DNA Molecules Research Articles

Next generation sequencing: Forensic applications and policy considerations

AbstractNext generation sequencing (NGS) or massively parallel sequencing (MPS) is a high‐throughput technology that can be used to analyze DNA and RNA molecules with greater sensitivity, scalability, and speed than traditional Sanger sequencing. NGS systems are slowly gaining popularity in forensic science and may eventually become the future of forensic DNA analysis. The data generated can be utilized to conduct an array of forensic biology analyses such as short tandem repeats (STR) profiling, forensic genetic genealogy, predictive DNA phenotyping, and more. However, the adoption of NGS for forensic casework is associated with a lot of ethical, social, and legal concerns. This article provides a comprehensive review of NGS systems, data analysis, and forensic applications. It also provides policy considerations that aim to reduce harm and bias, while promoting informed consent, standardization, transparency, and accountability.This article is categorized under: Forensic Biology > Interpretation of Biological Evidence Forensic Biology > Ethical and Social Implications Forensic Biology > Forensic DNA Technologies

The unusual structural properties and potential biological relevance of switchback DNA

Synthetic DNA motifs form the basis of nucleic acid nanotechnology. The biochemical and biophysical properties of these motifs determine their applications. Here, we present a detailed characterization of switchback DNA, a globally left-handed structure composed of two parallel DNA strands. Compared to a conventional duplex, switchback DNA shows lower thermodynamic stability and requires higher magnesium concentration for assembly but exhibits enhanced biostability against some nucleases. Strand competition and strand displacement experiments show that component sequences have an absolute preference for duplex complements instead of their switchback partners. Further, we hypothesize a potential role for switchback DNA as an alternate structure in sequences containing short tandem repeats. Together with small molecule binding experiments and cell studies, our results open new avenues for switchback DNA in biology and nanotechnology.

Biodegradable silica nanoparticles for efficient linear DNA gene delivery

Targeting, safety, scalability, and storage stability of vectors are still challenges in the field of nucleic acid delivery for gene therapy. Silica-based nanoparticles have been widely studied as gene carriers, exhibiting key features such as biocompatibility, simplistic synthesis, and enabling easy surface modifications for targeting. However, the ability of the formulation to incorporate DNA is limited, which restricts the number of DNA molecules that can be incorporated into the particle, thereby reducing gene expression. Here we use polymerase chain reaction (PCR)-generated linear DNA molecules to augment the coding sequences of gene-carrying nanoparticles, thereby maximizing nucleic acid loading and minimizing the size of these nanocarriers. This approach results in a remarkable 16-fold increase in protein expression six days post-transfection in cells transfected with particles carrying the linear DNA compared with particles bearing circular plasmid DNA. The study also showed that the use of linear DNA entrapped in DNA@SiO2 resulted in a much more efficient level of gene expression compared to standard transfection reagents. The system developed in this study features simplicity, scalability, and increased transfection efficiency and gene expression over existing approaches, enabled by improved embedment capabilities for linear DNA, compared to conventional methods such as lipids or polymers, which generally show greater transfection efficiency with plasmid DNA. Therefore, this novel methodology can find applications not only in gene therapy but also in research settings for high-throughput gene expression screenings.

Outer membrane vesicles of Acinetobacter baumannii DS002 carry circular DNA similar to bovine meat and milk factors (BMMFs) and SPHINX 2.36 and probably play a role in interdomain lateral gene transfer.

Several independent studies have demonstrated the existence of replication competent circular DNA molecules of bacterial and viral origin in mammalian cells and tissues. However, studies about their origin and lateral mobility to mammalian cells are scarce. Our work describes the existence of circular DNA, similar to that of DNA molecules identified in mammalian cells, OMVs derived from soil isolate of A. baumannii DS002. Furthermore, the work also provides visual evidence that demonstrates the passage of labeled OMVs to different organs of experimental mice within hours after intravenously administering OMVs into experimental mice. Some of the labeled OMVs have even crossed the membrane of Neuro2A, suggesting the existence of interkingdom horizontal mobility between bacteria and mammals.

Solution of state transfer matrix based on DNA strand displacement circuits

In recent years, DNA strand displacement (DSD) circuits have been developed in leaps and bounds. The high storage and parallelism of DNA give it an inherent advantage in the field of DNA computations. DSD is widely used in analog computations and neural network computations. However, there are few studies on solving state transfer matrix using DSD circuits. Aiming at the above problems, a scheme for solution of state transfer matrix based on DSD circuits is proposed. In this paper, the summation, subtraction, multiplication, division and exponential reaction modules are built by the DSD reactions. Based on the reaction modules, DNA chemical reaction networks of state transfer matrix are constructed. DSD circuits are built by cascading the DNA chemical reaction networks. The solution of the state transfer matrix is implemented through the DSD circuits. The Visual DSD is used to verify the practicality of DSD circuits. This scheme may provide a reference to analog computations based on DSD circuits.

