Oligonucleotide Hybridization Research Articles

Rational design of metal-responsive functional DNA supramolecules

AbstractDNA molecules have excellent molecular recognition abilities through the complementary hydrogen-bonded base pairing. Since the hybridization of oligonucleotides can be programmed based on the sequences of the nucleobases, a great number of DNA supramolecular architectures have been constructed via self-assembly processes. The development of stimuli-responsive DNA supramolecules has attracted increasing interests because it will contribute to the construction of dynamic molecular systems such as molecular machines. Metal ions are considered as useful chemical stimuli, but the construction of metal-responsive DNA systems is still in the early stage. This review article describes current progress on the development of DNA supramolecules whose structure and function can be regulated in response to metal ions, with mainly focusing on our recent studies. The basic strategy is the introduction of unnatural metal ligands that form interstrand metal complexes in DNA structures. For example, artificial metal-mediated base pairs, formed through complexation between ligand-type nucleobase analogs and a bridging metal ion, were incorporated into known DNAzymes (catalytic DNA) to allosterically regulate their activity in a metal-responsive manner. Novel ligand-type nucleobases that form both metal-mediated and hydrogen-bonded base pairs have been recently devised as metal-responsive building blocks, and were used to construct a simple prototype of DNA molecular machines. Branched DNA structures bearing metal ligands at the junction core were also synthesized as novel structural motifs, with which metal-mediated structure transformation was demonstrated. These metal-responsive DNA supramolecules are expected to expand the toolbox of DNA-based supramolecular chemistry and nanotechnology.

Transient states during the annealing of mismatched and bulged oligonucleotides.

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 versus 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.

DNA-Nanostructure-Guided Assembly of Proteins into Programmable Shapes.

The development of methods to synthesize artificial protein complexes with precisely controlled configurations will enable diverse biological and medical applications. Using DNA to link proteins provides programmability that can be difficult to achieve with other methods. Here, we use DNA origami as an "assembler" to guide the linking of protein-DNA conjugates using a series of oligonucleotide hybridization and displacement operations. We constructed several isomeric protein nanostructures, including a dimer, two types of trimer structures, and three types of tetramer assemblies, on a DNA origami platform by using a C3-symmetric building block composed of a protein trimer modified with DNA handles. Our approach expands the scope for the precise assembly of protein-based nanostructures and will enable the formulation of functional protein complexes with stoichiometric and geometric control.

Kinetic Referencing Allows Identification of Epigenetic Cytosine Modifications by Single-Molecule Hybridization Kinetics and Superresolution DNA-PAINT Microscopy.

We develop a DNA origami-based internal kinetic referencing system with a colocalized reference and target molecule to provide increased sensitivity and robustness for transient binding kinetics. To showcase this, we investigate the subtle changes in binding strength of DNA oligonucleotide hybrids induced by cytosine modifications. These cytosine modifications, especially 5-methylcytosine but also its oxidized derivatives, have been increasingly studied in the context of epigenetics. Recently revealed correlations of epigenetic modifications and disease also render them interesting biomarkers for early diagnosis. Internal kinetic referencing allows us to probe and compare the influence of the different epigenetic cytosine modifications on the strengths of 7-nucleotide long DNA hybrids with one or two modified nucleotides by single-molecule imaging of their transient binding, revealing subtle differences in binding times. Interestingly, the influence of epigenetic modifications depends on their position in the DNA strand, and in the case of two modifications, effects are additive. The sensitivity of the assay indicates its potential for the direct detection of epigenetic disease markers.

Identification and functional characterization of a novel aldo-keto reductase from Aloe vera.

