Short DNA Segments Research Articles

Dynamic modulation of enhancer-promoter and promoter-promoter connectivity in gene regulation.

Enhancers are short segments of regulatory DNA that control when and in which cell-type genes should be turned on in response to a variety of extrinsic and intrinsic signals. At the molecular level, enhancers serve as a genomic scaffold that recruits sequence-specific transcription factors and co-activators to facilitate transcription from linked promoters. However, it remains largely unclear how enhancers communicate with appropriate target promoters in the context of higher-order genome topology. In this review, we discuss recent progress in our understanding of the functional interplay between enhancers, genome topology, and the molecular properties of transcription machineries in gene regulation. We suggest that the activities of transcription hubs are highly regulated through the dynamic rearrangement of enhancer-promoter and promoter-promoter connectivity during animal development.

Unraveling the Dual-Stretch-Mode Impact on Tension Gauge Tethers' Mechanical Stability.

Tension gauge tethers (TGTs), short DNA segments serving as extracellular tension sensors, are instrumental in assessing the tension dynamics in mechanotransduction. These TGTs feature an initial shear-stretch region and an unzip-stretch region. Despite their utility, no theoretical model has been available to estimate their tension-dependent lifetimes [τ(f)], restricting insights from cellular mechanotransduction experiments. We have now formulated a concise expression for τ(f) of TGTs, accommodating contributions from both stretch regions. Our model uncovers a tension-dependent energy barrier shift occurring when tension surpasses a switching force of approximately 13 pN for the recently developed TGTs, greatly influencing τ(f) profiles. Experimental data from several TGTs validated our model. The calibrated expression can predict τ(f) of TGTs at 37 °C based on their sequences with minor fold changes, supporting future applications of TGTs.

Statistical framework to determine indel-length distribution.

Insertions and deletions (indels) of short DNA segments, along with substitutions, are the most frequent molecular evolutionary events. Indels were shown to affect numerous macro-evolutionary processes. Because indels may span multiple positions, their impact is a product of both their rate and their length distribution. An accurate inference of indel-length distribution is important for multiple evolutionary and bioinformatics applications, most notably for alignment software. Previous studies counted the number of continuous gap characters in alignments to determine the best-fitting length distribution. However, gap-counting methods are not statistically rigorous, as gap blocks are not synonymous with indels. Furthermore, such methods rely on alignments that regularly contain errors and are biased due to the assumption of alignment methods that indels lengths follow a geometric distribution. We aimed to determine which indel-length distribution best characterizes alignments using statistical rigorous methodologies. To this end, we reduced the alignment bias using a machine-learning algorithm and applied an Approximate Bayesian Computation methodology for model selection. Moreover, we developed a novel method to test if current indel models provide an adequate representation of the evolutionary process. We found that the best-fitting model varies among alignments, with a Zipf length distribution fitting the vast majority of them. The data underlying this article are available in Github, at https://github.com/elyawy/SpartaSim and https://github.com/elyawy/SpartaPipeline.

A Novel Predictor for the Analysis and Prediction of Enhancers and Their Strength via Multi-View Features and Deep Forest

Enhancers are short DNA segments (50–1500 bp) that effectively activate gene transcription when transcription factors (TFs) are present. There is a correlation between the genetic differences in enhancers and numerous human disorders including cancer and inflammatory bowel disease. In computational biology, the accurate categorization of enhancers can yield important information for drug discovery and development. High-throughput experimental approaches are thought to be vital tools for researching enhancers’ key characteristics; however, because these techniques require a lot of labor and time, it might be difficult for researchers to forecast enhancers and their powers. Therefore, computational techniques are considered an alternate strategy for handling this issue. Based on the types of algorithms that have been used to construct predictors, the current methodologies can be divided into three primary categories: ensemble-based methods, deep learning-based approaches, and traditional ML-based techniques. In this study, we developed a novel two-layer deep forest-based predictor for accurate enhancer and strength prediction, namely, NEPERS. Enhancers and non-enhancers are divided at the first level by NEPERS, whereas strong and weak enhancers are divided at the second level. To evaluate the effectiveness of feature fusion, block-wise deep forest and other algorithms were combined with multi-view features such as PSTNPss, PSTNPdss, CKSNAP, and NCP via 10-fold cross-validation and independent testing. Our proposed technique performs better than competing models across all parameters, with an ACC of 0.876, Sen of 0.864, Spe of 0.888, MCC of 0.753, and AUC of 0.940 for layer 1 and an ACC of 0.959, Sen of 0.960, Spe of 0.958, MCC of 0.918, and AUC of 0.990 for layer 2, respectively, for the benchmark dataset. Similarly, for the independent test, the ACC, Sen, Spe, MCC, and AUC were 0.863, 0.865, 0.860, 0.725, and 0.948 for layer 1 and 0.890, 0.940, 0.840, 0.784, and 0.951 for layer 2, respectively. This study provides conclusive insights for the accurate and effective detection and characterization of enhancers and their strengths.

