Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators

Abstract

The ability to identify single-nucleotide mutations is critical for probing cell biology and for precise detection of disease. However, the small differences in hybridization energy provided by single-base changes makes identification of these mutations challenging in living cells and complex reaction environments. Here, we report a class of de novodesigned prokaryotic riboregulators that provide ultraspecific RNA detection capabilities in vivo and in cell-free transcription-translation reactions. These single-nucleotide-specific programmable riboregulators (SNIPRs) provide over 100-fold differences in gene expression in response to target RNAs differing by a single nucleotide in E. coli and resolve single epitranscriptomic marks in vitro. By exploiting the programmable SNIPR design, we implement an automated design algorithm to develop riboregulators for a range of mutations associated with cancer, drug resistance, and genetic disorders. Integrating SNIPRs with portable paper-based cell-free reactions enables convenient isothermal detection of cancer-associated mutations from clinical samples and identification of Zika strains through unambiguous colorimetric reactions.

Detection of Coronaviruses Using RNA Toehold Switch Sensors

“…Due to the flexibility to design a pair of toehold switch and its cognate trigger RNA, the toehold reaction has been widely used as an RNA recognition-based detection system for pathogen identification and microbiome profiling [ 12 , 13 , 14 ]. The toehold switch contains a stem structure around the start codon ( Figure 1 ) [ 15 , 16 ].…”

“…RNA toehold switch sensors are robust molecular switches that can be used to detect RNA molecules in a sequence-specific manner. This flexible switch had been combined with a paper-based cell-free molecular diagnostic platform to detect various target nucleic acids [ 12 , 13 , 14 ]. However, to enable the sensitive detection of nucleic acids, toehold reactions require an amplification step that multiplies target RNA.…”

“…When NASBA was used as an amplification step for the toehold reaction, the detection limit was 3 fM of Zika viral RNA [ 12 ]. In another report, when recombinase polymerase amplification (RPA) was connected to the toehold reaction, the detection limit was around 250 aM [ 14 ]. The sensitivity we achieved in this study indicates the modified RT-LAMP is an appropriate and compatible isothermal amplification method for the toehold reaction.…”

“…NASBA required 120 min to amplify 3 fM of the Zika virus [ 12 ]. RPA took 120–160 min to amplify target RNA [ 14 ]. Compared to these isothermal amplifications, the modified RT-LAMP demonstrated in this study amplified the 120 copies (12 copies/μL, 20 aM) of target RNA in 20 min, allowing the reduction of overall reaction time to 70 min.…”

Botulinum toxin as an ultrasensitive reporter for bacterial and SARS-CoV-2 nucleic acid diagnostics

“…Currently, novel biosensors based diagnostic strategies have been developed with an improved sensitivity and accuracy ( Ren et al, 2019 ; Song et al, 2019 ; Yoo and Lee, 2016 ; Zhang et al, 2020 ). Those sensors include ( Hong et al, 2020 ; Wan et al, 2018 ; Wang and Alocilja, 2015 ; Wu et al, 2015 ) enzyme-based reporters [horseradish peroxidase (HRP) ( Wen et al, 2016 ), alkaline phosphatase (ALP) ( Han et al, 2020 ), and catalase ( de la Rica and Stevens, 2012 )], fluorescent molecule-based reporters [Fluorescein probes ( Cardoso et al, 2011 ) and Molecular Beacons ( Cao et al, 2014 )] and nanomaterial-based reporters [magneto-DNA nanoparticle system ( Chung et al, 2013 ), Gold nanoparticles ( Yang et al, 2020 )]. Although enzyme-based reporters shortened analysis time in many experiments, their sensitivity is limited due to strong background response ( Seo et al, 2015 ).…”