Ultrasensitive Scaffold-Dependent Protease Sensors with Large

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Ultrasensitive Scaffold Dependent Protease Sensors with Large Dynamic Range Viktor Stein, Masuda Nabi, and Kirill Alexandrov ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00370 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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ACS Synthetic Biology

Title Ultrasensitive Scaffold Dependent Protease Sensors with Large Dynamic Range

Author Viktor Stein1*†, Masuda Nabi1, Kirill Alexandrov1*

Author Affiliations 1

Institute for Molecular Biosciences, The University of Queensland, QBP Building 80, QLD 4072

St Lucia, Australia.

†Present Address: Technical University of Darmstadt, Faculty of Biology, Schni4spahnstrasse 3, 64287 Darmstadt, Germany.

Corresponding Authors Viktor Stein: [email protected] Kirill Alexandrov: [email protected]

Key Words Protein Switches, Protein Engineering, Proteases, Synthetic Biology, Molecular Diagnostics

1 ACS Paragon Plus Environment


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Abstract The rational construction of synthetic protein switches with pre-defined input-output parameters constitutes a key goal of synthetic biology with many potential applications ranging from metabolic engineering to diagnostics. Yet, generally applicable strategies to construct tailor-engineered protein switches have so far remained elusive. Here, we use SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffolddependent protease sensors that exploit a combination of affinity targeting and proteaseinducible protein-protein interactions. We use this architecture to create a suite of integrated signal sensing and amplification circuits that can detect the activity of α-thrombin and prostate specific antigen with a dynamic range covering five orders of magnitude. We determine the key design features critical for signal transmission between protease-based sensors, transducers and actuators.


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Manuscript The rational design of artificial signaling modules constitutes a key goal in synthetic biology which has been predominantly realized in the construction of synthetic genetic circuits1,2 and more recently allosterically regulated RNA3,4. However, the majority of biological signaling events is relayed through tightly regulated networks of interacting proteins. Yet, the idiosyncratic relationship of protein sequence, structure and function has so far prevented formulation of generally applicable strategies for designing signaling proteins with tailored input-output functions and signaling motifs5. To address these challenges, we recently developed a toolbox based on artificially autoinhibited proteases that can be assembled into protease-inducible signal transducers, allosterically regulated protease receptors and proximity sensors6. The elementary protease sensors are based on autoinhibited protease modules derived from the NIa protease of Tobacco Vein Mottling Virus (TVMV) or the NS3 protease of Hepatitis C Virus (HCV). In these designs, the viral proteases are connected to an engineered competitive autoinhibition (AI) domain which binds and blocks their active site. The activity of proteases can be modulated by different molecular inputs through the receptor elements in the connecting linker. In terms of applications, tailored protein switches and thereof assembled signaling circuits can be developed into diagnostic agents to detect molecular analytes in an autonomous and integrated fashion similar to naturally occurring signal transduction circuits, and thus provide an alternative to complex multistep assays procedures such as enzyme linked immunosorbent assays (ELISAs). We thus exploit our modular signaling toolbox to engineer a set of proteasebased sensors and signal amplification circuits to monitor the activity of clinically important proteases. Specifically, we focus on α-thrombin and prostate specific antigen (PSA) which play a critical role in the blood coagulation cascade or serve as molecular biomarkers of prostate cancer. In case of PSA, monitoring its functional state – as opposed to absolute levels – has been suggested to improve the performance of PSA as a diagnostic biomarker of malignant prostate cancer. Utilizing PSA activity measurements is thus expected to improve the clinical outcome for



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prostate cancer patients by reducing false positives and the number of unnecessary prostatectomies7.

