Polymer mechanochemistry utilizes mechanical force to activate latent functionalities in macromolecules and widely relies on ultrasonication techniques. Fundamental constraints of frequency and power intensity have prohibited the application of the polymer mechanochemistry principles in a biomedical context up to now, although medical ultrasound is a clinically established modality. Here, a universal polynucleotide framework is presented that allows the binding and release of therapeutic oligonucleotides, both DNA- and RNA-based, as cargo by biocompatible medical imaging ultrasound. It is shown that the high molar mass, colloidal assembly, and a distinct mechanochemical mechanism enable the force-induced release of cargo and subsequent activation of biological function in vitro and in vivo. Thereby, this work introduces a platform for the exploration of biological questions and therapeutics development steered by mechanical force.

Monitoring the site-specific local structure and dynamics of polynucleotides and DNA is important for understanding their biological functions. However, structurally characterizing these biomolecules with high time resolution has been known to be experimentally challenging. In this work, several 5-silylethynyl-2\'-deoxynucleosides and 5-substituted phenylethynyl-2\'-deoxynucleosides on the basis of deoxycytidine (dC) and deoxythymidine (dT) were synthesized, in which the alkynyl group shows intensified CC stretching vibration with infrared transition dipole moment magnitude close to that of typical CO stretching, and exhibits structural sensitivities in both vibrational frequency and spectral width. In particular, 5-trimethylsilylethynyl-2\'-dC (TMSEdC, molecule 1a) was examined in detail using femtosecond nonlinear IR spectroscopy. The solvent dependent CC stretching frequency of 1a can be reasonably interpreted mainly as the hydrogen-bonding effect between the solvent and cytosine base ring structure. Transient 2D IR and pump-probe IR measurements of 1a carried out comparatively in two aprotic solvents (DMSO and THF) and one protic solvent (MeOH) further reveal solvent dependent ultrafast vibrational properties, including diagonal anharmonicity, spectral diffusion, vibrational relaxation and anisotropy dynamics. These observed sensitivities are rooted in an extended π-conjugation of the base ring structure in which the CC group is actively involved. Our results show that the intensified CC stretching vibration can potentially provide a site-specific IR probe for monitoring the equilibrium and ultrafast structural dynamics of polynucleotides.

A study of the plant Arabidopsis thaliana detected lower mutation rates in genomic regions where mutations are more likely to be deleterious, challenging the principle that mutagenesis is blind to its consequence. To examine the generality of this finding, we analyze large mutational data from baker\'s yeast and humans. The yeast data do not exhibit this trend, whereas the human data show an opposite trend that disappears upon the control of potential confounders. We find that the Arabidopsis study identified substantially more mutations than reported in the original data-generating studies and expected from Arabidopsis\' mutation rate. These extra mutations are enriched in polynucleotide tracts and have relatively low sequencing qualities so are likely sequencing errors. Furthermore, the polynucleotide \"mutations\" can produce the purported mutational trend in Arabidopsis. Together, our results do not support lower mutagenesis of genomic regions of stronger selective constraints in the plant, fungal, and animal models examined.

