Precise and Programmable Detection of Mutations Using Ultraspecific Riboregulators
Authors: Lida A Mina
Kaiyue Wu
Duo Ma
Fan Hong
Yan Liu
Hao Yan
Alexander A Green
Rebecca C Luiten
Journal: Cell (2020)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: colorimetric; diagnostic; isothermal; mutation; paper-based assay
Affiliations: Biodesign Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA; School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA., Hematology-Oncology Department, Banner MD Anderson Cancer Center, Gilbert, AZ 85234, USA., Genetics Department, Banner MD Anderson Cancer Center, Gilbert, AZ 85234, USA., Biodesign Center for Molecular Design and Biomimetics at the Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA; School of Molecular Sciences, Arizona State University, Tempe, AZ 85287, USA. Electronic address: .

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 novo-designed 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. 

Retracted: No

PubPeer comments:
 Fig. 6D of this paper may contain duplications in the shown photographs. The spots marked in the same color seem to be the exact same photographs. ![file] , 
 Thank you for your comments. We have carefully checked the original data from the experiments. The source photograph for panel 6D in the paper is shown in Figure 1 below with each combination of SNIPR and amplified Zika RNA tested in triplicate. As described in the Method Details of the paper, figures showing the visual performance of the SNIPRs were cropped and arranged prior to inclusion in figures. Upon examining panel 6D and comparing it to the source photograph, we were dismayed to find that some of the reaction wells included in the panel were duplicated. The orange, purple, red, green, and blue squares in Figure 1 show the wells that were used to generate the panel 6D array included in the paper. We believe this oversight occurred when the images were cropped and inadvertently assigned incorrect file names. This mistake led to the incorrect images being used when the files were imported into Adobe Illustrator when the figure was being composed. We sincerely regret the error and have contacted the journal to inform them of this issue. ![file] **Figure 1.** Panel 6D (left) and the source photograph used for showing paper-based strain-specific SNIPR reactions (right). Large white boxes with rounded corners indicate the triplicates used for each SNIPR tested and the three Zika strain target RNAs. Orange, purple, red, green, and blue rectangles indicate the wells used for the original version of panel 6D. White rectangles indicate the wells used for the corrected version of panel 6D (see Figure 2). To correct the error, we have composed an updated version of panel 6D containing the paper disks in the wells indicated by white squares in Figure 1. The updated version panel 6D is included below as Figure 2B with the original version included as Figure 2A for comparison. The corrected figure still demonstrates that the strain-specific Zika SNIPRs can distinguish different Zika RNA targets in color-based reactions. Thus, the error in panel 6D does not affect the findings and conclusions of the paper. ![file] **Figure 2.** (A) Original version of panel 6D containing the inadvertently duplicated paper disks. (B) The corrected version of panel 6D showing that the SNIPRs enable three Zika strains to be identified by eye. To allay any potential concerns regarding sensor performance, we show below results from a completely independent experiment with the strain-specific Zika SNIPRs (Figure 3). This image appeared as Figure 14C in a patent application describing SNIPRs that was filed in 2017 ([https: patents.google.com patent WO2018026762A1 en]). In this experiment using synthetic Zika target RNAs, we also found that the riboregulators could clearly distinguish between the three Zika strains. ![file] **Figure 3.** Zika strain identification image from Figure 14C of patent application WO2018026762A1 by Green and Hong. The original patent application is available here: [https: patents.google.com patent WO2018026762A1 en] , 
 Thank you for your detailed reply! The source data together with the amended panel clearly supports the statements of the paper. I wish authors would always take issues like this as seriously. ,