Results and Discussion By introducing a cleavage site for α-thrombin (LVPR’GV6) into the linker connecting the TVMV with its autoinhibitory, we obtained an α-thrombin biosensor. It displayed a >75-fold induction range is capable of detecting 0.01 nM thrombin (Figure 1A-B). In contrast, a TVMV-based protease sensor for prostate specific antigen (PSA) engineered by introducing the PSA-specific cleavage site HSSKLQ’SGAG8 into the linker could only detect 5 µg/mL PSA (Figure 2A-B). This is a comparatively low sensitivity especially as catalytically-active PSA was reported to be present in blood in the sub ng/mL range7. To enhance the sensitivity of the PSA biosensor, we decided to introduce additional molecular recognition motifs designed to facilitate recruitment of the analyte protease to the biosensor promoting its cleavage and activation. In nature and biotechnology, antibodies provide a versatile class of high affinity protein binders that can be developed towards potentially any molecular target. Yet, conventional antibodies are not easily produced or engineered as complex expression systems and their large size complicates their functionalization with additional domains. Single chain antibody fragments and binding domains that can be efficiently expressed in Escherichia coli

9,10

are more suitable for this purpose. We thus fused the PSA

biosensor to PSA-specific VHH single domain antibodies and produced the resulting fusion protein in E.coli cytoplasm11,12. While detailed structural information is not available for the VHH-PSA complex, we hypothesized that recruiting PSA into the proximity of the PSA-specific TVMV protease sensor will significantly increase its local concentration to accelerate its cleavage and activation. Indeed, the affinity targeted construct TVMVPSA-AI-VHH demonstrated 25-fold sensitivity improvement compared to the parental biosensor (Figure 2B-C). Nonetheless, it falls short of the sensitivity required for the detection of biologically relevant PSA levels. Hence, we sought to devise an integrated system that could amplify the biomolecular signal generated by the TVMV-based receptor. Motivated by our earlier observation that induced colocalization of two autoinhibited proteases yields more sensitive and specific sensors compared


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to single-component sensors6, we decided to design a signaling circuit based on proteaseinducible protein:protein interaction (PPI). A number of well-characterized protein:protein interaction modules are available as elementary building blocks including coiled-coils, PDZ and Src Homology 3 (SH3) domains that have previously been recombined with signaling switches and metabolic enzymes to rewire, reconfigure and reorganize cellular signaling and metabolic pathways13–17. We chose to build a protease-inducible protein:protein interaction module based on the SH3 domain as it comprises a small globular scaffold that binds proline rich peptides with affinity in the 100 nM to µM range18. In comparison, coiled-coils are less suitable as they display affinity in the low nM range which precludes their use at low concentrations without optimization of the binding strength of the autoinhibitory peptide. Similarly, the PDZ domain requires the presence of a free C-terminus thus preventing C-terminal extensions that are often required for introduction of affinity tags that facilitate purification of full-length sensor protein. To engineer a protease-inducible protein:protein interaction module, we fused an SH3 domain to its cognate peptide ligand18 through a protease-cleavable linker. Cleavage of the autoinhibitory peptide by a target protease in turn creates an interface for SH3-dependent PPIs that leads to the recruitment, cleavage and activation of SH3-tagged HCV-based signal amplifier (Figure 3A). The design was first validated for α-thrombin by incorporating its cleavage site LVPR’GV between the SH3 domain autoinhibitory SH3 peptide (Figure 3A). To obtain optimal signal-to-noise ratio without significantly compromising the response times, we employed a high-affinity, autoinhibitory SH3 peptide PPPPLPPKRRR18 (KD = 96 nM) in combination with a weaker affinity recruiting SH3 peptide PPPALPPKKR18 (KD = 1.90 µM) while keeping the concentration of the primary SH3Thr-TVMV sensor low at 20 nM (Figure S1). Optimization results also showed that a sensor with an uninhibited TVMV transducer SH3Thr-TVMV in association with a weaker recruiting SH3 peptide gave rise to equal or better signal-to-noise ratios when it was applied at 20 nM or less compared to a partially autoinhibited sensor SH3Thr-TVMVTEV-AI (Figure S1). Furthermore, TEV protease (which was applied at 5 µM to remove the AI-domain of TVMV and remained in the sensing reaction) did not give rise to additional non-specific signals demonstrating the specificity of the scaffold-dependent sensor for the target protease αthrombin. Furthermore, the reaction generally displayed a delay (during which the TVMV-based



