Biology subjects

Ding, D.

Publications and source records attributed to Ding, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Transient States During Oligonucleotides Hybridization: Insights from Annealing Kinetics of Mismatches and Bulges

Oligonucleotide hybridization is crucial in various biological, prebiotic and nanotechnological processes, including gene regulation, non-enzymatic primer extension and DNA nanodevice assembly. Although extensive research has focused on the thermodynamics and kinetics of nucleic acid hybridization, the behavior of complex mixtures and the outcome of competition for target binding remain less well understood. In this study, we investigate the impact of mismatches and bulges in a 12bp DNA or RNA duplex on its association (kon) and dissociation (koff) kinetics. We find that such defects have relatively small effects on the association kinetics, while the dissociation kinetics vary in a position-dependent manner by up to 6 orders of magnitude. Building upon this observation, we explored a competition scenario involving multiple oligonucleotides, and observed a transient low specificity of probe hybridization to fully vs. partially complementary targets in solution. We characterize these long-lived metastable states and their evolution toward equilibrium, and show that sufficiently long-lived mis-paired duplexes can serve as substrates for prebiotically relevant chemical copying reactions. Our results suggest that transient low accuracy states may spontaneously emerge within all complex nucleic acid systems comprising a large enough number of competing strands, with potential repercussions for gene regulation in the realm of modern biology and the prebiotic preservation of genetic information.

biochemistry↗

bioRxiv2023

Enhanced nonenzymatic RNA copying with in-situ activation of short oligonucleotides

The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World. We have previously shown that a potentially prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can lead to the efficient synthesis of 2-aminoimidazole activated mononucleotides when carried out under freeze-thaw cycling conditions. Such activated nucleotides react with each other to form 5'-5' 2-aminoimidazolium bridged dinucleotides, enabling template-directed primer extension to occur within the same reaction mixture. However, mononucleotides linked to oligonucleotides by a 5'-5' 2-aminoimidazolium bridge are superior substrates for nonenzymatic primer extension, due to their higher intrinsic reactivity and their higher template affinity. Here we show that eutectic phase phospho-Passerini chemistry efficiently activates short oligonucleotides and promotes the formation of monomer-bridged-oligonucleotide species during freeze-thaw cycles. We then demonstrate that in-situ generated monomer-bridged-oligonucleotides lead to efficient nonenzymatic template copying in the same reaction mixture. Our findings pave the way for future research into the activation of complex mixtures of mono- and oligonucleotides for the enhanced copying and potentially the replication of arbitrary RNA sequences.

biochemistry↗

bioRxiv2023

Experimental Tests of the Virtual Circular Genome Model for Non-enzymatic RNA Replication

The virtual circular genome (VCG) model was proposed as a means of going beyond template copying to indefinite cycles of nonenzymatic RNA replication during the origin of life. In the VCG model the protocellular genome is a collection of short oligonucleotides that map to both strands of a virtual circular sequence. Replication is driven by templated nonenzymatic primer extension on a subset of kinetically trapped partially base-paired configurations, followed by shuffling of these configurations to enable continued oligonucleotide elongation. Here we describe initial experimental studies of the feasibility of the VCG model for replication. We designed a small 12-nucleotide model VCG and synthesized all 247 oligonucleotides of length 2 to 12 corresponding to this genome. We experimentally monitored the fate of individual labeled primers in the pool of VCG oligonucleotides following the addition of activated nucleotides, and investigated factors such as oligonucleotide length, concentration, composition, and temperature on the extent of primer extension. We observe a surprisingly prolonged equilibration process in the VCG system that enables a considerable extent of reaction. We find that environmental fluctuations would be essential for continuous templated extension of the entire VCG system, since the shortest oligonucleotides can only bind to templates at low temperatures, while the longest oligonucleotides require high temperature spikes to escape from inactive configurations. Finally, we demonstrate that primer extension is significantly enhanced when the mix of VCG oligonucleotides is pre-activated. We discuss the necessity of ongoing in-situ activation chemistry for continuous and accurate VCG replication.

biochemistry↗

bioRxiv2022

Independent mutation effects enable design of combinatorial protein binding mutants

