TMR-DN

Guide (The following is our recommended solution. This solution is merely a guideline and should be modified according to your specific needs.)

1. Stock Solution Preparation
1.1 Solvents: DMSO or DMF.

2. Working Solution Preparation
2.1 Diluents: Live cell imaging solution (for bacterial samples); FluoroBrite DMEM medium supplemented with 2 mM GlutaMAX (for HeLa cell samples); 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl₂ (for RNA samples); 40 mM Tris, 5 mM MgCl₂, 100 mM KCl (pH 7.6) (for in vitro experiments); DPBS (for cell imaging).
2.2 Working concentrations: 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM.
2.3 Notes: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining Protocol
3.1 Sample Type Description
3.1.1 Adherent cells (HeLa cells)^[1]: Seed onto chamber coverslips, with cell confluency reaching 60%-80% before transfection.
3.1.2 Bacteria expressing aptamers (e.g., DNB/SRB-2)^[1][3]: If an inducible promoter is used, add 1 mM IPTG and continue shaking culture at 37°C for 2-3 hours.
Take 200 μL of bacterial culture, collect the bacterial cells by centrifugation, and resuspend them in 1 mL of imaging buffer (containing 5 mM MgSO₄).
3.1.3 Purified RNA samples (RhoBAST aptamer and its mutants)^[2].

3.2 Incubation Conditions
3.2.1 For adherent HeLa cells^[1]: Incubate with 0.5 μM TMR-DN at 37°C for approximately 30 min.
3.2.2 For suspended bacterial cells^[1][3]: Add an equal volume of dye working solution to achieve a final concentration of 0.5-1 μM, and incubate at 37°C for approximately 10 min.
3.2.3 For purified RNA samples (RhoBAST aptamer and its mutants)^[2]: Incubate with 0.5 μM TMR-DN in a buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM KCl, 10 mM MgCl₂.
3.2.4 For in vitro experiments^[3]: Incubate with 0.5 μM TMR-DN, together with RNA constructs and target analytes (if applicable), in a buffer containing 40 mM Tris, 5 mM MgCl₂, 100 mM KCl (pH 7.6).

3.3 Washing Steps
3.3.1 Since free TMR-DN shows extremely low background, washing is generally not required after incubation, and imaging can be performed directly. If background is relatively high, gently wash 1-2 times with pre-warmed buffer.

4. Control Setup
4.1 Negative Controls
4.1.1 For bacterial experiments^[1][3]: Use bacteria without target-binding aptamers^[3].
4.1.2 For HeLa cell experiments^[1]: Use cells without target-binding aptamers.
4.1.3 For in vitro experiments^[3]: Set up experiments without target analytes.
4.2 Positive Controls^[3]: Cells expressing target-binding ratiometric sensors with known target concentrations; in vitro experiments supplemented with target analytes of known concentrations.
4.3 Blank Controls^[3]: In vitro experiments containing only TMR-DN (without RNA aptamers).

5. Detection and Analysis
5.1 Instrument Types: Fluorescence microscope; multi-mode microplate reader; fluorescence spectrophotometer; confocal microscope, etc.
5.2 Excitation/Emission Wavelengths: 560/40 nm excitation, 630/60 nm emission; 555 nm excitation, 582 nm emission.
5.3 Result Analysis
5.3.1 Changes in fluorescence intensity: The fluorescence turn-on fold of TMR-DN after binding to SRB-2 is approximately 17-fold; the fluorescence intensity of cells expressing SRB-2 is significantly higher than that of negative control cells^[1]; the fluorescence intensity of TMR-DN increases after binding to RhoBAST^[2]; the fluorescence of free TMR-DN is quenched, and gets activated after binding to DNB aptamer; the fluorescence intensity is further enhanced when the target analyte binds to the ratiometric sensor^[3]; non-target analogs only induce extremely weak fluorescence activation, confirming the specificity of the sensor^[3].
5.3.2 Fluorescence localization: Orange fluorescence localizes to cellular regions where SRB-2-tagged RNA is present, including bacterial cytoplasm/poles, HeLa cell nuclei and cytoplasm, as well as HeLa cell cytoplasm where membrane-targeted mRNA resides; whole-cell distributed fluorescence is observed in bacterial cells, and target analytes may show polar aggregation.
5.3.3 Fluorescence color: Orange fluorescence is produced after binding to SRB-2^[1]; red fluorescence is produced after binding to DNB aptamer^[3].
5.3.4 Molecular interaction analysis: TMR-DN binds to the semi-open pocket at the end of the asymmetric "A"-shaped structure of RhoBAST, with the xanthene group of TMR inserting into the binding core, and the DN quencher stacking with the benzene ring of TMR^[2].