Recognition Site Research Articles

Identification of Oligosaccharide Isomers Using Electrostatically Asymmetric OmpF Nanopore.

Glycans, unlike uniformly charged DNA and compositionally diverse peptides, are typically uncharged and exhibit rich stereoisomeric diversity in the glycosidic bonds between two monosaccharide units. This heterogeneity of charge and the structural complexity present significant challenges for accurate analysis. Herein, we developed a novel single-molecule oligosaccharide sensor, OmpF nanopore. The natural electroosmotic flow within OmpF provides robust driving force for unlabeled neutral oligosaccharides, accomplishing a detection concentration as low as 6.4 μM. Furthermore, the asymmetric constriction zone of OmpF was employed to construct a stereoselective recognition site, enabling sensitive identification of glycosidic bond differences in cell lysate samples. Assisted by machine learning algorithms, a recognition accuracy of 99.9% for tetrasaccharides differing in only one glycosidic bond was achieved. This nanopore sensor offers a highly sensitive analytical tool with a broad dynamic range for the chiral recognition of oligosaccharides at low concentrations, applicable to both low-abundance and practical samples.

Precise Electron-Withdrawing Strength Regulation of π-Conjugate Bridge-Boosted Specific Detection toward α-Methyltryptamine.

The specific fluorescent detection of α-methyltryptamine (AMT) presents a great challenge because similar amine groups and benzene rings exist in a variety of amines. Here, we show the precise modulation of the electron-withdrawing strength of the π-conjugate bridge in aldehyde-containing Schiff base-based fluorescent probes for ultratrace AMT discrimination. It is found that different electron-withdrawing groups -C6H4, -C6H2N2, and -C6H2Br2 as the π-conjugate bridge of the 2-dicyanomethylidene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran (TCF)-based probes can classify and identify organic amines with different amine nucleophilicities. Notably, the probe with -C6H2Br2 as the π-conjugate bridge, denoted as BrFS-TCF, which has the highest electrophilicity of the recognition site, shows a superior nM-level limit of detection (LOD) and an instant response time (<0.1 s) toward AMT. Especially, it shows an excellent selectivity facing the secondary amines, tertiary amines, aromatic amines, and even primary amines. The present strategy would provide a new pathway for chemical substances with similar structures and properties and especially for fighting against illegal drugs.

Alteration of the Catalytic Properties of the Epoxyalcohol Synthase CYP443D1 (NvEAS) of the Starlet Sea Anemone Nematostella vectensis as a Result of a Single Amino Acid Substitution.

Cytochromes of the P450 superfamily are widespread in nature; they were found in all studied aerobic organisms. Although the degree of similarity between cytochromes P450 of different families is low, all enzymes of this superfamily have similar tertiary structures. In addition, all cytochromes P450, including enzymes of the CYP74 clan, contain substrate recognition sites in their sequences, which form the catalytic center. Initially, CYP74 enzymes were discovered in plants, where they are widespread and play an important role in the lipoxygenase cascade. Later, CYP74-like enzymes of other families were identified in different taxa, including animals. Based on the results of phylogenetic studies, structures, and catalytic mechanisms, they were combined along with the CYP74 family into the CYP74 clan. One of the CYP74 clan enzymes is the epoxyalcohol synthase NvEAS (CYP443D1) of the starlet sea anemone Nematostella vectensis. A mutant form of NvEAS with a P93G substitution, that acquired additional hydroperoxide lyase activity, was obtained by site-directed mutagenesis. Before this work, only the results of site-directed mutagenesis of enzymes of the CYP74 family, but not of the CYP74 clan, were described. Moreover, in this work, the transformation of epoxyalcohol synthase into hydroperoxide lyase is described for the first time. These results confirm the previously stated assumption about the evolution of CYP74 enzymes, namely the epoxyalcohol synthase - hydroperoxide lyase - allene oxide synthase - divinyl ether synthase pathway.