Recent developments in protein design have adapted large neural networks with up to 100s of millions of parameters to learn complex sequence-function mappings. However, it is unclear which dependencies between residues are critical for determining protein function, and a better empirical understanding could enable high quality models that are also more data- and resource-efficient. Here, we observe that the per residue amino acid preferences - without considering interactions between mutations are sufficient to explain much, and sometimes virtually all of the combinatorial mutation effects across 7 datasets (R2 [~] 78-98%), including one generated here. These preference parameters (20*N, where N is the number of mutated residues) can be learned from as few as [~]5*20*N observations to predict a much larger number (potentially up to 20N) of combinatorial variant effects with high accuracy (Pearson r > 0.8). We hypothesized that the local structural dependencies surrounding a residue could be sufficient to learn these required mutation preferences, and developed an unsupervised design approach, which we term CoVES for Combinatorial Variant Effects from Structure. We show that CoVES outperforms not just model free sampling approaches but also complicated, high-capacity autoregressive neural networks in generating functional and diverse sequence variants for two example proteins. This simple, biologically-rooted model can be an effective alternative to high-capacity, out of domain models for the design of functional proteins.

bioengineering↗

bioRxiv2022

Testis-specific H2BFWT disrupts nucleosome integrity through reductions of DNA-histone interactions

During spermatogenesis, multiple testis-specific histone variants are involved in the dynamic chromatin transitions. H2BFWT is a primate testis-specific H2B variant with hitherto unclear functions, and SNPs of H2BFWT are closely associated with male non-obstructive infertility. Here, we found that H2BFWT is preferentially localized in the sub-telomeric regions and the promoters of genes highly expressed in testis from differentiated spermatogonia to early spermatocytes. Cryo-EM structural analysis shows that H2BFWT nucleosomes are defined by weakened interactions between H2A-H2BFWT dimer and H4, and between histone octamer and DNA. Furthermore, one of its SNPs, H2BFWTH100R further destabilizes nucleosomes and increases the nucleosome unwrapping rate by interfering with the interaction with H4K91. Our results suggest that H2BFWT may be necessary for the regulation of spermatogenesis-related gene expression by decreasing transcriptional barriers, and that H2BFWTH100R overdrives its nucleosome-destabilizing effects which causes infertility.

biochemistry↗

bioRxiv2022

MPZ-T124M mouse model replicates human axonopathy and suggest alteration in axo-glia communication

Myelin is essential for rapid nerve impulse propagation and axon protection. Accordingly, defects in myelination or myelin maintenance lead to secondary axonal damage and subsequent degeneration. Studies utilizing genetic (CNPase-, MAG-, and PLP-null mice) and naturally occurring neuropathy models suggest that myelinating glia also support axons independently from myelin. Myelin protein zero (MPZ or P0), which is expressed only by Schwann cells, is critical for myelin formation and maintenance in the peripheral nervous system. Many mutations in MPZ are associated with demyelinating neuropathies (Charcot-Marie-Tooth disease type 1B [CMT1B]). Surprisingly, the substitution of threonine by methionine at position 124 of P0 (P0T124M) causes axonal neuropathy (CMT2J) with little to no myelin damage. This disease provides an excellent paradigm to understand how myelinating glia support axons independently from myelin. To study this, we generated targeted knock-in P0T124M mutant mice, a genetically authentic model of T124M-CMT2J neuropathy. Similar to patients, these mice develop axonopathy between 2 and 12 months of age, characterized by impaired motor performance, normal nerve conduction velocities but reduced compound motor action potential amplitudes, and axonal damage with only minor compact myelin modifications. Mechanistically, we detected metabolic changes that could lead to axonal degeneration, and prominent alterations in non-compact myelin domains such as paranodes, Schmidt-Lanterman incisures, and gap junctions, implicated in Schwann cell-axon communication and axonal metabolic support. Finally, we document perturbed mitochondrial size and distribution along P0T124M axons suggesting altered axonal transport. Our data suggest that Schwann cells in P0T124M mutant mice cannot provide axons with sufficient trophic support, leading to reduced ATP biosynthesis and axonopathy. In conclusion, the P0T124M mouse model faithfully reproduces the human neuropathy and represents a unique tool for identifying the molecular basis for glial support of axons.

neuroscience↗

bioRxiv2021

Structural insights into the mechanism of nucleotide regulation of pancreatic KATP channel

