Sample preparation and imaging procedures for super-resolution microscopy using Repeat DNA-PAINT.

The performance of DNA-PAINT in biological samples is often constrained by strong background signals and non-specic binding events, both of which are exacerbated by high imager concentrations. Here we describe the procedure for conducting Repeat Domain DNA-PAINT, a method that substantially reduces imager concentration and thus suppresses spurious signals


Introduction
Introduction: In DNA-PAINT a target epitope is chemically labelled with a 'docking' strand (DS), a short DNA oligonucleotide.A dye-modi ed strand, an 'imager', is complementary to the DS with ~9 base pair (bp) overlap and is introduced into solution.The transient interaction, facilitated by the hybridization, of the imager to the DS results in a 'blink', or single-molecule event detectable in a camera frame.DNA-PAINT bene ts from having the dye free in solution which essentially helps it to evade the effects of photobleaching.There are however a number of drawbacks which limit the performance of DNA-PAINT within biological samples.
By having the imagers free in solution, a diffuse background is created which can compromise event detection and localisation precision.The signal from free-imagers when imaging deep into biological tissues, where TIRF cannot be used, can also be severe.Additionally, data interpretation 1 and the detection of sparse targets 2 are further complicated by imagers often exhibiting substantial non-speci c binding in samples, especially within biological preparations.
By decreasing the imager concentration both imager-induced background and non-speci c events can be reduced.However, in DNA-PAINT, such a reduction prolongs the time it takes to acquire a well sampled super-resolution image.This often makes an experiment more complicated due to limitations in mechanical and chemical sample stability.Here we describe the protocol to conduct Repeat DNA-PAINT which uses docking domains repeated up to 10x on the same strand, enabling an order of magnitude decrease in imager concentration whilst maintaining the levels of speci c single-molecule events.This simple strategy can be easily implemented and adapted for existing DNA-PAINT setups.17.Monitor event rates.They should begin to fall to background levels as the displacer sequence removes the 1x RD strand from the anchor strand.

Reagents
18. Once event rates have fully decreased, give the sample a further 5 minutes before washing off the anchor and displacer strand complexes, along with any excess displacer strands using 1x RD buffer.Event rates should remain at background levels.We do several washes at each on-stage washing step.
19. Wash sample with 10x RD imaging buffer to reduce imager concentration.20.Add 0.5 µL of 100 µM 10x RD strand to the ~500 µL sample solution and gently mix.21.Event rates should increase by a small amount.Give the sample 10-15 minutes for the 10x RD strands to fully attach to the anchor strands.At this stage, event rates do not appear higher because of the excess 10x RD strands in solution outcompeting those attached to the anchor strand at the targets of interest.
22. Wash sample with 10x RD imaging buffer several times to remove excess 10x RD.Events should increase to a rate similar to that measured with 1x RD.
Troubleshooting -if the event rate does not increase to a similar level as when using the 1x RD, this is likely due to insu cient washing at one of the steps.There could still be excess 10x RD in solution, or the displacer sequence was not adequately removed.Wash the sample a few more times with 10x RD imaging buffer, if the event rate remains low repeat step 20 onwards.Also consider that the 1x RD likely had a number of non-speci c events occurring at locations where no docking strand was present.Therefore, rates will vary depending on the sample type, the location you are imaging and other possible mechanisms leading to non-speci c events.Note: We imagine most users will want to simply go straight to using the 10x RD strands, in which case one can add 0.5 µL of 100 µM 10x RD strand to the ~500 µL sample solution and gently mix immediately after Step 10 of the labelling procedure section above.Then simply remove the excess from solution with successive washes using 10x RD imaging buffer and enjoy the bene ts of conducting DNA-PAINT with an order of magnitude lower imager concentration resulting in reduced background and fewer nonspeci c events.

Troubleshooting Time Taken
Anticipated Results

2 .
Move samples to fresh PBS for 10 minutes.

3 .
Move samples to 10% sucrose in PBS for 1 hour, 4 °C.(6 g sucrose made up to 60 mL total volume in PBS) 4. Move samples to 20% sucrose in PBS for 1 hour, 4 °C. 5. Move samples to 30% sucrose in PBS overnight at 4 °C.Samples should sink.