Native Gel Research Articles

The Structural Role of RPN10 in the 26S Proteasome and an RPN2-Binding Residue on RPN13 Are Functionally Important in Arabidopsis

The ubiquitin receptors RPN10 and RPN13 harbor multiple activities including ubiquitin binding; however, solid evidence connecting a particular activity to specific in vivo functions is scarce. Through complementation, the ubiquitin-binding site-truncated Arabidopsis RPN10 (N215) rescued the growth defects of rpn10-2, supporting the idea that the ubiquitin-binding ability of RPN10 is dispensable and N215, which harbors a vWA domain, is fully functional. Instead, a structural role played by RPN10 in the 26S proteasomes is likely vital in vivo. A site-specific variant, RPN10-11A, that likely has a destabilized vWA domain could partially rescue the rpn10-2 growth defects and is not integrated into 26S proteasomes. Native polyacrylamide gel electrophoresis and mass spectrometry with rpn10-2 26S proteasomes showed that the loss of RPN10 reduced the abundance of double-capped proteasomes, induced the integration of specific subunit paralogues, and increased the association of ECM29, a well-known factor critical for quality checkpoints by binding and inhibiting aberrant proteasomes. Extensive Y2H and GST-pulldown analyses identified RPN2-binding residues on RPN13 that overlapped with ubiquitin-binding and UCH2-binding sites in the RPN13 C-terminus (246–254). Interestingly, an analysis of homozygous rpn10-2 segregation in a rpn13-1 background harboring RPN13 variants defective for ubiquitin binding and/or RPN2 binding supports the criticality of the RPN13–RPN2 association in vivo.

Methylglyoxal-Induced Modifications in Human Triosephosphate Isomerase: Structural and Functional Repercussions of Specific Mutations

Triosephosphate isomerase (TPI) dysfunction is a critical factor in diverse pathological conditions. Deficiencies in TPI lead to the accumulation of toxic methylglyoxal (MGO), which induces non-enzymatic post-translational modifications, thus compromising protein stability and leading to misfolding. This study investigates how specific TPI mutations (E104D, N16D, and C217K) affect the enzyme’s structural stability when exposed to its substrate glyceraldehyde 3-phosphate (G3P) and MGO. We employed circular dichroism, intrinsic fluorescence, native gel electrophoresis, and Western blotting to assess the structural alterations and aggregation propensity of these TPI mutants. Our findings indicate that these mutations markedly increase TPI’s susceptibility to MGO-induced damage, leading to accelerated loss of enzymatic activity and enhanced protein aggregation. Additionally, we observed the formation of MGO-induced adducts, such as argpyrimidine (ARGp), that contribute to enzyme inactivation and aggregation. Importantly, the application of MGO-scavenging molecules partially mitigated these deleterious effects, highlighting potential therapeutic strategies to counteract MGO-induced damage in TPI-related disorders.

Purification and characterization of α-Galactosidases from Penicillium griseoroseum for Efficient Soymilk Hydrolysis

Soybean utilization is limited by the presence of raffinose oligosaccharides (RFO), which are not digested by humans and cause gastrointestinal discomfort. This study explores the potential of α-galactosidases from Penicillium griseoroseum for RFO hydrolysis in soymilk. Two distinct α-galactosidase enzymes, designated α-Gal1 and α-Gal2, were purified using a combination of ion-exchange chromatography and native polyacrylamide gel electrophoresis. Both enzymes exhibited characteristics of multimeric proteins and displayed similar biochemical properties. Optimal activity was observed at a pH range of 4.5-5.0 and a temperature range of 40-45 °C. Notably, α-Gal1 demonstrated high thermostability with a half-life of 16 hours at 40 °C. The α-galactosidases displayed different substrate affinitiesfor the substrates ρ-NP-αGal, o-NP-αGal, rD-raffinose, D-stachyose, and mD-melibiose. The Michaelis-Menten constant (Km) values for α-Gal1 were 1.06, 1.31, 28.74, 19.88, and 4.77 mmol/L, respectively, while those for α-Gal2 were 0.8, 1.26, 30.46, 21.74 and 5.01 mmol/L, respectively. Both α-Gal1 and α-Gal2 were strongly inhibited by metal ions (Ag⁺, Cu2⁺, Fe2⁺, and Hg2⁺) and moderately inhibited by D-melibiose. Importantly, both enzymes efficiently hydrolyzed RFOs, achieving complete D-stachyose elimination from soymilk after a 6-hour incubation. These findings propose the promising application of these α-galactosidases in industrial soymilk production, potentially enhancing its nutritional value and alleviating gastrointestinal issues in consumers.