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transducer cleaves the HCV-based amplifier) before the fluorescent signal increased linearly (as all of the secondary HCV-based amplifier is being cleaved) and eventually flattened off (as all of the HCV substrate is consumed). The resulting signaling system displayed a dose dependent response to thrombin concentration with a limit of detection comparable to the single stage αthrombin sensor (Figure 3A and 1B). We therefore conjectured that above discussed high Km of α-thrombin constitutes a bottleneck in the system making it unsuitable for measuring low concentrations of the enzyme. To overcome this barrier and to enhance the sensitivity of the developed circuit, we introduced additional affinity targeting features for α-thrombin. Due to the limited availability of antibody fragments or single domain antibodies specific for α-thrombin, we extended the α-thrombin cleavage site with a naturally occurring competitive thrombin inhibitor KTAPPFDFEAIPEEYL. This peptide was derived from s-variegin of the tropical bone tick Amblyomma variegatum19–21 which has previously been shown to bind the exosite I of α-thrombin with high specificity (PDB: 3B23)19. Crucially, binding occurs in an N- to C-terminal orientation in extension of the thrombin substrate LVPR’GV which is ~7.5 Å away and can be bridged by three glycine residues (Figure S2). The resulting sensor SH3Var-TVMV could detect >0.01 nM α-thrombin – a two-fold improvement compared to SH3Thr-TVMV with no affinity targeting features. This further supports the notion that affinity capture of the analyte on the biosensor provides a generic approach for increasing the sensitivity of the biosensors by overcoming activation rates at low concentrations of the analyte. With this in mind, we set out to construct an integrated PSA sensing and signal amplification circuit (Figure 4A). To facilitate recombinant expression in E.coli and optimize the relative orientation of individual components, the SpyCatch protein conjugation system22–24 was used to post-translationally assemble the VHH -domain, the PSA-cleavable SH3 module featuring an optimized PSA cleavage site LRLSSYY’S25 and the TVMV-based signal transducer into a single polypeptide complex. Notably, SpyTag-dependent conjugation occurs via an isopeptide bond and thus yields four separate termini in close proximity24 that can be functionalized with the additional domains. In comparison, alternative protein conjugation systems such as Sortase A fuse polypeptides linearly through a trans-peptidation reaction26,27 and thus do not allow


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organizing proteins in a branched fashion around a single focal point. The resulting circuit could resolve a response to the analyte over five orders of magnitude and was able to detect up to 50 pg/mL PSA over background in the absence of PSA (Figure 4B and C). This is more than 10 times lower than the physiological level of catalytically active PSA7. Similar to the scaffold sensors for α-thrombin, the TVMV transducer was employed in uninhibited form following pre-treatment with TEV protease which did not elicit a non-specific response. Crucially, in the absence of the VHH-domain, the resulting PSA-cleavable SH3 module could only detect 50 ng/mL PSA, which is ~500-fold less sensitive compared to the affinity targeted sensors (Figure S3). This further highlights the importance of affinity targeting by means of high affinity protein binders to improve the sensitivity and specificity of the protease assay for PSA. In comparison, previously developed protease assays based on conventional fluorescent peptides with no affinity targeting features could only detect PSA (immunopurified from xenograft tumor homogenates) with a detection limit of 50 ng/mL8. In summary, we engineered synthetic multi-domain scaffold-dependent complexes that combine affinity targeting, protease-inducible scaffolding and signal amplification. These cascades are capable of detecting α-thrombin and PSA with high sensitivity and large dynamic range. While protease-cleavable PPIs in association with split-enzymes have previously been exploited for the detection of protease activity28, individual components had to be co-expressed in vitro and assayed directly in cell-free expression lysates. This was due to the limited stability of split-enzyme fragments that prevented their purification and storage. In contrast, our scaffold-dependent sensors are composed of structurally distinct affinity targeting, sensing, as well as signal transducing and amplifying domains. Given the high degree of modularity, individual units can be readily exchanged to optimize sensor performance or adapt it to alternative analytes. Our work shows that only minimal modifications are required for targeting these biosensing systems to different proteases. In addition, the availability of single domain antibodies against many clinically important proteases9,29 enables rapid assembly of new biosensors using the SpyTag system. This is complemented by an increasing range of autoinhibited reporter enzyme modules5 including light-emitting luciferases30, β-lactamases31–33 or dehydrogenases34 that can be employed as secondary amplifiers or actuators. In addition,



