The best application of STD-NMR is ligand/drug screening, to identify ligand(s) that interacts with the host from a compound pool (multiple compounds) . In this system, the host is usually larger molecules, such as proteins and nucleic acids.
Samples: (using a DNA sample and one ligand as an example)
- DNA sample alone. For reference spectrum only, so low concentration is OK.
- Ligand sample alone.For reference spectrum only, so low concentration is OK.
- DNA+Ligand. Suggested molar ratio =<1:20, such as 0.1mM:2mM.
* All samples should have the same buffer composition.
- Run standard proton 1Ds on all three samples. The spectrum of ligand alone should closely match the spectrum of DNA+ligand, since in the latter sample ligand is in large excess.
- Have sufficient number of scans (ns) to see all signals, especially for DNA signals in sample c.
- The reference spectra will give you an idea the distribution of ligand and DNA signals. Since ligands are usually small, their signals are fewer. Therefore, it is usually not difficult to select a DNA signal for irradiation (on resonance frequency), which should be far from ligand signal positions.
- Set up STD experiment for sample 3 (DNA+Ligand).
- Create a new data set, then “rpar STDDIFFESGP.3”. This is a STD experiment with water suppression by excitation sculpting pulse.
- Use command “getprosol 1H XX XXX” to set up all the pulse parameters.
- Check O1p, which may not be at 4.7 if organic solvent is in the buffer system.
- Saturation time D20 =2s by default. May need adjustment if necessary.
- SPW9 40-60dB. My experience is that 40dB worked better than 60dB.
- Number of scans ns should be high, >=64
- Create an irradiation frequency list and save in AcquPars/FQLIST/FQ2LIST, to define the irradiation frequency (on resonance frequency). The frequency should be on a DNA proton signal which is NOT close to any ligand signals.
The FQ2LIST file should have three numbers, magnet frequency, on resonance frequency and off resonance frequency.
- Run the STD experiment.
- Process the data using AU program “stdsplit”.
- The first 1D should look like a standard 1D, and the second one would show ligand signals if the STD experiment worked.
- If no ligand signals appeared after trying different irradiation frequencies, either the ligand may not interact with the host, or the affinity may be very strong. The strong affinity can be identified by comparing 1D spectra of the DNA alone with the DNA in the presence of the ligand.