The present investigation profoundly asserted the catalytic potential of plant-based aldo-ketoreductase, postulating its role in polyketide biosynthesis and providing new insights for tailored biosynthesis of vital plant polyketides for therapeutics. Plants hold great potential as a future source of innovative biocatalysts, expanding the possibilities within chemical reactions and generating a variety of benefits. The aldo-keto reductase (AKR) superfamily includes a huge collection of NAD(P)H-dependent oxidoreductases that carry out a variety of redox reactions essential for biosynthesis, detoxification, and intermediary metabolism. The present study involved the isolation, cloning, and purification of a novel aldo-ketoreductase (AvAKR) from the leaves of Aloe vera (Aloe barbadensis Miller) by heterologous gene expression in Escherichia coli based on the unigene sequences of putative ketoreductase and cDNA library screening by oligonucleotide hybridization. The in-silico structural analysis, phylogenetic relationship, and molecular modeling were outranged to approach the novelty of the sequence. Additionally, agroinfiltration of the candidate gene tagged with a green fluorescent protein (GFP) was employed for transient expression in the Nicotiana benthamiana to evaluate the sub-cellular localization of the candidate gene. The AvAKR preferred cytoplasmic localization and shared similarities with the known plant AKRs, keeping the majority of the conserved active-site residues in the AKR superfamily enzymes. The enzyme facilitated the NADPH-dependent reduction of various carbonyl substrates, including benzaldehyde and sugars, proclaiming a broad spectrum range. Our study successfully isolated and characterized a novel aldo-ketoreductase (AvAKR) from Aloe vera, highlighting its versatile NADPH-dependent carbonyl reduction proficiency therewith showcasing its potential as a versatile biocatalyst in diverse redox reactions.

Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction

BackgroundDirect cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses.MethodsCardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry.ResultsWe found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair.ConclusionsFibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential.Graphical

Cost-effective complete genome sequencing using the MinION platform for identification of recombinant enteroviruses.

Enteroviruses (EVs) are a group of viruses that cause various human illnesses. While the CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primer) method can generate VP1 gene fragments for enterovirus genotyping, it is unable to detect recombinant strains. Recent advances in viral genome sequencing using next-generation sequencing technologies have enabled comprehensive analyses. However, the high cost poses a challenge for widespread adoption. To address this issue, this study proposes a cost-effective approach for generating complete enterovirus genome sequences using the Oxford Nanopore MinION sequencer. This protocol not only facilitates the generation of accurate genome sequences for various enterovirus strains but also allows for the differentiation of co-infections from viral isolates. In addition, the method can generate polyprotein sequences as well as peptide sequences of the upstream ORF (uORF) whose expression can impact virus infection. Through the analysis of complete enterovirus genomes, this study successfully identified seven enterovirus A71 isolates obtained during the 2018 enterovirus outbreak in Malaysia and Taiwan as recombinants between enterovirus A71 and coxsackievirus A2. Furthermore, our study has made a significant discovery by establishing a strong correlation between uORF trees and the epidemics of EVA71. This finding highlights the potential of uORF sequences as valuable indicators for monitoring and understanding the spread of EVA71 infections. We also identified notable amino acid changes in the transmembrane domain of the uORF protein within a newly identified lineage. These findings provide crucial insights into the molecular characteristics and evolutionary dynamics of EVA71, offering valuable information for future research and intervention strategies. IMPORTANCE By employing a cost-effective approach for complete genome sequencing, the study has enabled the identification of novel enterovirus strains and shed light on the genetic exchange events during outbreaks. The success rate of genome sequencing and the scalability of the protocol demonstrate its practical utility for routine enterovirus surveillance. Moreover, the study's findings of recombinant strains of EVA71 and CVA2 contributing to epidemics in Malaysia and Taiwan emphasize the need for accurate detection and characterization of enteroviruses. The investigation of the whole genome and upstream ORF sequences has provided insights into the evolution and spread of enterovirus subgenogroups. These findings have important implications for the prevention, control, and surveillance of enteroviruses, ultimately contributing to the understanding and management of enterovirus-related illnesses.

Understanding Oligonucleotide Hybridization and the Role of Anchoring on the Single-Walled Carbon Nanotube Corona Phase for Viral Sensing Applications