ATP-sensitive potassium channels (KATP) are metabolic sensors that convert the intracellular ATP/ADP ratio to the excitability of cells. They are involved in many physiological processes and implicated in several human diseases. Here we present the cryo-EM structures of the pancreatic KATP channel in both the closed state and the pre-open state, resolved in the same sample. The nucleotides bind at the inhibitory sites of the Kir6.2 channel in the closed state but not in the pre-open state. Structural comparisons reveal the mechanism for ATP inhibition and Mg-ADP activation, two fundamental properties of KATP channels. Moreover, the structure also uncovers the activation mechanism of diazoxide-type KATP openers.

biophysics↗

bioRxiv2021

Non-canonical Function of a Hif-1α Splice Variant Contributes to the Sustained Flight of Locusts

The hypoxia inducible factor (Hif) pathway is functionally conserved across metazoans in modulating cellular adaptations to hypoxia. However, the functions of this pathway under aerobic physiological conditions are rarely investigated. Here, we show that Hif-12, a locust Hif-1 isoform, does not induce canonical hypoxic responses but functions as a specific regulator of locust flight, which is a completely aerobic physiological process. Two Hif-1 splice variants were identified in locusts, a ubiquitously expressed Hif-11 and a muscle-predominantly expressed Hif-12. Hif-11 that induces typical hypoxic responses upon hypoxia exposure, remains inactive during flight. By contrast, the expression of Hif-12, which lacks C-terminal transactivation domain, is less sensitive to oxygen-tension but induced extensively by flying. Hif-12 sustains flight endurance by promoting glucose oxidation while simultaneously maintaining redox homeostasis by upregulating the production of a reactive oxygen species (ROS) quencher, DJ-1. Overall, this study reveals a novel Hif-mediated mechanism underlying prolonged aerobic physiological activity.

zoology↗

bioRxiv2021

Coevolution of interacting proteins through non-contacting and non-specific mutations

Proteins often accumulate neutral mutations that do not affect current functions1 but can profoundly influence future mutational possibilities and functions2-4. Understanding such hidden potential has major implications for protein design and evolutionary forecasting5-7, but has been limited by a lack of systematic efforts to identify potentiating mutations8,9. Here, through the comprehensive analysis of a bacterial toxin-antitoxin system, we identified all possible single substitutions in the toxin that enable it to tolerate otherwise interface-disrupting mutations in its antitoxin. Strikingly, the majority of enabling mutations in the toxin do not contact, and promote tolerance non-specifically to, many different antitoxin mutations, despite covariation in homologs occurring primarily between specific pairs of contacting residues across the interface. In addition, the enabling mutations we identified expand future mutational paths that both maintain old toxin-antitoxin interactions and form new ones. These non-specific mutations are missed by widely used covariation and machine learning methods10,11. Identifying such enabling mutations will be critical for ensuring continued binding of therapeutically relevant proteins, such as antibodies, aimed at evolving targets12-14.

evolutionary biology↗

bioRxiv2021

Kinetic Explanations for the Sequence Biases Observed in the Nonenzymatic Copying of RNA Templates

The identification of nonenzymatic pathways for nucleic acid replication is a key challenge in understanding the origin of life. We have previously shown that nonenzymatic RNA primer extension using 2-aminoimidazole (2AI) activated nucleotides occurs primarily through an imidazolium-bridged dinucleotide intermediate. The reactive nature and preorganized structure of the intermediate increase the efficiency of primer extension but remain insufficient to drive extensive copying of RNA templates containing all four canonical nucleotides. To understand the factors that limit RNA copying, we synthesized all ten 2AI-bridged dinucleotide intermediates and measured the kinetics of primer extension in a model. The affinities of the ten dinucleotides for the primer/template/helper complexes vary by over 7,000-fold, consistent with nearest neighbor energetic predictions. Surprisingly, the reaction rates at saturating intermediate concentrations still vary by over 15-fold, with the most weakly binding dinucleotides exhibiting a lower maximal reaction rate. Certain noncanonical nucleotides can decrease sequence dependent differences in affinity and primer extension rate, while monomers bridged to short oligonucleotides exhibit enhanced binding and reaction rates. We suggest that more uniform binding and reactivity of imidazolium-bridged intermediates may lead to the ability to copy arbitrary template sequences under prebiotically plausible conditions.

