Journal: Nature Chemical Biology

Article Title: Programming cell-free biosensors with DNA strand displacement circuits

doi: 10.1038/s41589-021-00962-9

Figure Lengend Snippet: a , In the presence of IVT components, T7 RNAP can non-specifically bind to the toehold region of the DNA signal gate. When the toehold is on the 3’ end of the gate, this leads to transcription of unwanted RNA side products that can displace the quencher strand. This process is blocked when the toehold is on the 5’ end of the gate. b , The 3’ toehold DNA signal gate leads to fluorescence activation in the presence of T7 RNAP, while the 5’ toehold DNA signal gate does not. c , When the reaction products from b were extracted and run on an urea-polyacrylamide gel, RNA side products appear only from the 3’ toehold DNA signal gate. A negative control where no DNA signal gate is present in the reaction (–) was run alongside for both 3’ and 5’ toeholds. d , Modifying the DNA gate with 2’-O-methyl oligonucleotides prevents promoter-independent transcription by T7 RNAP. e , When the 3’ toehold DNA signal gate is modified with 2’-O-methyl oligonucleotides, no fluorescence activation is observed in the absence of a T7 RNAP-driven IVT template. f , When the reactions from e were run on an urea- polyacrylamide gel, no RNA side products were observed from the 2’-O-methyl DNA signal gate. A negative control where no DNA signal gate is present in the reaction (–) was run alongside for both unmodified and modified gates. Data shown in b and e are n = 3 independent biological replicates each plotted as a line with raw fluorescence standardized to MEF (μM fluorescein). Shading indicates the average of the replicates ± standard deviation. Data shown in c and f are a representative of n = 3 independent biological replicates. The uncropped, unprocessed gel image shown in c and f is available as Source Data .

Article Snippet: Homemade IVT reactions were set up by adding the following components listed at their final concentration: IVT buffer (40 mM Tris–HCl pH 8, 8 mM MgCl 2 , 10 mM dithiothreitol, 20 mM NaCl and 2 mM spermidine), 11.4 mM NTPs pH 7.5, 0.3 U of thermostable inorganic pyrophosphatase (New England Biolabs, catalog no. M0296S), transcription template, DNA gate(s) and MilliQ ultrapure H 2 O to a total volume of 20 µl.

Techniques: Fluorescence, Activation Assay, Negative Control, Modification, Standard Deviation

Journal: Nature Chemical Biology

Article Title: Programming cell-free biosensors with DNA strand displacement circuits

doi: 10.1038/s41589-021-00962-9

Figure Lengend Snippet: Unregulated reactions were lyophilized overnight with the addition of 50 mM sucrose and 250 mM D-mannitol as the lyoprotectants unless otherwise indicated. The lyophilized reactions were then vacuum-packaged in a light protective bag with a dri-card and kept in a cool, shaded area until usage (see Materials and Methods for the detailed protocol). Kinetic traces of rehydrated reactions after a , 1 day, b , 4 days and c , 7 days of storage are shown. There is a decrease in overall signal as well as in the response speed over time. To investigate the cause of the signal loss over time, the DNA signal gate alone was lyophilized overnight with or without the lyoprotectants, packaged and stored as described above. The DNA signal gate was rehydrated with the rest of the IVT components after d , 1 day, e , 4 days and f , 7 days. The response speed as well as the magnitude of the signal are maintained, indicating that the signal loss is likely due to instability of certain IVT components. To test this hypothesis, unregulated reactions with Tris-buffered NTPs, instead of NaOH-buffered NTPs, were lyophilized with the lyoprotectants, packaged and stored as described above. Kinetic traces of rehydrated reactions after g , 1 day, h , 4 days and i , 7 days of storage are shown. The signal loss is somewhat mitigated with Tris-buffered NTPs, but a similar degree of signal loss is observed for a long-term storage of lyophilized reactions. All data shown are n = 3 independent biological replicates each plotted as a line with raw fluorescence values standardized to MEF (μM fluorescein). Shadings indicate the average of the replicates ± standard deviation.

Article Snippet: Homemade IVT reactions were set up by adding the following components listed at their final concentration: IVT buffer (40 mM Tris–HCl pH 8, 8 mM MgCl 2 , 10 mM dithiothreitol, 20 mM NaCl and 2 mM spermidine), 11.4 mM NTPs pH 7.5, 0.3 U of thermostable inorganic pyrophosphatase (New England Biolabs, catalog no. M0296S), transcription template, DNA gate(s) and MilliQ ultrapure H 2 O to a total volume of 20 µl.

Techniques: Fluorescence, Standard Deviation