Semiconducting single-walled carbon nanotubes (SWCNTs) with tailored corona phases (CPs), or surface adsorbed molecules, have emerged as a promising interface for sensing applications. The adsorption of an analyte can be specifically transduced as a modulation of their band gap near-infrared (nIR) photoluminescence (PL). One such CP ideal for this purpose is single-stranded DNA (ssDNA), where subsequent sequence dependent hybridization can result in PL emission wavelength shifts. Due to ssDNA adsorption to the SWCNT surface, the resultant non-canonical hybridization and its effect on SWCNT photophysical properties are not well understood. In this work, we study 20- and 21-mer DNA and RNA hybridization on the complementary ssDNA-SWCNT CP in the context of nucleic acid sensing for SARS-CoV-2 sequences as model analytes. We found that the van’t Hoff transition enthalpy of hybridization on SWCNT CP was -11.9 kJ mol-1, much lower than that of hybridization in solution (-707 kJ mol-1). We used SWCNT solvatochromism to calculate the solvent exposed surface area to indicate successful hybridization. We found that having a 30-mer anchor region in addition to the complementary region significantly improved PL response sensitivity and selectivity, with (GT)15 anchor preferred for RNA targets. Coincubation of ssDNA-SWCNT with analyte at 37°C resulted in faster hybridization kinetics without sacrificing specificity. Other methods aimed to improve CP rearrangement kinetics such as bath sonication and surfactant additions were ineffective. We also determined that target sequence choice is important as secondary structure formation in target is negatively correlated with hybridization. Best performing CPs showed detection limits of 11 nM and 13 nM for DNA and RNA targets respectively. Finally, we simulated sensing conditions using saliva environment, showing sensor compatibility in biofluids. In total, this work elucidates key design features and processing to enable sequence specific hybridization on ssDNA-SWCNT CP.

Biochemical and Phylogenetic Analysis of Italian Phaseolus vulgaris Cultivars as Sources of α-Amylase and α-Glucosidase Inhibitors.

Phaseolus vulgaris α-amylase inhibitor (α-AI) is a protein that has recently gained commercial interest, as it inhibits mammalian α-amylase activity, reducing the absorption of dietary carbohydrates. Numerous studies have reported the efficacy of preparations based on this protein on the control of glycaemic peaks in type-2 diabetes patients and in overweight subjects. A positive influence on microbiota regulation has also been described. In this work, ten insufficiently studied Italian P. vulgaris cultivars were screened for α-amylase- and α-glucosidase-inhibiting activity, as well as for the absence of antinutritional compounds, such as phytohemagglutinin (PHA). All the cultivars presented α-glucosidase-inhibitor activity, while α-AI was missing in two of them. Only the Nieddone cultivar (ACC177) had no haemagglutination activity. In addition, the partial nucleotide sequence of the α-AI gene was identified with the degenerate hybrid oligonucleotide primer (CODEHOP) strategy to identify genetic variability, possibly linked to functional α-AI differences, expression of the α-AI gene, and phylogenetic relationships. Molecular studies showed that α-AI was expressed in all the cultivars, and a close similarity between the Pisu Grogu and Fasolu cultivars' α-AI and α-AI-4 isoform emerged from the comparison of the partially reconstructed primary structures. Moreover, mechanistic models revealed the interaction network that connects α-AI with the α-amylase enzyme characterized by two interaction hotspots (Asp38 and Tyr186), providing some insights for the analysis of the α-AI primary structure from the different cultivars, particularly regarding the structure-activity relationship. This study can broaden the knowledge about this class of proteins, fuelling the valorisation of Italian agronomic biodiversity through the development of commercial preparations from legume cultivars.

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization.

Chemical cell surface modification is a fast-growing field of research, due to its enormous potential in tissue engineering, cell-based immunotherapy, and regenerative medicine. However, engineering of bacterial tissues by chemical cell surface modification has been vastly underexplored and the identification of suitable molecular handles is in dire need. We present here, an orthogonal nucleic acid-protein conjugation strategy to promote artificial bacterial aggregation. This system gathers the high selectivity and stability of linkage to a protein Tag expressed at the cell surface and the modularity and reversibility of aggregation due to oligonucleotide hybridization. For the first time, XNA (xeno nucleic acids in the form of 1,5-anhydrohexitol nucleic acids) were immobilized via covalent, SNAP-tag-mediated interactions on cell surfaces to induce bacterial aggregation.