In Serratia marcescens JNB5-1, prodigiosin was highly produced at 30°C, but it was noticeably repressed at ≥37°C. Our initial results demonstrated that both the production and the stability of the O-methyl transferase (PigF) and oxidoreductase (PigN) involved in the prodigiosin pathway in S. marcescens JNB5-1 sharply decreased at ≥37°C. Therefore, in this study, we improved mRNA stability and protein production using de novo polynucleotide fragments (PNFs) and the introduction of disulfide bonds, respectively, and observed their effects on prodigiosin production. Our results demonstrate that adding PNFs at the 3\' untranslated regions of pigF and pigN significantly improved the mRNA half-lives of these genes, leading to an increase in the transcript and expression levels. Subsequently, the introduction of disulfide bonds in pigF improved the thermal stability, pH stability, and copper ion resistance of PigF. Finally, shake flask fermentation showed that the prodigiosin titer with the engineered S. marcescens was increased by 61.38% from 5.36 to 8.65 g/liter compared to the JNB5-1 strain at 30°C and, significantly, the prodigiosin yield increased 2.05-fold from 0.38 to 0.78 g/liter at 37°C. In this study, we revealed that the introduction of PNFs and disulfide bonds greatly improved the expression and stability of pigF and pigN, hence efficiently enhancing prodigiosin production with S. marcescens at 30 and 37°C. IMPORTANCE This study highlights a promising strategy to improve mRNA/enzyme stability and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. Moreover, PNFs could increase the relative intensity of target genes without affecting the expression of other genes; as a result, it could alleviate the cellular burden compared to other regulatory elements such as promoters. In addition, we obtained a PigF variant with improved activity and stability by the introduction of disulfide bonds into PigF. Collectively, we demonstrate here a novel approach for improving mRNA/enzyme stability using PNFs, which results in enhanced prodigiosin production in S. marcescens at 30°C.

MicroRNAs (miRNAs) are associated with various cellular processes and have been recognized as potential biomarkers for many human diseases. The sensitive and accurate determination of miRNA expression levels in biological specimens is highly significant for understanding their biological functions and clinical diagnosis. Mass spectrometry (MS) has shown its potential to study bioactive molecules, however, direct MS analysis of miRNAs is often hampered by limited sensitivity. For sensitive detection of miRNAs, indirect methods are generally employed through the use of DNA probes labeled with peptides or metal elements. In this work, we proposed a novel MS-based label-free strategy for miRNA quantification. A dual-amplification system was developed by using a padlock probe containing the poly(thymine) sequence in combination with rolling circle amplification (RCA). The specific recognition of target miRNA by the padlock probes produced long single-stranded DNAs containing poly (adenine) segments, which guaranteed the specificity of detection and realized primary amplification. Then the RCA products were extracted and treated with acid to release a large number of free adenines as reporter molecules for secondary signal amplification. Overall, the quantification of target miRNA was carried out by signal switching from high-molecular-weight RCA products to highly sensitive small molecule of adenine. The developed method achieved a linear detection range from 200 amol to 100 fmol for miRNA-21 with a limit of detection of 50 amol, and successfully applied to detect endogenous miRNA-21 levels from lung cancer cells. Overall, the present study provides a sensitive, specific MS-based method for miRNA detection and holds great potential for further application of MS technology to detect other biomarkers in biomedical research and early clinical diagnosis.

BACKGROUND: The reduced range of motion and pain are the most characteristic clinical features of osteoarthritis (OA). Hyaluronic acid (HA), which is one of the infiltrative therapies for OA treatment, and polynucleotides (PNs), which is a DNA-derived macromolecule favored cell growth and collagen production, are an ongoing debate in clinical effectiveness.
METHODS: We plan to perform a systematic review and meta-analysis of randomized clinical trial to evaluate efficacy of intra-articular polynucleotides associated with hyaluronic acid versus hyaluronic acid alone in the treatment of knee osteoarthritis. We will search PubMed, EMBASE, Cochrane Library using a comprehensive strategy. The related conference proceedings and reference lists of the included studies will also be checked to identify additional studies. Two reviewers will screen retrieved records, extract information and assess the risk of bias independently. Stata v15.1 software will be used to conduct data synthesis.
RESULTS: This study will be submitted to a peer-reviewed journal for publication.
CONCLUSIONS: We hope it will provide a relatively comprehensive reference for clinical practice and future relevant clinical trials.
BACKGROUND: Ethics approval and patient consent are not required, as this study is a systematic review and meta-analysis.
UNASSIGNED: CRD42020167678.

Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein\'s scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a β-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed β-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop\'s key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple β-α repeat proteins, primarily as a polynucleotide binding motif.