Facile preparation of toluidine blue-loaded DNA nanogels for anticancer photodynamic therapy.

Photodynamic therapy (PDT) provides an effective therapeutic option for different types of cancer in addition to surgery, radiation, and chemotherapy. The treatment outcome of PDT is largely determined by both the light and dark toxicity of photosensitizers (PSs), which can be technically improved with the assistance of a drug delivery system, especially the nanocarriers. Toluidine blue (TB) is a representative PS that demonstrates high PDT efficacy; however, its application is largely limited by the associated dark toxicity. Inspired by TB's noncovalent binding with nucleic acids, in this study, we demonstrated that DNA nanogel (NG) could serve as an effective TB delivery vehicle to facilitate anticancer PDT. The DNA/TB NG was constructed by the simple self-assembly between TB and short DNA segments using cisplatin as a crosslinker. Compared with TB alone, DNA/TB NG displayed a controlled TB-releasing behavior, effective cellular uptake, and phototoxicity while reducing the dark toxicity in breast cancer cells MCF-7. This DNA/TB NG represented a promising strategy to improve TB-mediated PDT for cancer treatments.

Weak tension accelerates hybridization and dehybridization of short oligonucleotides.

The hybridization and dehybridization of DNA subject to tension is relevant to fundamental genetic processes and to the design of DNA-based mechanobiology assays. While strong tension accelerates DNA melting and decelerates DNA annealing, the effects of tension weaker than 5 pN are less clear. In this study, we developed a DNA bow assay, which uses the bending rigidity of double-stranded DNA (dsDNA) to exert weak tension on a single-stranded DNA (ssDNA) target in the range of 2-6 pN. Combining this assay with single-molecule FRET, we measured the hybridization and dehybridization kinetics between a 15 nt ssDNA under tension and a 8-9 nt oligonucleotide, and found that both the hybridization and dehybridization rates monotonically increase with tension for various nucleotide sequences tested. These findings suggest that the nucleated duplex in its transition state is more extended than the pure dsDNA or ssDNA counterpart. Based on coarse-grained oxDNA simulations, we propose that this increased extension of the transition state is due to steric repulsion between the unpaired ssDNA segments in close proximity to one another. Using linear force-extension relations verified by simulations of short DNA segments, we derived analytical equationsfor force-to-rate conversion that are in good agreement with our measurements.

The evolutionary dynamics that retain long neutral genomic sequences in face of indel deletion bias: a model and its application to human introns.

Insertions and deletions (indels) of short DNA segments are common evolutionary events. Numerous studies showed that deletions occur more often than insertions in both prokaryotes and eukaryotes. It raises the question why neutral sequences are not eradicated from the genome. We suggest that this is due to a phenomenon we term border-induced selection. Accordingly, a neutral sequence is bordered between conserved regions. Deletions occurring near the borders occasionally protrude to the conserved region and are thereby subject to strong purifying selection. Thus, for short neutral sequences, an insertion bias is expected. Here, we develop a set of increasingly complex models of indel dynamics that incorporate border-induced selection. Furthermore, we show that short conserved sequences within the neutrally evolving sequence help explain: (i) the presence of very long sequences; (ii) the high variance of sequence lengths; and (iii) the possible emergence of multimodality in sequence length distributions. Finally, we fitted our models to the human intron length distribution, as introns are thought to be mostly neutral and bordered by conserved exons. We show that when accounting for the occurrence of short conserved sequences within introns, we reproduce the main features, including the presence of long introns and the multimodality of intron distribution.