biochemistry↗

bioRxiv2021

SARS-CoV-2 Variant Identification Using a Genome Tiling Array and Genotyping Probes

With over three million deaths worldwide attributed to the respiratory disease COVID-19 caused by the novel coronavirus SARS-CoV-2, it is essential that continued efforts be made to track the evolution and spread of the virus globally. We previously presented a rapid and cost-effective method to sequence the entire SARS-CoV-2 genome with 95% coverage and 99.9% accuracy. This method is advantageous for identifying and tracking variants in the SARS-CoV-2 genome when compared to traditional short read sequencing methods which can be time consuming and costly. Herein we present the addition of genotyping probes to our DNA chip which target known SARS-CoV-2 variants. The incorporation of the genotyping probe sets along with the advent of a moving average filter have improved our sequencing coverage and accuracy of the SARS-CoV-2 genome.

bioinformatics↗

bioRxiv2021

Destruction of DNA-binding proteins by programmable O'PROTAC: Oligonucleotide-based PROTAC

DNA-binding proteins including transcription factors (TFs) play essential roles in gene transcription and DNA replication and repair during normal organ development and pathogenesis of diseases such as cancer, cardiovascular disease and obesity, deeming to be a large repertoire of attractive therapeutic targets. However, this group of proteins are generally considered undruggable as they lack an enzymatic catalytic site or a ligand binding pocket. PROteolysis-TArgeting Chimera (PROTAC) technology has been developed by engineering a bifunctional small molecule chimera to bring a protein of interest (POI) to the proximity of an E3 ubiquitin ligase, thus inducing the ubiquitination of POI and further degradation through proteasome pathway. Here we report the development of Oligonucleotide-based PROTAC (OPROTACs), a class of noncanonical PROTACs in which a TF-recognizing double-stranded oligonucleotide is incorporated as a binding moiety of POI. We demonstrate that OPROTACs of ERG and LEF1, two highly cancer-related transcription factors selectively promote degradation of these proteins and inhibit their transcriptional activity in cancer cells. The programmable nature of OPROTACs indicates that this approach is applicable to destruct other TFs. OPROTACs not only can serve as a research tool, but also can be harnessed as a therapeutic arsenal to target DNA binding proteins for effective treatment of diseases such as cancer.

biochemistry↗

bioRxiv2021

Competition between bridged dinucleotides and activated mononucleotides determines the error frequency of nonenzymatic RNA primer extension

Nonenzymatic copying of RNA templates with activated nucleotides is a useful model for studying the emergence of heredity at the origin of life. Previous experiments with defined-sequence templates have pointed to the poor fidelity of primer extension as a major problem. Here we examine the origin of mismatches during primer extension on random templates in the simultaneous presence of all four 2-aminoimidazole-activated nucleotides. Using a deep sequencing approach that reports on millions of individual template-product pairs, we are able to examine correct and incorrect polymerization as a function of sequence context. We have previously shown that the predominant pathway for primer extension involves reaction with imidazolium-bridged dinucleotides, which form spontaneously by the reaction of two mononucleotides with each other. We now show that the sequences of correctly paired products reveal patterns that are expected from the bridged dinucleotide mechanism, whereas those associated with mismatches are consistent with direct reaction of the primer with activated mononucleotides. Increasing the ratio of bridged dinucleotides to activated mononucleotides, either by using purified components or by using isocyanide-based activation chemistry, reduces the error frequency. Our results point to testable strategies for the accurate nonenzymatic copying of arbitrary RNA sequences.

molecular biology↗

bioRxiv2020

Acute noise causes down-regulation of ECM protein expression in guinea pig cochlea