Favourable HDL composition in endurance athletes is not associated with changes in HDL in vitro antioxidant and endothelial anti-inflammatory function.

Given the failure of high-density lipoprotein (HDL) raising therapies to reduce cardiovascular disease risk, attention has turned towards HDL composition and vascular protective functions. In individuals with insulin resistance, exercise interventions recover HDL function. However, the effect of exercise on HDL in otherwise healthy individuals is unknown. This cross-sectional study aimed to measure HDL composition and antioxidant / endothelial anti-inflammatory function in insulin sensitive endurance athlete and healthy control men. HDL was isolated using density gradient ultracentrifugation. HDL composition was measured using microplate assays for apolipoprotein A-I, total cholesterol content and apolipoprotein M. HDL protein composition was measured using nano-liquid chromatography tandem mass spectrometry. HDL subclass distribution was measured by native gel electrophoresis. HDL in vitro antioxidant function was measured by paraoxonase-1 activity assay and anti-inflammatory function assessed in endothelial cells. Compared to controls, endurance athlete HDL had higher apolipoprotein A-1 (1.65 ± 0.62 mg/mL vs 1.21 ± 0.34 mg/mL, p = 0.028) and higher total cholesterol content (1.54 ± 0.33 mmol/L vs 2.09 ± 0.44 mmol/L, p < 0.001). Proteomics revealed higher apolipoprotein A-II, A-IV and D and transthyretin in endurance athlete HDL versus controls. There was no difference observed in in vitro HDL antioxidant or anti-inflammatory functions between controls and endurance athletes. Despite a more favourable composition, endurance athlete HDL did not have higher in vitro antioxidant or anti-inflammatory function. It is possible that HDL has a ceiling of function, i.e. that healthy HDL function cannot be enhanced by endurance exercise.

MecE, MecB, and MecC proteins orchestrate methyl group transfer during dichloromethane fermentation.

Dichloromethane (DCM), a common hazardous industrial chemical, is anaerobically metabolized by four bacterial genera: Dehalobacter, Dehalobacterium, Ca. Dichloromethanomonas, and Ca. Formimonas. However, the pivotal methyltransferases responsible for DCM transformation have remained elusive. In this study, we investigated the DCM catabolism of Dehalobacterium formicoaceticum strain EZ94, contained in an enriched culture, using a combination of biochemical approaches. Initially, enzymatic assays were conducted with cell-free protein extracts, after protein separation by blue native polyacrylamide gel electrophoresis. In the slices with the highest DCM transformation activity, a high absolute abundance of the methyltransferase MecC was revealed by mass spectrometry. Enzymatic activity assays with heterologously expressed MecB, MecC, and MecE from strain EZ94 showed complete DCM transformation only when all three enzymes were present. Our experimental results, coupled with the computational analysis of MecB, MecC, and MecE sequences, enabled us to assign specific roles in DCM transformation to each of the proteins. Our findings reveal that both MecE and MecC are zinc-dependent methyltransferases responsible for DCM demethylation and re-methylation of a product, respectively. MecB functions as a cobalamin-dependent shuttle protein transferring the methyl group between MecE and MecC. This study provides the first biochemical evidence of the enzymes involved in the anaerobic metabolism of DCM.IMPORTANCEDichloromethane (DCM) is a priority regulated pollutant frequently detected in groundwater. In this work, we identify the proteins responsible for the transformation of DCM fermentation in Dehalobacterium formicoaceticum strain EZ94 using a combination of biochemical approaches, heterologous expression of proteins, and computational analysis. These findings provide the basis to apply these proteins as biological markers to monitor bioremediation processes in the field.

