Double-Stranded DNA: Microscopic Aspects of Early Base-Pair Opening Events and the Role of Water.

Despite its rigid structure, DNA is a remarkably flexible molecule. Flexibility is essential for biological functions (such as transcription and gene repair), which require large-amplitude structural changes such as bubble formation. The bubbles thus formed are required to have a certain stability of their own and survive long on the time scale of molecular motions. A molecular understanding of fluctuations leading to quasi-stable structures is not available. Through extensive atomistic molecular dynamics simulations, we identify a sequence of microscopic events that culminate in local bubble formation, which is initiated by base-pair (BP) opening, resulting from the cleavage of native BP hydrogen bonds (HBs). This is followed by the formation of mismatched BPs with non-native contacts. These metastable structures can either revert to their original forms or undergo a flipping transition to form a local bubble that can span across 3-4 BPs. A substantial distortion of the DNA backbone and a disruption of BP stacking are observed because of the structural changes induced by these local perturbations. We also explored how water helps in the entire process. A small number of water molecules undergo rearrangement to stabilize the intermediate states by forming HBs with DNA bases. Water thus acts as a lubricant that counteracts the enthalpic penalty suffered from the loss of native BP contacts. Although the process of bubble formation is reversible, the sequence of steps involved poses an entropic barrier, preventing it from easily retracing the path to the native state.

DNA Logic Gates for Small Molecule Activation Circuits in Cells.

DNA-based devices such as DNA logic gates self-assemble into supramolecular structures, as dictated by the sequences of the constituent oligonucleotides and their predictable Watson-Crick base pairing interactions. The programmable nature of DNA-based devices permits the design and implementation of DNA circuits that interact in a dynamic and sequential manner capable of spatially arranging disparate DNA species. Here, we report the application of an activatable fluorescence reporter based on a proximity-driven inverse electron demand Diels-Alder (IEDDA) reaction and its robust integration with DNA strand displacement circuits. In response to specific DNA input patterns, sequential strand displacement reactions are initiated and culminate in the hybridization of two modified DNA strands carrying probes capable of undergoing an IEDDA reaction between a vinyl-ether-caged fluorophore and its reactive partner tetrazine, leading to the activation of fluorescence. This approach provides a major advantage for DNA computing in mammalian cells since circuit degradation does not induce fluorescence, in contrast to traditional fluorophore-quencher designs. We demonstrate the robustness and sensitivity of the reporter by testing its ability to serve as a readout for DNA logic circuits of varying complexity inside cells.

Investigation of the stability of D5SIC-DNAM-incorporated DNA duplex in Taq polymerase binary system: a systematic classical MD approach.

DNA polymerases are fundamental enzymes that play a crucial role in processing DNA with high fidelity and accuracy ensuring the faithful transmission of genetic information. The recognition of unnatural base pairs (UBPs) by polymerases, enabling their replication, represents a significant and groundbreaking discovery with profound implications for genetic expansion. Romesberg et al. examined the impact of DNA containing 2,6-dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN) UBPs bound to T. aquaticus DNA polymerase (Taq) through crystal structure analysis. Here, we have used polarizable and nonpolarizable classical molecular dynamics (MD) simulations to investigate the structural aspects and stability of Taq in complex with a DNA duplex including a DS-DN pair in the terminal 3' and 5' positions. Our results suggest that the flexibility of UBP-incorporated DNA in the terminal position is arrested by the polymerase, thus preventing fraying and mispairing. Our investigation also reveals that the UBP remains in an intercalated conformation inside the active site, exhibiting two distinct orientations in agreement with experimental findings. Our analysis pinpoints particular residues responsible for favorable interactions with the UBP, with some relying on van der Waals interactions while other on Coulombic forces.

Investigation of dynamical flexibility of D5SIC-DNAM inside DNA duplex in aqueous solution: a systematic classical MD approach.

Incorporation of artificial 3rd base pairs (unnatural base pairs, UBPs) has emerged as a fundamental technique in pursuit of expanding the genetic alphabet. 2,6-Dimethyl-2H-isoquiniline-1-thione: D5SIC (DS) and 2-methoxy-3-methylnaphthalene: DNAM (DN), a potential unnatural base pair (UBP) developed by Romesberg and colleagues, has been shown to have remarkable capability for replication within DNA. Crystal structures of a Taq polymerase/double-stranded DNA (ds-DNA) complex containing a DS-DN pair in the 3' terminus showed a parallelly stacked geometry for the pre-insertion, and an intercalated geometry for the post-insertion structure. Unconventional orientations of DS-DN inside a DNA duplex have inspired scientists to investigate the conformational orientations and structural properties of UBP-incorporated DNA. In recent years, computational simulations have been used to investigate the geometry of DS-DN within the DNA duplex; nevertheless, unresolved questions persist owing to inconclusive findings. In this work, we investigate the structural and dynamical properties of DS and DN inside a ds-DNA strand in aqueous solution considering both short and long DNA templates using polarizable, and non-polarizable classical MD simulations. Flexible conformational change of UBP with major populations of Watson-Crick-Franklin (WCF) and three distinct non-Watson-Crick-Franklin (nWCFP1, nWCFP2, nWCFO) conformations through intra and inter-strand flipping have been observed. Our results suggest that a dynamical conformational change leads to the production of diffierent conformational distribution for the systems. Simulations with a short ds-DNA duplex suggest nWCF (P1 and O) as the predominant structures, whereas long ds-DNA duplex simulations indicate almost equal populations of WCF, nWCFP1, nWCFO. DS-DN in the terminal position is found to be more flexible with occasional mispairing and fraying. Overall, these results suggest flexibility and dynamical conformational change of the UBP as well as indicate varied conformational distribution irrespective of starting orientation of the UBP and length og DNA strand.

Site-specific incorporation of a fluorescent nucleobase analog enhances i-motif stability and allows monitoring of i-motif folding inside cells.

The i-motif is an intriguing non-canonical DNA structure, whose role in the cell is still controversial. Development of methods to study i-motif formation under physiological conditions in living cells is necessary to study its potential biological functions. The cytosine analog 1,3-diaza-2-oxophenoxazine (tCO) is a fluorescent nucleobase able to form either hemiprotonated base pairs with cytosine residues, or neutral base pairs with guanines. We show here that when tCO is incorporated in the proximity of a G:C:G:C minor groove tetrad, it induces a strong thermal and pH stabilization, resulting in i-motifs with Tm of 39ºC at neutral pH. The structural determination by NMR methods reveals that the enhanced stability is due to a large stacking interaction between the guanines of the tetrad with the tCO nucleobase, which forms a tCO:C+ in the folded structure at unusually-high pHs, leading to an increased quenching in its fluorescence at neutral conditions. This quenching is much lower when tCO is base-paired to guanines and totally disappears when the oligonucleotide is unfolded. By taking profit of this property, we have been able to monitor i-motif folding in cells.