Elucidation of mode of anticancerous activity of metal guanidinobenzimidazoles: A computational and experimental screening

DNA‐binding agents often exhibits dual behavior of simultaneous binding with protein associated with cancerous cell development pathways. This simultaneous protein binding if contributed to malfunctioning of pro‐apoptotic proteins will reduce anticancerous potential of drugs. The elucidation of binding target for anticancerous agent can give an insight into the cancer cell apoptotic pathway. Therefore, anticancerous Zn(II) and Ni(II) complexes of 2‐guanidinobenzimidazole (2GBz) were subjected to DNA‐binding mode assay along with protein‐binding interference assay. The stoichiometry Zn‐2GBz and Ni‐2GBz were determined through density functional theory (DFT) and UV–Vis spectroscopy, followed by structural verification by X‐ray crystallography. The 2GBz and Ni‐2GBz were found to bind with grooves of DNA while groove binding of Zn‐2GBz induced unwinding of DNA through interactive pi‐stacking. Binding constant revealed the M‐2GBz to be a strong binder of DNA molecule with the effect of enhanced cell killing potential of M‐2GBz against MCF‐7 cell lines. Protein‐binding assay revealed that despite of significant interaction of M‐2GBz with chick serum albumin, DNA binding was not altered by simultaneous protein binding. The most potent Zn‐2GBz was found to be sphingosine 1 kinase 2 (SPhK2) which can be a cause of enhanced cancer cell apoptosis with lesser normal cell apoptosis than standard fluorouracil in MCF‐7 cell lines. Co‐administered Zn‐2GBz increased cancer cell sensitivity towards fluorouracil as potent anticancerous agent.

MEILB2-BRME1 forms a V-shaped DNA clamp upon BRCA2-binding in meiotic recombination

DNA double-strand break repair by homologous recombination has a specialised role in meiosis by generating crossovers that enable the formation of haploid germ cells. This requires meiosis-specific MEILB2-BRME1, which interacts with BRCA2 to facilitate loading of recombinases onto resected DNA ends. Here, we report the crystal structure of the MEILB2-BRME1 2:2 core complex, revealing a parallel four-helical assembly that recruits BRME1 to meiotic double-strand breaks in vivo. It forms an N-terminal β-cap that binds to DNA, and a MEILB2 coiled-coil that bridges to C-terminal ARM domains. Upon BRCA2-binding, MEILB2-BRME1 2:2 complexes dimerize into a V-shaped 2:4:4 complex, with rod-like MEILB2-BRME1 components arranged at right-angles. The β-caps located at the tips of the MEILB2-BRME1 limbs are separated by 25 nm, allowing them to bridge between DNA molecules. Thus, we propose that BRCA2 induces MEILB2-BRME1 to function as a DNA clamp, connecting resected DNA ends or homologous chromosomes to facilitate meiotic recombination.

Virtual reality inspection of chromatin 3D and 2D data

Understanding the packing of long DNA strands into chromatin is one of the ultimate challenges in genomic research. An intrinsic part of this complex problem is studying the chromatin’s spatial structure. Biologists reconstruct 3D models of chromatin from experimental data, yet the exploration and analysis of such 3D structures is limited in existing genomic data visualization tools. To improve this situation, we investigated the current options of immersive methods and designed a prototypical VR visualization tool for 3D chromatin models that leverages virtual reality to deal with the spatial data. We showcase the tool in three primary use cases. First, we provide an overall 3D shape overview of the chromatin to facilitate the identification of regions of interest and the selection for further investigation. Second, we include the option to export the selected regions and elements in the BED format, which can be loaded into common analytical tools. Third, we integrate epigenetic modification data along the sequence that influence gene expression, either as in-world 2D charts or overlaid on the 3D structure itself. We developed our application in collaboration with two domain experts and gathered insights from two informal studies with five other experts.

Universal Targeted Haplotyping by Droplet Digital PCR Sequencing and Its Applications in Noninvasive Prenatal Testing and Pharmacogenetics Analysis.

