An Ultrasensitive, One-Pot RNA Detection Method Based on Rationally Engineered Cas9 Nickase-Assisted Isothermal Amplification Reaction.

RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized molecular diagnostics by offering versatile Cas effectors. We previously developed an isothermal amplification reaction method using Cas9 nickase (Cas9 nAR) to detect genomic DNA. However, slow dissociation of Cas9n from nicked double-stranded DNA (dsDNA) substrates dramatically hampers the cooperation between Cas9n and DNA polymerase, leading to low amplification efficiency. Here, we use structure-guided protein engineering to generate a Cas9n variant with faster kinetics and enhanced targeting specificity, and apply it to develop Cas9 nAR version 2 (Cas9 nAR-v2) by deftly merging reverse transcription with nicking-extension-displacement-based amplification for isothermal, one-pot RNA detection. This assay is validated by detecting Salmonella typhimurium 16S rRNA, Escherichia coli O157:H7 16S rRNA, synthetic SARS-CoV-2 genes, and HIV virus RNA, showing a quantitative analysis over a wide, linear range and a detection limit as low as fewer than ten copies of RNA molecules per reaction (20 µL volume). It also shows an excellent nucleotide-mutation discrimination capability in detecting SARS-CoV-2 variants. Furthermore, Cas9 nAR-v2 is compatible with low-cost point-of-care (POC) tests based on fluorescence and lateral-flow readouts. In summary, this method provides a new paradigm for sensitive, direct RNA detection and would spur the exploration of engineered Cas effectors with improved properties for a wide range of biological applications.

Structural analysis of cysteine-free Nt.BspD6 nicking endonuclease and its functional features.

Nicking endonuclease Nt.BspD6I (Nt.BspD6I) is the large subunit of the heterodimeric restriction endonuclease R.BspD6I. It recognizes the short specific DNA sequence 5´'- GAGTC and cleaves only the top strand in dsDNA at a distance of four nucleotides downstream the recognition site toward the 3´'-terminus. A mechanism of interaction of this protein with DNA is still unknown. Here we report the crystal structure of Cysteine-free Nt.BspD6I, with four cysteine residues (11, 160, 508, 578) substituted by serine, which was determined with a resolution of 1.93 Å. A comparative structural analysis showed that the substitution of cysteine residues induced marked conformational changes in the N-terminal recognition and the C-terminal cleavage domains. As a result of this changes were formed three new hydrogen bonds and the electrostatic field in these regions changed compared with wild type Nt.BspD6I. The substitution of cysteine residues did not alter the nicking function of Cysteine-free Nt.BspD6I but caused change in the activity of Cysteine-free heterodimeric restriction endonuclease R.BspD6I due to a change in the interaction between its large and small subunits. The results obtained contribute to the identification of factors influencing the interactions of subunits in the heterodimeric restriction enzyme R.BspD6I.

Comparison of biosimilar Tigerase and Pulmozyme in long-term symptomatic therapy of patients with cystic fibrosis and severe pulmonary impairment (subgroup analysis of a Phase III randomized open-label clinical trial (NCT04468100)).

BACKGROUND: Patients with cystic fibrosis (CF) need costly medical care and adequate therapy with expensive medicinal products. Tigerase® is the first biosimilar of dornase alfa, developed by the lead Russian biotechnology company GENERIUM. The aim of the manuscript to present post hoc sub-analysis of patients' data with cystic fibrosis and severe pulmonary impairment of a larger comparative study (phase III open label, prospective, multi-centre, randomized study (NCT04468100)) of a generic version of recombinant human DNase Tigerase® to the only comparable drug, Pulmozyme®. METHODS: In the analyses included subgroup of 46 severe pulmonary impairment patients with baseline FEV1 level 40-60% of predicted (23 patients in each treatment group) out of 100 patients registered in the study phase III open label, prospective, multi-center, randomized study (NCT04468100), and compared efficacy endpoints (FEV1, FVC, number and time of exacerbations, body weight, St.George's Respiratory Questionnaire) as well as safety parameters (AEs, SAEs, anti-drug antibody) within 24 treatment weeks. RESULTS: All outcomes were comparable among the studied groups. In the efficacy dataset, the similar mean FEV1 and mean FVC changes for 24 weeks of both treatment groups were observed. The groups were also comparable in safety, all the secondary efficacy parameters and immunogenicity. CONCLUSIONS: The findings from this study support the clinical Tigerase® biosimilarity to Pulmozyme® administered in CF patients with severe impairment of pulmonary function.

Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease.

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1's ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5'-TTTTT/AA-3'. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN's sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes 'compression' near the cleavage site. This provides multiple advantages for L1-EN's role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

Integrated nicking enzyme-powered numerous-legged DNA walker prepared by rolling circle amplification for fluorescence detection of microRNA.

MicroRNAs (miRNAs) have been accepted as promising non-invasive biomarkers for cancer early diagnosis. Developing amplified sensing strategies for detecting ultralow concentration of miRNAs in clinical samples still requires much effort. Herein, an integrated fluorescence biosensor using nicking enzyme-powered numerous-feet DNA walking machine was developed for ultrasensitive detection of miRNA. A long numerous-feet walker produced by target-triggered rolling circle amplification autonomously moves along the defined DNA tracks on gold nanorods (AuNRs) with the help of nicking enzyme, leading to the recovery of fluorescence. This results in an amplified fluorescence signal, typically measured at 518 nm emission wavelength. Benefiting from the long walker that dramatically improves movement range, the homogenous and one-step strategy realizes ultrahigh sensitivity with a limit of detection of 0.8 fM. Furthermore, this walking machine has been successfully used to quantification of miRNA in clinical serum samples. The consistency of the gained results between of the developed strategy and reverse transcription quantitative polymerase chain reaction (RT-qPCR) shows that the sensing method has great promise for tumor diagnostics based on nucleic acid. Schematic representation of the fluorescent biosensing strategy, numerous-legged DNA walker prepared by rolling circle amplification on gold nanorods (AuNRs) for microRNA analysis, which can be applied in real samples with good results.

Actin-Resistant DNase1L2 as a Potential Therapeutics for CF Lung Disease.

In cystic fibrosis (CF), the accumulation of viscous lung secretions rich in DNA and actin is a major cause of chronic inflammation and recurrent infections leading to airway obstruction. Mucolytic therapy based on recombinant human DNase1 reduces CF mucus viscosity and promotes airway clearance. However, the marked susceptibility to actin inhibition of this enzyme prompts the research of alternative treatments that could overcome this limitation. Within the human DNase repertoire, DNase1L2 is ideally suited for this purpose because it exhibits metal-dependent endonuclease activity on plasmid DNA in a broad range of pH with acidic optimum and is minimally inhibited by actin. When tested on CF artificial mucus enriched with actin, submicromolar concentrations of DNase1L2 reduces mucus viscosity by 50% in a few seconds. Inspection of superimposed model structures of DNase1 and DNase1L2 highlights differences at the actin-binding interface that justify the increased resistance of DNase1L2 toward actin inhibition. Furthermore, a PEGylated form of the enzyme with preserved enzymatic activity was obtained, showing interesting results in terms of activity. This work represents an effort toward the exploitation of natural DNase variants as promising alternatives to DNase1 for the treatment of CF lung disease.

Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering.

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor.

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.

Injectable Drug-Conjugated DNA Hydrogel for Local Chemotherapy to Prevent Tumor Recurrence.

Considering the high rate of postsurgical tumor recurrence due to the possible residual cancer cells and the non-negligible toxicity of postsurgical systemic chemotherapy, we designed an injectable DNA hydrogel assembled by chemodrug-grafted DNA strands for localized chemotherapy. First, a multitude of camptothecin was successfully grafted on backbones of the phosphorothioate DNAs, which could be assembled into two types of Y-shaped building blocks and then hierarchically associated together to form drug-containing hydrogels. The injectable feature of drug-containing DNA hydrogels enables a minimally invasive approach for local drug administration. Owing to the enzymatic degradation, the hydrogel can gradually disassemble into nanosized particles, allowing its good permeation into the residual tumor tissue and efficient uptake by cells. Together with its sustained and responsive drug release behaviors, the drug-containing DNA hydrogel can significantly inhibit the regrowth of tumor cells and prevent cancer recurrence. Compared to the control groups, mice treated with our drug-containing DNA hydrogel show the lowest tumor relapse rate (1/3) and substantial slow tumor progression. Despite the long-term local embedding, negligible systemic toxicity and organ damages are observed after the treatment with our drug-grafted DNA hydrogel. With excellent antitumor efficacy and low side effects in vivo, our DNA-drug conjugate (DDC)-based hydrogel represents a promising candidate for local adjuvant therapy in cancer treatment.

