Synthetic molecular switches driven by DNA-modifying enzymes.

Taking inspiration from natural systems, in which molecular switches are ubiquitous in the biochemistry regulatory network, we aim to design and construct synthetic molecular switches driven by DNA-modifying enzymes, such as DNA polymerase and nicking endonuclease. The enzymatic treatments on our synthetic DNA constructs controllably switch ON or OFF the sticky end cohesion and in turn cascade to the structural association or disassociation. Here we showcase the concept in multiple DNA nanostructure systems with robust assembly/disassembly performance. The switch mechanisms are first illustrated in minimalist systems with a few DNA strands. Then the ON/OFF switches are realized in complex DNA lattice and origami systems with designated morphological changes responsive to the specific enzymatic treatments.

Synthesis and DNase I Inhibitory Properties of New Squaramides.

Three new monosquaramides (3a-c) were synthesized, characterized by IR, NMR and X-ray, and evaluated for inhibitory activity against deoxyribonuclease I (DNase I) and xanthine oxidase (XO) in vitro. The target compounds inhibited DNase I with IC50 values below 100 µM, being at the same time more potent DNase I inhibitors than crystal violet, used as a positive control. 3-Ethoxy-4-((1-(pyridin-3-yl)propan-2-yl)amino)cyclobut-3-ene-1,2-dione (3c) stood out as the most potent compound, exhibiting a slightly better IC50 value (48.04 ± 7.98 µM) compared to the other two compounds. In order to analyze potential binding sites for the studied compounds with DNase I, a molecular docking study was performed. Compounds 3a-c are among the most potent small organic DNase I inhibitors tested to date.

Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA.

RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies1,2. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development3. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain4. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.

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.

Repurposing of 8-Hydroxyquinoline-Based Butyrylcholinesterase and Cathepsin B Ligands as Potent Nonpeptidic Deoxyribonuclease I Inhibitors.

A library of 31 butyrylcholinesterase (BChE) and cathepsin B (CatB) inhibitors was screened in vitro for inhibition of deoxyribonuclease I (DNase I). Compounds 22, 8 and 7 are among the most potent synthetic non-peptide DNase I inhibitors reported to date. Three 8-hydroxyquinoline analogues inhibited both DNase I and BChE with IC50 values below 35 µM and 50 nM, respectively, while two nitroxoline derivatives inhibited DNase I and Cat B endopeptidase activity with IC50 values below 60 and 20 µM. Selected derivatives were screened for various co-target binding affinities at dopamine D2 and D3 , histamine H3 and H4 receptors and inhibition of 5-lipoxygenase. Compound 8 bound to the H3 receptor and is highlighted as the most promising multifunctional ligand with a favorable pharmacokinetic profile and one of the most potent non-peptide DNase I inhibitors. The present study demonstrates that 8-hydroxyquinoline is a structural fragment critical for DNase I inhibition in the presented series of compounds.

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.

Comparison of the secretory murine DNase1 family members expressed in Pichia pastoris.

Soluble nucleases of the deoxyribonuclease 1 (DNase1) family facilitate DNA and chromatin disposal (chromatinolysis) during certain forms of cell differentiation and death and participate in the suppression of anti-nuclear autoimmunity as well as thrombotic microangiopathies caused by aggregated neutrophil extracellular traps. Since a systematic and direct comparison of the specific activities and properties of the secretory DNase1 family members is still missing, we expressed and purified recombinant murine DNase1 (rmDNase1), DNase1-like 2 (rmDNase1L2) and DNase1-like 3 (rmDNase1L3) using Pichia pastoris. Employing different strategies for optimizing culture and purification conditions, we achieved yields of pure protein between ~3 mg/l (rmDNase1L2 and rmDNase1L3) and ~9 mg/l (rmDNase1) expression medium. Furthermore, we established a procedure for post-expressional maturation of pre-mature DNase still bound to an unprocessed tri-N-glycosylated pro-peptide of the yeast α-mating factor. We analyzed glycosylation profiles and determined specific DNase activities by the hyperchromicity assay. Additionally, we evaluated substrate specificities under various conditions at equimolar DNase isoform concentrations by lambda DNA and chromatin digestion assays in the presence and absence of heparin and monomeric skeletal muscle α-actin. Our results suggest that due to its biochemical properties mDNase1L2 can be regarded as an evolutionary intermediate isoform of mDNase1 and mDNase1L3. Consequently, our data show that the secretory DNase1 family members complement each other to achieve optimal DNA degradation and chromatinolysis under a broad spectrum of biological conditions.

