De Novo Discovery of Cysteine Frameworks for Developing Multicyclic Peptide Libraries for Ligand Discovery.

Conserved cysteine frameworks are essential components of disulfide-rich peptides (DRPs), which dominantly define the structural diversity of both naturally occurring and de novo-designed DRPs. However, there are only very limited numbers of conserved cysteine frameworks, and general methods enabling de novo discovery of cysteine frameworks with robust foldability are still not available. Here, we devised a "touchstone"-based strategy that relies on chasing oxidative foldability between two individual disulfide-rich folds on the phage surface to discover new cysteine frameworks from random sequences. Unique cysteine frameworks with a high degree of compatibility with phage display systems and broad sequence tolerance were successfully identified, which were subsequently exploited for the development of multicyclic DRP libraries, enabling the rapid discovery of new peptide ligands with low-nanomolar and picomolar binding affinity. This study provides an unprecedented method for exploring and exploiting the sequence and structure space of DRPs that is not readily accessible by existing strategies, holding the potential to revolutionize the study of DRPs and significantly advance the design and discovery of multicyclic peptide ligands and drugs.

Selection and evolution of disulfide-rich peptides via cellular protein quality control.

Disulfide-rich peptides (DRPs) are an interesting and promising molecular format for drug discovery and development. However, the engineering and application of DRPs rely on the foldability of the peptides into specific structures with correct disulfide pairing, which strongly hinders the development of designed DRPs with randomly encoded sequences. Design or discovery of new DRPs with robust foldability would provide valuable scaffolds for developing peptide-based probes or therapeutics. Herein we report a cell-based selection system leveraging cellular protein quality control (termed PQC-select) to select DRPs with robust foldability from random sequences. By correlating the foldability of DRPs with their expression levels on the cell surface, thousands of sequences that can fold properly have been successfully identified. We anticipated that PQC-select will be applicable to many other designed DRP scaffolds in which the disulfide frameworks and/or the disulfide-directing motifs can be varied, enabling the generation of a variety of foldable DRPs with new structures and superior potential for further developments.

Disulfide-Directed Multicyclic Peptide Libraries for the Discovery of Peptide Ligands and Drugs.

Multicyclic peptides with stable 3D structures are a kind of novel and promising peptide formats for drug design and discovery as they have the potential to combine the best characteristics of small molecules and proteins. However, the development of multicyclic peptides is largely limited to naturally occurring products. It remains a big challenge to develop multicyclic peptides with new structures and functions without recourse to the existing natural scaffolds. Here, we report a general and robust method relying on the utility of new disulfide-directing motifs for designing and discovering diverse multicyclic peptides with potent protein-binding capability. These peptides, referred to as disulfide-directed multicyclic peptides (DDMPs), are tolerant to extensive sequence manipulations and variations of disulfide-pairing frameworks, enabling the development of de novo DDMP libraries useful for ligand and drug discovery. This study opens a new avenue for creating a new generation of multicyclic peptides in sequence and structure space inaccessible by natural scaffolds, thus would greatly benefit the field of peptide drug discovery.

Redox-Regulated Conformational Change of Disulfide-Rich Assembling Peptides.

Disulfide bond formation is a common mechanism for regulating conformational changes in proteins during oxidative folding. Despite extensive studies of the use of multiple disulfide bonds to constrain peptide conformation, few studies have explored their usage in developing self-assembling peptides. Herein, we report that a thiol-rich peptide could fold into an amphiphilic ß-hairpin conformation through the formation of two hetero-disulfide bonds upon oxidation, subsequently self-assembling into a mechanically rigid hydrogel. Breaking disulfide bonds under reductive condition, the hydrogel exhibited a transition from hydrogel to solution. Molecular simulation revealed that intermolecular interaction between two tryptophan residues was indispensable for hydrogelation. This work is the first case of the use of multiple disulfide bonds to control conformational change and self-assembly, and provides a cell-compatible hydrogel material for potential biomedical application.

