Table 1.

Troubleshooting table.

Step	Problem	Possible reason	Solution
6	Transcription yields are low	Yields of the PCR products are low	Check PCR product by agarose gel electrophoresis before transcription, proceed only after visualization
		Transcription reagents are old	Use freshly prepared reagents such as DTT, which degrades after multiple freeze-thaw cycles
7B(vi), 9A(vii)	Multiple banding on gels because of RNA contamination or degradation	RNase contamination	Check monomers on denaturing gel, resynthesize RNA if necessary
		RNA degradation because of the presence of divalent ions (Mg2+)	Keep assemblies on ice to avoid degradation via nonspecific cleavage in the presence of metal ions
9A(iv)	RNA does not migrate or is stuck in the wells	Wells were not washed well enough	Wells have particulate in them: filter the reagents for the gel, clean the comb well with water and ethanol and let it air dry
		Prefolding of the strategy 1 monomers was not achieved	For ring assemblies, heat to 95 °C for 2–3 min and snap cool on ice for 3 min
9A(vii)	No RNA assembly products on PAGE gel	Absence of divalent ions (Mg2+)	Verify whether magnesium salt was added to assembly buffer, gel and gel buffer
	Multiple assembly products on native PAGE gel	Monomer single-stranded RNAs tend to stick to the tube walls	Vortex individual monomers well before assembly
		Initial concentrations were not measured correctly	Remeasure concentrations of monomers before assembly
		Alternative folding may prevent the accessibility of kissing loops	Check sequences for complementary kissing loops and/or secondary structure
9B(iv)	Broad DLS spectra or spectra with multiple peaks	Sample is polydispersed because of contamination or poor assembly	Remeasure concentrations of monomers before assembly; centrifuge your sample
12	Assemblies are not processed by Dicer	Sequence design is incorrect	Verify the RNA design so that annealed strands result in 2-nt 3′ overhangs
		Assembly problems	Anneal a slight excess (~ 5%) of duplex strands to the NPs
		Inactivation of the enzyme	Check the freshness or activity of Dicer by dicing with short 27/25-nt duplexes43