A carbon nanoparticle and DNase I-Assisted amplified fluorescent biosensor for miRNA analysis.

Nucleic acid-based biosensors have become powerful tools in biomedical applications. But the stability issue seriously limits their wide applications. Fortunately, the emergence of carbon nanoparticles (CNPs), which can effectively protect DNA probes from enzymatic digestion and unspecific protein binding, provides a good solution. In this work, a DNase I-aided cyclic enzymatic amplification method (CEAM) for microRNA analysis has been developed based on the coupling use of nucleic acid probes with specific molecular recognition ability as well as CNPs with excellent biostability. The method is simple and sensitive, with a detection limit down to 3.2 pM. Furthermore, satisfactory results are achieved for miRNA analysis in breast cancer cell lysate, demonstrating the applicability in disease diagnosis. The ingenious combination of CNPs and nucleic acid probes can open a new chapter in the development of versatile analytical strategies that holds great potentials for clinical diagnosis, food safety, and environmental monitoring.

Peptide nucleic acid based tension sensor for cellular force imaging with strong DNase resistance.

DNA is a versatile biomaterial with well-defined mechanical and biochemical properties. It has been broadly adopted to synthesize tension sensors that calibrate and visualize cellular forces at the cell-matrix interface. Here we showed that DNA-based tension sensors are vulnerable to deoxyribonucleases (DNases) which cells may express on cell membrane or secret to the culture environment. These DNases can damage the sensors, lower signal-to-noise ratio or even produce false signal in cellular force imaging. To address this issue, we tested peptide nucleic acid (PNA), chemically modified RNA and their hybrids with DNA as alternative biomaterials for constructing tension sensors. Four duplexes: double-stranded DNA (dsDNA), PNA/DNA, dsRNA (modified RNA) and PNA/RNA, were tested and evaluated in terms of DNase resistance, cellular force imaging ability and material robustness. The results showed that all PNA/DNA, dsRNA and PNA/RNA exhibited strong resistance to both soluble DNase I and membrane-bound DNase on cells. However, PNA/RNA-based tension sensor had low signal-to-noise ratio in cellular force imaging, and dsRNA-based tension sensor exhibited strong non-specific signal unrelated to cellular forces. Only PNA/DNA-based tension sensor reported cellular forces with highest signal-to-noise ratio and specificity. Collectively, we confirmed that PNA/DNA hybrid is an accessible material for the synthesis of DNase-resistant tension sensor that retains the force-reporting capability and remains stable in DNase-expressing cells. This new class of tension sensors will broaden the application of tension sensors in the study of cell mechanobiology.

A biodegradable poly(amido amine) based on the antimicrobial polymer polyhexamethylene biguanide for efficient and safe gene delivery.

Inspired by the excellent membrane affinity of antimicrobial polymers, we synthesized a novel biodegradable poly(amino amine) polymer with pendent side chains that mimic the widely used biocide polyhexamethylene biguanide (PHMB) for gene delivery. Michael addition polymerization was utilized to form the polymer scaffold between N,N'-cystaminebisacrylamide (CBA) and N-Boc-1,6-diaminohexane (Boc-DAH) followed by N-Boc deprotection. Then the exposed primary amino groups were partly (about 75%) transformed into biguanide by an addition reaction with dicyandiamide to obtain the final product CBA-DAH-biguanide (CBA-DAH-BG). The polymer CBA-DAH-BG was able to condense plasmid DNA (pDNA) into nano-sized (<200 nm), positively-charged (>35 mV) polyplexes that were well resistant to heparin and DNase I. Rapid DNA release was observed in the presence of dithiothreitol (DTT), indicating that CBA-DAH-BG was equipped with biodegradability by the cleavage of disulfide bonds, which was helpful for unpacking DNA and decreasing cytotoxicity. CBA-DAH-BG/pDNA polyplexes were characterized by efficient cellular uptake efficacy, extremely low cytotoxicity, and high transfection efficiency in two cell lines (i.e., NIH/3T3 and U87 MG), compared to 25 kDa polyethyleneimine (PEI) and the intermediate product CBA-DAH that were both devoid of biguanide groups. Of note, clathrin-mediated endocytosis and lipid rafts played an important role in the internalization of the polyplexes. Taken together, this strategy described herein may represent an innovative avenue for the design of more advanced nonviral gene vectors with high transfection efficiency and biocompatibility.