The analysis of haplotypes of variants is important for pharmacogenomics analysis and noninvasive prenatal testing for monogenic diseases. However, there is a lack of robust methods for targeted haplotyping. We developed digital PCR haplotype sequencing (dHapSeq) for targeted haplotyping of variants, which is a method that compartmentalizes long DNA molecules into droplets. Within one droplet, 2 target regions are PCR amplified from one template molecule, and their amplicons are fused together. The fused products are then sequenced to determine the phase relationship of the single nucleotide polymorphism (SNP) alleles. The entire haplotype of 10s of SNPs can be deduced after the phase relationship of individual SNPs are determined in a pairwise manner. We applied dHapSeq to noninvasive prenatal testing in 4 families at risk for thalassemia and utilized it to detect NUDT15 diplotypes for predicting drug tolerance in pediatric acute lymphoblastic leukemia (72 cases and 506 controls). For SNPs within 40 kb, phase relation can be determined with 100% accuracy. In 7 trio families, the haplotyping results for 97 SNPs spanning 185 kb determined by dHapSeq were concordant with the results deduced from the genotypes of both parents and the fetus. In 4 thalassemia families, a 19.3-kb Southeast Asian deletion was successfully phased with 97 downstream SNPs, enabling noninvasive determination of fetal inheritance using relative haplotype dosage analysis. In the NUDT15 analysis, the variant status and phase of the variants were successfully determined in all cases and controls. The dHapSeq represents a robust and scalable haplotyping approach with numerous clinical and research applications.

Enhancing siRNA efficacy in vivo with extended nucleic acid backbones.

Therapeutic small interfering RNA (siRNA) requires sugar and backbone modifications to inhibit nuclease degradation. However, metabolic stabilization by phosphorothioate (PS), the only backbone chemistry used clinically, may be insufficient for targeting extrahepatic tissues. To improve oligonucleotide stabilization, we report the discovery, synthesis and characterization of extended nucleic acid (exNA) consisting of a methylene insertion between the 5'-C and 5'-OH of a nucleoside. exNA incorporation is compatible with common oligonucleotide synthetic protocols and the PS backbone, provides stabilization against 3' and 5' exonucleases and is tolerated at multiple oligonucleotide positions. A combined exNA-PS backbone enhances resistance to 3' exonuclease by ~32-fold over the conventional PS backbone and by >1,000-fold over the natural phosphodiester backbone, improving tissue exposure, tissue accumulation and efficacy in mice, both systemically and in the brain. The improved efficacy and durability imparted by exNA may enable therapeutic interventions in extrahepatic tissues, both with siRNA and with other oligonucleotides such as CRISPR guide RNA, antisense oligonucleotides, mRNA and tRNA.

Mrc1 regulates parental histone segregation and heterochromatin inheritance

Chromatin-based epigenetic memory relies on the symmetric distribution of parental histones to newly synthesized daughter DNA strands, aided by histone chaperones within the DNA replication machinery. However, the mechanism of parental histone transfer remains elusive. Here, we reveal that in fission yeast, the replisome protein Mrc1 plays a crucial role in promoting the transfer of parental histone H3-H4 to the lagging strand, ensuring proper heterochromatin inheritance. In addition, Mrc1 facilitates the interaction between Mcm2 and DNA polymerase alpha, two histone-binding proteins critical for parental histone transfer. Furthermore, Mrc1's involvement in parental histone transfer and epigenetic inheritance is independent of its known functions in DNA replication checkpoint activation and replisome speed control. Instead, Mrc1 interacts with Mcm2 outside of its histone-binding region, creating a physical barrier to separate parental histone transfer pathways. These findings unveil Mrc1 as a key player within the replisome, coordinating parental histone segregation to regulate epigenetic inheritance.

Protein-Induced DNA Dumbbell Amplification (PINDA) and its Applications to Food Hazards Detection

Quantification of trace amounts of proteins is technically challenging because proteins cannot be directly amplified like nucleic acids. To improve the analytical sensitivity and to complement conventional protein analysis methods, we developed a highly sensitive and homogeneous detection strategy called Protein-Induced DNA Dumbbell Amplification (PINDA). PINDA combines protein recognition with exponential nucleic acid amplification by using protein binding probes made of DNA strands conjugated to protein affinity ligands. When a pair of probes bind to the same target protein, complementary nucleic acid sequences that are conjugated to each probe are brought into close proximity. The increased local concentration of the probes results in the formation of a stable dumbbell structure of the nucleic acids. The DNA dumbbell is readily amplifiable exponentially using techniques such as loop-mediated isothermal amplification. The PINDA assay eliminates the need for washing or separation steps, and is suitable for on-site applications. Detection of the model protein, thrombin, has a linear range of 10 fM-100 pM and detection limit of 10 fM. The PINDA technique is successfully applied to the analysis of dairy sample for the detection of β-lactoglobulin, a common food allergen, and Salmonella enteritidis, a foodborne pathogenic bacterium. The PINDA assay can be easily modified to detect other targets by changing the affinity ligands used to bind to the specific targets.

