TABLE 3.

Troubleshooting table.

Step	Problem	Possible reason	Solution
R5 labeling
7	Precipitation when spin label is added to the nucleic acid solution	Nucleic acid (generally 4100 nt) crashed out of solution	Reduce the amount of acetonitrile
	Phase separation	Poor mixing between the aqueous solution and the acetonitrile solution of nitroxide	Constant and gentle vortexing during incubation
8	Broad elution profile without clearly distinguishable peaks	Nucleic acid forms secondary structures that interfere with HPLC separation	Purify using denaturing gel electrophoresis
	Doublet in the HPLC traces, all elute earlier than the unlabeled species	Separation of the phosphorothioate diastereomers in the labeled oligonucleotide	Collect all peaks and confirm R5 labeling by mass spectrometry and continuous-wave-electron paramagnetic resonance (cw-EPR); All R5-labeled fractions may be combined in subsequent steps
	Multiple peaks in HPLC traces, some of them elute at the same position as the unlabeled species	Incomplete R5 labeling	Verify the presence of phosphorothioate in the oligonucleotide by mass spectrometry, I2 cleavage and nuclease digestion
			Increase incubation time at Step 7
			Increase the amount of compound 1
DEER data acquisition
14	The temperature does not decrease	Presence of an obstruction in the system	Check that the needle valve which controls the flow of liquid helium is open
			If the needle valve works well, disassemble the transfer line, warm up to room temperature and purge with dry air or nitrogen
			In rare instances, the obstruction may be in the cryostat. If purging the transfer line does not solve the issue, warm up the entire system and purge it carefully before reassembly
15	The ‘dip’ cannot be located	The system is over- or undercoupled	Adjust the coupling while scanning the frequency to locate the ‘dip’
		The system has automatically entered the pulse mode to protect the hardware	Enter the FT Bridge window and use the Bridge/Standard Configuration tab to select the cw mode
16	The slight shift in frequency is not observed when the sample is inserted in the cryostat	The capillary may not be positioned correctly in the cavity	Remove the sample and insert it again
	Ice forms on the capillary	Long exposure of the frozen capillary to air	Keep capillary immersed in liquid N2 until loading
	Capillary stuck to the resonator	Ice introduced into the resonator	Warm up and purge the system
	‘Dip’ disappears	Ice introduced into the resonator	Warm up and purge the system
	Capillary shatters	Capillary not properly sealed	Prepare multiple sample capillaries
21	Echo signal is clipped	Video gain value is too high	Display the signal without averaging and set the video gain so that the echo is not clipped
	SpecJet Display gives an error message	One or more of the timings may be incorrect	Verify that all the time intervals are set correctly
	Refocused echo and Hahn echo are weak or cannot be detected	Settings are not optimized properly	Verify that center field and field position are set properly; Readjust settings
		Sample not in the active volume	Reinsert the sample
		Spin concentration may be too low	Collect a cw-EPR spectrum to verify spin concentration. Relabel as needed
	Strong Hahn echo, but weak or non-existence of the refocused echo	Sample has short-phase memory time	Try to increase phase memory time by lowering temperature or changing solvent (D2O instead of H2O)
23	Only mono-exponential echo decay is observed	Low concentration of interacting spin pairs	Check and adjust labeling and assembling procedures to ensure sufficient presence of double-labeled sample
NASNOX-W
38	The NASNOX-W Applet fails to run	Errors in the PDB coordinates; incorrect atom names in the PDB file	Check structure correctness using a graphics program; check atoms names, particularly of phosphates
39	Corrupted structure in ‘data001lig.pdb’	Errors in writing the output structure due to variations in the PDB input file (e.g., modified nucleotides)	Check the input PDB file format. However, in most such cases, distance information in the ‘data.add’ file is preserved