Intracellular expression of a fluorogenic DNA aptamer using retron Eco2
- Department of Chemistry and Chemical Biology, TU Dortmund University, Germany
Figures
Figure 1
Eco2 retron locus and RT-DNA architecture.
Schematic of the Eco2 gene locus, which encodes the non-coding RNA (ncRNA, blue) and the Eco2 reverse transcriptase gene (Eco2RT, green). The msd (yellow) region of the ncRNA is reverse transcribed into msd DNA, which is linked to the remaining msr via a 2'–5' linkage conserved guanosine close to the 3’-end of the msr, as indicated in the inset.
Figure 2 with 1 supplement
Recombinant expression of Eco2 RT-DNA.
(A) Illustration of primer target sites for qPCR experiments to determine RT-DNA abundance. The black arrows indicate the forward primer that pairs either with the purple reverse primer (amplify both RT-DNA and plasmid DNA) or the blue reverse primer that only amplified the plasmid DNA. (B) Fold enrichment of the RT-DNA/plasmid template over the plasmid alone upon induction, as measured by qPCR; Unpaired t-test, induced versus uninduced: p<0.0001; n=3 biological replicates. (C) A TBE-Urea polyacrylamide gel, stained with SYBR Gold showing RT-DNA corresponding to retron Eco1 (90 nt) and retron Eco2 (70 nt).
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Figure 2—source data 1
Raw data corresponding to Figure 2B.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig2-data1-v1.zip
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Figure 2—source data 2
PDF file containing original PAGE gel indicating relevant bands, corresponding to Figure 2C.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig2-data2-v1.zip
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Figure 2—source data 3
Original image file of PAGE gel displayed in Figure 2C.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig2-data3-v1.zip
Figure 2—figure supplement 1
Determination of half-life of RT-DNA.
(A) OD600 measurements fitted with the equation for logistic growth. (B) Fold change of RT-DNA/plasmid amplicon over plasmid alone, at different time points. The data corresponds to n=5 biological replicates with error bars indicating s.e.m.
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Figure 2—figure supplement 1—source data 1
Raw data corresponding to Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig2-figsupp1-data1-v1.zip
Figure 3 with 5 supplements
Expression of Lettuce-Eco2 fusion constructs in E. coli cells.
(A) Schematic of Lettuce aptamer. The arrows indicate the length to which the P1 stem can be shortened without significant loss of FLAP functionality. (B) Schematic of insertion of Lettuce aptamer sequence in 4 distinct positions in the msd region of retron Eco2. The ssDNA structure was simulated using Vienna RNA fold with DNA parameters. Lettuce aptamer sequence was inserted at single-stranded and/or loop regions to minimize interference with the native fold of the msd sequence. (C) DFHBI-1T stained TBE urea-PAGE showing fluorescence of different oligonucleotides mimicking the Lettuce-Eco2 (LE) fusion constructs with varying P1 lengths: 8LE v1-v4 (134 nt), 11LE (140 nt), and FLE (166 nt). Free Lettuce with either the 4 nt P1 stem (4 L) or full-length P1 (FL) served as positive controls. (D) SYBR stained TBE urea-PAGE of extracted Eco2 wild type and Eco2-Lettuce RT-DNA fusions after expression in E. coli. In all variants, the lettuce aptamer was inserted into position v4 of the msd scaffold. (E) DFHBI-1T stained TBE urea-PAGE showing fluorescence of 8LEv4 RT-DNA purified from E. coli cells. Lanes with different loading amounts in pmol of the synthetic RT-DNA standards are shown as comparison.
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Figure 3—source data 1
PDF file containing original PAGE gel indicating relevant bands, corresponding to Figure 3C.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-data1-v1.zip
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Figure 3—source data 2
Original image file of PAGE gel displayed in Figure 3C.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-data2-v1.zip
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Figure 3—source data 3
PDF file containing original PAGE gel indicating relevant bands, corresponding to Figure 3D.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-data3-v1.zip
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Figure 3—source data 4
Original image file of PAGE gel displayed in Figure 3D.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-data4-v1.zip
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Figure 3—source data 5
PDF file containing original PAGE gel indicating relevant bands, corresponding to Figure 3E.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-data5-v1.zip
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Figure 3—source data 6
Original image file of PAGE gel displayed in Figure 3E.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-data6-v1.zip
Figure 3—figure supplement 1
DFBI-1T staining of 4LE oligonucleotides.
(A) In-gel DFBI-1T staining of 4LE oligonucleotides mimicking the 4Lettuce length variants in Eco2, with free 4Lettuce and full Lettuce aptamer as positive controls. (B) SYBR-stained gel showing expression of 4LEv1-4.
