A Rare Case Report of Fanconi Anemia-Associated Nuclease 1 Mutation Causing Nephrotic Syndrome.

A 25-year-old African male patient presented with a history of frothy urination for one month. He had a significant family history of early onset chronic kidney disease (CKD) in his older brother. On evaluation, he was found to have deranged renal function and nephrotic-range proteinuria of 6152 mg/day. Urine examination revealed proteinuria and glycosuria. Viral serology and autoimmune screening results were negative. Ultrasonography revealed contracted kidneys that were not amenable to biopsy. Genetic analysis revealed a Fanconi anemia-associated nuclease 1 (FAN 1) mutation in exon 4 (c.1399G>A) and exon 12 (c.2786A>C). The patient was managed conservatively with a maximum dose of angiotensin receptor blockers with a reduction in proteinuria on follow-up. This case report highlights the rare manifestation of FAN 1 mutation and its variable effects on the kidney.

Enzymatic characterization and dominant sites of foot-and-mouth disease virus 2C protein.

Foot-and-mouth disease virus (FMDV) 2C protein is a conserved non-structural protein and crucial for replication of the virus. In this study, FMDV 2C protein was prepared and the enzymatic activities were investigated in detail. The protein could digest ssDNA or ssRNA into a small fragment at about 10 nt, indicating that the protein has nuclease activity. But it did not show digestion to blunt-end dsDNA or dsRNA. The nuclease activity of 2C protein could be inhibited in 2 mM Zn2+ or Ca2+ while enhanced by Mg2+ or Mn2+. FMDV 2C protein exhibited unwinding activity to all the three kinds of dsDNA and dsRNA (5' protruded, 3' protruded, and blunt-end). The unwinding velocity to 5' protruded dsRNA was higher than to the blunt-end dsRNA. 2C protein only showed unwinding activity in high concentration of Mg2+, but no unwinding activity in physiological concentrations of Mg2+ and Ca2+, as well as in cell lysate. The 2C protein could catalyze two structured ssRNA to form double strand, thus it was proved to have RNA chaperone activity. The Mg2+ and ATP in different concentrations did not show promotion to the RNA chaperone activity. Finally, six mutant proteins (K116A, D160A, D170A, N207A, R226A, and F316A) were constructed and the enzymatic activities were analyzed. All the six mutations reduced the ATPase activity, D170A and F361A could inactivate the nuclease activity, while the N207A and F316A could inactivate the helicase activity. Our study provides a comprehensive understanding of the enzymatic activities of FMDV 2C protein.

Observing one-divalent-metal-ion-dependent and histidine-promoted His-Me family I-PpoI nuclease catalysis in crystallo.

Metal-ion-dependent nucleases play crucial roles in cellular defense and biotechnological applications. Time-resolved crystallography has resolved catalytic details of metal-ion-dependent DNA hydrolysis and synthesis, uncovering the essential roles of multiple metal ions during catalysis. The histidine-metal (His-Me) superfamily nucleases are renowned for binding one divalent metal ion and requiring a conserved histidine to promote catalysis. Many His-Me family nucleases, including homing endonucleases and Cas9 nuclease, have been adapted for biotechnological and biomedical applications. However, it remains unclear how the single metal ion in His-Me nucleases, together with the histidine, promotes water deprotonation, nucleophilic attack, and phosphodiester bond breakage. By observing DNA hydrolysis in crystallo with His-Me I-PpoI nuclease as a model system, we proved that only one divalent metal ion is required during its catalysis. Moreover, we uncovered several possible deprotonation pathways for the nucleophilic water. Interestingly, binding of the single metal ion and water deprotonation are concerted during catalysis. Our results reveal catalytic details of His-Me nucleases, which is distinct from multi-metal-ion-dependent DNA polymerases and nucleases.

Engineering IscB to develop highly efficient miniature editing tools in mammalian cells and embryos.

