A Dual‐Response DNA Probe for Simultaneously Monitoring Enzymatic Activity and Environmental pH Using a Nanopore

Abstract

Both protease overexpression and local pH changes are key signatures of cancer.However,the sensitive detection of protease activities and the accurate measurement of pH in at umor environment remain challenging.H ere,w ed evelop ad ual-response DNAp robe that can simultaneously monitor protease activities and measure the local pH by translocation through a-hemolysin (aHL). The DNAp robe bears as hort peptide containing phenylalanine at ap redesigned position. Enzymatic cleavage of the peptide either exposes or removes the N-terminal phenylalanine that can form ac omplex with cucurbit[7]uril. Translocation of the DNAh ybrid through aHL generates current signatures that can be used to quantify protease activities.Furthermore,the current signatures possess ap H-dependent pattern that reflects the local pH. Our results demonstrate that the versatile DNAp robe mayb ef urther explored for simultaneously measuring multiple parameters of ac omplex system such as single cells in the future.

DNA Origami-Enabled Biosensors

“…The pulse modulations in ionic current indicate the target translocation through the nanopore [90]. Nanopores are typically divided into two categories including biological (fabricated from proteins [91]) and solid-state (formed by inorganic or organic materials) nanopores [92]. The sizes of solid state nanopores are tunable, but the controllability of surface functionality (precisely positioning single binding sites for analytes) limits their development.…”

Cucurbiturils in nucleic acids research

“…Since 2015, H.-C. Wu and co-workers are developing a versatile method for nanopore sensing of biologically related analytes based on DNA-CB [7] host-guest probes. [75][76][77][78][79][80][81][82][83] In the method developed by H.-C. Wu and co-workers, introduction of the analyte leads to the release or in situ formation of the single-stranded DNA (ssDNA) bearing a ferrocene-CB [7] or adamantane-CB [7] complex, referred to as a DNA probe. Threading of the DNA probe through the a-hemolysin (aHL) nanopore results in the dissociation of the host-guest complex and liberation of CB [7] (Fig.…”

“…Recently, the method was used for simultaneous monitoring of protease activities and local pH values. 82 Finally, this approach has been applied to determine the binding constants of the host-guest interactions of CB [6] and CB [7] with small molecules and proteins. 83 However, the resulting binding constants were not fully consistent with the previously reported data making this method suitable for qualitative analysis only.…”

Measuring Enzymatic Activities with Nanopores

“…Our group have been using DNA probes for nanopore sensing for more than one decade. By exploiting the current signal changes caused by DNA conformational changes or DNA modifications during their translocation through protein nanopores, we have developed a series of approaches for sensing a wide variety of different analytes . To measure enzymatic activities, we attached the peptide substrate to a DNA molecule to form a hybrid probe.…”

“…By exploiting the current signal changes caused by DNA conformational changes or DNA modifications during their translocation through protein nanopores, we have developed a series of approaches for sensing a wide variety of different analytes. [45][46][47][48][49][50][51][52] To measure enzymatic activities, we attached the peptide substrate to a DNA molecule to form a hybrid probe. We managed to amplify the current difference before and after enzymatic cleavage by the introduction of cucurbit [7]uril (CB [7]) which is known to have strong affinities with peptides containing N-terminal phenylalanine ( Figure 5A).…”