Rapid and specific detection of Enterococcus faecalis with a visualized isothermal amplification method.

Enterococcus faecalis is a serious problem for hospitals and can spread from patient to patient. Most of the current detection methods are associated with limitations associated with the need for trained personnel; they are also time-consuming. Thus, it is necessary to develop rapid and accurate detection methods to control the spread of E. faecalis. In this study, we developed a rapid and accurate detection method for E. faecalis using recombinase polymerase amplification (RPA) combined with a lateral flow strip (LFS). This method could be completed in approximately 35 min at 37°C. The limit of detection was 10 CFU/µL, irrespective of whether the templates were pure or complex. This method also showed good specificity and compatibility. In total, 278 clinical samples were tested using the RPA-LFS method; the detection accuracy was equal to that of the conventional qPCR method. This visualized isothermal amplification method could be useful for the future on-site detection of E. faecalis.

Structure-guided design of CPPC-paired disulfide-rich peptide libraries for ligand and drug discovery.

Peptides constrained through multiple disulfides (or disulfide-rich peptides, DRPs) have been an emerging frontier for ligand and drug discovery. Such peptides have the potential to combine the binding capability of biologics with the stability and bioavailability of smaller molecules. However, DRPs with stable three-dimensional (3D) structures are usually of natural origin or engineered from natural ones. Here, we report the discovery and identification of CPPC (cysteine-proline-proline-cysteine) motif-directed DRPs with stable 3D structures (i.e., CPPC-DRPs). A range of new CPPC-DRPs were designed or selected from either random or structure-convergent peptide libraries. Thus, for the first time we revealed that the CPPC-DRPs can maintain diverse 3D structures by taking advantage of constraints from unique dimeric CPPC mini-loops, including irregular structures and regular α-helix and ß-sheet folds. New CPPC-DRPs that can specifically bind the receptors (CD28) on the cell surface were also successfully discovered and identified using our DRP-discovery platform. Overall, this study provides the basis for accessing an unconventional peptide structure space previously inaccessible by natural DRPs and computational designs, inspiring the development of new peptide ligands and therapeutics.

Fast and sensitive graphene oxide-DNAzyme-based biosensor for Vibrio alginolyticus detection.

DNAzymes have been widely and effectively used for the detection of pathogenic bacteria, which pose a serious public health threat. However, the rapid and cost-effective detection of such bacteria remains a major challenge. In this study, we successfully selected Vibrio alginolyticus-specific DNAzymes. The activity of the candidates was assessed via fluorescence intensity and gel electrophoresis. The DNAzyme DT1 had a detection limit of 31 CFU/ml for V. alginolyticus and exhibited high specificity. Graphene oxide (GO) was used to develop a DNAzyme-based fluorescent sensor for the detection of V. alginolyticus, which significantly improved detection performance and shortened the reaction time as little as 10 s. The proposed method was then validated using crab, shrimp, fish, clam, and oyster samples. This study thus provides a new method for the rapid and sensitive detection of V. alginolyticus.

Crystal structure of the polyketide cyclase from Mycobacterium tuberculosis.

About 40% of proteins are classified as conserved hypothetical proteins in Mycobacterium tuberculosis (TB). Identification and characterization of these proteins are beneficial to understand the pathogenesis of TB and exploiting novel drugs for TB treatments. The polyketide cyclase, a protein from M. tuberculosis ( MtPC) has been annotated as a hypothetical protein in Uniprot database. Sequence analysis shows that the MtPC belongs to the NTF2-like superfamily proteins with diverse functions. Here, we determined the crystal structure of MtPC at a resolution of 2.4 Šand measured backbone relaxation parameters for the MtPC protein. MtPC exists as a dimer in solution, and each subunit contains a six-stranded mixed ß-sheet and three α helixes which are arranged in the order α1-α2-ß1-ß2-α3-ß3-ß4-ß5-ß6. The NMR dynamics analysis showed that the overall structure of MtPC is highly rigid on ps-ns time scales. Furthermore, we predicted the potential function of MtPC based on the crystal structure. Our results lay the basis for further exploiting and mechanistically understanding the biological functions of MtPC.