The IscB proteins, as the ancestors of Cas9 endonuclease, hold great promise due to their small size and potential for diverse genome editing. However, their activity in mammalian cells is unsatisfactory. By introducing three residual substitutions in IscB, we observed an average 7.5-fold increase in activity. Through fusing a sequence-non-specific DNA-binding protein domain, the eIscB-D variant achieved higher editing efficiency, with a maximum of 91.3%. Moreover, engineered ωRNA was generated with a 20% reduction in length and slightly increased efficiency. The engineered eIscB-D/eωRNA system showed an average 20.2-fold increase in activity compared with the original IscB. Furthermore, we successfully adapted eIscB-D for highly efficient cytosine and adenine base editing. Notably, eIscB-D is highly active in mouse cell lines and embryos, enabling the efficient generation of disease models through mRNA/ωRNA injection. Our study suggests that these miniature genome-editing tools have great potential for diverse applications.

Structural determinants of DNA cleavage by a CRISPR HNH-Cascade system.

Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity.

Electrochemical Biosensing Platform Based on Toehold-Mediated Strand Displacement Reaction and DSN Enzyme-Assisted Amplification for Two-Target Detection.

Simultaneous detection of multiple targets is of great significance for accurate disease diagnosis. Herein, based on duplex-specific nuclease (DSN) assisted signal amplification and the toehold-mediated strand displacement reaction (TSDR), we constructed an electrochemical biosensor with high sensitivity and high specificity for dual-target detection. MiRNA-141 and miRNA-133a were used as the targets, and ferrocene (Fc) and methylene blue (MB) with significant peak potential differentiation were used as the electrochemical signal probes. The elaborately designed hairpin probe H1, which was fixed on the electrode surface, could be hybridized with the target miRNA-141 to perform signal amplification by the DSN-assisted enzyme cleavage cycle; thus, miRNA-141 could be detected by Fc signal changes at 0.41 V. The hairpin H1 can also combine with the MB-labeled signal probe (SP) output from miRNA-133a-induced TSDR, and the detection of miRNA-133a can be realized according to the response signal generated by MB at -0.26 V. The two sensing lines are independent of each other, and there is no mutual interference in the detection process. Therefore, two independent detection lines could be connected in series, and the simultaneous detection of two targets can be achieved on a single electrode. This novel detection strategy provides a new way to simultaneously detect different biomarkers.

Unusual Guide-binding Pockets in RNA-targeting pAgo Nucleases.

Argonaute nucleases use small nucleic acid guides to recognize and degrade complementary nucleic acid targets. Most prokaryotic Argonautes (pAgos) recognize DNA targets and may play a role in cell immunity against invader genetic elements. We have recently described two related groups of pAgo nucleases that have distinct specificity for DNA guides and RNA targets (DNA > RNA pAgos). Here, we describe additional pAgos from the same clades of the pAgo tree and demonstrate that they have the same unusual nucleic acid specificity. The two groups of DNA > RNA pAgos have non-standard guide-binding pockets in the MID domain and differ in the register of guide DNA binding and target cleavage. In contrast to other pAgos, which coordinate the 5'-end of the guide molecule by their C-terminal carboxyl, DNA > RNA pAgos have an extended C-terminus located away from the MID pocket. We show that modifications of the C-terminus do not affect guide DNA binding, but inhibit cleavage of complementary and mismatched RNA targets by some DNA > RNA pAgos. Our data suggest that the unique C-terminus found in DNA > RNA pAgos can modulate their catalytic properties and can be used as a target for pAgo modifications.

Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the binding affinity of the pre-crRNA, while deletion of an upstream sequence has no significant effect. After processing, the mature crRNA remains very tightly bound to Cas12a with a comparable affinity. Strikingly, the affinity contribution of the guide region increases to 600-fold after processing, suggesting that additional contacts are formed and may pre-order the crRNA for efficient DNA target recognition. Using a direct competition assay, we find that pre-crRNA binding specificity is robust to changes in the guide sequence, addition of a 3' extension, and secondary structure within the guide region. However, stable secondary structure in the guide region can strongly inhibit DNA targeting, indicating that care should be taken in crRNA design. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in genome editing applications.

Rational Design of Chimeric Antisense Oligonucleotides on a Mixed PO-PS Backbone for Splice-Switching Applications.

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson-Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO's pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO-PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs.

Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

Superlarge, Rigidified DNA Tetrahedron with a Y-Shaped Backbone for Organizing Biomolecules Spatially and Maintaining Their Full Bioactivity.

A major impediment to the clinical translation of DNA tiling nanostructures is a technical bottleneck for the programmable assembly of DNA architectures with well-defined local geometry due to the inability to achieve both sufficient structural rigidity and a large framework. In this work, a Y-backbone was inserted into each face to construct a superlarge, sufficiently rigidified tetrahedral DNA nanostructure (called RDT) with extremely high efficiency. In RDT, the spatial size increased by 6.86-fold, and the structural rigidity was enhanced at least 4-fold, contributing to an ∼350-fold improvement in the resistance to nucleolytic degradation even without a protective coating. RDT can be mounted onto an artificial lipid-bilayer membrane with molecular-level precision and well-defined spatial orientation that can be validated using the fluorescence resonance energy transfer (FRET) assay. The spatial orientation of Y-shaped backbone-rigidified RDT is unachievable for conventional DNA polyhedrons and ensures a high level of precision in the geometric positioning of diverse biomolecules with an approximately homogeneous environment. In tests of RDT, surface-confined horseradish peroxidase (HRP) exhibited nearly 100% catalytic activity and targeting aptamer-immobilized gold nanoparticles showed 5.3-fold enhanced cellular internalization. Significantly, RDT exhibited a 27.5-fold enhanced structural stability in a bodily environment and did not induce detectable systemic toxicity.

Viral Genomic DNA Packaging Machinery.

Tailed double-stranded DNA bacteriophage employs a protein terminase motor to package their genome into a preformed protein shell-a system shared with eukaryotic dsDNA viruses such as herpesviruses. DNA packaging motor proteins represent excellent targets for antiviral therapy, with Letermovir, which binds Cytomegalovirus terminase, already licensed as an effective prophylaxis. In the realm of bacterial viruses, these DNA packaging motors comprise three protein constituents: the portal protein, small terminase and large terminase. The portal protein guards the passage of DNA into the preformed protein shell and acts as a protein interaction hub throughout viral assembly. Small terminase recognises the viral DNA and recruits large terminase, which in turn pumps DNA in an ATP-dependent manner. Large terminase also cleaves DNA at the termination of packaging. Multiple high-resolution structures of each component have been resolved for different phages, but it is only more recently that the field has moved towards cryo-EM reconstructions of protein complexes. In conjunction with highly informative single-particle studies of packaging kinetics, these structures have begun to inspire models for the packaging process and its place among other DNA machines.

DSN signal amplification strategy based nanochannels biosensor for the detection of miRNAs.

MiRNA-21 is recognized as an important biological marker for the diagnosis, treatment, and prognosis of breast cancer. Here, we have created a nanochannel biosensor utilizing the duplex-specific nuclease (DSN) signal amplification strategy to achieve the detection of miRNAs. In this system, DNA as the capture probe was covalently immobilized on the surface of nanochannels, which hybridized with the target miRNA and forms RNA/DNA duplexes. DSN could cleave the probe DNA in RNA/DNA duplexes, recycling target miRNA, which may again hybridized with other DNA probes. After N cycles, most of the DNA probes had been cleaved, and the content of miRNA could be quantified by detecting changes in surface charge density. This biosensor can distinguish miR-21 from non-complementary miRNAs and one-base mismatched miRNAs, with reliable detection limits as low as 1 fM in PBS. In addition, we had successfully applied this method to analysis of total RNA samples in MCF-7 cells and HeLa cells, and the nanochannels had also shown excellent responsiveness and strong anti-interference ability. This new method is expected to contribute to miRNA detection in clinical diagnostics, providing a unique approach to detecting and distinguishing disease-associated molecules.

Targeted Modification of Grain Dormancy Genes in Barley.

Transgenesis technologies, such as overexpression or RNA interference-mediated suppression, have often been used to alter the activity of target genes. More recently developed targeted genome modification methods using customizable endonucleases allow for the regulation or knockout mutation of target genes without the necessity of integrating recombinant DNA. Such approaches make it possible to create novel alleles of target genes, thereby significantly contributing to crop improvement. Among these technologies, the Cas9 endonuclease-based method is widely applied to several crops, including barley (Hordeum vulgare). In this chapter, we describe an Agrobacterium-based approach to the targeted modification of grain dormancy genes in barley using RNA-guided Cas9 nuclease.

