In Situ Polymerization-Mediated Antigen Presentation.

Activating antigen-presenting cells is essential to generate adaptive immunity, while the efficacy of conventional activation strategies remains unsatisfactory due to suboptimal antigen-specific priming. Here, in situ polymerization-mediated antigen presentation (IPAP) is described, in which antigen-loaded nanovaccines are spontaneously formed and efficiently anchored onto the surface of dendritic cells in vivo through co-deposition with dopamine. The resulting chemically bound nanovaccines can promote antigen presentation by elevating macropinocytosis-based cell uptake and reducing lysosome-related antigen degradation. IPAP is able to prolong the duration of antigen reservation in the injection site and enhance subsequent accumulation in the draining lymph nodes, thereby eliciting robust antigen-specific cellular and humoral immune responses. IPAP is also applicable for different antigens and capable of circumventing the disadvantages of complicated preparation and purification. By implementation with ovalbumin, IPAP induces a significant protective immunity against ovalbumin-overexpressing tumor cell challenge in a prophylactic murine model. The use of the SARS-CoV-2 Spike protein S1 subunit also remarkably increases the production of S1-specific immunoglobulin G in mice. IPAP offers a unique strategy for stimulating antigen-presenting cells to boost antigen-specific adaptive responses and proposes a facile yet versatile method for immunization against various diseases.

An electrochemical PNA-based sensor for the detection of the SARS-CoV-2 RdRP by using surface-initiated-reversible-addition-fragmentation-chain-transfer polymerization technique.

Coronavirus disease 2019 is one of the global health problems. Herein, a highly sensitive electrochemical biosensor has been designed to detect the RNA-dependent RNA polymerase (RdRP) of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) (SARS-CoV-2 RdRP). Herein, the surface-initiated reversible-addition-fragmentation-chain-transfer polymerization was used to amplify the electrochemical signal. To do that, the thiol-terminated peptide nucleic acid (PNA) probes were first immobilized on the surface of a screen-printed electrode modified with reduced graphene oxide-gold nanocomposite and then the fixed concentration of the SARS-CoV-2 RdRP was added to the electrode surface to interact with PNA probes. Subsequently, the Zr 4+ ions were added to interact with the phosphate groups of the SARS-CoV-2 RdRP. It allowed us to polymerase the ferrocenylmethyl methacrylate (FcMMA) and 4-cyano-4-(phenylcarbonothioylthio)-pentanoic acid on the SARS-CoV-2 RdRP chain. Since the poly-FcMMA has an electrochemical signal, the response of the PNA-based sensor to SARS-CoV-2 RdRP was increased in the range of 5-500 aM. The limit of detection was calculated to be 0.8 aM which is lower than the previous sensor for SARS-CoV-2 RdRP detection. The proposed PNA-based sensor showed high selectivity to the SARS-CoV-2 RdRP in the presence of the gene fragments of influenza A and Middle East respiratory syndrome coronavirus.

COVID-19 patient fibrinogen produces dense clots with altered polymerization kinetics, partially explained by increased sialic acid.

BACKGROUND: Thrombogenicity is a known complication of COVID-19, resulting from SARS-CoV-2 infection, with significant effects on morbidity and mortality. OBJECTIVE: We aimed to better understand the effects of COVID-19 on fibrinogen and the resulting effects on clot structure, formation, and degradation. METHODS: Fibrinogen isolated from COVID-19 patients and uninfected subjects was used to form uniformly concentrated clots (2 mg/ml), which were characterized using confocal microscopy, scanning electron microscopy, atomic force microscopy, and endogenous and exogenous fibrinolysis assays. Neuraminidase digestion and subsequent NANA assay were used to quantify sialic acid residue presence; clots made from the desialylated fibrinogen were then assayed similarly to the original fibrinogen clots. RESULTS: Clots made from purified fibrinogen from COVID-19 patients were shown to be significantly stiffer and denser than clots made using fibrinogen from noninfected subjects. Endogenous and exogenous fibrinolysis assays demonstrated that clot polymerization and degradation dynamics were different for purified fibrinogen from COVID-19 patients compared with fibrinogen from noninfected subjects. Quantification of sialic acid residues via the NANA assay demonstrated that SARS-CoV-2-positive fibrinogen samples contained significantly more sialic acid. Desialylation via neuraminidase digestion resolved differences in clot density. Desialylation did not normalize differences in polymerization, but did affect rate of exogenous fibrinolysis. DISCUSSION: These differences noted in purified SARS-CoV-2-positive clots demonstrate that structural differences in fibrinogen, and not just differences in gross fibrinogen concentration, contribute to clinical differences in thrombotic features associated with COVID-19. These structural differences are at least in part mediated by differential sialylation.

