Temperature dependence of DNA elasticity: An all-atom molecular dynamics simulation study.

We used all-atom molecular dynamics simulation to investigate the elastic properties of double-stranded DNA (dsDNA). We focused on the influences of temperature on the stretch, bend, and twist elasticities, as well as the twist-stretch coupling, of the dsDNA over a wide range of temperature. The results showed that the bending and twist persistence lengths, together with the stretch and twist moduli, decrease linearly with temperature. However, the twist-stretch coupling behaves in a positive correction and enhances as the temperature increases. The potential mechanisms of how temperature affects dsDNA elasticity and coupling were investigated by using the trajectories from atomistic simulation, in which thermal fluctuations in structural parameters were analyzed in detail. We analyzed the simulation results by comparing them with previous simulation and experimental data, which are in good agreement. The prediction about the temperature dependence of dsDNA elastic properties provides a deeper understanding of DNA elasticities in biological environments and potentially helps in the further development of DNA nanotechnology.

Characterization of Z-DNA Using Circular Dichroism.

The B-DNA to Z-DNA transition is a remarkable conformational change in DNA, which was originally observed in poly-GC DNA in the presence of high salt concentration. This eventually prompted the observation of the crystal structure of Z-DNA, a left-handed double-helical DNA, at atomic resolution. Despite advances in Z-DNA research, the application of circular dichroism (CD) spectroscopy as the fundamental technique to characterize this unique DNA conformation has remained constant. In this chapter, we describe a CD spectroscopic method for characterizing the B-DNA to Z-DNA transition of a CG-repeat double-stranded DNA fragment formed from a protein or chemical inducer.

Characterization of Z-DNA by Infrared Spectroscopy.

Infrared spectrum stems from the matter's absorption of light in the infrared (IR) light region. Generally, this infrared light absorption is due to the transition of vibrational and rotational energy levels of the involved molecule. Since different molecules have different structures and vibration modes, infrared spectroscopy can therefore be widely applied to analyze the chemical compositions and structures of molecules. Here we describe the method of application of infrared spectroscopy in the investigation of Z-DNA in cells, as infrared spectroscopy can distinguish DNA secondary structures sensitively and the band at 930 cm-1 is specifically attributed to the Z-form DNA. Based on the curve fitting, the relative content of Z-DNA in the cells may be evaluated.

BZ Junctions and Its Application as Probe (2AP) to Detect Z-DNA Formation and Its Effector.

The left-handed Z-DNA is surrounded by right-handed canonical B-DNA, and thus the junction between B- and Z-DNA has been occurred during temporal Z-DNA formation in the genome. The base extrusion structure of the BZ junction may help detect Z-DNA formation in DNAs. Here we describe the BZ junction structural detection by using 2-aminopurine (2AP) fluorescent probe. BZ junction formation can be measured in solution by this method.

Crystallization of Z-DNA in Complex with Chemical and Z-DNA Binding Z-Alpha Protein.

The molecular basis of Z-DNA recognition and stabilization is mostly discovered via X-ray crystallography. The sequences composed with alteration of purine and pyrimidine are known to adopt Z-DNA conformation. Due to the energy penalty for forming Z-DNA, the small molecular stabilizer or Z-DNA-specific binding protein is required for DNA to adopt Z conformation prior to crystallizing Z-DNA. Here we described the methods ranging from preparation of DNA and Z-alpha protein to crystallization of Z-DNA in detail.

Methods to Study Z-DNA-Induced Genetic Instability.

Alternative DNA structures that differ from the canonical B-DNA double helix, including Z-DNA, have received much attention recently due to their impact on DNA metabolic processes, including replication, transcription, and genome maintenance. Non-B-DNA-forming sequences can also stimulate genetic instability associated with disease development and evolution. Z-DNA can stimulate different types of genetic instability events in different species, and several different assays have been established to detect Z-DNA-induced DNA strand breaks and mutagenesis in prokaryotic and eukaryotic systems. In this chapter, we will introduce some of these methods including Z-DNA-induced mutation screening and detection of Z-DNA-induced strand breaks in mammalian cells, yeast, and mammalian cell extracts. Results from these assays should provide better insight into the mechanisms of Z-DNA-related genetic instability in different eukaryotic model systems.