The biochemical characterization of a TatD nuclease from Thermus thermophilus.

Nucleases play pivotal roles in DNA repair and apoptosis. Moreover, they have various applications in biotechnology and industry. Among nucleases, TatD has been characterized as an exonuclease with various biological functions in different organisms. Here, we biochemically characterized the potential TatD nuclease from Thermus thermophilus. The tatD gene from T. thermophilus was cloned, then the recombinant TatD nuclease was expressed and purified. Our results revealed that the TthTatD nuclease could degrade both single-stranded and double-stranded DNA, and its activity is dependent on the divalent metal ions Mg2+ and Mn2+. Remarkably, the activity of TthTatD nuclease is highest at 37 °C and decreases with increasing temperature. TthTatD is not a thermostable enzyme, even though it is from a thermophilic bacterium. Based on the sequence similarity and molecular docking of the DNA substrate into the modeled TthTatD structure, several key conserved residues were identified and their roles were confirmed by analyzing the enzymatic activities of the site-directed mutants. The residues E86 and H149 play key roles in binding metal ions, residues R124/K126 and K211/R212 had a critical role in binding DNA substrate. Our results confirm the enzymatic properties of TthTatD and provide a primary basis for its possible application in biotechnology.

Napthalimide-based nuclease inhibitor: A multifunctional therapeutic material to bolster MRSA uptake by macrophage-like cells and mitigate pathogen adhesion on orthopaedic implant.

The healthcare burden rendered by methicillin-resistant Staphylococcus aureus (MRSA) warrants the development of therapeutics that offer a distinct benefit in the clinics as compared to conventional antibiotics. The present study describes the potential of napthalimide-based synthetic ligands (C1-C3) as inhibitors of the staphylococcal nuclease known as micrococcal nuclease (MNase), a key virulence factor of the pathogen. Amongst the ligands, the most potent MNase inhibitor C1 rendered non-competitive inhibition, reduced MNase turnover number (Kcat) and catalytic efficiency (Kcat/Km) with an IC50 value of ~950 nM. CD spectroscopy suggested distortion of MNase conformation in presence of C1. Flow cytometry and confocal microscopy indicated that C1 restored the ability of activated THP-1 cells to engulf DNA-entrapped MRSA cells. Interestingly, C1 could inhibit MRSA adhesion onto collagen. For potential application, C1-loaded pluronic F-127 micellar nanocarrier (C1-PMC) was generated, wherein the anti-adhesion activity of the pluronic carrier (PMC) and C1 was harnessed in tandem to deter MRSA cell adhesion onto collagen. MRSA biofilm formation was hindered on C1-PMC-coated titanium (Ti) wire, while eluates from C1-PMC-coated Ti wires were non-toxic to HEK 293, MG-63 and THP-1 cells. The multifunctional C1 provides a blueprint for designing therapeutic materials that hold translational potential for mitigation of MRSA infections.

Two-metal ion mechanism of DNA cleavage by activated, filamentous SgrAI.

Enzymes that form filamentous assemblies with modulated enzymatic activities have gained increasing attention in recent years. SgrAI is a sequence specific type II restriction endonuclease that forms polymeric filaments with accelerated DNA cleavage activity and expanded DNA sequence specificity. Prior studies have suggested a mechanistic model linking the structural changes accompanying SgrAI filamentation to its accelerated DNA cleavage activity. In this model, the conformational changes that are specific to filamentous SgrAI maximize contacts between different copies of the enzyme within the filament and create a second divalent cation binding site in each subunit, which in turn facilitates the DNA cleavage reaction. However, our understanding of the atomic mechanism of catalysis is incomplete. Herein, we present two new structures of filamentous SgrAI solved using cryo-EM. The first structure, resolved to 3.3 Å, is of filamentous SgrAI containing an active site mutation that is designed to stall the DNA cleavage reaction, which reveals the enzymatic configuration prior to DNA cleavage. The second structure, resolved to 3.1 Å, is of WT filamentous SgrAI containing cleaved substrate DNA, which reveals the enzymatic configuration at the end of the enzymatic cleavage reaction. Both structures contain the phosphate moiety at the cleavage site and the biologically relevant divalent cation cofactor Mg2+ and define how the Mg2+ cation reconfigures during enzymatic catalysis. The data support a model for the activation mechanism that involves binding of a second Mg2+ in the SgrAI active site as a direct result of filamentation induced conformational changes.