OligoHydrogelArray (OHA) for Parallelized Solid‐Phase Extraction of Oligonucleotides

AbstractImmobilization of oligonucleotides on solid surfaces is an important step in many experimental workflows in biology, such as gene expression analysis, genotyping, and drug discoveries. Capturing oligonucleotides in a highly efficient miniaturized format is still challenging. In this work, preparation of functionalized miniaturized acrylamide hydrogels on a droplet microarray (DMA) chip is reported. By further modification of these hydrogels through the Cu‐catalyzed alkyne‐azide cycloaddition, oligonucleotides are attached covalently, creating the OligoHydrogelArray (OHA). OHA enables the hybridization of oligonucleotides with defined sequences in a parallelized, miniaturized, and high‐throughput manner with an overall loading capacity and extraction efficiency suitable for most biological and chemical applications. This system enables the parallelized solid‐phase extraction of target oligonucleotides out of a mixture for purification and enrichment of DNA/RNA/PNA for application in fields such as transcriptomics. In the current study, are establish the chemical procedure for reproducible preparation of OHA and determine key characteristics, such as height, diameter, and shape of miniaturized gel pads as well as the loading capacity of oligonucleotides within the material. Finally, the ability to extract complementary oligonucleotide strands out of solution through hybridization is demonstrated to set the course for future applications.

Synthetic host-guest pairs as novel bioorthogonal tools for pre-targeting

Due to its simplicity, high efficiency, and chemo-selectivity, bioorthogonal chemistry has shown a great application potential in pre-targeting. Currently, four bioorthogonal pairs as targeting tools, including (strept)avidin/biotin, antibody/antigen, oligonucleotide hybridization and IEDDA tools, have been developed and applied in targeted delivery. Nevertheless, all of these tools still suffer from some limitations, such as difficult modification, biochemical fragility and larger molecular weight for biological association tools, as well as chemical instability for IEDDA tools. Synthetic host-guest pairs with relatively small molecular sizes not only possess strong chemical stability, but also have the features of fast conjugation rate, tunable binding affinity, easy modification, and high chemo-selectivity. Consequently, they can be used as a novel non-covalent bioorthogonal tool for pre-targeting. In order to further promote the development of host-guest pairs as novel bioorthogonal tools for pre-targeted delivery, we firstly calculate their conversion rate to make researcher aware of their unique advantages; next, we summarize the recent research progress in this area. The future perspectives and limitations of these unique tools will be discussed. This review will provide a systemic overview of the development of synthetic host-guest pairs as novel bioorthogonal tools for pre-targeting, and may serve as a “go for” resort for researchers who are interested in searching for new synthetic tools to improve pre-targeting.

Analysis of genotyping features of bovine cattle individuals at the CSN2 locus using ACRS-PCR methods

In the context of solving the problem of obtaining high quality dairy products from livestock, the issue of determining the type of beta-casein (A1 and A2) in the protein fraction of milk becomes essential. Purpose – to analyse the use of ACRS-PCR methods for differentiation of A1 and A2 alleles of bovine beta-casein locus. Genotyping features were analysed using the artificially created restriction site polymerase chain reaction method utilising TaqI and DdeI restriction endonucleases. The electrophoretic distribution of DNA fragments in agarose gels of various concentrations was used to analyse restriction patterns. Based on the results of bioinformatic analysis of the nucleotide reference sequences of the experimental fragment of the beta-casein gene, it was found that the primer system for the ACRS-PCR DdeI method is characterised by higher parameters of flanking efficiency of the target DNA site compared to the ACRS-PCR TaqI system due to significantly greater effectiveness of hybridisation of oligonucleotides on the target DNA. Based on the results of laboratory tests of both methods, it is proposed to use an additional procedure for analysing the fluorescence intensity of individual elements of restriction patterns, which allows reducing the number of false genotyping that occurs in both cases (based on the results of using both methods) due to the appearance of non-specific amplification/restriction fragments within the size of target restrictions. The application of the ACRS-PCR DdeI method provides more differentiated patterns of the corresponding genotypes in agarose gel compared to the ACRS-PCR TaqI method, but leads to higher material costs for conducting research. These disadvantages of using primer systems for ACRS-PCR of the beta-casein locus determine the relevance of developing alternative methods for typing A1 and A2 alleles which include allele-specific PCR. The use of results is promising for solving the problems of genotyping cattle individuals of different breeds by A1 and A2 alleles of the beta-casein locus

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.

