PARIS2, optimized photochemistry method to study dynamic in vivo RNA structuromes and interactomes


 Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods.


Introduction
We systematically investigated the basic physics and chemistry of the crosslink-ligation principle; and developed next generation of the PARIS method (PARIS2). In particular, we report amotosalen as a more e cient crosslinker compared to the commonly used psoralen AMT. We discover that crosslinking increases RNA hydrophobicity, rendering it unextractable using the classical AGPC (acid guanidine thiocyanate phenol chloroform) aqueous-organic phase separation method (commercially known as TRIzol, etc.) or silica-based solid phase extraction methods 1,2 . We invent a generally applicable method, TNA (total nucleic acid extraction), to purify crosslinked RNA, enabling targeted analysis of RNAs with antisense enrichment. Given the low e ciency crosslinking, several methods have been developed to enrich crosslinked fragments, including native-denatured two-dimension (ND2D) gel, biotin-tagging and RNase R treatment, however, these approaches are often expensive and ine cient 3 . We develop a denatured-denatured 2D (DD2D) gel system to isolate pure crosslinked RNA without the need for tagging the crosslinker. We also introduce chemical and enzymatic approaches to prevent and bypass photochemical damages to RNA, a fundamental problem in RNA research. Together, these optimizations in PARIS2 resulted in >4000-fold increased e ciency, and importantly, the deep mechanistic insights into photochemistry, RNA chemistry and enzymology for individual improvements are also broadly applicable in RNA studies.
14. Centrifuge tube lters, 0.45 μm (e.g., Sigma-Aldrich Corning Costar Spin-X Swirl the plates every 10 mins and make sure that they are horizontal. 5) Remove cross-linking solution after cross-linking and wash cells twice with 1x PBS. (see Note 1) 2. TNA (total nucleic acid) extraction from psoralen crosslinked cells: 6) For each 10 cm dish cells, add 100 μL of 6 M GuSCN, lyse cells with vigorous manual shaking for 1 min. The cells should be lysed into a nearly homogenous solution, which may not be entirely clear. Be careful, as the 6 M GuSCN is highly corrosive. 7) Then to each tube add 12 μL of 500 mM EDTA, 60 μL of 10x PBS, and bring the volume to 600 μL with water. This dilution of the sample will lead to some insoluble material. Then pass the sample through a 25G or 26G needle about 20 times to further break the insoluble material. 8) Add proteinase K to 1 mg/ml (30 μL from the 20 mg/mL stock), mix well and incubate at 37 o C for 1 hour on a shaker (eg: Thermomixer C), at 600-900 RPM. Manually shake the tubes a few times during the incubation to facilitate mixing. 9) After PK digestion, add 60 μL of 3 M sodium acetate (pH 5.3), 600 μL of water-saturated phenol (pH 6.7), mix well divide into two tubes and then to each tube add 600 μL of pure isopropanol. (see Note 2) 10) Spin down the precipitate at 15000 rpm for 20 min at 4 o C and remove supernantant (dispose of phenol waster properly). 11) Wash the precipitate with 70% ethanol twice to remove residual phenol and other contaminants. In each wash, mix well and shake vigorously before spinning down. 12) Combine the TNA pellets from two tubes and resuspend in 300 μL od nuclease-free water for each 10 cm plate of cells. 22) Prepare the 8% 1.5 mm thick denatured rst dimension gel using the UreaGel system. For 10 mL gel solution, use 3.2 mL of UreaGel concentrate, 5.8 mL of UreaGel diluent, 1 mL of UreaGel buffer, 4 μL of TEMED, and 80 μL of 10% APS. Add TEMED and APS right before pouring the gel. 23) Use 15-well combs so that each lane is narrower and the second dimension has a higher resolution.

24)
To each 10 μL sample add 10 μL GBLII loading dye. Load 200 ng dsRNA ladder as molecular weight marker. Run the rst dimension gel at 30 W for 7~8 mins in 0.5X TBE. 25) After electrophoresis nishes, stain the gel with 2 μL of SYBR Gold in 20 mL 0.5X TBE, incubate for 5 min. Image the gel using 300 nm transillumination (not the 254 nm epi-illumination, which reverses the psoralen cross-linking). Excise each lane between 50 nt to topside from the rst dimension gel. The second dimension gel can usually accommodate three gel splices.