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faster primary transducers with higher kcat and Km values (making protease activation exclusively dependent on molecular proximity to the analyte) combined with tightly autoinhibited secondary amplifiers should accelerate the response of the system by several orders of magnitude.

Methods

DNA Cloning: Individual DNA constructs were generated using a combination of commercial gene synthesis services (Protein CT, GenScript and IDT gBlocks) and custom DNA cloning. A complete list of

constructs used in this study and their amino acid sequences is provided in the supporting information.

Protein Expression, Purification and Assembly: Different protease constructs were generally expressed as fusion proteins with maltose binding protein (MBP) which served as a solubility enhancing factor to assist in the functional expression of the different protease constructs, but was autoproteolytically separated inside the cell35,36. Autoinhibited protease sensors were generally purified by means of His-tag affinity chromatography while the VHH construct was affinity purified by means of Strep-tag affinity chromatography. Affinity purification tags were generally located at the C-terminus to ensure purification of full length protein. Posttranslational assembly of the VHH, TVMV and SH3 modules was performed post-translationally using the SpyTag/Catch protein conjugation system23,24.

Protease Assays: TVMV and HCV protease functions were assayed using commercially synthesized

(Mimotopes

Australia)

quenched

fluorescent

peptide

substrates

ANA-

GETVRFQSDT(164)-NH2 and ANA-DDVTPCSMS(164)-NH2 (ANA: 5-amino-2-nitrobenzoyl; 164: lysine coupled 7-methoxycoumarinyl-4-acetyl). Individual protease sensors were employed at the concentrations indicated. Protease reactions were assayed in 50 mM Tris-HCl, 50 mM NaCl, 50 µg/mL bovine serum albumin (BSA) pH 8.0 and monitored with 5 µM protease substrate. Cleavage was monitored by increasing in fluorescence at 330 nm following excitation at 405


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nm. The enzymatically active target protease analytes α-thrombin (Sigma-Aldrich, Catalogue No. T7009) and PSA (MerckMillipore/Calbiochem, Catalogue No. 539834) were obtained commercially and included in individual assays as indicated.

A detailed summary of materials and methods and sequences of individual constructs is provided in the supporting information.

Associated Content Please see supporting information for detailed experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information

Corresponding Authors: Viktor Stein: [email protected] Kirill Alexandrov: [email protected]

Conflict of Interest: V.S. and K.A. are co-inventors on a patent application that covers aspects of the protease-based biosensor technology described in this publication. K.A. holds equity in Molecular Warehouse Ltd that in possession of a license to the above patent.

Funding This work was funded by the Australian Research Council Discovery Project Grant DP1094080 to KA and in part by National Breast Cancer Foundation Innovator Grant. This research was also supported by New Concept Grant awarded through the It’s a Bloke’s Thing initiative and Prostate Cancer Foundation of Australia to KA and VS. We thank Marinna Nilsson for excellent technical assistance. The authors are grateful to Frank Gardiner for discussions and insights into prostate cancer diagnostics.



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Figure Captions

Figure 1. Protease-based sensors of α-thrombin. (A) Principle organization of the elementary thrombin sensor TVMVThr-AI based on autoinhibited TVMV modules with a cleavage site for αthrombin separating TVMV from its autoinhibition (AI) domain. (B) Titration of α-thrombin to 500 nM solution of the biosensor demonstrates a limit of detection >0.01 nM for α-thrombin. Reaction progress was monitored for in six hours using fluorescently quenched TVMV peptide substrate that gains fluorescence at 405 nm upon cleavage.