Tailoring Chirality and Optimizing Enantioselective Recognition in Strategic Defect Engineering of Chiral Metal-Organic Frameworks.

Introducing chiral molecules into metal-organic frameworks (MOFs) to obtain chiral MOFs (CMOFs), the tunability of their structures makes them a highly anticipated class of chiral materials for electrochemical sensing. However, the structure of CMOFs is often limited by synthesis challenges, and introducing chiral molecules into MOFs often leads to a decrease in their internal space. This study introduces a defect engineering strategy into the synthesis of CMOFs to obtain CMOFs with defects, which is an efficient synthesis method. The two CMOFs constructed with different structures not only have more chiral recognition sites but also greatly increase the substrate capacity due to the defects, making them have a wide range of substrates and enhancing the enantioselective recognition effect of the two defective CMOFs. In addition, using MOF as a chiral carrier greatly overcomes the problem of low conductivity of chiral molecules. Based on the advantages of defective CMOFs, we have designed a novel chiral electrochemical sensor with an excellent enantiomer recognition performance. This study provides a simple and scalable synthetic method for constructing CMOFs with defects and high stability.

Solid-liquid Extraction of Lithium Chloride with a Simple and Flexible Heteroditopic Receptor

Abstract The global demand for lithium has surged in recent years due to its wide applications in industry and medicine. We have achieved selective solid-liquid extraction of LiCl using a heteroditopic receptor with an ether linker as a cation recognition site and two urea functionalities as cooperative anion recognition sites. The receptor exhibits high selectivity for LiCl from 1H NMR spectra by complexation in CD3CN and CDCl3. The solid-liquid extraction, followed by stripping to neutral aqueous phase using with the receptor revealed recovery of LiCl about 90% by ion chromatography and ICP-MS analyses, with a slight competition with MgCl2. LiCl was also recovered from the artificially prepared salt mixture and natural one from the Salar de Uyuni by the solid-liquid extraction with less competitive recovery of MgCl2.

A highly sensitive and fast-response fluorescence nanoprobe for in vivo imaging of hypochlorous acid.

Fluorescent probes for in vivo hypochlorous acid (HClO) imaging often face challenges of low selectivity and high cytotoxicity, largely due to poor analyte recognition and water-insoluble aromatic skeletons. To address this, we synthesized fluorescein hydrazide by introducing a spiro-lactam unit into fluorescein, which offers high emission intensity and molar absorption. The five-membered heterocycle in fluorescein hydrazide is selectively disrupted by HClO, enhancing the conjugated system and electron delocalization of the fluorophore, resulting in highly sensitive fluorescence detection of HClO. For in vivo imaging, fluorescein hydrazide was covalently grafted onto the surface of silica nanoparticles via nucleophilic substitution reaction, avoiding complex modifications. This fluorescent nanoprobe (Si-FL) leverages the high-density fluorophores and hydroxyl groups on the silica surface to enrich low-concentrations of HClO through weak supramolecular interactions, thereby accelerating the reaction between HClO and the recognition sites. Compared to other molecular probes, Si-FL demonstrates a superior response speed (within 20 s) and a lower detection limit (72 nM), alongside excellent biocompatibility and water solubility. The nanoprobe Si-FL was successfully applied for HClO detection in living cells, zebrafish, and plants, significantly improving the stability of fluorescence imaging.

One-Step Ethylene Purification from Ternary Mixture through Adaptive Recognition Sites.