Second dimension gel:
26) Prepare the 16% 1.5 mm thick urea denatured second dimension gel using the UreaGel system. For 20 mL gel solution, use 12.8 mL UreaGel concentrate, 5.2 mL UreaGel diluent, 2 mL UreaGel buffer, 8 μL TEMED, and 160 μL 10% APS. 27) To make the second dimension gel, put the square plate horizontally and arrange gel slices in a "head-to-toe" manner with 2-5 mm gap between them. Leave 1 cm space at the top of the notched plate so that the second dimension gel would completely encapsulate the rst dimension gel slices. 28) Apply 20-50 μL 0.5X TBE buffer on each gel slice to avoid air bubbles when placing the notched plate on top of the gel slices. 29) Remove the excess TBE buffer after the cassette is assembled, and leave 2 mm space at the bottom of the notched plate to facilitate pouring the second dimension gel. 30) Pour and gel solution from the bottom of the plates, while slightly tilting the plates to one side to avoid air bubbles building up between the plates. If there are air bubbles, use the thin loading tips to draw them out. 31) Use ~60 o C prewarmed 0.5X TBE buffer to ll the electrophoresis chamber to facilitate denaturation of the cross-linked RNA. Run the second dimension at 30 W for 50 min to maintain high temperature and promote denaturation. The voltage starts around 300 V and gradually increases to 500 V, while the current starts around 100 mA and gradually decreases to 60 mA. 32) After electrophoresis, stain the gel with SYBR Gold the same as the rst dimension gel. 36) Transfer ~0.5 mL gel slurry to Spin-X 0.45 μm column. Spin at room temperature, 3400X g for 1 min. Continue until all gel slurry is ltered. 37) Aliquot 500 μL of the ltered RNA sample to an Amicon 10 k 0.5 mL column. Spin at 12,000 X g for 5 min. Repeat until all of the ltered RNA sample owed through the column. 38) Wash the column with 300 μL water and spin the column at 12,000X g for 5 min. 39) Invert and place the column in a new collection tube, and spin at 6000 X g for 5 min. Recover ~85 μL RNA from each column (~170 μL total from two columns). 40) Precipitate the RNA using the standard ethanol precipitation method, with glycogen as a carrier. Alternatively, the RNA can be puri ed using the Zymo RNA clean and concentrator-5 columns. 41) Reconstitute RNA in 11 μL water and dilute 1 μL RNA sample for Bioanalyzer analysis. The RNA sample should have a broad size distribution between 40 and 150 nt in the Bioanalyzer trace. The yield is typically 0.1-0.5% from 10 μg input RNA. 2. The PK digestion should clarify the solutions to some extent and greatly reduce turbidity. The addition of isopropanol should clarify the solution, resulting in obvious compact and stringy precipitates that contain both DNA and RNA, but little protein.
3. TNA sample: Most of the TNA sample should be soluble. If there is still some insoluble material, spin down and remove it. The cross-linked samples yield 60-70% of TNA compared to controls, and the A260/A280 ratios are usually in around 1.90, in the middle between the ratios for DNA and RNA. The A260/A230 ratios for the controls samples are usually above 2.1 and the ratios for crosslinked samples are usually below 1.9.
The Tape Station pro le for the TNA from cross-linked samples should show an obvious smear across the entire size range, while controls show three major peaks, namely the small RNAs, the 18S and 28S rRNAs. The controls should have a RIN number close to 10 while the cross-linked ones have a RIN number below 8. Alternatively use bioanalyzer to check size distribution.
4. Puri cation of RNA using Trizol/chloroform will lose ~50% of cross-linked RNAs. It is better to extract RNA directly using phenol/isopropanol precipitation method.
5. After 5 mins of short cut digestion, reaction need to be stopped as soon as possible. Longer reaction time will reduce the RNA fragments size.
6. Typically, the AMT cross-linked samples have a stronger tail above 100 nt than the control samples. 7. Mth RNA ligases will tightly bind target RNAs, affecting RNA recovery e ciency. Proteinase K treatment will remove Mth RNA ligases before puri cation, and increase the recovery e ciency. 8. UV irradiation will produce heavy RNA damage, such as cyclobutene pyrimidine dimer (CPD) and  lesion. Absorption of UV photons produce RNA singlet and triplet excited states. In this respect, the characterization of the RNA singlet states are mainly responsible for the formation of pyrimidine photoproducts. Triplet excited states only play a limited role (less than 10%) (1-2). Acridine dyes bind to double strand RNA by intercalating between adjacent base pairs or by exterior ionic bonding, and inhibit the pyrimidine dimer formation. Energy transfer from RNA to acridine is important in the reduction of dimer yields. And singlet states of RNA are responsible for this transfer (3). 9. Denature treatment and 10% of DMSO will unfold the RNA duplexes and enhance the adapter ligation e ciency.
10. Due to the modular design of PARIS2, users can apply some of the improvements while not using others. For example, if amotosalen is not available to the user, AMT can be used, in combination with all other improvements. In this case, other improvements will remain effective.