Chiroptical Properties of Z-DNA Using Ionic Porphyrins and Metalloporphyrins.

The non-covalent interaction of achiral porphyrins with nucleic acids has been extensively studied, and various macrocycles have been indeed utilized as reporters of different sequences of DNA bases. Nevertheless, few studies have been published on the capability of these macrocycles to discriminate among the various nucleic acid conformations. Circular dichroism spectroscopy allowed to characterize the binding of several cationic and anionic mesoporphyrins and metallo derivatives with Z-DNA, in order to exploit the functionality of these systems as probes, storing system, and logic gate.

Human Heme Oxygenase-1 Promoter Activity Is Mediated by Z-DNA Formation.

In recent years, it has been shown that Z-DNA formation in DNA plays functionally significant roles in nucleic acid metabolism, such as gene expression, chromosome recombination, and epigenetic regulation. The reason for the identification of these effects is mainly due to the advancement of Z-DNA detection methods in target genome regions in living cells.The heme oxygenase-1 (HO-1) gene encodes an enzyme that degrades an essential prosthetic heme, and environmental stimuli, including oxidative stress, lead to robust induction of the HO-1 gene. Many DNA elements and transcription factors are involved in the induction of the HO-1 gene, and Z-DNA formation in the thymine-guanine (TG) repetitive sequence in the human HO-1 gene promoter region is required for maximum gene induction.Here, we describe a detailed protocol for Z-DNA detection in the human HO-1 gene promoter region based on chromatin immunoprecipitation with quantitative PCR. We also provide some control experiments to consider in routine lab procedures.

Oligonucleotide Containing 8-Trifluoromethyl-2'-Deoxyguanosine as a Z-DNA Probe.

Z-DNA structure is a noncanonical left-handed alternative form of DNA, which has been suggested to be biologically important and is related to several genetic diseases and cancer. Therefore, investigation of Z-DNA structure associated with biological events is of great importance to understanding the functions of these molecules. Here, we described the development of a trifluoromethyl labeled deoxyguanosine derivative and employed it as a 19F NMR probe to study Z-form DNA structure in vitro and in living cells.

Detection of Z-DNA Structures in Supercoiled Genome.

Z-DNAs are nucleic acid secondary structures that form within a special pattern of nucleotides and are promoted by DNA supercoiling. Through Z-DNA formation, DNA encodes information by dynamic changes in its secondary structure. A growing body of evidence indicates that Z-DNA formation can play a role in gene regulation; it can affect chromatin architecture and demonstrates its association with genomic instability, genetic diseases, and genome evolution. Many functional roles of Z-DNA are yet to be discovered highlighting the need for techniques to detect genome-wide folding of DNA into this structure. Here, we describe an approach to convert linear genome into supercoiled genome sponsoring Z-DNA formation. Applying permanganate-based methodology and high-throughput sequencing to supercoiled genome allows genome-wide detection of single-stranded DNA. Single-stranded DNA is characteristic of the junctions between the classical B-form of DNA and Z-DNA. Consequently, analysis of single-stranded DNA map provides snapshots of the Z-DNA conformation over the whole genome.

ChIP-Seq Strategy to Identify Z-DNA-Forming Sequences in the Human Genome.

Different from the canonical right-handed B-DNA, a left-handed Z-DNA forms an alternating syn- and anti-base conformations along the double-stranded helix under physiological conditions. Z-DNA structure plays a role in transcriptional regulation, chromatin remodeling, and genome stability. To understand the biological function of Z-DNA and map the genome-wide Z-DNA-forming sites (ZFSs), a ChIP-Seq strategy is applied, which is a combination of chromatin immunoprecipitation (ChIP) and high-throughput DNA sequencing analysis. Cross-linked chromatin is sheared and its fragments associated with Z-DNA-binding proteins are mapped onto the reference genome sequence. The global information of ZFSs positioning can provide a useful resource for better understanding of DNA structure-dependent biological mechanism.

Single-Molecule Visualization of B-Z Transition in DNA Origami Using High-Speed AFM.