One-step adsorptive purification of ethylene (C2H4) from ternary mixture comprising of acetylene (C2H2), ethylene (C2H4) and carbon dioxide (CO2) is a great challenge in the chemical industry. Herein, a microporous metal-organic framework (FJI-H38) has been reported, whichpossesses a high density of electronegative O/N binding sites and appropriate pore size. Notably, at 0.01 bar and 298 K FJI-H38 shows excellent trapping capability for C2H2 (1.64 mmol/g) and CO2 (2.33 mmol/g), while the uptake of C2H4 is only 0.41 mmol/g, which endows FJI-H38 high C2H2/C2H4 selectivity and top-level CO2/C2H4 simultaneously. Polymer-grade C2H4 (≥99.95%) with record-high productivity can be successfully obtained from ternary C2H2/CO2/C2H4 mixture in one step under various conditions. Even at 318 K, the separation performance has no obvious decrease. Such excellent separation performance is due to the adaptive recognition of C2H2 and CO2 by FJI-H38 through the synergistic effect of appropriate pore size and the match of electrostatic potentials, where C2H2 and CO2 can be stabilized by the O/N and aromatic ring sites.

One‐Step Ethylene Purification from Ternary Mixture through Adaptive Recognition Sites

One‐step adsorptive purification of ethylene (C2H4) from ternary mixture comprising of acetylene (C2H2), ethylene (C2H4) and carbon dioxide (CO2) is a great challenge in the chemical industry. Herein, a microporous metal‐organic framework (FJI‐H38) has been reported, which possesses a high density of electronegative O/N binding sites and appropriate pore size. Notably, at 0.01 bar and 298 K FJI‐H38 shows excellent trapping capability for C2H2 (1.64 mmol/g) and CO2 (2.33 mmol/g), while the uptake of C2H4 is only 0.41 mmol/g, which endows FJI‐H38 high C2H2/C2H4 selectivity and top‐level CO2/C2H4 simultaneously. Polymer‐grade C2H4 (≥99.95%) with record‐high productivity can be successfully obtained from ternary C2H2/CO2/C2H4 mixture in one step under various conditions. Even at 318 K, the separation performance has no obvious decrease. Such excellent separation performance is due to the adaptive recognition of C2H2 and CO2 by FJI‐H38 through the synergistic effect of appropriate pore size and the match of electrostatic potentials, where C2H2 and CO2 can be stabilized by the O/N and aromatic ring sites.

The Role of nsp5 in SARS-CoV-2

SARS-CoV-2, the causative agent of the COVID-19 pandemic, is a single-stranded positive-sense RNA virus belonging to the coronavirus family. It emerged at the end of 2019 and has swiftly disseminated globally, presenting an unparalleled threat to public health worldwide. The non-structural protein 5 (nsp5) is a key component in the SARS-CoV-2 replication process, also known as the main protease (Mpro or 3CLpro), which is essential for cleaving viral polyproteins into functional proteins required for replication and transcription. The conserved characteristics and essential role of nsp5 render it an attractive target for antiviral pharmacological development. Recent developments in nsp5-targeted treatments, like Paxlovid, exhibit significant effectiveness in decreasing COVID-19-associated hospitalizations and fatalities. However, the development of nsp5 inhibitors continues to face challenges due to viral mutations and specific administration requirements. This review explores the structural and biochemical properties of nsp5, with particular attention to its catalytic dyad, substrate recognition sites, and three-domain architecture that underpin its role in viral replication. It highlights the potential impact of nsp5 research on advancing antiviral strategies, aiding in the fight against current SARS-CoV-2 infections, and laying the groundwork for future coronavirus outbreak preparedness.

Programmable DNA Nanoswitch-Regulated Plasmonic CRISPR/Cas12a-Gold Nanostars Reporter Platform for Nucleic Acid and Non-Nucleic Acid Biomarker Analysis Assisted by a Spatial Confinement Effect.

CRISPR/Cas 12a system based nucleic acid and non-nucleic acid targets detection faces two challenges including (1) multiple crRNAs are needed for multiple biomarkers detection and (2) insufficient sensitivity resulted from photobleaching of fluorescent dyes and the low kinetic cleavage rate for a traditional single-strand (ssDNA) reporter. To address these limitations, we developed a programmable DNA nanoswitch (NS)-regulated plasmonic CRISPR/Cas12a-gold nanostars (Au NSTs) reporter platform for detection of nucleic acid and non-nucleic acid biomarkers with the assistance of the spatial confinement effect. Through simply programming the target recognition sequence in NS, only one crRNA is required to detect both nucleic acid and non-nucleic acid biomarkers. The detection limit decreased by ∼196-fold for miRNA-375 and 122-fold for prostate-specific antigen (PSA), respectively. Moreover, versatile evaluation of miRNA-375 and PSA in clinical urine samples can also be achieved, according to which prostate cancer and healthy groups can be well identified.