Figure 2. Biosensors of prostate specific antigen (PSA). (A) The elementary PSA sensors TVMVPSA-AI are based on autoinhibited TVMV modules with a cleavage site for PSA separating TVMV from its autoinhibition (AI) domain. (B) Experiment demonstrating that the elementary sensor TVMVPSA-AI can detect 5 µg/mL PSA. (C) Introduction of a PSA-specific, VHH-based antibody domain yields TVMVPSA-AI-VHH that recruits PSA thereby increasing its effective concentration and accelerating cleavage and activation of the biosensor. (D) As in B but using TVMVPSA-AI-VHH. This experiment demonstrates that introduction of the affinity domain improves the sensitivity of the primary sensor approximately 25-fold allowing detection of 40 ng/mL PSA. The activity of TVMV was monitored as in Figure 1.


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Figure 3. Scaffold-dependent protease sensors of α-thrombin. (A) Schematic representation of the signal sensing and amplification circuit where α-thrombin cleaves the SH3-based protein:protein interaction module. Activated module in turn recruits an SH3-peptide tagged secondary amplifier based on autoinhibited HCV into the proximity of the TVMV transducer resulting in cleavage and activation of the HCV-based amplifier. PS and PW denote autoinhibitory and recruiting SH3 peptides with strong (KD = 96 nM) and weak binding affinities (KD = 1.9 µM). (B) Titration of the indicated amounts of SH3Thr-TVMV and HCVTVMV-AI-SH3 with increasing concentrations of α-thrombin. This experiment demonstrates that the scaffold-dependent sensor SH3Thr-TVMV allow the detection of >0.01 nM α-thrombin. (C) Extending the thrombin cleavage site with an exosite I binder of s-variegin yields SH3Var-TVMV and improves the limit of detection by approximately two-fold. The progress of the reaction was monitored with a fluorescently quenched, HCV-specific peptide substrate that upon cleavage gains fluorescence at 405 nm.

Figure 4. Scaffold-dependent protease sensors for PSA. (A) Schematic representation of the signal sensing and amplification circuit operation: First, PSA is recruited into the vicinity of the SH3 protein interaction module through a high affinity PSA-specific VHH binder (KD = 100 pM) enabling PSA to cleave the SH3 protein:protein interaction module. Activation of the SH3 module in turn recruits an SH3-peptide tagged HCV secondary amplifier into the proximity of the TVMV transducer resulting in accelerated cleavage and activation of the HCV-based amplifier. PS and PW denote autoinhibitory and recruiting SH3 peptides with strong (KD = 96 nM) and weak binding affinities (KD = 1.9 µM) respectively. (B) Reaction progress for the PSA-specific scaffold sensor TVMV-Spy-VHH-SH3PSA-SH3Pep which can detect the proteolytic activity of PSA over five orders of magnitude in a range from 50 pg/mL to 500 ng/mL. (C) To facilitate comparisons between repeat experiments, reaction traces were scaled between 0 and 50 ng/mL PSA covering the clinically relevant range, and the fluorescent response measured at 720 min before the signal saturated. This confirmed a detection limit of 50 pg/mL PSA, which elicited a fluorescent response of 6.5% + 1.3% relative to the signal observed for 50 ng/mL PSA. The error bars denote the standard error of the mean from three independent titration



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experiments. The activity of HCV was monitored as in Figure 3. The progress of the reaction was monitored with a fluorescently quenched, HCV-specific peptide substrate that upon cleavage gains fluorescence at 405 nm.