To study the physical properties of molecules and their reaction processes, direct visualization of target molecules is one of the straightforward methods. Atomic force microscopy (AFM) enables the direct imaging of biomolecules under physiological conditions at nanometer-scale spatial resolution. In addition, using the DNA origami technology, the precise placement of target molecules in a designed nanostructure has been achieved, and the detection of the molecules at the single-molecule level has been realized. DNA origami is applied for visualizing the detailed movement of molecules combining with high-speed AFM (HS-AFM), which enables the analysis of the dynamic movement of biomolecules in a subsecond time resolution.Here, we describe the combination of the DNA origami system with HS-AFM for the imaging of rotation of dsDNA originated from B-Z transition. The rotation of dsDNA during B-Z transition is directly visualized in a DNA origami using the HS-AFM. These target-oriented observation systems serve to the detailed analysis of DNA structural changes in real time at molecular resolution.

Thermogenomic Analysis of Left-Handed Z-DNA Propensities in Genomes.

The initial discovery of left-handed Z-DNA was met with great excitement as a dramatic alternative to the right-handed double-helical conformation of canonical B-DNA. In this chapter, we describe the workings of the program ZHUNT as a computational approach to mapping Z-DNA in genomic sequences using a rigorous thermodynamic model for the transition between the two conformations (the B-Z transition). The discussion starts with a brief summary of the structural properties that differentiate Z- from B-DNA, focusing on those properties that are particularly relevant to the B-Z transition and the junction that splices a left- to right-handed DNA duplex. We then derive the statistical mechanics (SM) analysis of the zipper model that describes the cooperative B-Z transition and show that this analysis very accurately simulates this behavior of naturally occurring sequences that are induced to undergo the B-Z transition through negative supercoiling. A description of the ZHUNT algorithm and its validation are presented, followed by how the program had been applied for genomic and phylogenomic analyses in the past and how a user can access the online version of the program. Finally, we present a new version of ZHUNT (called mZHUNT) that has been parameterized to analyze sequences that contain 5-methylcytosine bases and compare the results of the ZHUNT and mZHUNT analyses on native and methylated yeast chromosome 1.

Self-assembled DNA nanoparticles enable cascade circuits for mRNA detection and imaging in living cells.

Fluorescence molecular probes have been regarded as a valuable tool for RNA detection and imaging. However, the pivotal challenge is how to develop an efficient fluorescence imaging platform for accurate identification of RNA molecules with low expression in complicated physiological environments. Herein, we construct the DNA nanoparticles to glutathione (GSH)-responsive controllable release of hairpin reactants for catalytic hairpin assembly (CHA)-hybridization chain reaction (HCR) cascade circuits, which enables the analysis and imaging of low-abundance target mRNA in living cells. The aptamer-tethered DNA nanoparticles are constructed via the self-assembly of single-stranded DNAs (ssDNAs), exhibiting sufficient stability, cell-specific penetration, and precise controllability. Moreover, the in-depth integration of different DNA cascade circuits shows the improved sensing performance of DNA nanoparticles in live cell analysis. Therefore, through the combination of multi-amplifiers and programmable DNA nanostructure, the developed strategy enables accurately triggered release of hairpin reactants and further achieves sensitive imaging and quantitative evaluation of survivin mRNA in carcinoma cells, which provides a potential platform to facilitate RNA fluorescence imaging applications in early clinical cancer theranostics.

Public and family support and concerns for providing DNA to law enforcement in long-term missing person cases.

The identification of long-term missing persons and unidentified human remains is a global challenge. Many people stay on missing persons registers, with unidentified human remains stored for extended periods in mortuaries around the world. Research exploring public and/or family support for providing DNA in long-term missing persons cases is scarce. The aims of this study were to examine whether trust in police predicted the level of support for providing DNA and explore public/family support and concerns for providing DNA in such cases. Trust in police was measured through two widely used empirical attitude scales; "The Measures of Police Legitimacy and Procedural Justice". Support and concerns for giving DNA were measured through four hypothetical missing persons case scenarios. The results showed more positive attitudes towards police legitimacy and procedural justice significantly predicted support, with the percentage level of positive support across the four case types as follows: cases involving a long-term missing child (89%), elderly adult with dementia (83%), young adult with a history of runaway (76%), with the lowest level of support for an adult with an estranged family (73%). Participants also reported more concerns about providing DNA when the missing person circumstances involved family estrangement. Understanding levels of public/family support and concerns around providing DNA to police in missing persons cases is vital to ensure that DNA collection practices reflect what the public/family support and, wherever possible, alleviate public concerns.