A dual-cycling fluorescence scheme for ultrasensitive DNA detection through signal amplification and target regeneration.

In this work, we propose an ultrasensitive fluorescence strategy for DNA detection. This method utilizes a molecular beacon (MB), a hairpin probe (HP), and an enzyme to trigger dual-cycling reactions (cycles I and II). In cycle I, the target is repeatedly used to amplify the fluorescence emission through hybridizations with the MB and cleavage reactions achieved by the enzyme. In cycle II, hybridization reactions between the HP and a segment of the MB continuously regenerate the target to trigger more cycle I reactions, leading to an enhanced fluorescent signal. The detection limit of the method is determined to be as low as 50 fM within 45 min, which is 2 to 3 orders of magnitude lower than that of the conventional fluorescence strategies. The method also shows a high selectivity over mismatched and random DNA sequences. The signal amplification mechanism of the strategy offers insights into constructing efficient and ultrasensitive biosensors for various applications.

New Nuclease from Extremely Psychrophilic Microorganism Psychromonas ingrahamii 37: Identification and Characterization.

Nucleases are an important group of hydrolases that degrade nucleic acids, with broad spectrum of applications in science and industry. In this paper, we report the identification and characterization of the nuclease from extremely psychrophilic bacterium Psychromonas ingrahamii that grows exponentially at 5 °C, but may also grow at even lower temperatures (down to - 12 °C). The putative endonuclease I gene, identified in the genome of P. ingrahamii, was cloned and expressed in Pichia pastoris. The recombinant protein was purified and its nucleolytic features were studied. The new enzyme, named by us as PinNuc, displays the features characteristic for the nonselective endonucleases, and has the ability to degrade different forms of nucleic acids. It is very active at room temperature in low ion-strength buffer and in the presence of low concentrations of magnesium ions. The enzyme, which possesses six cysteine residues, the most likely all engaged in disulphide bridges, is active only in oxidized form, and can be efficiently inactivated by the addition of low amounts of a reducing agent. According to our knowledge, it is the first nuclease, belonging to endonuclease I family, isolated from such extremely psychrophilic organism.

Assembly of DNA Probes into Superstructures for Dramatically Enhancing Enzymatic Stability and Signal-to-Background Ratio.

DNA fluorescent probes are versatile tools that are widely used for biological detection in tubes. Using DNA probes in living systems, however, represents a significant challenge because of the endogenous nuclease-induced DNA degradation and strong background fluorescence in complex biological environments. Here, we show that assembling DNA probes into core-satellite gold nanoparticle (AuNP) superstructures could unprecedentedly enhance enzymatic stability and reduce background interference. The embedded DNA probes are protected from interaction with nuclease, eliminating the enzymatic degradation. In the meantime, the AuNP superstructures show extremely high quenching efficiency (>98%) toward the embedded DNA probes, whose fluorescence can be instantly turned on by the target binding, resulting in high signal-to-background ratio. To demonstrate these distinct properties, we made use of the assembled nanoprobes to monitor the ATP levels under different stimuli in living cells. The assembly strategy leads to a new opportunity for accurately sensing targets in living systems.

Pathway of Actin Folding Directed by the Eukaryotic Chaperonin TRiC.

The hetero-oligomeric chaperonin of eukarya, TRiC, is required to fold the cytoskeletal protein actin. The simpler bacterial chaperonin system, GroEL/GroES, is unable to mediate actin folding. Here, we use spectroscopic and structural techniques to determine how TRiC promotes the conformational progression of actin to the native state. We find that actin fails to fold spontaneously even in the absence of aggregation but populates a kinetically trapped, conformationally dynamic state. Binding of this frustrated intermediate to TRiC specifies an extended topology of actin with native-like secondary structure. In contrast, GroEL stabilizes bound actin in an unfolded state. ATP binding to TRiC effects an asymmetric conformational change in the chaperonin ring. This step induces the partial release of actin, priming it for folding upon complete release into the chaperonin cavity, mediated by ATP hydrolysis. Our results reveal how the unique features of TRiC direct the folding pathway of an obligate eukaryotic substrate.

Clinical Application of Polysialylated Deoxyribonuclease and Erythropoietin.