Synthesis and analysis of 4-oxothiazolidines as potential dual inhibitors of deoxyribonuclease I and xanthine oxidase.

In this study, seven new 4-oxothiazolidine derivatives were synthesized and assayed, along 7 known derivatives, for inhibitory properties against deoxyribonuclease I (DNase I) and xanthine oxidase (XO) in vitro. Among tested compounds, (5Z)-Ethyl-2-(2-(cyanomethylene)-4-oxothiazolidin-5-yliden)acetate (6) exhibited inhibitory activity against both enzymes (DNase I IC50 = 67.94 ± 5.99 µM; XO IC50 = 98.98 ± 13.47 µM), therefore being the first reported dual inhibitor of DNase I and XO. Observed DNase I inhibition qualifies compound 6 as the most potent small organic DNase I inhibitor reported so far. Derivatives of 2-alkyliden-4-oxothiazolidinone (1) inhibited DNase I below 200 µM, while the other tested 4-oxothiazolidine derivatives remained inactive against both enzymes. The molecular docking and molecular dynamics simulations into the binding sites of DNase I and XO enzyme allowed us to clarify the binding modes of this 4-oxothiazolidine derivative, which might aid future development of dual DNase I and XO.

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.

The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence.

Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.

CEPZ: A Novel Predictor for Identification of DNase I Hypersensitive Sites.

DNase I hypersensitive sites (DHSs) have proven to be tightly associated with cis-regulatory elements, commonly indicating specific function on the chromatin structure. Thus, identifying DHSs plays a fundamental role in decoding gene regulatory behavior. While traditional experimental methods turn to be time-consuming and expensive, computational techniques promise to be practical to discovering and analyzing regulatory factors. In this study, we applied an efficient model that considered composition information and physicochemical properties and effectively selected features with a boosting algorithm. CEPZ, our predictor, greatly improved a Matthews correlation coefficient and accuracy of 0.7740 and 0.9113 respectively, more competitive than any predictor before. This result suggests that it may become a useful tool for DHSs research in the human and other complex genomes. Our research was anchored on the properties of dinucleotides and we identified several dinucleotides with significant differences in the distribution of DHS and non-DHS samples, which are likely to have a special meaning in the chromatin structure. The datasets, feature sets and the relevant algorithm are available at https://github.com/YanZheng-16/CEPZ_DHS/.

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.

Pseudomonas putida MPE, a manganese-dependent endonuclease of the binuclear metallophosphoesterase superfamily, incises single-strand DNA in two orientations to yield a mixture of 3'-PO4 and 3'-OH termini.

Pseudomonas putida MPE exemplifies a novel clade of manganese-dependent single-strand DNA endonuclease within the binuclear metallophosphoesterase superfamily. MPE is encoded within a widely conserved DNA repair operon. Via structure-guided mutagenesis, we identify His113 and His81 as essential for DNA nuclease activity, albeit inessential for hydrolysis of bis-p-nitrophenylphosphate. We propose that His113 contacts the scissile phosphodiester and serves as a general acid catalyst to expel the OH leaving group of the product strand. We find that MPE cleaves the 3' and 5' single-strands of tailed duplex DNAs and that MPE can sense and incise duplexes at sites of short mismatch bulges and opposite a nick. We show that MPE is an ambidextrous phosphodiesterase capable of hydrolyzing the ssDNA backbone in either orientation to generate a mixture of 3'-OH and 3'-PO4 cleavage products. The directionality of phosphodiester hydrolysis is dictated by the orientation of the water nucleophile vis-à-vis the OH leaving group, which must be near apical for the reaction to proceed. We propose that the MPE active site and metal-bound water nucleophile are invariant and the enzyme can bind the ssDNA productively in opposite orientations.

Structural basis for two metal-ion catalysis of DNA cleavage by Cas12i2.

To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2-crRNA-DNA complexes and a Cas12i2-crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1-8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9-15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.

Nuclease Degradation Analysis of DNA Nanostructures Using Gel Electrophoresis.