Detection of tomato leaf curl New Delhi virus DNA using U-bent optical fiber-based LSPR probes

The detection of Tomato leaf curl New Delhi virus (ToLCNDV) DNA has been presented using U-bent fiber optic sensor (FOS) by an evanescent wave absorption method in the visible region. The localized surface plasmon resonance (LSPR) based FOS was developed by immobilizing gold nanoparticles on the amine-functionalized surface and the receptor probe was prepared by attaching a short segment of the complementary DNA (cDNA) on this surface. Further, the cDNA attachment was confirmed by monitoring the real-time change in LSPR absorption spectra. The conjugation protocol of single-strand DNA (ssDNA) from the viral genome of ToLCNDV was established like antibody-antigen-based assays by observing the real-time change in absorption kinetics. Consequently, a linear correlation between ssDNA concentration and the conjugation-dependent change in refractive index (RI) has been observed. The limit of detection (LOD) using two different cDNA immobilized FOS probes was determined to be 50±10 ng/μL and the change in RI was found 12.5±1.3×10−4 RIU/Abs. From this systematic binding study, a straightforward target-specific sensor has been constructed using a digital electronic logic gate analogy. This device has significant commercial potential for the development of a portable LSPR sensor for real-time detection of plant pathogens in addition to the possibility of multi-target environments.

Enhancer-LSTMAtt: A Bi-LSTM and Attention-Based Deep Learning Method for Enhancer Recognition.

Enhancers are short DNA segments that play a key role in biological processes, such as accelerating transcription of target genes. Since the enhancer resides anywhere in a genome sequence, it is difficult to precisely identify enhancers. We presented a bi-directional long-short term memory (Bi-LSTM) and attention-based deep learning method (Enhancer-LSTMAtt) for enhancer recognition. Enhancer-LSTMAtt is an end-to-end deep learning model that consists mainly of deep residual neural network, Bi-LSTM, and feed-forward attention. We extensively compared the Enhancer-LSTMAtt with 19 state-of-the-art methods by 5-fold cross validation, 10-fold cross validation and independent test. Enhancer-LSTMAtt achieved competitive performances, especially in the independent test. We realized Enhancer-LSTMAtt into a user-friendly web application. Enhancer-LSTMAtt is applicable not only to recognizing enhancers, but also to distinguishing strong enhancer from weak enhancers. Enhancer-LSTMAtt is believed to become a promising tool for identifying enhancers.

In Silico Investigation of the Biological Implications of Complex DNA Damage with Emphasis in Cancer Radiotherapy through a Systems Biology Approach.

Different types of DNA lesions forming in close vicinity, create clusters of damaged sites termed as “clustered/complex DNA damage” and they are considered to be a major challenge for DNA repair mechanisms resulting in significant repair delays and induction of genomic instability. Upon detection of DNA damage, the corresponding DNA damage response and repair (DDR/R) mechanisms are activated. The inability of cells to process clustered DNA lesions efficiently has a great impact on the normal function and survival of cells. If complex lesions are left unrepaired or misrepaired, they can lead to mutations and if persistent, they may lead to apoptotic cell death. In this in silico study, and through rigorous data mining, we have identified human genes that are activated upon complex DNA damage induction like in the case of ionizing radiation (IR) and beyond the standard DNA repair pathways, and are also involved in cancer pathways, by employing stringent bioinformatics and systems biology methodologies. Given that IR can cause repair resistant lesions within a short DNA segment (a few nm), thereby augmenting the hazardous and toxic effects of radiation, we also investigated the possible implication of the most biologically important of those genes in comorbid non-neoplastic diseases through network integration, as well as their potential for predicting survival in cancer patients.

Forensic genomics research on microhaplotypes.