An evolution-inspired strategy to design disulfide-rich peptides tolerant to extensive sequence manipulation.

Natural disulfide-rich peptides (DRPs) are valuable scaffolds for the development of new bioactive molecules and therapeutics. However, there are only a limited number of topologically distinct DRP folds in nature, and most of them suffer from the problem of in vitro oxidative folding. Thus, strategies to design DRPs with new constrained topologies beyond the scope of natural folds are desired. Herein we report a general evolution-inspired strategy to design new DRPs with diverse disulfide frameworks, which relies on the incorporation of two cysteine residues and a random peptide sequence into a precursor disulfide-stabilized fold. These peptides can spontaneously fold in redox buffers to the expected tricyclic topologies with high yields. Moreover, we demonstrated that these DRPs can be used as templates for the construction of phage-displayed peptide libraries, enabling the discovery of new DRP ligands from fully randomized sequences. This study thus paves the way for the development of new DRP ligands and therapeutics with structures not derived from natural DRPs.

Fast and Selective Reaction of 2-Benzylacrylaldehyde with 1,2-Aminothiol for Stable N-Terminal Cysteine Modification and Peptide Cyclization.

N-terminal cysteine (Cys)-specific reactions have been exploited for protein and peptide modifications. However, existing reactions for N-terminal Cys suffer from low reaction rate, unavoidable side reactions, or poor stability for reagents or products. Herein we report a fast, efficient, and selective conjugation between 2-benzylacrylaldehyde (BAA) and 1,2-aminothiol, which involves multistep reactions including aldimine condensation, Michael addition, and reduction of imine by NaBH3CN. This conjugation proceeds with a rate constant of ∼2700 M-1 s-1 under neutral condition at room temperature to produce a pair of seven-membered ring diastereoisomers, which are stable under neutral and acidic conditions. This method enables the selective modifications of the N-terminal Cys residue without interference from the internal Cys and lysine residues, providing a useful alternative to existing approaches for site-specific peptide or protein modifications and synthesis of cyclic peptides.

1H, 13C, 15N backbone and side-chain chemical shift assignments of the polyketide cyclase from Mycobacterium tuberculosis.

Polyketide cyclase from Mycobacterium tuberculosis (MtPC) is related to the formation of sterol derivatives, which may play a role in immune escape in the initial stage of macrophage infection by Mycobacterium tuberculosis. However, the structure and specific functions of MtPC are still unknown. Here we report the backbone and side-chain NMR resonance assignments for the MtPC. Most resonances were assigned and the secondary structure was predicted according to the assigned backbone resonances by TALOS-N and PECAN. These NMR assignments represent a first step towards researching the structure and function of MtPC.

Rapid detection of Pseudomonas aeruginosa using a DNAzyme-based sensor.

In the present study, a DNAzyme was screened in vitro through the use of a DNA library and crude extracellular mixture (CEM) of Pseudomonas aeruginosa. Following eight rounds of selection, a DNAzyme termed PAE-1 was obtained, which displayed high rates of cleavage with strong specificity. A fluorescent biosensor was designed for the detection of P. aeruginosa in combination with the DNAzyme. A detection limit as low as 1.2 cfu/ml was observed. Using proteases and filtration, it was determined that the target was a protein with a molecular weight of 10 kDa-50 kDa. The DNAzyme was combined with a polystyrene board to construct a simple indicator plate sensor which produced a color that identified the target within 10 min. The results were reliable when tap water and food samples were tested. The present study provides a novel experimental strategy for the development of sensors based on a DNAzyme to rapidly detect P. aeruginosa in the field.

Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor.