A replisome-associated histone H3-H4 chaperone required for epigenetic inheritance

Faithful transfer of parental histones to newly replicated daughter DNA strands is critical for inheritance of epigenetic states. Although replication proteins that facilitate parental histone transfer have been identified, how intact histone H3-H4 tetramers travel from the front to the back of the replication fork remains unknown. Here, we use AlphaFold-Multimer structural predictions combined with biochemical and genetic approaches to identify the Mrc1/CLASPIN subunit of the replisome as a histone chaperone. Mrc1 contains a conserved histone-binding domain that forms a brace around the H3-H4 tetramer mimicking nucleosomal DNA and H2A-H2B histones, is required for heterochromatin inheritance, and promotes parental histone recycling during replication. We further identify binding sites for the FACT histone chaperone in Swi1/TIMELESS and DNA polymerase α that are required for heterochromatin inheritance. We propose that Mrc1, in concert with FACT acting as a mobile co-chaperone, coordinates the distribution of parental histones to newly replicated DNA.

Assessment of DNA quality for whole genome library preparation

In recent years, more sophisticated DNA technologies for genotyping have enabled considerable progress in various fields such as clinical genetics, archaeogenetics and forensic genetics. DNA samples previously rejected as too challenging to analyze due to low amounts of degraded DNA can now provide useful information. To increase the chances of success with the new methodologies, it is crucial to know the fragment size of the template DNA molecules, and whether the DNA in a sample is mostly single or double stranded. With this knowledge, an appropriate library preparation method can be chosen, and the DNA shearing parameters of the protocol can be adjusted to the DNA fragment size in the sample. In this study, we first developed and evaluated a user-friendly fluorometry-based protocol for estimation of DNA strandedness. We also evaluated different capillary electrophoresis methods for estimation of DNA fragmentation levels. Next, we applied the developed methodologies to a broad variety of DNA samples processed with different DNA extraction protocols. Our findings show that both the applied DNA extraction method and the sample type affect the DNA strandedness and fragmentation. The established protocols and the gained knowledge will be applicable for future sequencing-based high-density SNP genotyping in various fields.

Motor domain of condensin and step formation in extruding loop of DNA.

During the asymmetric loop extrusion of DNA by a condensin complex, one domain of the complex stably anchors to the DNA molecule, and another domain reels in the DNA strand into a loop. The DNA strand in the loop is fully relaxed, or there is no tension in the loop. Just outside of the loop, there is a tension that resists the extrusion of DNA. To maintain the extrusion of the DNA loop, the condensin complex must have a domain capable of generating a force to overcome the tension outside of the loop. This study proposes that the groove-shaped HEAT repeat domain Ycg1 plays the role of a molecular motor. A DNA molecule may bind to the groove electrostatically, and the weak binding force facilitates the random thermal motion of DNA molecules. A mechanical model that random collisions between DNA and the nonparallel inner surfaces of the groove may generate a directional force which is required for the loop extrusion to sustain. The hinge domain binds to the DNA molecule and acts as an anchor during asymmetric DNA loop extrusion. When the effects of ATP hydrolysis and the viscous drag of the fluid environment are considered, the motor-anchor model for the condensin complex and the mechanical model might explain the asymmetric loop extrusion, the formation of steps, the step size distribution in the loop extrusion, the tension-dependent extrusion speed, the interaction between coexisting loops on the DNA strand, and untying the knots during extrusion. This model can also explain the observed formation of the Z-loop.

Repair of genomic interstrand crosslinks

Genomic interstrand crosslinks (ICLs) are formed by reactive species generated during normal cellular metabolism, produced by the microbiome, and employed in cancer chemotherapy. While there are multiple options for replication dependent and independent ICL repair, the crucial step for each is unhooking one DNA strand from the other. Much of our insight into mechanisms of unhooking comes from powerful model systems based on plasmids with defined ICLs introduced into cells or cell free extracts. Here we describe the properties of exogenous and endogenous ICL forming compounds and provide an historical perspective on early work on ICL repair. We discuss the modes of unhooking elucidated in the model systems, the concordance or lack thereof in drug resistant tumors, and the evolving view of DNA adducts, including ICLs, formed by metabolic aldehydes.

Understanding the relationship between sequences and kinetics of DNA strand displacements.