NADH-bound AIF activates the mitochondrial CHCHD4/MIA40 chaperone by a substrate-mimicry mechanism.

Mitochondrial metabolism requires the chaperoned import of disulfide-stabilized proteins via CHCHD4/MIA40 and its enigmatic interaction with oxidoreductase Apoptosis-inducing factor (AIF). By crystallizing human CHCHD4's AIF-interaction domain with an activated AIF dimer, we uncover how NADH allosterically configures AIF to anchor CHCHD4's β-hairpin and histidine-helix motifs to the inner mitochondrial membrane. The structure further reveals a similarity between the AIF-interaction domain and recognition sequences of CHCHD4 substrates. NMR and X-ray scattering (SAXS) solution measurements, mutational analyses, and biochemistry show that the substrate-mimicking AIF-interaction domain shields CHCHD4's redox-sensitive active site. Disrupting this shield critically activates CHCHD4 substrate affinity and chaperone activity. Regulatory-domain sequestration by NADH-activated AIF directly stimulates chaperone binding and folding, revealing how AIF mediates CHCHD4 mitochondrial import. These results establish AIF as an integral component of the metazoan disulfide relay and point to NADH-activated dimeric AIF as an organizational import center for CHCHD4 and its substrates. Importantly, AIF regulation of CHCHD4 directly links AIF's cellular NAD(H) sensing to CHCHD4 chaperone function, suggesting a mechanism to balance tissue-specific oxidative phosphorylation (OXPHOS) capacity with NADH availability.

Nup107 contributes to the maternal to zygotic transition by preventing the premature nuclear export of pri-miRNA 427.

Emerging evidence suggests that the nuclear pore complex can have unique compositions and distinct nucleoporin functions in different cells. Here, we show that Nup107, a key component of the NPC scaffold, varies in expression over development: it is expressed at higher levels in the blastula compared to the gastrula suggesting a critical role prior to gastrulation. We find depletion of Nup107 affects the differentiation of the early germ layers leading to an expansion of the ectoderm at the expense of endoderm and mesoderm. By analyzing an RNAseq time course, we observed that depletion of Nup107 affects the maternal-zygotic transition by delaying the degradation of maternal transcripts that occurs as zygotic transcription begins. The transcripts are enriched in miR427 recognition sites, a conserved microRNA that destabilizes maternal transcripts including REST, which encodes a Kruppel-type zinc finger transcription factor that we demonstrate is critical for ectodermal cell fates. Mechanistically, we show that Nup107 is required to prevent the premature export of pri-miR427 transcript before processing. Nup107 depletion leads to the reduced production of mature miR427 and maternal transcript stabilization. We conclude that high levels of Nup107 in the early embryo are critical for the nuclear retention and subsequent processing of pri-miR427 transcripts that is required for timely maternal RNA clearance to enable gastrulation.

Surprising features of nuclear receptor interaction networks revealed by live-cell single-molecule imaging.

Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

The Construction of Heterothallic Strains of Komagataella kurtzmanii Using the I-SceI Meganuclease.