Understanding Oligonucleotide Hybridization and the Role of Anchoring on the Single-Walled Carbon Nanotube Corona Phase for Viral Sensing Applications

Semiconducting single-walled carbon nanotubes (SWCNTs) with tailored corona phases (CPs), or surface-adsorbed molecules, have emerged as a promising interface for sensing applications. The adsorption of an analyte can be specifically transduced as a modulation of their band-gap near-infrared (nIR) photoluminescence (PL). One such CP ideal for this purpose is single-stranded DNA (ssDNA), where subsequent sequence-dependent hybridization can result in PL emission wavelength shifts. Due to ssDNA adsorption to the SWCNT surface, the resultant noncanonical hybridization and its effect on SWCNT photophysical properties are not well understood. In this work, we study 20- and 21-mer DNA and RNA hybridization on the complementary ssDNA-SWCNT CP in the context of nucleic acid sensing for SARS-CoV-2 sequences as model analytes. We found that the van’t Hoff transition enthalpy of hybridization on SWCNT CP was −11.9 kJ mol–1, much lower than that of hybridization in solution (−707 kJ mol–1). We used SWCNT solvatochromism to calculate the solvent-exposed surface area to indicate successful hybridization. We found that having a 30-mer anchor region in addition to the complementary region significantly improved PL response sensitivity and selectivity, with a (GT)15 anchor preferred for RNA targets. Coincubation of ssDNA-SWCNTs with an analyte at 37 °C resulted in faster hybridization kinetics without sacrificing specificity. Other methods aimed to improve CP rearrangement kinetics such as bath sonication and surfactant additions were ineffective. We also determined that the target sequence choice is important as secondary structure formation in the target is negatively correlated with hybridization. Best-performing CPs showed detection limits of 11 and 13 nM for DNA and RNA targets, respectively. Finally, we simulated sensing conditions using the saliva environment, showing sensor compatibility in biofluids. In total, this work elucidates key design features and processing to enable sequence-specific hybridization on ssDNA-SWCNT CPs.

Evaluation of Thermal Stability of DNA Oligonucleotide Structures Embedded in Hydrogels

Understanding the self-assembly and hybridization properties of DNA oligonucleotides in confined spaces can help to improve their applications in biotechnology and nanotechnology. This study investigates the effects of spatial confinement in the pores of hydrogels on the thermal stability of DNA oligonucleotide structures. The preparation of oligonucleotides embedded in agarose gels was simple, whereas the preparation of oligonucleotides embedded in polyacrylamide gels was required to remove unpolymerized monomers. In the latter case, a method for rehydrating a washed dry gel with a buffer solution containing oligonucleotides was developed. Fluorescence measurements of oligonucleotides bearing fluorescent probes revealed no significant influence of the internal environment of the gel pores on the stability of DNA duplex, hairpin, and G-quadruplex structures. Moreover, the effects of poly(ethylene glycol) on the stability of DNA structures in the gels were similar to those in solutions. It is likely that the oligonucleotides are not strongly constrained in the gels and may be preferentially located in a water-rich environment in the gel matrix. The gel preparation was also applied to the assessment of the stability of DNA structures under the conditions of a reduced number of water molecules. The studies using hydrogels provide insights into the ability of self-assembly and hybridization of oligonucleotides in confined environments and under low-water-content conditions.

Detection of SARS-CoV-2 Virus by Triplex Enhanced Nucleic Acid Detection Assay (TENADA)

SARS-CoV-2, a positive-strand RNA virus has caused devastating effects. The standard method for COVID diagnosis is based on polymerase chain reaction (PCR). The method needs expensive reagents and equipment and well-trained personnel and takes a few hours to be completed. The search for faster solutions has led to the development of immunological assays based on antibodies that recognize the viral proteins that are faster and do not require any special equipment. Here, we explore an innovative analytical approach based on the sandwich oligonucleotide hybridization which can be adapted to several biosensing devices including thermal lateral flow and electrochemical devices, as well as fluorescent microarrays. Polypurine reverse-Hoogsteen hairpins (PPRHs) oligonucleotides that form high-affinity triplexes with the polypyrimidine target sequences are used for the efficient capture of the viral genome. Then, a second labeled oligonucleotide is used to detect the formation of a trimolecular complex in a similar way to antigen tests. The reached limit of detection is around 0.01 nM (a few femtomoles) without the use of any amplification steps. The triplex enhanced nucleic acid detection assay (TENADA) can be readily adapted for the detection of any pathogen requiring only the knowledge of the pathogen genome sequence.