Vibrio vulnificus is an important pathogenic bacterium that is often associated with seafood-borne illnesses. Therefore, to detect this pathogen in aquatic products, a DNAzyme-based fluorescent sensor was developed for the in vitro detection of V. vulnificus. After screening and mutation, a DNAzyme that we denominated "RFD-VV-M2" exhibited the highest activity, specificity, and sensitivity. The limit of detection was 2.2 × 103 CFU/ml, and results could be obtained within 5-10 min. Our findings suggested that the target of DNAzyme RFD-VV-M2 was a protein with a molecular weight between 50 and 100 kDa. The proposed biosensor exhibited an excellent capacity to detect marine products contaminated with V. vulnificus. Therefore, our study established a rapid, simple, sensitive, and highly specific detection method for V. vulnificus in aquatic products.

Rapid Detection of Enterocytozoon hepatopenaei Infection in Shrimp With a Real-Time Isothermal Recombinase Polymerase Amplification Assay.

Enterocytozoon hepatopenaei (EHP) infection has become a significant threat in shrimp farming industry in recent years, causing major economic losses in Asian countries. As there are a lack of effective therapeutics, prevention of the infection with rapid and reliable pathogen detection methods is fundamental. Molecular detection methods based on polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) have been developed, but improvements on detection speed and convenience are still in demand. The isothermal recombinase polymerase amplification (RPA) assay derived from the recombination-dependent DNA replication (RDR) mechanism of bacteriophage T4 is promising, but the previously developed RPA assay for EHP detection read the signal by gel electrophoresis, which restricted this application to laboratory conditions and hampered the sensitivity. The present study combined fluorescence analysis with the RPA system and developed a real-time RPA assay for the detection of EHP. The detection procedure was completed in 3-7 min at 39°C and showed good specificity. The sensitivity of 13 gene copies per reaction was comparable to the current PCR- and LAMP-based methods, and was much improved than the RPA assay analyzed by gel electrophoresis. For real clinical samples, detection results of the real-time RPA assay were 100% consistent with the industrial standard nested PCR assay. Because of the rapid detection speed and the simple procedure, the real-time RPA assay developed in this study can be easily assembled as an efficient and reliable on-site detection tool to help control EHP infection in shrimp farms.

Rapid Detection of Helicobacter pylori by the Naked Eye Using DNA Aptamers.

Helicobacter pylori was first isolated from gastritis patients by Barry J. Marshall and J. Robin Warren in 1982, and more than 90% of duodenal ulcers and about 80% of gastric ulcers are caused by H. pylori infection. Most detection methods require sophisticated instruments and professional operators, making detection slow and expensive. Therefore, it is critical to develop a simple, fast, highly specific, and practical strategy for the detection of H. pylori. In this study, we used H. pylori as a target to select unique aptamers that can be used for the detection of H. pylori. In our study, we used random ssDNA as an initial library to screen nucleic acid aptamers for H. pylori. We used binding rate and the fluorescence intensity to identify candidate aptamers. One DNA aptamer, named HPA-2, was discovered through six rounds of positive selection and three rounds of negative selection, and it had the highest affinity constant of all aptamers tested (K d = 19.3 ± 3.2 nM). This aptamer could be used to detect H. pylori and showed no specificity for other bacteria. Moreover, we developed a new sensor to detect H. pylori with the naked eye for 5 min using illumination from a hand-held flashlight. Our study provides a framework for the development of other aptamer-based methods for the rapid detection of pathogenic bacteria.

Biophysical and functional characterizations of recombinant RimI acetyltransferase from Mycobacterium tuberculosis.