G-quadruplex forming regions in GCK and TM6SF2 are targets for differential DNA methylation in metabolic disease and hepatocellular carcinoma patients

The alarming increase in global rates of metabolic diseases (MetDs) and their association with cancer risk renders them a considerable burden on our society. The interplay of environmental and genetic factors in causing MetDs may be reflected in DNA methylation patterns, particularly at non-canonical (non-B) DNA structures, such as G-quadruplexes (G4s) or R-loops. To gain insight into the mechanisms of MetD progression, we focused on DNA methylation and functional analyses on intragenic regions of two MetD risk genes, the glucokinase (GCK) exon 7 and the transmembrane 6 superfamily 2 (TM6SF2) intron 2-exon 3 boundary, which harbor non-B DNA motifs for G4s and R-loops.Pyrosequencing of 148 blood samples from a nested cohort study revealed significant differential methylation in GCK and TM6SF2 in MetD patients versus healthy controls. Furthermore, these regions harbor hypervariable and differentially methylated CpGs also in hepatocellular carcinoma versus normal tissue samples from The Cancer Genome Atlas (TCGA). Permanganate/S1 nuclease footprinting with direct adapter ligation (PDAL-Seq), native polyacrylamide DNA gel electrophoresis and circular dichroism (CD) spectroscopy revealed the formation of G4 structures in these regions and demonstrated that their topology and stability is affected by DNA methylation. Detailed analyses including histone marks, chromatin conformation capture data, and luciferase reporter assays, highlighted the cell-type specific regulatory function of the target regions. Based on our analyses, we hypothesize that changes in DNA methylation lead to topological changes, especially in GCK exon 7, and cause the activation of alternative regulatory elements or potentially play a role in alternative splicing.Our analyses provide a new view on the mechanisms underlying the progression of MetDs and their link to hepatocellular carcinomas, unveiling non-B DNA structures as important key players already in early disease stages.

Formulation and Ex Vivo Evaluation of Membrane Permeation Properties of a Mixed Acetaminophen Gel for Rectal Administration

The purpose of this work was to evaluate the permeation of a mixed native starch-based gel of Ipomoea batatas (Convolvulaceae), using acetaminophen as a tracer through a rat rectal membrane (ex vivo method). The formulated gel was composed of a 10 g solution of poloxamer 407 at 20% and 2.5 g of glycerolized potato starch. The gel obtained was a smooth, homogeneous mixed gel with no foam, air bubbles or lumps, there was no characteristic odor, and the gel was whitish in color. This gel was characterized at the physicochemical and rheological level by means of the viscosimeter KINEXUS, pH- meter EUTECH and the study of the permeation was carried out by means of the Ussing chamber of horizontal type (the dual chamber). The formulated mixed gel is thermogelling, rheofluidifying and viscoelastic with a gelling temperature of 23.83; the permeation study gave a relatively low permeation percentage of 0.16% but which can be improved. The different viscoelastic, rheofluidizing and thermogelling characteristics contained in this mixed gel as well as the pH did not influence the permeation of the active ingredient (AP) through the rat rectal mucosa.

Characterization of the SARS-CoV-2 Genome 3'-Untranslated Region Interactions with Host MicroRNAs.