The methylotrophic yeast Komagataella kurtzmanii belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typically contain a mixture of sexually dimorphic cells that are prone to self-diploidisation and spore formation accompanied by genetic rearrangements. These characteristics pose a significant challenge to the development of genetically stable producers for industrial use. In the present study, we constructed heterothallic strains of K. kurtzmanii, ensuring a constant mating type by unifying the genetic sequences in the active and silent MAT loci. To obtain such strains, we performed site-directed inactivation of one of the two yeast MAT loci, replacing its sequence with a selective HIS4 gene surrounded by I-SceI meganuclease recognition sites. We then used transient expression of the SCE1 gene, encoding a recombinant I-SceI meganuclease, to induce site-specific cleavage of HIS4, followed by damage repair by homologous recombination in mutant cells. As a result, heterothallic strains designated 'Y-727-2(alpha)' and 'Y-727-9(a)', which correspond to the α and a mating type, respectively, were obtained. The strains demonstrated a loss of the ability to self-diploidize. The results of PCR and whole genome analysis confirmed the identity of the contents of the MAT loci. Analysis of the genomes of the final strains, however, revealed a fusion of chromosome 3 and chromosome 4 in strain Y-727-2(alpha)-1. This finding was subsequently confirmed by pulsed-field gel electrophoresis of yeast chromosomes. However, the ability of the Y-727-2(alpha)-derived producers to efficiently secrete recombinant β-galactosidase was unaffected by this genomic rearrangement.

Cholesterol Attenuates the Pore-Forming Capacity of CARC-Containing Amphipathic Peptides.

Artificial peptides P4, A1 and A4 are homologous to amphipathic α-helical fragments of the influenza virus M1 protein. P4 and A4 contain the cholesterol recognition sequence CARC, which is absent in A1. As shown previously, P4 and A4 but not A1 have cytotoxic effects on some eukaryotic and bacterial cells. This might be caused by the dysfunction of cholesterol-dependent cellular structures, inhibition of the respiratory chain, or disruption of the membrane. Here, we analyzed the latter hypothesis by studying the uncoupling effect of the peptides on asolectin membranes. The influence of A4 on Δψ pre-formed either by the valinomycin-dependent K+ diffusion or by the activity of membrane-built cytochrome c oxidase (CcO) was studied on (proteo)liposomes. Also, we investigated the effect of P4, A1 and A4 on liposomes loaded with calcein. It is found that A4 in a submicromolar range causes an immediate and complete dissipation of diffusion Δψ across the liposomal membrane. Uncoupling of the CcO-containing proteoliposomes requires an order of magnitude of higher peptide concentration, which may indicate the sorption of A4 on the enzyme. The presence of cholesterol in the membrane significantly weakens the uncoupling. Submicromolar A4 and P4 cause the release of calcein from liposomes, indicating the formation of membrane pores. The process develops in minutes and is significantly decelerated by cholesterol. Micromolar A1 induces pore formation in a cholesterol-independent manner. We conclude that the peptides P4, A4 and, in higher concentrations, A1 form pores in the asolectin membrane. The CARC-mediated interaction of A4 and P4 with cholesterol impedes the peptide oligomerization necessary for pore formation. The rapid uncoupling effect of A4 is apparently caused by an increase in the proton conductivity of the membrane without pore formation.

Machine Learning Recognition of Artificial DNA Sequence with Quantum Tunneling Nanogap Junction.

Artificially synthesized DNA holds significant promise in addressing fundamental biochemical questions and driving advancements in biotechnology, genetics, and DNA digital data storage. Rapid and precise electric identification of these artificial DNA strands is crucial for their effective application. Herein, we present a comprehensive investigation into the electric recognition of eight artificial synthesized DNA (xDNA and yDNA) nucleobases using quantum tunneling transport and machine learning (ML) techniques. By embedding these nucleobases within a solid-state nanogap junction, we calculated their fingerprint transmission and current readouts and also analyzed the influence of electronic coupling and molecular orbital delocalization on these properties. The trained ML model achieved a predictive basecalling accuracy of up to 100% for xDNA nucleobases and 99.80% for yDNA transmission readout data sets. ML explainability study revealed that normalized descriptors have a greater impact on nucleobase prediction than the original transmission function, proving more effective in disentangling overlapping artificial DNA nucleobase signals. Quaternary classification results highlighted higher recognition accuracy for xDNA nucleobases than for yDNA nucleobases. Furthermore, precise calling of complementary, purine, and pyrimidine base pair combinations was demonstrated with high sensitivity and an F1 score. Our findings reveal the feasibility of highly sensitive and precise electrical recognition of artificial DNA nucleobases, which can transform genetic research and spur advancements in genetic data storage, synthetic biology, and diagnostics.