Microhaplotype loci (microhaplotype, MHs), defined by two or more closely linked single nucleotide polymorphisms, are a type of molecular marker within a short segment of DNA. As emerging forensic genetic markers, MHs have no stutter artefacts and higher polymorphism, and permit the design of smaller amplicons. In order to identify the markers from a genome wide perspective and explore their potential application further, we constructed the most comprehensive MH dataset to date, based on the whole genome sequencing data of 105 Han individuals in Southern China from 1000 Genomes Project. The results showed that there were 9,490,075 MH loci in the range of 350 bp in the human genome, and the distribution density of microhaplotypes suggests gene variation. Polymorphism analysis of MHs from various base spans showed that the polymorphism of MHs could reach or exceed common short tandem repeat sites. In addition, based on their flexible assembly, a scheme to build the public database of microhaplotypes was proposed.

Mechanisms of Sporicidal Activity Induced by Ionized Hydrogen Peroxide in the Spores of Bacillus atrophaeus.

Introduction: Ionized hydrogen peroxide (iHP) is a new technology used for the decontamination of surfaces or laboratory areas. It utilizes a low concentration of hydrogen peroxide (H2O2) mixed with air and ionized through a cold plasma arc. This technology generates reactive oxygen species as a means of decontamination. Objectives: The purpose of this study is to review the effects of iHP on the structure of the spores of Bacillus atrophaeus by observing its effects using transmission electron microscopy (TEM) and also by evaluating the existence of DNA damage by fluorescence-based quantitative polymerase chain reaction (qPCR). Methods: Spore samples of B. atrophaeus decontaminated using iHP at different exposure times (Control, 1, 2, 6, and 12 h) were fixed for TEM. In addition, DNA was extracted for evaluation of DNA damages using fluorescence-based qPCR assays. Results: Damages to the spore structures of B. atrophaeus caused by the decontamination process with iHP at different exposure times (Control, 1, 2, 6, and 12 h) can be observed in micrographs. The effects of the decontamination to short DNA segment (132 base pairs [bp]) of the yaaH gene using qPCR present a linear degradation, and for the long DNA segment (680 bp), it presents a biphasic mode. Conclusion: The results of the qPCR analysis show two initial stages of damage to DNA with very noticeable damage at 12 h contact time, which confirms the observations of the TEM micrographs for the B. atrophaeus spores. The study demonstrates damage to the spore core DNA.

Multi-Indel: A Microhaplotype Marker Can Be Typed Using Capillary Electrophoresis Platforms.

Since the concept of microhaplotypes was proposed by Kidd in 2013, various microhaplotype markers have been investigated for various forensic purposes, such as individual identification, deconvolution of DNA mixtures, or forensic ancestry inference. In our opinion, various compound markers are also regarded as generalized microhaplotypes, encompassing two or more variants in a short segment of DNA (e.g., 200 bp). That is, a set of variants (referred to herein as multi-variants) within a certain length includes single nucleotide polymorphisms (SNP), insertion/deletion polymorphisms (Indels), or short tandem repeat polymorphisms (STRs). At present, multi-variant is mainly aimed at multi-SNPs. However, the haplotype genotyping of multi-variants relies on single-strand analysis, mainly using massively parallel sequencing (MPS). Here, we describe a method based on a capillary electrophoresis (CE) platform that can directly obtain haplotypes of individuals. Several microhaplotypes consisting of three or more Indels with different insertion or deletion lengths in the range of less than 200 bp were screened out, each of which had at least three haplotypes. As a result, the haplotype of an individual was reflected by the length of its polymorphism. Finally, we established a multiplex amplification system containing 18 multi-Indel markers that could identify haplotypes on each chromosome of an individual. The combined power of discrimination (CPD) and the cumulative probability of exclusion (CPE) were 0.999999999997234 and 0.9984, respectively.