Ensemble inequivalence and negative extensibility in a strongly stretched wormlike chain with fluctuating bending stiffness.

Many semiflexible polymers exhibit fluctuations in the local bending stiffness along their contour. This may be due to intrinsic conformational changes (e.g., denaturation bubble formation in double stranded DNA or helix-coil transition in polypeptides) or the reversible adsorption and desorption of molecules from the polymer's environment (e.g., DNA-protein interactions or hybridization of oligonucleotides). In this article, we analyze the tensile elasticity of a strongly stretched wormlike chain, which consists of N concatenated segments, where each segment can be in one of two states, A or B, which differ in bending stiffness. We call this model the reversible wormlike chain (rWLC) model. In the Gibbs (fixed-force, isotensional) ensemble, we obtain analytic expressions for the force-extension relation and the mean fraction of B segments. We show that, under certain conditions, there is a tension-induced crossover from a mostly A to a mostly B rWLC. In the Helmholtz (fixed-extension, isometric) ensemble, we obtain analytic expressions up to a summation. We show that, for finite N, there is marked ensemble inequivalence. Remarkably, in the Helmholtz ensemble, the rWLC can exhibit negative extensibility and multiple peaks.

Structural Characteristics of the 5'-Terminal Region of Mouse p53 mRNA and Identification of Proteins That Bind to This mRNA Region.

A mouse model has often been used in studies of p53 gene expression. Detailed interpretation of functional studies is, however, hampered by insufficient knowledge of the impact of mouse p53 mRNA’s structure and its interactions with proteins in the translation process. In particular, the 5′-terminal region of mouse p53 mRNA is an important region which takes part in the regulation of the synthesis of p53 protein and its N-truncated isoform Δ41p53. In this work, the spatial folding of the 5′-terminal region of mouse p53 mRNA and its selected sub-fragments was proposed based on the results of the SAXS method and the RNAComposer program. Subsequently, RNA-assisted affinity chromatography was used to identify proteins present in mouse fibroblast cell lysates that are able to bind the RNA oligomer, which corresponds to the 5′-terminal region of mouse p53 mRNA. Possible sites to which the selected, identified proteins can bind were proposed. Interestingly, most of these binding sites coincide with the sites determined as accessible to hybridization of complementary oligonucleotides. Finally, the high binding affinity of hnRNP K and PCBP2 to the 5′-terminal region of mouse p53 mRNA was confirmed and their possible binding sites were proposed.

Redesign of a rapid, low-cost HPV typing assay to support risk-based cervical screening and management.

Accelerated cervical cancer control will require widespread human papillomavirus (HPV) vaccination and screening. For screening, sensitive HPV testing with an option of self‐collection is increasingly desirable. HPV typing predicts risk of precancer/cancer, which could be useful in management, but most current typing assays are expensive and/or complicated. An existing 15‐type isothermal amplification assay (AmpFire, Atila Biosystems, USA) was redesigned as a 13‐type assay (ScreenFire) for public health use. The redesigned assay groups HPV types into four channels with differential cervical cancer risk: (a) HPV16, (b) HPV18/45, (c) HPV31/33/35/52/58 and (d) HPV39/51/56/59/68. Since the assay will be most useful in resource‐limited settings, we chose a stratified random sample of 453 provider‐collected samples from a population‐based screening study in rural Nigeria that had been initially tested with MY09‐MY11‐based PCR with oligonucleotide hybridization genotyping. Frozen residual specimens were masked and retested at Atila Biosystems. Agreement on positivity between ScreenFire and prior PCR testing was very high for each of the channels. When we simulated intended use, that is, a hierarchical result in order of clinical importance of the type groups (HPV16 > 18/45 > 31/33/35/52/58 > 39/51/56/59/68), the weighted kappa for ScreenFire vs PCR was 0.90 (95% CI: 0.86‐0.93). The ScreenFire assay is mobile, relatively simple, rapid (results within 20‐60 minutes) and agrees well with reference testing particularly for the HPV types of greatest carcinogenic risk. If confirmed, ScreenFire or similar isothermal amplification assays could be useful as part of risk‐based screening and management.