High Selectivity Fluorescence and Electrochemical Dual-Mode Detection of Glutathione in the Serum of Parkinson's Disease Model Mice and Humans.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by the loss of dopaminergic neurons and the accumulation of alpha-synuclein. Glutathione (GSH), a key antioxidant, is significantly depleted in PD patients. This study presents a dual-mode detection strategy for selectively determining GSH using a single probe. A series of "turn-on" electrochemical and fluorescent probes were developed, with resorufin (Re) serving as the reporting unit and featuring specific GSH recognition sites. Among these, the 7-(3,5-dinitrophenoxy)-3H-phenoxazin-3-one (Re-DNP) probe was selected for its high selectivity as both a fluorescent and electrochemical probe. Its response to GSH was superior in comparison to that observed for hydrogen sulfide (H2S) and cysteine (Cys). For electrochemical detection using screen-printed carbon electrode (SPCE)/carbon nanotube (CNT) modified electrodes, the detection limit for GSH was 5 μM, with a linear range of 25-500 μM. In fluorescence detection, the probe produced a 78-fold increase in emission at 630 nm in the presence of GSH, with a strong linear correlation between fluorescence intensity and GSH concentration in the range of 10-700 μM, and a detection limit of 2 μM. When applied to real clinical serum samples, the probe demonstrated significantly lower GSH levels in both PD mice and human patients compared to healthy controls. This dual-mode detection method provides a sensitive and accurate tool for GSH detection, with potential applications in understanding GSH's role in PD and related neurodegenerative diseases.

De novo design of ribosomally synthesized and post-translationally modified peptides.

In nature, peptides are enzymatically modified to constrain their structure and introduce functional moieties. De novo peptide structures could be built by combining enzymes from different pathways, but determining the rules of their use is difficult. We present a biophysical model to combine enzymes sourced from bacterial ribosomally synthesized and post-translationally modified peptide (RiPP) gene clusters. Using a pipeline to evaluate more than 1,000 peptides, the model was parameterized under uniform conditions in Escherichia coli for enzymes from different classes (graspetide, spliceotide, pantocin, cyanobactin, glycocin, lasso peptide and lanthipeptide). Synthetic leader peptides with recognition sequences for up to three enzymes were designed to modify core sequences sharing no identity to natural RiPPs. Empirically, RiPPs with the desired modifications constituted 7-67% of the total peptides produced, and 6 of our 8 peptide designs were successfully modified. This work is an example of the design of enzyme-modified peptides and libraries, using a framework that can be expanded to include new enzymes and chemical moieties.

Accurate sequence-to-affinity models for SH2 domains from multi-round peptide binding assays coupled with free-energy regression.

Short linear peptide motifs play important roles in cell signaling. They can act as modification sites for enzymes and as recognition sites for peptide binding domains. SH2 domains bind specifically to tyrosine-phosphorylated proteins, with the affinity of the interaction depending strongly on the flanking sequence. Quantifying this sequence specificity is critical for deciphering phosphotyrosine-dependent signaling networks. In recent years, protein display technologies and deep sequencing have allowed researchers to profile SH2 domain binding across thousands of candidate ligands. Here, we present a concerted experimental and computational strategy that improves the predictive power of SH2 specificity profiling. Through multi-round affinity selection and deep sequencing with large randomized phosphopeptide libraries, we produce suitable data to train an additive binding free energy model that covers the full theoretical ligand sequence space. Our models can be used to predict signaling network connectivity and the impact of missense variants in phosphoproteins on SH2 binding.

Flexibility in PAM Recognition Expands DNA Targeting in xCas9.

xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.