Polymerized porin as a novel delivery platform for coronavirus vaccine.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), seriously threatens human life and health. The correct folding and polymerization of the receptor-binding domain (RBD) protein of coronavirus in Escherichia coli may reduce the cost of SARS-CoV-2 vaccines. In this study, we constructed this nanopore by using the principle of ClyA porin polymerization triggered by the cell membrane. We used surfactants to "pick" the ClyA-RBD nanopore from the bacterial outer membrane. More importantly, the polymerized RBD displayed on the ClyA-RBD polymerized porin (RBD-PP) already displays some correct spatial conformational epitopes that can induce neutralizing antibodies. The nanostructures of RBD-PP can target lymph nodes and promote antigen uptake and processing by dendritic cells, thereby effectively eliciting the production of anti-SARS-CoV-2 neutralizing antibodies, systemic cellular immune responses, and memory T cells. We applied this PP-based vaccine platform to fabricate an RBD-based subunit vaccine against SARS-CoV-2, which will provide a foundation for the development of inexpensive coronavirus vaccines. The development of a novel vaccine delivery system is an important part of innovative drug research. This novel PP-based vaccine platform is likely to have additional applications, including other viral vaccines, bacterial vaccines, tumor vaccines, drug delivery, and disease diagnosis.

Fluorescent bioassay for SARS-CoV-2 detection using polypyrene-g-poly(ε-caprolactone) prepared by simultaneous photoinduced step-growth and ring-opening polymerizations.

The construction of a rapid and easy immunofluorescence bioassay for SARS-CoV-2 detection is described. We report for the first time a novel one-pot synthetic approach for simultaneous photoinduced step-growth polymerization of pyrene (Py) and ring-opening polymerization of ε-caprolactone (PCL) to produce a graft fluorescent copolymer PPy-g-PCL that was conjugated to SARS-CoV-2-specific antibodies using EDC/NHS chemistry. The synthesis steps and conjugation products were fully characterized using standard spectral analysis. Next, the PPy-g-PCL was used for the construction of a dot-blot assay which was calibrated for applications to human nasopharyngeal samples. The analytical features of the proposed sensor showed a detection range of 6.03-8.7 LOG viral copy mL-1 (Ct Scores: 8-25), the limit of detection (LOD), and quantification (LOQ) of 1.84 and 6.16 LOG viral copy mL-1, respectively. The repeatability and reproducibility of the platform had a coefficient of variation (CV) ranging between 1.2 and 5.9%. The fluorescence-based dot-blot assay was tested with human samples. Significant differences were observed between the fluorescence intensity of the negative and positive samples, with an overall correct response of 93.33%. The assay demonstrated a high correlation with RT-PCR data. This strategy opens new insights into simplified synthesis procedures of the reporter molecules and their high potential sensing and diagnosis applications.

Sulfur atom modification on thymine improves the specificity and sensitivity of DNA polymerization and detection.

Although accurate base-pairing ensures specificity of molecular recognition, DNA polymerization and DNA amplification, there are many non-specific pairings that arise from mismatched pairs, such as the T/G wobble pair. We have found that by using 2-S-TTP (STTP), we can minimize T/G mismatch, improve the DNA polymerization specificity and enhance the detection sensitivity (up to 20 fold), without significantly compromising the polymerization efficiency (the extension rate ratio of TTP vs.STTP is 1.08). With the STTP strategy, DNA polymerization is more specific and allows the detection of pathogens (such as COVID-19) in single digits (up to 5 copies), which is not possible with conventional RT-PCR. We have discovered that STTP can generally promote much higher specificity and sensitivity in DNA polymerization and nucleic acid detection than canonical TTP.