The 2019 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has marked the spread of a novel human coronavirus. SARS-CoV-2 has exhibited increased disease severity and immune evasion across its variants, and the molecular mechanisms behind these phenomena remain largely unknown. Conserved elements of the viral genome, such as secondary structures within the 3'-untranslated region (UTR), could prove crucial in furthering our understanding of the host-virus interface. Analysis of the SARS-CoV-2 viral genome 3'-UTR revealed the potential for host microRNA (miR) binding sites, allowing for sequence-specific interactions. In this study, we demonstrate that the SARS-CoV-2 genome 3'-UTR binds the host cellular miRs miR-34a-5p, miR-34b-5p, and miR-760-3p in vitro. Native gel electrophoresis and steady-state fluorescence spectroscopy were utilized to biophysically characterize the binding of these miRs to their predicted sites within the SARS-CoV-2 genome 3'-UTR. Additionally, we investigated 2'-fluoro-d-arabinonucleic acid (FANA) analogs as competitive binding inhibitors for these interactions. These miRs modulate the translation of granulin (GRN), interleukin-6 (IL-6), and the IL-6 receptor (IL-6R), all of which are key modulators and activators of JAK/STAT3 signaling and are implicated in regulation of the immune response. Thus, we propose that hijacking of these miRs by SARS-CoV-2 could identify a mechanism of host immune modulation by the virus. The mechanisms detailed in this study have the potential to drive the development of antiviral treatments for SARS-CoV-2, through direct targeting of the virus-host interface.

Effects of sodium dodecyl sulfate, Sarkosyl and sodium lauroyl glutamate on the structure of proteins monitored by agarose native gel electrophoresis and circular dichroism

We have studied binding properties of three detergents, i.e., sodium dodecyl sulfate (SDS), Sarkosyl and sodium lauroyl glutamate (SLG), to model proteins based on their effects on electrophoretic mobilities of the proteins using agarose native gel electrophoresis and circular dichroism (CD). This simple technology can evaluate the dissociative properties of bound detergents from the proteins and their effects on protein structure. SDS influenced the electrophoretic mobilities of all model proteins more strongly than the other two detergents, implying a stronger inclination for protein binding and subsequent alterations in protein structure or reductions in activity, which are supported by CD analysis. On the contrary, Sarkosyl and SLG showed weaker binding and interfered less with the structure and biological activities, indicating that these detergents may be useful for protein purification and analysis. It appeared that SLG was weaker in protein binding than Sarkosyl, although the effects of these two detergents appeared to depend on the proteins.

Polysaccharide as a Separation Medium for Gel Electrophoresis

Gel electrophoresis and size exclusion chromatography (SEC) are vital techniques in biochemical research, employing gel matrix structures made of polysaccharides or synthetic polymers like polyacrylamide for the analysis and separation of macromolecules. Polysaccharides, such as agarose, offer safer alternatives to acrylamide. Polysaccharide gels, notably agarose, facilitate the analysis and purification of proteins and nucleic acids through a molecular sieving mechanism. Gel electrophoresis for proteins is mainly divided into denaturing and native methods. Denaturing electrophoresis with sodium dodecyl sulfate (SDS) simplifies protein migration but disrupts molecular interactions. Conversely, native gel electrophoresis, without SDS, allows proteins to migrate based on the running pH and the isoelectric point of the proteins, while nucleic acids consistently migrate toward the anode. The electrophoresis of proteins with variable charges presents complexes. This review focuses on the use of polysaccharides, particularly agarose, for native gel electrophoresis, highlighting their applications in separating macromolecules. It also discusses the applications and limitations of agarose gels when used as a matrix for electrophoresis. Such information should help in designing electrophoresis experiments using polysaccharides.

Oligomerization of protein arginine methyltransferase 1 and its effect on methyltransferase activity and substrate specificity.

Proper protein arginine methylation by protein arginine methyltransferase 1 (PRMT1) is critical for maintaining cellular health, while dysregulation is often associated with disease. How the activity of PRMT1 is regulated is therefore paramount, but is not clearly understood. Several studies have observed higher order oligomeric species of PRMT1, but it is unclear if these exist at physiological concentrations and there is confusion in the literature about how oligomerization affects activity. We therefore sought to determine which oligomeric species of PRMT1 are physiologically relevant, and quantitatively correlate activity with specific oligomer forms. Through quantitative western blotting, we determined that concentrations of PRMT1 available in a variety of human cell lines are in the sub-micromolar to low micromolar range. Isothermal spectral shift binding data were modeled to a monomer/dimer/tetramer equilibrium with an EC50 for tetramer dissociation of ~20 nM. A combination of sedimentation velocity and Native polyacrylamide gel electrophoresis experiments directly confirmed that the major oligomeric species of PRMT1 at physiological concentrations would be dimers and tetramers. Surprisingly, the methyltransferase activity of a dimeric PRMT1 variant is similar to wild type, tetrameric PRMT1 with some purified substrates, but dimer and tetramer forms of PRMT1 show differences in catalytic efficiencies and substrate specificity for other substrates. Our results define an oligomerization paradigm for PRMT1, show that the biophysical characteristics of PRMT1 are poised to support a monomer/dimer/tetramer equilibrium in vivo, and suggest that the oligomeric state of PRMT1 could be used to regulate substrate specificity.

