Out-of-Plane Aptamer Functionalization of RNA Three-Helix Tiles

Co-transcriptionally folding RNA nanostructures have great potential as biomolecular scaffolds, which can be used to organize small molecules or proteins into spatially ordered assemblies. Here, we develop an RNA tile composed of three parallel RNA double helices, which can associate into small hexagonal assemblies via kissing loop interactions between its two outer helices. The inner RNA helix is modified with an RNA motif found in the internal ribosome entry site (IRES) of the hepatitis C virus (HCV), which provides a 90° bend. This modification is used to functionalize the RNA structures with aptamers pointing perpendicularly away from the tile plane. We demonstrate modifications with the fluorogenic malachite green and Spinach aptamers as well with the protein-binding PP7 and streptavidin aptamers. The modified structures retain the ability to associate into larger assemblies, representing a step towards RNA hybrid nanostructures extending in three dimensions.

concentrated using Eppendorf Concentrator 530f and the concentration was estimated by denaturing polyacrylamide gel electrophoresis (denaturing PAGE).
Polyacrylamide gel electrophoresis (PAGE) -8% Acrylamide: Bis (29:1) gel containing 8 M urea and 1X TBE buffer (denaturing PAGE) was used to analyze the purified RNA tiles and estimate their concentration. The gel was pre-run at 50 °C for 30 min. The samples and RiboRuler Low Range Ladder (Thermofisher scientific) were prepared in 1:1 ratio with 2X RNA loading dye (Thermofisher scientific) and incubated at 80 °C for 10 min prior to loading onto the gel. The gels were run at 80 V for 1 h 45 min. After electrophoresis, the gels were stained with SYBR™ Green II RNA Gel Stain (Thermofisher scientific) for 10 min and scanned on a Typhoon FLV 9000 laser scanner and analyzed with the software package Fiji [1].
Native PAGE -8% Acrylamide: Bis (29:1) gel containing 1X TBE buffer and 2 mM (MgOAc)2 was used to analyze the proper folding of purified folded RNA tiles. The gel was pre-run at 4 °C for 30 min. The tiles were subjected to a heat denaturation/renaturation protocol (95° for 3 min and then snap-cooled to 4 °C for 5 min) and buffer was added (1X buffer = Tris-Borate 1X (pH 8.1), 2 mM magnesium acetate (Mg(OAc)2), 50mM potassium chloride (KCl), 50 mM sodium chloride (NaCl)). The samples were either kept on ice or further incubated for 1 h at 37 °C prior to loading onto the gel. The gel was run at 80 V for 1 h 30 min at 4 °C. After electrophoresis, the gels were stained with 10 µ M Malachite green for 30 min and scanned on a Typhoon FLV 9000 laser scanner. Additionally, the gel was stained with SYBR™ Green II RNA Gel Stain (Thermofisher scientific) for 10 min and scanned on a Typhoon FLV 9000 laser scanner. The gel was analyzed with the software package Fiji [1].

3H-AE-Spinach with Streptavidin Aptamer
2% agarose gel in 1X TBE and 2 mM MgCl2 were used to determine the shift in RNA tile migration after being bound to streptavidin via its RNA aptamer. The gel was run in ice water bath at 80 V for 2 h. 100 nM RNA tiles were subjected to a heat denaturation/renaturation protocol as mentioned previously and then incubated with different concentrations of streptavidin (NEB, 1mg/mL, MW: 52.8KDa, ~19 µ M) for 10-15 min in presence of 1X AFM buffer and Murine RNase Inhibitor (NEB) at room temperature. 40% sucrose was used for loading the samples onto the gel. After EMSA, the gel was stained with SYBR™ Green II RNA Gel Stain (Thermofisher scientific) for 30 min and destained. It was scanned on a Typhoon FLV 9000 laser scanner and analyzed with the software package Fiji [1].
1.1.2. 3H-AE-Spinach with PP7 RNA Aptamer 2% agarose gel in 1X Tris-Glycine buffer (25 mM Tris, 192 mM Glycine, 10 mM KCl, 2 mM MgCl2, pH 8.1) was used to determine the shift in RNA tile migration after being bound to PCP-mCherry via its PP7 RNA aptamer. The gel was run in ice-water bath at 200 V for 35 min. The gel was pre-run at 10 V for 30 min. 100 nM RNA tiles were subjected to a heat denaturation/renaturation protocol as mentioned previously and then incubated with different concentrations of PCP-mCherry (~4 µ M) (details about expression and purification are provided in the next section) for 10-15 min in presence of 1X AFM buffer and Murine RNase Inhibitor (NEB) at RT. 40% sucrose was used for loading the samples onto the gel. After EMSA, the gel was scanned first at wavelength corresponding to mCherry fluorescence, stained with SYBR™ Green II RNA Gel Stain (Thermofisher scientific) for 30 min and destained. It was scanned on a Typhoon FLV 9000 laser scanner and analyzed with the software package Fiji [1].