Cooperative Binding between Distant Transcription Factors is a Hallmark of Active Enhancers

Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short MNase-protected DNA segments indicates that the majority of enhancers harbor two or more TF binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. We integrate MNase-seq, methylation accessibility profiling, and CUT&RUN chromatin profiling as a comprehensive strategy to characterize co-binding of the Trithorax-like (TRL) DNA binding protein and multiple other TFs. We identify states where an enhancer is bound by no TF, by either single factor, by multiple factors, or where binding sites are occluded by nucleosomes. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at a majority of active enhancers. Factor cooperativity occurs predominantly between sites spaced 50 bp apart in active enhancers, indicating that most TF cooperativity in cells occurs without apparent protein-protein interactions. Our findings suggest a mechanism for nucleosomes to promote cooperativity by requiring co-binding to effectively clear nucleosomes and promote enhancer function.

Targeted mutagenesis of Mycoplasma gallisepticum using its endogenous CRISPR/Cas system

New, more efficient methods are needed to facilitate studies of gene function in the mycoplasmas. CRISPR/Cas systems, which provide bacteria with acquired immunity against invading nucleic acids, have been developed as tools for genomic editing in a wide range of organisms. We explored the potential for using the endogenous Mycoplasma gallisepticum CRISPR/Cas system to introduce targeted mutations into the chromosome of this important animal pathogen. Three constructs carrying different CRISPR arrays targeting regions in the ksgA gene (pK1-CRISPR, pK-CRISPR-1 and pK-CRISPR-2) were assembled and introduced into M. gallisepticum on an oriC plasmid. The loss of KsgA prevents ribosomal methylation, which in turn confers resistance to the aminoglycoside antimicrobial kasugamycin, enabling selection for ksgA mutants. Analyses of the complete sequence of the ksgA gene in 78 resistant transformants revealed various modifications of the target region, presumably caused by the directed CRISPR/Cas activity of M. gallisepticum. The analyses suggested that M. gallisepticum may utilize a non-homologous end joining (NHEJ) repair system, which can result in deletion or duplication of a short DNA segment in the presence of double-stranded breaks. This study has generated an improved understanding of the M. gallisepticum CRISPR/Cas system, and may also facilitate further development of tools to genetically modify this important pathogen.

Three-dimensional nanodosimetric characterisation of proton track structure

This work presents a detailed investigation into the nanodosimetric properties of the track structure of protons in water at energies relevant for proton radiotherapy. The ionization component of the tracks had been simulated in previous work using Geant4-DNA for proton start energies between 1 MeV and 100 MeV. From the simulation results, the frequency distribution of ionization clusters formed in nanometric target volumes was obtained in dependence of the impact parameter of the proton trajectory with respect to the target center. In the track core, targets of cylindrical shape and a size comparable to a short segment of DNA were used for scoring ionization cluster size distributions. For the penumbra region, three different options for defining the cylinder shell sectors were investigated, with each cylinder shell sector volume the same as the cylinder target volume. The radial distributions were numerically integrated to obtain the effective track cross sections with respect to different nanodosimetric parameters. Graphically displaying the radial dependence in a similar way as microdosimetric distributions allowed elucidating the contribution of different radial distances to the overall radial integral of the quantities under consideration. Furthermore, it was tested how well the radial dependence of the nanodosimetric parameters could be fitted with a model function derived in literature for the radial energy deposition in proton tracks. It was found that this model function allows describing only the radial dependence of the total frequency of nanometric targets where ionizations occur. The deviation from a 1/r2-dependence of the radial dependence of the frequency of targets receiving more than a minimum number of ionizations (exceeding one) includes a second peak centred around 10 nm radial distance from the proton trajectory.

Foreign DNA detection by high-throughput sequencing to regulate genome-edited agricultural products

Although the advent of several new breeding techniques (NBTs) is revolutionizing agricultural production processes, technical information necessary for their regulation is yet to be provided. Here, we show that high-throughput DNA sequencing is effective for the detection of unintended remaining foreign DNA segments in genome-edited rice. A simple k-mer detection method is presented and validated through a series of computer simulations and real data analyses. The data show that a short foreign DNA segment of 20 nucleotides can be detected and the probability that the segment is overlooked is 10−3 or less if the average sequencing depth is 30 or more, while the number of false hits is less than 1 on average. This method was applied to real sequencing data, and the presence and absence of an external DNA segment were successfully proven. Additionally, our in-depth analyses also identified some weaknesses in current DNA sequencing technologies. Hence, for a rigorous safety assessment, the combination of k-mer detection and another method, such as Southern blot assay, is recommended. The results presented in this study will lay the foundation for the regulation of NBT products, where foreign DNA is utilized during their generation.