Structure, characterization, and application of a novel thermoreversible emulsion gel fabricated by citrate agar

A novel thermoreversible emulsion gel was successfully prepared with citrate agar (CA) as the sole emulsifier. Compared with native agar gel emulsion, CA gel emulsion (CAGE) formed a stable emulsion gel when the CA concentration was increased to 1.25 % (w/w). Results of time-temperature scanning experiments showed that the emulsion gel rapidly transformed into liquid emulsion when heated to 40–50 °C and then solidified into emulsion gel after cooling to the critical temperature of solidification. The emulsion gel had stable sol–gel transformation ability after seven cycles repeated heating–cooling treatment (HCT) at 85 °C and 4 °C. However, the stability of emulsion gels gradually decreased because of the large-droplet formation during heating, which affected the CA molecular-reconfiguration network structure in cooling. The conjunction analysis of microstructure and properties of the emulsion gel indicated that its stability depended primarily on the spatial repulsion and electrostatic repulsion provided by CA gel, and the main factor driving thermal reversibility was the temperature-responsive gelation performance of CA. The retention of quercetin was >90.23 % after seven HCTs because CAGEG enhanced the homogeneity and stability of the droplets.

What are the main proteins in the hemolymph of Haemaphysalis flava ticks?

Haemaphysalis flava is a notorious parasite for humans and animals worldwide. The organs of H. flava are bathed in hemolymph, which is a freely circulating fluid. Nutrients, immune factors, and waste can be transported to any part of the body via hemolymph. The main soluble components in hemolymph are proteins. However, knowledge of the H. flava proteome is limited. The hemolymph was collected from fully engorged H. flava ticks by leg amputation. Hemolymph proteins were examined by both blue native polyacrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulfate PAGE (SDS-PAGE). Proteins extracted from the gels were further identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two bands (380 and 520 kDa) were separated from tick hemolymph by BN-PAGE and were further separated into four bands (105, 120, 130, and 360 kDa) by SDS-PAGE. LC-MS/MS revealed that seven tick proteins and 13 host proteins were present in the four bands. These tick proteins mainly belonged to the vitellogenin (Vg) family and the α-macroglobulin family members. In silico structural analysis showed that these Vg family members all had common conserved domains, including the N-terminus lipid binding domain (LPD-N), the C-terminus von Willebrand type D domain (vWD), and the domain of unknown function (DUF). Additionally, two of the Vg family proteins were determined to belong to the carrier protein (CP) by analyzing the unique N-terminal amino acid sequences and the cleaving sites. These findings suggest that the Vg family proteins and α-macroglobulin are the primary constituents of the hemolymph in the form of protein complexes. Our results provide a valuable resource for further functional investigations of H. flava hemolymph effectors and may be useful in tick management.

Mettl3-m6A-YTHDF1 axis promotion of mitochondrial dysfunction in metabolic dysfunction–associated steatotic liver disease