Thousands of years of Malay and Chinese population history in Indonesia and its implication on Paternity Index in DNA paternity testing.

The existence of the Chinese population in the predominantly Malay population in Indonesia can be traced back thousands of years, and it has been suspected that it played an essential role in the history of the Malay population origin in Maritime South East Asia. With the fact that the Malay-Indonesian population is currently predominant compared to the Chinese population in Indonesia (Chinese-Indonesian), the selection of the origin of the STRs allele frequency panel population becomes an issue in DNA profiling, including in paternity testing. This study analyses the genetic relationship between the Chinese-Indonesian and Malay-Indonesian populations and how this affects the Paternity Index (PI) ​​calculation in paternity test cases. The study of the relationship between populations was carried out using neighbour-joining (NJ) tree analysis and multidimensional scaling (MDS) on the allele frequency panel of 19 autosomal STRs loci of Malay-Indonesian (n = 210) and Chinese-Indonesian (n = 78) populations. Four population groups were used as references: Malay-Malaysian, Filipino, Chinese, and Caucasian. An MDS analysis was also performed based on the pairwise FST calculation. The combined Paternity Index (CPI) calculation was carried out on 132 paternity cases from the Malay-Indonesian population with inclusive results using a panel of allele frequencies from the six populations. The pairwise FST MDS indicates a closer relationship between the Chinese-Indonesian and Malay-Indonesian compared to the Chinese population, which is in line with the CPIs comparison test. The outcome suggests that the alternative use of allele frequency database between Malay-Indonesian and Chinese-Indonesian for CPI calculations is not very influential. These results can also be considered in studying the extent of genetic assimilation between the two populations. In addition, these results support the robustness claim of multivariate analysis to represent phenomena that phylogenetic analyses may not be able to demonstrate, especially for massive panel data.

Prospects and feasibility of synergistic therapy with radiotherapy, immunotherapy, and DNA methyltransferase inhibitors in non-small cell lung cancer.

The morbidity and mortality of lung cancer are increasing, seriously threatening human health and life. Non-small cell lung cancer (NSCLC) has an insidious onset and is not easy to be diagnosed in its early stage. Distant metastasis often occurs and the prognosis is poor. Radiotherapy (RT) combined with immunotherapy, especially with immune checkpoint inhibitors (ICIs), has become the focus of research in NSCLC. The efficacy of immunoradiotherapy (iRT) is promising, but further optimization is necessary. DNA methylation has been involved in immune escape and radioresistance, and becomes a game changer in iRT. In this review, we focused on the regulation of DNA methylation on ICIs treatment resistance and radioresistance in NSCLC and elucidated the potential synergistic effects of DNA methyltransferases inhibitors (DNMTis) with iRT. Taken together, we outlined evidence suggesting that a combination of DNMTis, RT, and immunotherapy could be a promising treatment strategy to improve NSCLC outcomes.

A Target-Triggered Emission Enhancement Strategy Based on a Y-Shaped DNA Fluorescent Nanoprobe with Aggregation-Induced Emission Characteristic for microRNA Imaging in Living Cells.

DNA self-assembled fluorescent nanoprobes have been developed for bio-imaging owing to their high resistance to enzyme degradation and great cellular uptake capacity. In this work, we designed a new Y-shaped DNA fluorescent nanoprobe (YFNP) with aggregation-induced emission (AIE) characteristic for microRNA imaging in living cells. With the modification of the AIE dye, the constructed YFNP had a relatively low background fluorescence. However, the YFNP could emit a strong fluorescence due to the generation of microRNA-triggered AIE effect in the presence of target microRNA. Based on the proposed target-triggered emission enhancement strategy, microRNA-21 was detected sensitively and specifically with a detection limit of 122.8 pM. The designed YFNP showed higher bio-stability and cell uptake than the single-stranded DNA fluorescent probe, which has been successfully applied for microRNA imaging in living cells. More importantly, the microRNA-triggered dendrimer structure could be formed after the recognition of target microRNA, achieving a reliable microRNA imaging with a high spatiotemporal resolution. We expect that the proposed YFNP will become a promising candidate for bio-sensing and bio-imaging.