BACKGROUND: While protein therapeutics are invaluable in managing numerous diseases, many require frequent injections to maintain therapeutically effective concentrations, due to their short half-life in circulation. PolyXen™, a platform and patented technology employing biodegradable, non-immunogenic and hydrophilic Polysialic Acids (PSA) for drug delivery, is being utilized to overcome such limitations, thereby potentially enabling the clinical utility of a broad range of protein therapeutics. Here, we report the recent progress on two development candidates, polysialylated deoxyribonuclease I (PSA-DNase) and polysialylated erythropoietin (PSA-EPO). METHODS AND RESULTS: Chemical polysialylation of DNase I (DNase) using PSA with different chain length at various conjugation sites led to improved stability against proteases and thermal stress, and slightly reduced enzymatic activity. Polysialylation of EPO resulted in retention of protein structure and PSA-EPO remained biologically active. PSA-EPO had a significantly prolonged circulating half-life (e.g. t1/2 of PSA-EPO = ~400 h in patients after subcutaneous administration, aimed for once monthly administration, vs. t1/2 of EPO = ~22 h; administered twice or thrice weekly), and retained in vivo efficacy. CONCLUSION: This approach has been clinically validated in phase I (in healthy volunteers) and II studies of PSA-EPO [for managing anemia in patients with chronic kidney disease (CKD)].

Polydopamine Nanosphere/Gold Nanocluster (Au NC)-Based Nanoplatform for Dual Color Simultaneous Detection of Multiple Tumor-Related MicroRNAs with DNase-I-Assisted Target Recycling Amplification.

A novel fluorescence resonance energy transfer (FRET)-based platform using polydopamine nanospheres (PDANSs) as energy acceptors and dual colored Au NCs as energy donors for simultaneous detection of multiple tumor-related microRNAs with DNase-I-assisted target recycling amplification was developed for the first time. On the basis of monitoring the change of the recovered fluorescence intensity at 445 and 575 nm upon the addition of targets miRNA-21 and let-7a, these two microRNAs (miRNAs) can be simultaneously quantitatively detected, with detection limits of 4.2 and 3.6 pM (3σ) for miRNA-21 and let-7a, which was almost 20 times lower than that without DNase I. Additionally, semiquantitative determination of miRNA-21 and let-7a can also be realized through photovisualization. Most importantly, serums from normal and breast cancer patients can be visually and directly discriminated without any sample pretreatment by confocal microscope experiments, demonstrating promising potential for auxiliary clinical diagnosis.

Fluorescence and photoacoustic dual-mode imaging of tumor-related mRNA with a covalent linkage-based DNA nanoprobe.

We developed a dual-mode DNA nanoprobe based on covalent linkage for fluorescence imaging and photoacoustic imaging of tumor-related mRNA.

Molecular basis for the integration of environmental signals by FurB from Anabaena sp. PCC 7120.

FUR (Ferric uptake regulator) proteins are among the most important families of transcriptional regulators in prokaryotes, often behaving as global regulators. In the cyanobacterium Anabaena PCC 7120, FurB (Zur, Zinc uptake regulator) controls zinc and redox homeostasis through the repression of target genes in a zinc-dependent manner. In vitro, non-specific binding of FurB to DNA elicits protection against oxidative damage and avoids cleavage by deoxyribonuclease I. The present study provides, for the first time, evidence of the influence of redox environment in the interaction of FurB with regulatory zinc and its consequences in FurB-DNA-binding affinity. Calorimetry studies showed that, in addition to one structural Zn(II), FurB is able to bind two additional Zn(II) per monomer and demonstrated the implication of cysteine C93 in regulatory Zn(II) coordination. The interaction of FurB with the second regulatory zinc occurred only under reducing conditions. While non-specific FurB-DNA interaction is Zn(II)-independent, the optimal binding of FurB to target promoters required loading of two regulatory zinc ions. Those results combined with site-directed mutagenesis and gel-shift assays evidenced that the redox state of cysteine C93 conditions the binding of the second regulatory Zn(II) and, in turn, modulates the affinity for a specific DNA target. Furthermore, differential spectroscopy studies showed that cysteine C93 could also be involved in heme coordination by FurB, either as a direct ligand or being located near the binding site. The results indicate that besides controlling zinc homeostasis, FurB could work as a redox-sensing protein probably modifying its zinc and DNA-binding abilities depending upon environmental conditions.