BackgroundN6-methyladenosine (m6A) mRNA modification and mitochondrial function hold paramount importance in the advancement of metabolic dysfunction-associated steatotic liver disease (MASLD). AimThe aim of this study was to elucidate the impact of m6A on hepatic mitochondrial dysfunction and provide a novel perspective for a more comprehensive understanding of the pathogenesis of MASLD. MethodsHigh-throughput screening methods were used to identify the underlying transcriptome and proteome changes in MASLD model mice. Western blotting, blue native gel electrophoresis (BNGE), dot blot, and Seahorse analyses were conducted to identify and validate the underlying regulatory mechanisms of m6A on mitochondria. ResultsIn vivo, abnormal m6A modification in MASLD was attributed to the upregulation of methyltransferase like 3 (Mettl3) and the downregulation of YTH N6-methyladenosine RNA binding protein 1 (YTHDF1) induced by high-fat foods. In vitro, knockdown of Mettl3 inhibited hepatic oxidative phosphorylation (OXPHOS) and the mitochondrial respiratory chain (MRC), while overexpression of Mettl3 promoted these processes. However, knockout of the reader protein YTHDF1, which plays a crucial role in the m6A modification process, counteracted the effect of Mettl3 and suppressed mitochondrial OXPHOS. ConclusionsIn MASLD, damage to the MRC may be regulated by the Mettl3-m6A-YTHDF1 axis, particularly by the role of YTHDF1. Modulation of the Mettl3-m6A-YTHDF1 axis has the potential to improve mitochondrial function, alleviate MASLD symptoms, and decrease the likelihood of disease progression.

Mitoregulin self-associates to form likely homo-oligomeric pore-like structures.

We and others previously found that a misannotated long noncoding RNA encodes for a conserved mitochondrial transmembrane microprotein named Mitoregulin (Mtln). Beyond an established role for Mtln in lipid metabolism, Mtln has also been shown to more broadly influence mitochondria, boosting respiratory efficiency and Ca 2+ retention capacity, while lowering ROS, yet the underlying mechanisms remain unresolved. Prior studies have identified possible Mtln protein interaction partners; however, a lack of consensus persists, and no claims have been made about Mtln's structure. We previously noted two key published observations that seemingly remained overlooked: 1) endogenous Mtln co-immunoprecipitates with epitope-tagged Mtln at high efficiency, and 2) Mtln primarily exists in a ∼66 kDa complex. To investigate if Mtln may self-oligomerize into higher-order complexes, we performed co-immunoprecipitation, protein modeling simulations, and native gel assessments of Mtln-containing complexes in cells and tissues, as well as tested whether synthetic Mtln protein itself forms oligomeric complexes. Our combined results provide strong support that Mtln self-associates and likely forms a hexameric pore-like structure.

Conjugation with the Carrier Helped to Reveal acidification-Induced Structural Shift in the Peptide from Phospholipase Domain of Parvovirus B19.

Spectroscopic studies on domains and peptides of large proteins are complicated because of the tendency of short peptides to form oligomers in aquatic buffers, but conjugation of a peptide with a carrier protein may be helpful. In this study we approved that a fragment of SK30 peptide from phospholipase A2 domain of VP1 Parvovirus B19 capsid protein (residues: 144-159; 164; 171-183; sequence: SAVDSAARIHDFRYSQLAKLGINPYTHWTVADEELLKNIK) turns from random coil to alpha helix in the acidic medium only in case if it had been conjugated with BSA (through additional N-terminal Cys residue, turning it into CSK31 peptide, and SMCC linker) according to CD-spectroscopy results. In contrast, unconjugated SK30 peptide does not undergo such shift because it forms stable oligomers connected by intermolecular antiparallel beta sheet, according to IR-spectroscopy, CD-spectroscopy, blue native gel electrophoresis and centrifugal ultrafiltration, as, probably, the whole isolated phospholipase domain of VP1 protein does. However, being a part of the long VP1 capsid protein, phospholipase domain may change its fold during the acidification of the medium in the endolysosome by the way of the formation of contacts between protonated His153 and Asp175, promoting the shift from random coil to alpha helix in its N-terminal part. This study opens up a perspective of vaccine development, since rabbit polyclonal antibodies against the conjugate of CSK31 peptide with BSA, in which the structure of the second alpha helix from the phospholipase A2 domain should be reproduced, can bind epitopes of the complete recombinant unique part of VP1 Parvovirus B19 capsid (residues: 1-227).

Accelerated Screening of Alternative DNA Base‐Organic Molecule‐Base Architectures via Integrated Theory and Experiment

AbstractOrganic molecule‐mediated noncanonical DNA self‐assembly expands the standard DNA base‐pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base‐pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub‐stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule‐incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA‐molecule architectures beyond Watson–Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA‐based delivery system and probe design for sensitive detection of certain molecules.

