Date: 28th November 2019
The diagnostic field is rapidly evolving, driven by factors including the growing incidence of chronic and infectious diseases. We are also beginning to see the increased uptake of automation and artificial intelligence, and together with advances in synthetic biology, this is fuelling a revolution.
In recent months we have reported AI vocal diagnostics of cognitive & mental disorders, the use of CRISPR nuclease as a diagnostic tool to rapidly detect RNA/DNA, and a CRISPR-Chip biosensor able to detect genetic mutations leading to the world’s first DNA search engine, The Genome Sensor™. The latest report came just a few days ago with DNA circuits detecting the molecular signatures on cancer cells.
Now scientists from The University of Toronto, Canada, and Arizona State University, US, have combined cell-free synthetic biology with state-of-the-art nanostructured electrodes to generate an electronic output from a biological system. The system is able to detect specific target RNA molecules indicative of antibiotic resistance and pathogenic states and paves the way for advanced, portable multiplexing diagnostic tools.
It is the first example of a gene circuit being directly coupled to electrodes and has the potential to accelerate diagnostics within both healthcare and agriculture.
The paper, published in Nature Chemistry, describes the design, evolution and use of a system comprised of several parts:
Toehold switch RNA sensors: Programmable synthetic riboregulators that controlled the translation of genes once activated by the binding of their specific trigger RNA. The switches contained a hairpin structure that blocked gene translation by sequestering the ribosome binding site and start codon. When the switch was bound by the trigger RNA, sequestering was relieved, and gene translation could occur. This in turn allowed expression of a number of corresponding restriction-enzyme based reporters.
Restriction-enzyme based reporters: Activation allowed translation of specific restriction enzymes, which could then cleave distinct annealed reporter DNA. The enzyme reporters were expressed in several ways; by DNA expression constructs, or by ligating to each set of toehold switches.
Annealed reporter DNA: DNA labelled with a redox reporter such as methylene blue, and which was free-floating in the cell-free reactions. These could be recruited to the microelectrodes and were designed as DNA duplexes, with one strand containing the full-length reporter DNA and the other a complementary inhibitor DNA which prevented the uncleaved duplex binding to the electrode.
DNA-functionalised nanostructured microelectrodes: Capture DNA, conjugated to nanostructured microelectrodes, which, when recruited the redox-active reporter DNA, could generate an electrochemical signal.
Setting up the system
Several experiments were performed to determine the most suitable restriction enzymes and to confirm the specificity of the system.
The authors showed that the restriction-enzyme-mediated electrochemical signal could be detected in as little as 20 min after transcription initiation.
Furthermore scalability and reporter validation were performed showing the system was specific. Each reporter-DNA and capture-DNA system was activated by its respective restriction-enzyme reporter, with little cross-talk between the alternative systems. Furthermore, a subset of these restriction enzymes (five of them) generated clear electrochemical signals on-chip when co-expressed in a single reaction mixture.
Detection using toehold switches
The authors had previously demonstrated that toehold switches could be designed to recognise specific RNA sequences and that these RNA sensors could be used to identify the presence of pathogens. This was used effectively to detect Zika virus.
To explore the potential for using toehold switches to detect specific RNAs here, the authors needed to first determine the specificity of the switches. To do this they synthesised six toehold switches designed to recognise distinct synthetic RNA sequences which would control different restriction enzyme reporters. When they looked at the electrochemical output for each reporter they observed electrochemical increases between 7- and 30-fold over the background, which were shown to be only activated when the respective trigger RNA sequence was added.
Furthermore, using a ligand-inducible gene circuit to control the restriction-enzyme reporter, the system was shown to be ligand-dependent adding another level of potential control to the system.
Detection of antibiotic resistance
With antibiotic resistance becoming an ever-increasing risk in healthcare, the authors then wanted to test the system’s ability to detect resistance genes responsible for colistin antibiotic resistance.
Toehold switches against four colistin-resistant genes were designed (mobilized colistin resistance genes mcr-1, mcr-2, mcr-3 and mcr-4) and top performing switches were selected. Detection of the four mcr RNAs was determined by adding each of these mcr RNAs to this electrochemical system on-chip and the output was monitored.
The data showed that addition of each mcr RNA saw a ~20-fold signal enhancement of the electrochemical response. Furthermore, multiplexing was demonstrated, enabling the specific and simultaneous detection of RNA sequences from mcr-3 and mcr-4.
Whilst the results from the previous experiments were promising, the real test was to expose the system to a more complex sample. In order to achieve this, E.coli expressing constitutively active promoter-driven mcr-4 was used. Mcr-4 RNA from whole cell RNA was electrochemically detected by the platform and led to a resulting signal that was specific to mcr-4 RNA, and provided a distinct signal when compared to negative controls and so vlidating the system in a biologically significant sample.
The authors conclude ‘we have presented here a series of proof-of-concept experiments for a direct and scalable interface between engineered gene circuits and electronics’.
Future applications
One of the really exciting features of this electrochemical interface for gene-circuit-based sensors is achieving an electronic output from a biological system. In the future this interface is primed to connect with computers, tablets and smartphones, giving this system great flexibility and scope. Whether it is bedside, home-based or out in the field it is able to interface with sophisticated and interactive applications.
In this respect, its applications are likely to evolve and develop. Here, antibiotic resistance genes were detected, but the authors have already shown in previous work, virus detection is also possible. It is likely, in addition to pathogen detection, screening for other diseases and infections would be possible.
Furthermore, the system would not be limited to healthcare and other market verticals also stand to potentially benefit. For example, in agriculture, it is not difficult to imagine such a system being utilised for pathogen detection in crops or determining appropriate livestock antibiotic treatments.
Wherever this new set of synthetic biology tools takes the field will be an interesting one. The transversal nature of this system means it has the potential to translate to areas that the authors did not anticipate, however one thing they are adamant about is that it will accelerate the healthcare field and has great capacity to improve lives. We will be keeping a close eye out for future applications.
For more information please read the press release from Arizona State University
Sadat Mousavi, P., S. J. Smith, J. B. Chen, M. Karlikow, A. Tinafar, C. Robinson, W. Liu, D. Ma, A. A. Green, S. O. Kelley and K. Pardee (2019). “A multiplexed, electrochemical interface for gene-circuit-based sensors.” Nature Chemistry.
https://doi.org/10.1038/s41557-019-0366-y