Precisely modulating the kinetics of toehold-mediated DNA strand displacements (TMSD) is essential for its application in DNA nanotechnology. The sequence in the toehold region significantly influences the kinetics of TMSD. However, due to the large sample space resulting from various arrangements of base sequences and the resulted complex secondary structures, such a correlation is not intuitive. Herein, machine learning was employed to reveal the relationship between the kinetics of TMSD and the toehold sequence as well as the correlated secondary structure of invader strands. Key factors that influence the rate constant of TMSD were identified, such as the number of free hydrogen bonding sites in the invader, the number of free bases in the toehold, and the number of hydrogen bonds in intermediates. Moreover, a predictive model was constructed, which successfully achieved semi-quantitative prediction of rate constants of TMSD even with subtle distinctions in toehold sequence.

DNA nanosensor based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly for sensitive and fast TK1 mRNA detection.

Hyperproliferative diseases are the first step for tumor formation; thymidine kinase 1 (TK1) mRNA is closely related to cell proliferation. Therefore, the risk of malignant proliferation can be identified by sensitively detecting the variance in TK1 mRNA concentration, which can be used for tumor auxiliary diagnosis and monitoring tumor treatment. Owing to the low abundance and instability of TK1 mRNA in real samples, the development of a sensitive and fast mRNA detection method is necessary. A DNA nanosensor that can be used for detecting TK1 mRNA based on bipedal 3D DNA walker-driven proximal catalytic hairpin assembly (P-CHA) was developed. P-CHA hairpins were hybridized to a linker DNA strand coupled with magnetic nanoparticles to increase their local concentrations. The bipedal DNA walking on the surface of NPs accelerates reaction kinetics using the proximity effect. Taking advantage of the signal amplification of P-CHA as well as the rapid reaction rate of the DNA walker in 80min, the proposed sensor detects TK1 mRNA with a low detection limit of 14pM and may then be applied toclinical diagnosis.

Effects of imidazolium-based ionic liquids on the elasticity of DNA revealed by magnetic tweezers

The functions of DNA are related to its mechanical properties, including elasticity and conformational transitions. Ionic liquids, known as green solvents, are used for DNA separation and as a solvent medium for long-term DNA storage. This paper elucidated DNA interaction mechanisms with two imidazolium-based ionic liquids: 1-butyl-3-methylimi-dazolium chloride ([bmim]Cl) and 1-octyl-3-methylimidazolium chloride ([omim]Cl), via magnetic tweezers, bulk techniques and molecular dynamic simulation (MD). By stretching and twisting individual DNA molecules, we measured the elasticity of DNA in the presence of ILs ([bmim]Cl and [omim]Cl). As results, the persistence length, stretch modulus, torsional stiffness and twist-stretch coupling exhibited a significant decrease compared to the DNA without ILs binding. Isothermal titration calorimetry analysis indicated that the ILs in the presence of DNA were thermodynamically favored driven by enthalpy and entropy. DNA underwent size transition and conformational change induced by ILs, and the charges of DNA were neutralized by the added ILs. The binding of ILs induced unwinding and softening of DNA. Circular dichroism spectra and MD results indicated that DNA maintained B-conformation after interacting with ILs. Compared to [bmim]+, [omim]+ showed stronger binding affinity to DNA and tended to aggregate on the DNA surface. ILs binding reduced Na+ and water binding on DNA, which likely prevented the hydrolytic reactions that denatured DNA and helped stabilize DNA for the long term. This research provides a critical theoretical foundation for the utilization of ionic liquids in life sciences.

Mechanistic Insights into How the Single Point Mutation Change the Autoantibody Repertoire.

A recent study showed that just one point mutation F33 to Y in the complementarity-determining region 1 of heavy chain (H-CDR1) could lead to the auto-antibody losing its DNA binding ability. However, the potential molecular mechanisms have not been well elucidated. In this study, we investigated how the antibody lost the DNA binding ability caused by mutation F33 to Y in the H-CDR1. We found that the electrostatic force was not the primary driving force for the interaction between anti-DNA antibodies and the antigen single strand DNA (ssDNA), and that the H-CDR2 largely contributed to the binding of antigen ssDNA, even larger than H-CDR1. The H-F33Y mutation could increase the hydrogen-bond interaction but impair the pi-pi stacking interaction between the antibody and ssDNA. We further found that F33H, W98H and Y95L in the wiletype antibody could form the stable pi-pi stacking interaction with the nucleotide bases of ssDNA. However, the Y33 in mutant could not form the parallel sandwich pi-pi stacking interaction with the ssDNA. To further confirm the importance of pi-pi stacking, the wildtype antibody and the mutants (F33YH, F33AH, W98AH and Y95AL) were experimentally expressed in CHO cells and purified, and the results from ELISA clearly showed that all the mutants lost the ssDNA binding ability. Taken together, our findings may not only deepen the understanding of the underlying interaction mechanism between autoantibody and antigen, but also broad implications in the field of antibody engineer.