Accelerated Screening of Alternative DNA Base-Organic Molecule-Base Architectures via Integrated Theory and Experiment.

Organic molecule-mediated noncanonical DNA self-assembly expands the standard DNA base-pairing alphabets. However, only a very limited number of small molecules have been recognized as mediators because of the tedious and complicated experiments like crystallization and microscopy imaging. Here we present an integrative screening protocol incorporating molecular dynamics (MD) for fast theoretical simulation and native polyacrylamide gel electrophoresis for convenient experimental validation. Melamine, the molecule that was confirmed mediating noncanonical DNA base-pairing, and 38 other candidate molecules were applied to demonstrate the feasibility of this protocol. We successfully identified seven stable noncanonical DNA duplex structures, and another eight novel structures with sub-stability. In addition, we discovered that hairpins at both ends can significantly stabilize the noncanonical DNA structures, providing a guideline to design small organic molecule-incorporated DNA structures. Such an efficient screening protocol will accelerate the design of alternative DNA-molecule architectures beyond Watson-Crick pairs. Considering the wide range of potential mediators, it will also facilitate applications such as noncovalent, highly dense loading of drug molecules in DNA-based delivery system and probe design for sensitive detection of certain molecules.

Enzymatic Dissociation of DNA-Histone Condensates in an Electrophoretic Setting: Modulating DNA Patterning and Hydrogel Viscoelasticity.

Development of an energy-driven self-assembly process is a matter of interest for understanding and mimicking diverse ranges of biological and environmental patterns in a synthetic system. In this article, first we demonstrate transient and temporally controlled self-assembly of a DNA-histone condensate where trypsin (already present in the system) hydrolyzes histone, resulting in disassembly. Upon performing this dynamic self-assembly process in a gel matrix under an electric field, we observe diverse kinds of DNA patterning across the gel matrix depending on the amount of trypsin, incubation time of the reaction mixture, and gel porosity. Notably, here, the micrometer-sized DNA-histone condensate does not move through the gel and only free DNA can pass; therefore, transport and accumulation of DNA at different zones depend on the release rate of DNA by trypsin. Furthermore, we show that the viscoelasticity of the native gel increases in the presence of DNA and a pattern over gel viscoelasticity at different zones can be achieved by tuning the amount of enzyme, i.e., the dissociation rate of the DNA-histone condensate. We believe enabling spatiotemporally controlled DNA patterning by applying an electric field will be potentially important in designing different kinds of spatiotemporally distinct dynamic materials.

Impact of Deformability and Rigidity of Starch Granules on Linear and Non-Linear Rheological Behavior of Waxy Rice Starch Gels and Applicability for Food End Uses.

The objective of this study was to investigate granule size and distribution and deformability of granules and their effect on the rheological properties of waxy starch gels. Native (granular) waxy rice gels (10%) were prepared, and their response in oscillatory shear was investigated in the linear and non-linear viscoelastic regime. The results show the gels were mainly composed of aggregated and deformed swollen granules. Significance of granule size and its distribution, deformability of granules, and the molecular characteristics of amylopectin (AP) on storage modulus of those gels was demonstrated. A low degree of deformability of granules, typical for small granules with a broad size distribution and small molecular size of AP with short external chains, resulted in rigid and brittle gels. Highly deformed granules and high AP leachates, however, yielded soft gels. It was found that the transition of elastic to plastic behavior in the non-linear regime (LAOS) was gradual when AP had long external chains, but an abrupt transition was observed with the gel with short exterior chains of AP. Differences in rheological properties of cohesive waxy starch gels appear to be mainly impacted by the varying degrees of granule deformability and rigidity, which is further attributed to a combination of factors, including granule size, particle size distribution, molecular size, the external chain length of amylopectin (AP), and lipid content. The significance of this study is that it will assist the food industry in selecting suitable waxy rice starches to gain desired textural properties of end products.