ObjectiveTo study the differences in cochlea protein expression before and after noise exposure using proteomics to reveal the pathological mechanism of noise-induced hearing loss (NIHL). MethodsA guinea pig NIHL model was established to test the ABR thresholds before and after noise exposure. The proteomics technology was used to study the mechanism of differential protein expression in the cochlea by noise stimulation. ResultsThe average hearing threshold of guinea pigs on the first day after noise exposure was 57.00{+/-}6.78dB SPL, high-frequency hearing loss was more severe than low frequency; the average hearing threshold on the seventh day after noise exposure was 45.83{+/-}6.07dB SPL, and the 4k Hz hearing threshold has the best recovery. The proteomics technology identified 3122 different inner ear proteins, of which six proteins related to the hearing were down-regulation: TenascinC, Collagen type XI alpha two chains, Collagen type II alpha one chain, Thrombospondin 2, Collagen type XI alpha one chain and Ribosomal protein L38, and are enriched in protein absorption, focal adhesion, and extracellular matrix receptor pathways. ConclusionImpulse noise can affect the expression of differential proteins through focal adhesion pathways. This data can provide an experimental basis for the research on the prevention and treatment of NIHL.

immunology↗

bioRxiv2020

NTR 2.0: a rationally-engineered prodrug converting enzyme with substantially enhanced efficacy for targeted cell ablation

Heterologously-expressed bacterial nitroreductase (NTR) enzymes sensitize eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell ablation paradigms that have expanded studies of cell function and regeneration in vertebrate systems. However, first-generation NTRs require confoundingly toxic prodrug treatments (e.g. 10 mM MTZ) and some cell types have proven resistant. We used rational engineering and cross-species screening to develop a NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy. Toxicity tests in zebrafish showed no deleterious effects of prolonged MTZ treatments of [≤]1 mM. NTR 2.0 therefore enables sustained cell loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and enable modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic resources to facilitate dissemination of NTR 2.0 to the research community.

cell biology↗

bioRxiv2020

Large-scale Phenotypic Drug Screen Identifies Neuroprotectants in Zebrafish and Mouse Models of Retinitis Pigmentosa

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in zebrafish and mouse RP models, reasoning drugs effective across species may translate better clinically. We first performed a large-scale phenotypic drug screen using a larval zebrafish model of inducible RP. 2,934 compounds, mostly human-approved drugs, were tested across six concentrations. Statistically, 113 compounds achieved "hit" status. Secondary tests of 42 high-priority hits confirmed eleven lead compounds. Nine leads were then evaluated in mouse RP models, with six exhibiting neuroprotective effects. An analysis of potential mechanisms of action suggested complementary activities. Paired lead compound assays in zebrafish showed additive neuroprotective effects for the majority. These results highlight the value of cross-species phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for patients with RP and IRDs.

neuroscience↗

bioRxiv2019

Uncovering the subtype-specific temporal order of cancer pathway dysregulation

Cancer is driven by genetic mutations that dysregulate pathways important for proper cell function. Therefore, discovering these cancer pathways and their dysregulation order is key to understanding and treating cancer. However, the heterogeneity of mutations between different individuals makes this challenging and requires that cancer progression is studied in a subtype-specific way. To address this challenge, we provide a mathematical model, called Subtype-specific Pathway Linear Progression Model (SPM), that simultaneously captures cancer subtypes and pathways and order of dysregulation of the pathways within each subtype. Experiments with synthetic data indicate the robustness of SPM to problem specifics including noise compared to an existing method. Moreover, experimental results on glioblastoma multiforme and colorectal adenocarcinoma show the consistency of SPMs results with the existing knowledge and its superiority to an existing method in certain cases. The implementation of our method is available at https://github.com/Dalton386/SPM.

bioinformatics↗

bioRxiv2018

A novel histone H4 variant regulates rDNA transcription in breast cancer

Histone variants, present in various cell types and tissues, are known to exhibit different functions. For example, histone H3.3 and H2A.Z are both involved in gene expression regulation, whereas H2A.X is a specific variant that responds to DNA double-strand breaks. In this study, we characterized H4G, a novel hominidae-specific histone H4 variant. H4G expression was found in a variety of cell lines and was particularly overexpressed in the tissues of breast cancer patients. H4G was found to localize primarily to the nucleoli of the cell nucleus. This localization was controlled by the interaction of the alpha helix 3 of the histone fold motif with the histone chaperone, nucleophosphomin 1. In addition, we found that H4G nucleolar localization increased rRNA levels, protein synthesis rates, and cell cycle progression. Furthermore, micrococcal nuclease digestion of H4G-containing nucleosomes reconstituted in vitro indicated that H4G destabilizes the nucleosome, which may serve to alter nucleolar chromatin in a way that enhances rDNA transcription in breast cancer tissues.

molecular biology↗