Custom-built DNA nanostructures are now used in applications such as biosensing, molecular computation, biomolecular analysis, and drug delivery. While the functionality and biocompatibility of DNA makes DNA nanostructures useful in such applications, the field faces a challenge in making biostable DNA nanostructures. Being a natural material, DNA is most suited for biological applications, but is also easily degraded by nucleases. Several methods have been employed to study the nuclease degradation rates and enhancement of nuclease resistance. This protocol describes the use of gel electrophoresis to analyze the extent of nuclease degradation of DNA nanostructures and to report degradation times, kinetics of nuclease digestion, and evaluation of biostability enhancement factors. © 2020 Wiley Periodicals LLC. Basic Protocol: Timed analysis of nuclease degradation of DNA nanostructures Support Protocol: Calculating biostability enhancement factors.

Molecular Structure and Functional Analysis of Pyocin S8 from Pseudomonas aeruginosa Reveals the Essential Requirement of a Glutamate Residue in the H-N-H Motif for DNase Activity.

Multidrug resistance (MDR) is a serious threat to public health, making the development of new antimicrobials an urgent necessity. Pyocins are protein antibiotics produced by Pseudomonas aeruginosa strains to kill closely related cells during intraspecific competition. Here, we report an in-depth biochemical, microbicidal, and structural characterization of a new S-type pyocin, named S8. Initially, we described the domain organization and secondary structure of S8. Subsequently, we observed that a recombinant S8 composed of the killing subunit in complex with the immunity (ImS8) protein killed the strain PAO1. Furthermore, mutation of a highly conserved glutamic acid to alanine (Glu100Ala) completely inhibited this antimicrobial activity. The integrity of the H-N-H motif is probably essential in the killing activity of S8, as Glu100 is a highly conserved residue of this motif. Next, we observed that S8 is a metal-dependent endonuclease, as EDTA treatment abolished its ability to cleave supercoiled pUC18 plasmid. Supplementation of apo S8 with Ni2+ strongly induced this DNase activity, whereas Mn2+ and Mg2+ exhibited moderate effects and Zn2+ was inhibitory. Additionally, S8 bound Zn2+ with a higher affinity than Ni2+ and the Glu100Ala mutation decreased the affinity of S8 for these metals, as shown by isothermal titration calorimetry (ITC). Finally, we describe the crystal structure of the Glu100Ala S8 DNase-ImS8 complex at 1.38 Å, which gave us new insights into the endonuclease activity of S8. Our results reinforce the possibility of using pyocin S8 as an alternative therapy for infections caused by MDR strains, while leaving commensal human microbiota intact.IMPORTANCE Pyocins are proteins produced by Pseudomonas aeruginosa strains that participate in intraspecific competition and host-pathogen interactions. They were first described in the 1950s and since then have gained attention as possible new antibiotics. However, there is still only scarce information about the molecular mechanisms by which these molecules induce cell death. Here, we show that the metal-dependent endonuclease activity of pyocin S8 is involved with its antimicrobial action against strain PAO1. We also describe that this killing activity is dependent on a conserved Glu residue within the H-N-H motif. The potency and selectivity of pyocin S8 toward a narrow spectrum of P. aeruginosa strains make this protein an attractive antimicrobial alternative for combatting MDR strains, while leaving commensal human microbiota intact.

Construction of Specific and Reversible Nanoreceptors for Proteins via Sequential Surface-Imprinting Strategy.

Molecular recognition of proteins is critical for study and manipulation of protein-related biological processes. However, design and synthesis of abiotic receptors for precise recognition of proteins still remains a challenging task. Herein, we developed a universal sequential surface-imprinting strategy that integrated two different types of imprinting reactions to construct artificial protein receptors with high selectivity. Employing dopamine self-polymerization and boronate/diol complexation as the first-step and second-step imprinting reactions, respectively, we synthesized surface-imprinted magnetic nanocomposites against two different enzyme proteins: deoxyribonuclease I (DNase I) and apurinic/apyrimidinic endonuclease/redox effector factor 1 (APE1). The obtained nanocomposites both showed strong and specific binding toward their respective template proteins. Moreover, the bound enzymes could be totally recovered with high activity under mild buffer conditions. These antibody-like specific and reversible binding properties enabled effective purification and enrichment of the low-abundance target proteins from complex serum samples. Compared to existing one-pot or one-step imprinting methods, the proposed sequential surface-imprinting approach offers a more flexible combination of different functional monomers and greatly enhances the performance and biocompatibility of the imprinted materials. The generality and simplicity of the sequential imprinting strategy would make it an appealing and competitive method to prepare artificial protein receptors.