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Figure for Table of Content 80x53mm (300 x 300 DPI)

ACS Paragon Plus Environment


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Figure 1. Protease-based sensors of α-thrombin. (A) Principle organization of the elementary thrombin sensor TVMVThr-AI based on autoinhibited TVMV modules with a cleavage site for α-thrombin separating TVMV from its autoinhibition (AI) domain. (B) Titration of α-thrombin to 500 nM solution of the biosensor demonstrates a limit of detection >0.01 nM for α-thrombin. Reaction progress was monitored for in six hours using fluorescently quenched TVMV peptide substrate that gains fluorescence at 405 nm upon cleavage. 104x40mm (300 x 300 DPI)


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Figure 2. Biosensors of prostate specific antigen (PSA). (A) The elementary PSA sensors TVMVPSA-AI are based on autoinhibited TVMV modules with a cleavage site for PSA separating TVMV from its autoinhibition (AI) domain. (B) Experiment demonstrating that the elementary sensor TVMVPSA-AI can detect 5 µg/mL PSA. (C) Introduction of a PSA-specific, VHH-based antibody domain yields TVMVPSA-AI-VHH that recruits PSA thereby increasing its effective concentration and accelerating cleavage and activation of the biosensor. (D) As in B but using TVMVPSA-AI-VHH. This experiment demonstrates that introduction of the affinity domain improves the sensitivity of the primary sensor approximately 25-fold allowing detection of 40 ng/mL PSA. The activity of TVMV was monitored as in Figure 1. 85x73mm (300 x 300 DPI)



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Figure 3. Scaffold-dependent protease sensors of α-thrombin. (A) Schematic representation of the signal sensing and amplification circuit where α-thrombin cleaves the SH3-based protein:protein interaction module. Activated module in turn recruits an SH3-peptide tagged secondary amplifier based on autoinhibited HCV into the proximity of the TVMV transducer resulting in cleavage and activation of the HCV-based amplifier. PS and PW denote autoinhibitory and recruiting SH3 peptides with strong (KD = 96 nM) and weak binding affinities (KD = 1.9 µM). (B) Titration of the indicated amounts of SH3Thr-TVMV and HCVTVMV-AISH3 with increasing concentrations of α-thrombin. This experiment demonstrates that the scaffolddependent sensor SH3Thr-TVMV allow the detection of >0.01 nM α-thrombin. (C) Extending the thrombin cleavage site with an exosite I binder of s-variegin yields SH3Var-TVMV and improves the limit of detection by approximately two-fold. The progress of the reaction was monitored with a fluorescently quenched, HCVspecific peptide substrate that upon cleavage gains fluorescence at 405 nm. 171x127mm (300 x 300 DPI)


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Figure 4. Scaffold-dependent protease sensors for PSA. (A) Schematic representation of the signal sensing and amplification circuit operation: First, PSA is recruited into the vicinity of the SH3 protein interaction module through a high affinity PSA-specific VHH binder (KD = 100 pM) enabling PSA to cleave the SH3 protein:protein interaction module. Activation of the SH3 module in turn recruits an SH3-peptide tagged HCV secondary amplifier into the proximity of the TVMV transducer resulting in accelerated cleavage and activation of the HCV-based amplifier. PS and PW denote autoinhibitory and recruiting SH3 peptides with strong (KD = 96 nM) and weak binding affinities (KD = 1.9 µM) respectively. (B) Reaction progress for the PSA-specific scaffold sensor TVMV-Spy-VHH-SH3PSA-SH3Pep which can detect the proteolytic activity of PSA over five orders of magnitude in a range from 50 pg/mL to 500 ng/mL. (C) To facilitate comparisons between repeat experiments, reaction traces were scaled between 0 and 50 ng/mL PSA covering the clinically relevant range, and the fluorescent response measured at 720 min before the signal saturated. This confirmed a detection limit of 50 pg/mL PSA, which elicited a fluorescent response of 6.5% + 1.3% relative to the signal observed for 50 ng/mL PSA. The error bars denote the standard error of the mean from three independent titration experiments. The activity of HCV was monitored as in Figure 3. The progress of the reaction was monitored with a fluorescently quenched, HCV-specific peptide substrate that upon cleavage gains fluorescence at 405 nm. 172x124mm (300 x 300 DPI)


Ultrasensitive Scaffold-Dependent Protease Sensors with Large

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