Highly polymerized proanthocyanidins (PAC) components from blueberry leaf and stem significantly inhibit SARS-CoV-2 infection via inhibition of ACE2 and viral 3CLpro enzymes.

With the current worldwide pandemic of COVID-19, there is an urgent need to develop effective treatment and prevention methods against SARS-CoV-2 infection. We have previously reported that the proanthocyanidin (PAC) fraction in blueberry (BB) leaves has strong antiviral activity against hepatitis C virus (HCV) and human T-lymphocytic leukemia virus type 1 (HTLV-1). In this study, we used Kunisato 35 Gou (K35) derived from the rabbit eye blueberry (Vaccinium virgatum Aiton), which has a high PAC content in the leaves and stems. The mean of polymerization (mDP) of PAC in K35 was the highest of 7.88 in Fraction 8 (Fr8) from the stems and 12.28 of Fraction 7 (Fr7) in the leaves. The composition of BB-PAC in K35 is that most are B-type bonds with a small number of A-type bonds and cinchonain I as extension units. A strong antiviral effect was observed in Fr7, with a high polymerized PAC content in both the leaves and stems. Furthermore, when we examined the difference in the action of BB-PAC before and after SARS-CoV-2 infection, we found a stronger inhibitory effect in the pre-infection period. Moreover, BB-PAC Fr7 inhibited the activity of angiotensin II converting enzyme (ACE2), although no effect was observed in a neutralization test of pseudotyped SARS-CoV-2. The viral chymotrypsin-like cysteine protease (3CLpro) of SARS-CoV-2 was also inhibited by BB-PAC Fr7 in leaves and stems. These results indicate that BB-PAC has at least two different inhibitory effects, and that it is effective in suppressing SARS-CoV-2 infection regardless of the time of infection.

Is amyloid fibrillation related to 3D domain swapping for the C-terminal domain of SARS-CoV main protease?

The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. Here, we report that Mpro-C can also form amyloid fibrils under the 3D domain-swappable conditions in vitro, and the fibrils are not formed through runaway/propagated domain swapping. It is found that there are positive correlations between the rates of domain swapping dimerization and amyloid fibrillation at different temperatures, and for different mutants. However, some Mpro-C mutants incapable of 3D domain swapping can still form amyloid fibrils, indicating that 3D domain swapping is not essential for amyloid fibrillation. Furthermore, NMR H/D exchange data and molecular dynamics simulation results suggest that the protofibril core region tends to unpack at the early stage of 3D domain swapping, so that the amyloid fibrillation can proceed during the 3D domain swapping process. We propose that 3D domain swapping makes it possible for the unpacking of the amyloidogenic fragment of the protein and thus accelerates the amyloid fibrillation process kinetically, which explains the well-documented correlations between amyloid fibrillation and 3D domain swapping observed in many proteins.

RNA polymerization actuating nucleic acid membrane (RANAM)-based biosensing for universal RNA virus detection.

The coronavirus disease (COVID-19) pandemic has shown the importance of early disease diagnosis in preventing further infection and mortality. Despite major advances in the development of highly precise and rapid detection approaches, the time-consuming process of designing a virus-specific diagnostic kit has been a limiting factor in the early management of the pandemic. Here, we propose an RNA polymerase activity-sensing strategy utilizing an RNA polymerization actuating nucleic acid membrane (RANAM) partially metallized with gold for colorimetric RNA virus detection. Following RANAM-templated amplification of newly synthesized RNA, the presence of the RNA polymerase was determined by visualization of the inhibition of an oxidation/reduction (redox) reaction between 3,3',5,5'-tetramethylbenzidine (TMB) and blocked Au3+. As a proof of concept, a viral RNA-dependent RNA polymerase (RdRP), which is found in various RNA virus-infected cells, was chosen as a target molecule. With this novel RANAM biosensor, as little as 10 min of RdRP incubation could significantly reduce the colorimetric signal. Further development into an easy-to-use prototype kit in viral infection diagnosis detected RdRP present at levels even as low as 100 aM. Color formation based on the presence of RdRP could be simply and clearly confirmed through smartphone-assisted color imaging of the prototype kit. This study provides a non-PCR-based RNA virus detection including its variants using RdRP-mediated polymerization.