Nα-acetylation is a universal protein modification related to a wide range of physiological processes in eukaryotes and prokaryotes. RimI, an Nα-acetyltransferase in Mycobacterium tuberculosis, is responsible for the acetylation of the α-amino group of the N-terminal residue in the ribosomal protein S18. Despite growing evidence that protein acetylation may be correlated with the pathogenesis of tuberculosis, no structural information is yet available for mechanistically understanding the MtRimI acetylation. To enable structural studies for MtRimI, we constructed a serial of recombinant MtRimI proteins and assessed their biochemical properties. We then chose an optimal construct MtRimIC21A4-153 and expressed and purified the truncated high-quality protein for further biophysical and functional characterizations. The 2D 1H-15N heteronuclear single quantum coherence spectrum of MtRimIC21A4-153 exhibits wider chemical shift dispersion and favorable peak isolation, indicating that MtRimIC21A4-153 is amendable for further structural determination. Moreover, bio-layer interferometry experiments showed that MtRimIC21A4-153 possessed similar micromolar affinity to full-length MtRimI for binding the hexapeptide substrate Ala-Arg-Tyr-Phe-Arg-Arg. Enzyme kinetic assays also exhibited that MtRimIC21A4-153 had almost identical enzymatic activity to MtRimI, indicating insignificant influence of the recombinant variations on enzymatic functions. Furthermore, binding sites of the peptide were predicted by molecular docking approach, suggesting that this substrate binds to MtRimI primarily through electrostatic and hydrogen bonding interactions. Our results lay a foundation for the further structural determination and dynamics detection of MtRimI.

Solution structure and backbone dynamics for S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis.

The pro-drug pyrazinamide is hydrolyzed to pyrazinoic acid (POA) in its use for the treatment of tuberculosis. As a molecule with bactericidal activity, POA binds to the C-terminal S1 domain of ribosomal protein S1 from Mycobacterium tuberculosis (MtRpsACTD_S1) to inhibit trans-translation. Trans-translation is a critical component of protein synthesis quality control, and is mediated by transfer-messenger RNA. Here, we have determined the solution structure of MtRpsACTD_S1(280-368), and analyzed its structural dynamics by NMR spectroscopy. The solution structure of MtRpsACTD_S1(280-368) mainly consists of five anti-parallel ß strands, two α helices, and two 310 helices. Backbone dynamics reveals that the overall structure of MtRpsACTD_S1(280-368) is rigid, but segment L326-V333 undergoes large amplitude fluctuations on picosecond to nanosecond time scales. In addition, residues V321, H322, V331 and D335 with large Rex values exhibit significant chemical or conformational exchange on microsecond to millisecond time scale. Titration of the truncated MtRpsACTD_S1(280-368) with POA shows similar characteristics to titration of MtRpsACTD_S1(280-438) with POA. In addition, diverse length fragments of MtRpsACTD_S1 show various HN resonance signals, and we find that the interaction of MtRpsA(369-481) with MtRpsACTD_S1(280-368) [Kd = (4.25 ± 0.15) mM] is responsible for the structural difference between MtRpsACTD_S1(280-368) and MtRpsACTD_S1. This work may shed light on the underlying molecular mechanism of MtRpsACTD recognizing and binding POA or mRNA, as well as the detailed mechanism of interactions between MtRpsACTD_S1(280-368) and the additional C-terminal MtRpsA(369-481).

Selection of DNAzymes for Sensing Aquatic Bacteria: Vibrio Anguillarum.

Vibrio anguillarum is a bacterial pathogen that causes serious damage to aquatic fish, and its rapid detection and prevention are critical. DNAzymes are DNA-based catalysts with excellent stability. In this study, in vitro selection of DNAzymes was performed using the crude extracellular matrix (CEM) of V. Anguillarum as the target. Different from previous selections targeting bacterial CEM, this work used an unmodified DNA library, allowing easier adoption of the technology. After seven rounds of selection, a DNAzyme named VAE-2 with high activity and specificity was obtained. It showed the highest activity toward V. Anguillarum among eight types of tested bacterial strains. Polyvalent metal ions are needed for its activity. Protease treatment of the CEM and filtration studies indicated that the target is a protein with a molecular weight between 50 k and 100 k Da. A fluorescent biosensor was designed for V. anguillarum with a detection limit down to 4000 cfu/mL, and detection was demonstrated for real fish tissue and feeding water samples. Being the first work of DNAzyme-based sensing of aquatic bacteria, this study indicates that unmodified DNA can be used for targeting bacterial CEM, and it provides a new framework for developing other RNA-cleaving DNAzymes for rapid detection of pathogenic bacteria and water pollution.