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Figure 3—figure supplement 1—source data 1
PDF file containing original PAGE gel indicating relevant bands, corresponding to Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
Original image file of PAGE gel displayed in Figure 3—figure supplement 1B.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-figsupp1-data2-v1.zip
Figure 3—figure supplement 2
Fluorescence titration experiments using chemically synthesized Lettuce-Eco2 v4 variants.
Solid lines represent plots of Equation 1 using the best fit parameters. Fluorescence was measured at a fixed concentration of 1 µM DFHBI-1T and increasing DNA concentrations. Experimental data points are averages from n=3 biological replicates with error bars indicating standard deviations. Beset-fit KD values are: 4LE-v4 6 µM (4.93, 7.18, 95% CI); 8LE-v4 0.04 µM (0.01, 0.12, 95% CI); 11LE-v4 0.01 µM (0.01, 0.07, 95% CI); FLE-v4 0.07 µM (0.01, 0.16, 95% CI); 4L 1.28 µM (0.9, 1.6, 95% CI); FL 0.12 µM (0.01, 0.25, 95% CI).
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Figure 3—figure supplement 2—source data 1
Raw data corresponding to Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-figsupp2-data1-v1.zip
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Figure 3—figure supplement 2—source code 1
Source code used for fitting data from Figure 3—figure supplement 2.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-figsupp2-code1-v1.zip
Figure 3—figure supplement 3
Detection of extracted RT-DNA constructs.
SYBR-stained TBE urea-PAGE showing TRIzol extracted RT-DNA corresponding to Eco2 (90 nt) and 8LEv1-4 (134 nt). The total extracted RNA was quantified using Qubit and an amount corresponding to 2000 ng RNA was loaded on the gel.
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Figure 3—figure supplement 3—source data 1
PDF file containing original PAGE gel indicating relevant bands, corresponding to Figure 3—figure supplement 3.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-figsupp3-data1-v1.zip
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Figure 3—figure supplement 3—source data 2
Original image file of PAGE gel displayed in Figure 3—figure supplement 3.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-figsupp3-data2-v1.zip
Figure 3—figure supplement 4
Bulk in vivo fluorescence measurements of 8LEv1-4 with DFHBI-1T.
Experimental data points are averages from n=3 biological replicates ± standard deviations.
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Figure 3—figure supplement 4—source data 1
Raw data corresponding to Figure 3—figure supplement 4.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-figsupp4-data1-v1.zip
Figure 3—figure supplement 5
Comparison of fluorogenic activity of Lettuce (DNA) and Broccoli (RNA) aptamers.
(A) Emission spectra of equivalent concentrations (10 µM) of DNA Lettuce aptamer and RNA Broccoli aptamer, with equimolar DFHBI-1T, upon excitation at 462 nm. (B) Fluorescence output of Lettuce and Broccoli aptamer upon excitation at 462 nm, recorded at 510 nm. Experimental data points are averages from n=3 biological replicates with error bars indicating standard deviations.
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Figure 3—figure supplement 5—source data 1
Raw data corresponding to Figure 3—figure supplement 5.
- https://cdn.elifesciences.org/articles/99554/elife-99554-fig3-figsupp5-data1-v1.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Escherichia coli) | Ec67 (Eco2) | https://doi.org/10.1016/j.cell.2020.09.065 | Ec67 (pAA11) | Supplementary file 1a |
| Strain, strain background (E. coli) | BL21(AI) | Thermo Fisher Scientific | Cat. #: C607003 | Chemically competent cells |
| Chemical compound, drug | Ready-Lyse Lysozyme Solution | Lucigen, Biozym | Cat. #:R1804M | |
| Chemical compound, drug | TRIzol Reagent | Thermo Fisher Scientific | Cat. #:15596026 | |
| Commercial assay or kit | Luna Universal qPCR Master Mix | New England Biolabs | Cat. #:M3003L | |
| Commercial assay or kit | Monarch Spin Plasmid Miniprep Kit | New England Biolabs | Cat. #:T1110L | |
| Chemical compound, drug | DFHBI-1T | Lucerna | Cat. #:410-10mg | Fluorophore |
| Commercial assay or kit | ssDNA/RNA Clean & Concentrator | Zymo | Cat. #:D7011 | |
| Chemical compound, drug | SYBR Gold Nucleic Acid Gel Stain (10,000X Concentrate in DMSO) | Invitrogen | Cat. #:S11494 |
Additional files
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Supplementary file 1
Nucleic acid sequences used in this work.
- https://cdn.elifesciences.org/articles/99554/elife-99554-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/99554/elife-99554-mdarchecklist1-v1.pdf
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Intracellular expression of a fluorogenic DNA aptamer using retron Eco2
eLife 13:RP99554.
https://doi.org/10.7554/eLife.99554.3