Gibson Assembly and Protein Expression
A DNA template encoding the PCP coat protein (insert) was ordered as gBlocks from IDT and cloned into the pET28 backbone containing mCherry fluorescent protein by Gibson Assembly (NEB E2611S) following the manufacturer's protocol. Primer sequences along with the corresponding conditions used for PCR used for Gibson Assembly are listed in Tables S5 and S6 in the supplementary information. The plasmid maps and other relevant sequences are provided in Figure  S10 and Table S7. The product of Gibson assembly was electroporated in electro competent Dh5 E. coli cells. Plasmid was isolated from an overnight culture of Dh5 containing the pET28-PCP-mCherry plasmid and sequenced for the correctness of the insert. Further, purified pET28-PCP-mCherry plasmid was electroporated in BL21DE3star E. coli for protein expression.
100 mL LB medium was inoculated with an overnight culture of BL21DE3star E. coli containing pET28-PCP-mCherry plasmid and was induced with 1 mM IPTG at OD = 0.6. The culture was allowed to grow for 3-4 h and pelleted. The pellet was resuspended in lysis buffer (50 mM Tris-NaOH, 500 mM NaCl, 5 mM imidazole, 1 mM Benzamidine and 0.1% PMSF) and sonicated (Amplitude: 50%, Pulse: 30 s, time: 10 min) on ice. It was further centrifuged and the supernatant was collected and loaded onto Ni-NTA column for purification by AKTA as PCP-mCherry protein included a His-tag (6xHis). The protein was eluted at ~400 mM imidazole concentration. The fractions collected from AKTA were run on a SDS-PAGE gel. 2X Laemmli buffer was added to the fractions and they were incubated at 95 °C for 3 min. 12% SDS-PAGE gel was run at 250 V for 25 min in 1X SDS buffer as running buffer. The protein was washed in equilibration buffer (50 mM Tris-NaOH, 500 mM NaCl, 1 mM EDTA) 3-4 times at 4000 rcf for 10 min at 4 °C using 10 kD cut off filters from EMD millipore. 10% glycerol was added to make the stocks and kept at −80 °C. The concentration of the protein was determined by the absorbance measured by UV-Vis spectrophotometer and the protein parameters determined by Expasy [3].

Sample Preparation and Atomic Force Microscopy (AFM)
Freshly-cleaved mica affixed to a metal disk was placed inside an empty tip box containing a wet Kimwipe and water (to maintain the humidity in the chamber). A 60 µ L drop of 1.25X AFM buffer (1X AFM buffer = Tris-Borate 1X (pH 8.1), 2 mM magnesium acetate (Mg(OAc)2), 50 mM potassium chloride (KCl), 50 mM sodium chloride (NaCl)) was added to the mica and incubated at 37 °C. Purified RNA tiles (after Freeze and Squeeze gel extraction) were diluted in pure water at a concentration calibrated to give coverage, but not large aggregates, on the mica surface (~75-100 nM for 3H tiles). Tiles were next subjected to a heat denaturation/renaturation protocol (95° for 3 min and then snap-cooled to 4 °C for 5 min). 15 µ L of RNA sample was added to the mica on top of the preheated (37 °C) buffer drop and incubated for 45 min to 1 h to allow coverage of the surface and formation of hexagonal lattices. AFM images were collected in tapping mode under buffer using Asylum research's high resolution fast scanning AFM Cypher S. Olympus BL-AC40TS-C2 silicon nitride probes with a spring constant of ~0.09 N/m and at a resonant frequency of ~20-30 kHz was typically used for imaging, with a drive frequency of ~5-8 kHz.

Inmobilization of RNA Tiles on Streptavidin-coated Beads and Imaging
For studying the arrangement of RNA tiles on the surface of microbeads, we used 20 µ m streptavidin polystyrene coated beads (Spherotech). Beads were washed three times for 10 min with nuclease free water and 5 µ L were transferred to 30 µ L transcription reactions and incubated for 1 h at 37 °C. Transcription reactions were performed as previously described for 6-8 h. Samples were washed once with 1x TX reaction to remove the unspecific binding and resuspended in 20 µ L transcription buffer with 100 µ M DFHBI (40 mM Tris-HCl pH 7.9, 100 mM MgCl2, 125 nM KCl, 1 mM DTT, 2 mM spermidine). Samples were imaged with a Nikon Ti-Eclipse epi-fluorescence microscope controlled with NIS-Elements Imaging Software. The microscope was equipped with a SOLA SM II LED light source, a sCMOS camera (Zyla, Andor), a motorized stage (Prior Scientific, Cambridge, UK), and a perfect focus system                  Steps Vector Insert