Aberrant Function of the C-Terminal Tail of HIST1H1E Accelerates Cellular Senescence and Causes Premature Aging

Histones mediate dynamic packaging of nuclear DNA in chromatin, a process that is precisely controlled to guarantee efficient compaction of the genome and proper chromosomal segregation during cell division and to accomplish DNA replication, transcription, and repair. Due to the important structural and regulatory roles played by histones, it is not surprising that histone functional dysregulation or aberrant levels of histones can have severe consequences for multiple cellular processes and ultimately might affect development or contribute to cell transformation. Recently, germline frameshift mutations involving the C-terminal tail of HIST1H1E, which is a widely expressed member of the linker histone family and facilitates higher-order chromatin folding, have been causally linked to an as-yet poorly defined syndrome that includes intellectual disability. We report that these mutations result in stable proteins that reside in the nucleus, bind to chromatin, disrupt proper compaction of DNA, and are associated with a specific methylation pattern. Cells expressing these mutant proteins have a dramatically reduced proliferation rate and competence, hardly enter into the S phase, and undergo accelerated senescence. Remarkably, clinical assessment of a relatively large cohort of subjects sharing these mutations revealed a premature aging phenotype as a previously unrecognized feature of the disorder. Our findings identify a direct link between aberrant chromatin remodeling, cellular senescence, and accelerated aging.

Large distances separate coregulated genes in living Drosophila embryos

Transcriptional enhancers are short segments of DNA that switch genes on and off in response to a variety of cellular signals. Many enhancers map quite far from their target genes, on the order of tens or even hundreds of kilobases. There is extensive evidence that remote enhancers are brought into proximity with their target promoters via long-range looping interactions. However, the exact physical distances of these enhancer-promoter interactions remain uncertain. Here, we employ high-resolution imaging of living Drosophila embryos to visualize the distances separating linked genes that are coregulated by a shared enhancer. Cotransvection assays (linked genes on separate homologs) suggest a surprisingly large distance during transcriptional activity: at least 100-200 nm. Similar distances were observed when a shared enhancer was placed into close proximity with linked reporter genes in cis. These observations are consistent with the occurrence of "transcription hubs," whereby clusters (or condensates) of multiple RNA polymerase II complexes and associated cofactors are periodically recruited to active promoters. The dynamics of this process might be responsible for rapid fluctuations in the distances separating the transcription of coregulated reporter genes during transvection. We propose that enhancer-promoter communication depends on a combination of classical looping and linking models.

A 5'-to-3' strand exchange polarity is intrinsic to RecA nucleoprotein filaments in the absence of ATP hydrolysis.

RecA is essential to recombinational DNA repair in which RecA filaments mediate the homologous DNA pairing and strand exchange. Both RecA filament assembly and the subsequent DNA strand exchange are directional. Here, we demonstrate that the polarity of DNA strand exchange is embedded within RecA filaments even in the absence of ATP hydrolysis, at least over short DNA segments. Using single-molecule tethered particle motion, we show that successful strand exchange in the presence of ATP proceeds with a 5′-to-3′ polarity, as demonstrated previously. RecA filaments prepared with ATPγS also exhibit a 5′-to-3′ progress of strand exchange, suggesting that the polarity is not determined by RecA disassembly and/or ATP hydrolysis. RecAΔC17 mutants, lacking a C-terminal autoregulatory flap, also promote strand exchange in a 5′-to-3′ polarity in ATPγS, a polarity that is largely lost with this RecA variant when ATP is hydrolyzed. We propose that there is an inherent strand exchange polarity mediated by the structure of the RecA filament groove, associated by conformation changes propagated in a polar manner as DNA is progressively exchanged. ATP hydrolysis is coupled to polar strand exchange over longer distances, and its contribution to the polarity requires an intact RecA C-terminus.