Acridine homodimer

Guidance (The following is our recommended protocol. This protocol is for reference only and should be modified according to your specific needs).

1. Stock solution preparation
1.1 Solvent: For most dyes, an organic solvent such as anhydrous DMSO is typically used for dissolution.
1.2 Recommended concentration: It is generally recommended to prepare a high-concentration stock solution of 1-10 mM.

2. Working solution preparation
2.1 Diluent: Prepare Acridine homodimer in 0.2 M acetate buffer at pH 5.0.
2.2 Working concentration: Use 2 × 10^-7 M Acridine homodimer (Compound IIA) for fluorescence measurements.
2.3 Note: Adjust the working solution concentration as needed; prepare fresh before use.

3. Staining procedure
3.1 Sample types: Use double-stranded DNA in solution, including synthetic poly[d (A-T)] and natural DNA; use covalently closed circular PM2 DNA for unwinding measurements and sonicated DNA for viscosity measurements.
3.2 Incubation conditions: Incubate at 20°C in 0.2 M acetate buffer at pH 5.0.
3.2.1 For fluorescence titration, use 0.55 μg/mL poly[d (A-T)] and vary the dye/DNA-P ratio.
3.2.2 For DNA unwinding measurements, use 150 μg/mL DNA in 0.2 M acetate buffer at pH 5.0 and increase the dye concentration.

5. Detection and analysis
5.1 Instrument types: Use a photon-counting spectrofluorometer for fluorescence measurements, time-correlated single photon counting instrumentation for fluorescence lifetime measurements, and viscometry for DNA unwinding and length change measurements.
5.2 Wavelength settings: For fluorescence measurements of DNA-bound Acridine homodimer, use 460 nm excitation and 495 nm emission.
5.3 Result analysis: The fluorescence lifetime of DNA-bound Acridine homodimer remains constant at 28.4 ns throughout the titration.
5.3.1 The fluorescence intensity of DNA-bound Acridine homodimer varies with the fourth power of the A-T fraction, thereby allowing discrimination between synthetic poly[d (A-T)] and crab poly[d (A-T)].
5.3.2 Binding is sequence-independent, as shown by fluorescence measurements on DNA mixtures of different base compositions.
5.3.3 The DNA unwinding angle is 38°, and bis-intercalation is observed below r = 0.125.