Exponential Amplification Using Photoredox Autocatalysis.

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here, we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a nonfluorescent eosin Y derivative (EYH3-) under green light. The deactivated photocatalyst is stable and rapidly activated under low-intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH3- is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e-/H+ transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH3- degradation, we successfully improved EYH3--to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photoinduced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19.

Selenium atom on phosphate enhances specificity and sensitivity of DNA polymerization and detection.

DNA polymerization is of high specificity in vivo. However, its specificity is much lower in vitro, which limits advanced applications of DNA polymerization in ultrasensitive nucleic acid detection. Herein, we report a unique mechanism of single selenium-atom modified dNTP (dNTPαSe) to enhance polymerization specificity. We have found that both dNTPαSe (approximately 660 fold) and Se-DNA (approximately 2.8 fold) have lower binding affinity to DNA polymerase than canonical ones, and the Se-DNA duplex has much lower melting-temperature (Tm) than the corresponding canonical DNA duplex. The reduced affinity and Tm can destabilize the substrate-primer-template-enzyme assembly, thereby largely slowing down the mismatch of DNA polymerization and enhancing the amplification specificity and in turn detection sensitivity. Furthermore, the Se-strategy enables us to develop the selenium enhanced specific isothermal amplification (SEA) for nucleic acid detection with high specificity and sensitivity (up to detection of single-digit copies), allowing convenient detection of clinical HPV and COVID-19 viruses in the low-copy number. Clearly, we have discovered the exciting mechanism for enhancing DNA polymerization accuracy, amplification specificity and detection sensitivity by SEA, up to two orders of magnitude higher.

Selenium-atom-modified thymidine enhances the specificity and sensitivity of DNA polymerization and detection.

Nucleobase mismatches can jeopardize DNA polymerization specificity, causing mutations and errors in DNA replication and detection. Herein we report the first synthesis of novel 2-Se-thymidine triphosphate (SeTTP), describe the single-selenium atom-specific modification strategy (SAM) against T/G mismatches, and demonstrate SAM-assisted polymerization and detection with much higher specificity and sensitivity. SAM can effectively suppress the formation of non-specific products in DNA polymerization and detection. Thus, SAM enhances the specificity of DNA synthesis by approximately 10 000 fold, and in turn, it allows the detection of clinical COVID-19 viral RNA in low copy numbers (single-digit copies), while the conventional RT-qPCR does not.

Specificity Enhancement of Deoxyribonucleic Acid Polymerization for Sensitive Nucleic Acid Detection.

Specificity of DNA polymerization plays a critical role in DNA replication and storage of genetic information. Likewise, biotechnological applications, such as nucleic acid detection, DNA amplification, and gene cloning, require high specificity in DNA synthesis catalyzed by DNA polymerases. However, errors in DNA polymerization (such as mis-incorporation and mis-priming) can significantly jeopardize the specificity. Herein, we report our discovery that the specificity of DNA enzymatic synthesis can be substantially enhanced (up to 100-fold higher) by attenuating DNA polymerase kinetics via the phosphorothioate dNTPs. This specificity enhancement allows convenient and sensitive nucleic acid detection, polymerization, PCR, and gene cloning with complex systems (such as human cDNA and genomic DNA). Further, we found that the specificity enhancement offered higher sensitivity (up to 50-fold better) for detecting nucleic acids, such as COVID-19 viral RNAs. Our findings have revealed a simple and convenient strategy for facilitating specificity and sensitivity of nucleic acid detection, amplification, and gene cloning.