The generalized energy-based fragmentation (GEBF) approach has been applied to study the THz spectra and vibrational modes of base pair cocrystals under periodic boundary conditions (denoted as PBC-GEBF). Results of vibrational mode reveal that hydrogen bonds play a pivotal role in the pairing process of base crystals, where most NH and CH bonds stretch to some extent. We also found that hydrogen bonds of a self-made A:T cocrystal completely break in a transition from liquid to the solid state, while self-made C:G cocrystal is different and easier to form a cocrystal, as confirmed by X-ray diffraction (XRD) and terahertz (THz) spectra. Furthermore, we have studied DNA polynucleotides (in both A and B forms) found that the vibrational modes changed a lot during the process of their forming double strand. Despite the key role played by hydrogen bonds, the key contribution originates from collective motions of the main skeleton. A comparative study of the spectra of some stranded fragments suggests that different sequences or forms have similar spectra in THz band. They distinguish from each other mainly in the low-frequency regions, especially below 1 THz. This study would make great contributions to the molecular dynamics model based DNA long-chain structure simulation in the future study.

In keratitis, keratocytes play a vital role by releasing inflammatory cytokines and expressing intercellular cell adhesion molecule-1(ICAM-1). GC31 is a peptide derived from thrombomodulin, an endogenous protein with potential anti-inflammation properties. We evaluated the protective effect of GC31 in LPS- or poly(I:C)-induced corneal fibroblasts. Cultured keratocytes were treated with either LPS or poly(I:C); The mRNA and protein expressions of IL-6, IL-8, MCP-1, and IFN-γ were determined by real-time RT-PCR and ELISA. The expression level of ICAM-1 was estimated by real-time RT-PCR, immunofluorescence, and western blot. The underlying pathways were investigated by detecting NF-κB p65 translocation and phosphorylation of IκBα, p65, p38, JNK, and ERK. The MTS assay was used to measure cell viability of keratocytes after GC31 incubation. The elevation of IL-6, IL-8, MCP-1, and IFN-γ expression induced by LPS or poly(I:C) was significantly inhibited by GC31 in a dose-dependent manner at both mRNA and protein levels. GC31 also reduced the expression of ICAM-1 in keratocytes after LPS or poly(I:C) stimulation. LPS or poly(I:C) induced p65 translocation and phosphorylation of IκBα, p65, p38, and JNK were suppressed by GC31.GC31 is not only an effective inhibitor of LPS-induced inflammatory response, but it also inhibits poly(I:C)-induced release of inflammatory cytokines and ICAM-1 expression by blocking the NF-κB and MAPK (p38 and JNK) pathways. This suggested that GC31 may exert a protective effect in attenuating corneal inflammation by suppressing the immune response of the fibroblasts.

The safe and effective delivery of therapeutic genes into target cell interiors is of great importance in gene therapy. Chitosan has been extensively studied as a gene delivery carrier due to its good biocompatibility and biodegradability. Understanding the atomic interaction mechanism between chitosan and DNA is important in the design and application of chitosan-based drug and gene delivery systems. In this work, the interactions between single-stranded polynucleotides and different types of chitosan were systematically investigated by using molecular dynamics (MD) simulation. Our results demonstrate that the functional groups of chitosan, the types of base and length of polynucleotides regulate the interaction behavior between chitosan and polynucleotides. The encapsulation capacity of polynucleotide by chitosan is mainly balanced by two factors: the strength of polynucleotide binding to chitosan and the tendency of self-aggregation of polynucleotide in the solution. For -NH3+ chitosan, due to the strong electrostatic interaction, especially the H-bond between -NH3+ groups in chitosan and phosphate groups in polynucleotide, the aggregation effect could be partially eliminated. The good dispersal capacity of polynucleotides may improve the encapsulation of polynucleotides by chitosan, and hence increase the delivery and transfection efficiency of chitosan-based gene carrier.