Plancitoxin-1 mediates extracellular trap evasion by the parasitic helminth Trichinella spiralis.

BACKGROUND: Trichinella spiralis (T. spiralis) is a parasitic helminth that causes a globally prevalent neglected zoonotic disease, and worms at different developmental stages (muscle larvae, adult worms, newborn larvae) induce immune attack at different infection sites, causing serious harm to host health. Several innate immune cells release extracellular traps (ETs) to entrap and kill most pathogens that invade the body. In response, some unicellular pathogens have evolved a strategy to escape capture by ETs through the secretion of nucleases, but few related studies have investigated multicellular helminths. RESULTS: In the present study, we observed that ETs from neutrophils capture adult worms of T. spiralis, while ETs from macrophages trap muscle larvae and newborn larvae, and ETs had a killing effect on parasites in vitro. To defend against this immune attack, T. spiralis secretes plancitoxin-1, a DNase II-like protein, to degrade ETs and escape capture, which is essential for the survival of T. spiralis in the host. CONCLUSIONS: In summary, these findings demonstrate that T. spiralis escapes ET-mediated capture by secreting deoxyribonuclease as a potential conserved immune evasion mechanism, and plancitoxin-1 could be used as a potential vaccine candidate.

Advances in structural-guided modifications of siRNA.

To date, the US Food and Drug Administration (FDA) has approved six small interfering RNA (siRNA) drugs: patisiran, givosiran, lumasiran, inclisiran, vutrisiran, and nedosiran, serving as compelling evidence of the promising potential of RNA interference (RNAi) therapeutics. The successful implementation of siRNA therapeutics is improved through a combination of various chemical modifications and diverse delivery approaches. The utilization of chemically modified siRNA at specific sites on either the sense strand (SS) or antisense strand (AS) has the potential to enhance resistance to ribozyme degradation, improve stability and specificity, and prolong the efficacy of drugs. Herein, we provide comprehensive analyses concerning the correlation between chemical modifications and structure-guided siRNA design. Various modifications, such as 2'-modifications, 2',4'-dual modifications, non-canonical sugar modifications, and phosphonate mimics, are crucial for the activity of siRNA. We also emphasize the essential strategies for enhancing overhang stability, improving RISC loading efficacy and strand selection, reducing off-target effects, and discussing the future of targeted delivery.

Deep learning with a small dataset predicts chromatin remodelling contribution to winter dormancy of apple axillary buds.

Epigenetic changes serve as a cellular memory for cumulative cold recognition in both herbaceous and tree species, including bud dormancy. However, most studies have discussed predicted chromatin structure with respect to histone marks. In the present study, we investigated the structural dynamics of bona fide chromatin to determine how plants recognize prolonged chilling during the initial stage of bud dormancy. The vegetative axillary buds of the 'Fuji' apple, which shows typical low temperature-dependent, but not photoperiod, dormancy induction, were used for the chromatin structure and transcriptional change analyses. The results were integrated using a deep-learning model and interpreted using statistical models, including Bayesian estimation. Although our model was constructed using a small dataset of two time points, chromatin remodelling due to random changes was excluded. The involvement of most nucleosome structural changes in transcriptional changes and the pivotal contribution of cold-driven circadian rhythm-dependent pathways regulated by the mobility of cis-regulatory elements were predicted. These findings may help to develop potential genetic targets for breeding species with less bud dormancy to overcome the effects of short winters during global warming. Our artificial intelligence concept can improve epigenetic analysis using a small dataset, especially in non-model plants with immature genome databases.