Target-activated transcription for the amplified sensing of protease biomarkers

Abstract

Signal amplification is an effective way to achieve sensitive analysis of biomarkers, exhibiting great promise in biomedical research and clinical diagnosis. Inspired by the transcription process, here we present a versatile strategy that enables effective amplification of proteolysis into nucleic acid signal outputs in a homogeneous system. In this strategy, a protease-activatable T7 RNA polymerase is engineered as the signal amplifier and achieves 3 orders of magnitude amplification in signal gain. The versatility of this strategy has been demonstrated by the development of sensitive and selective assays for protease biomarkers, such as matrix metalloproteinase-2 (MMP-2) and thrombin, with sub-picomole sensitivity, which is 4.3 Â 10 3 -fold lower than that of the standard peptide-based method. Moreover, the proposed assay has been further applied in the detection of MMP-2 secreted by cancer cells, as well as in the assessment of MMP-2 levels in osteosarcoma tissue samples, providing a general approach for the monitoring of protease biomarkers in clinical diagnosis.

Modular Combination of Proteolysis-Responsive Transcription and Spherical Nucleic Acids for Smartphone-Based Colorimetric Detection of Protease Biomarkers

“…In the previous work, we have constructed the plasmids for the expression of PR MMP‑2 and PR Thr . Here, we constructed the plasmid for the expression HCV NS3/4A protease based on the same protocol.…”

“…Aiming at the rational design and fabrication of artificial biological systems to perform programmable functions, synthetic biology has provided an arsenal of useful biorecognition elements for the highly specific target recognition in biosensing. − One major advantage of these synthetic recognition elements is that they can transduce biorecognition events into programmable nucleic acid information that can be readily amplified into detectable signals. , For instances, riboswitches and allosteric transcription factors (aTFs) have been demonstrated as effective elements to sense a series of chemical contaminants, including metal ions, antibiotics, and toxins. ,,, Recently, the strategy to engineer RNA polymerase (RNAP) with target-responsive capabilities has aroused much research interest because such a strategy would provide a more direct approach to accurately control synthetic genetic circuits than the methods using riboswitches and aTFs. − With the use of target-responsive RNAPs (PR), researchers can use light, small molecules, or proteins as the triggers to execute regulatory functions. ,,, Especially, the protease-PR is able to transduce each proteolytic cleavage event into programmable RNA sequences, providing a powerful biorecognition element to sense protease biomarkers . Based on the modularity and programmability of PR, the rational combination of PR with other sensing techniques is expected to generate a new paradigm for the detection of protease biomarkers.…”

Peptide probes for proteases – innovations and applications for monitoring proteolytic activity

“…Enhanced delivery to the tumour tissue was enabled by the directing peptide, where the acidic pH in the cancer microenvironment (and during uptake in the endosome) triggered the release of DOX from the construct while caspase-3 cleaved the peptide resulting in a fluorescent signal. Transcriptiondependent fluorogenic probes have been used for specific protease dependent activation or amplification of signal, 67,68 while analysis kits for plasma and peripheral blood testing have been developed using polymer immobilization of FRET-based protease substrates. 69 1.1.2 Ratiometric.…”

Construction of Stable T7 Expression System in Saccharomyces cerevisiae by Improving Nuclear Membrane Permeability with Viroporin HIV-1 Vpu

“…T7 expression system, derived from T7 bacteriophage, consisting of T7 promoter and T7 RNA polymerase (T7 RNAP), is simple, efficient, orthogonal [ 1 ], which make it be an enabling tool in synthetic biology [ 1 , 2 ], metabolic engineering [ 3 , 4 ], biomedicine [ 5 , 6 ], and enzyme engineering [ 7 , 8 ]. T7 expression system has been extensively applied to synthesize protein in prokaryote and eukaryote.…”