RdRp assay for DENV4 NS5Pol

DENV4 RdRp assay protocol (biotin-labeled)

Part I. Core Reagents and Buffers

                                                               Table 1 Reagents information

★This compound has been discontinued. For replacement, Biotin-16-UTP (Sigma-Aldrich 11388908910) may be used.

                                                                              Table 2 Buffers

★Do not pre-mix RdRp reaction buffer B with manganese(II) solution

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Part II. Experimental Procedure

1. Prerequisites

1.1 This protocol is designed for the RdRp activity assay of DENV4 NS5Pol protein. For the characterization of RdRp activities of other flavivirus NS5 proteins, optimization may be required, and the protocol may not be suitable for the investigation of an RdRp which does not originate from members of the Flavivirus genus.

1.2 To perform the DENV4 RdRp assay, The NS5Pol or full-length NS5 protein should be expressed, purified and stored as single-use aliquots at -80°C in advance. The preparation of template RNA is another prerequisite. Properly purified In vitro transcribed RNA can be stored at -80°C for at least one year. In this protocol, the RNA corresponding to the first 5′-160 nt of DENV4 genome, or the mini-genomic DENV4 RNA, which contains both 5′ and 3′ ends of DENV4, is utilized as the templates.

1.3 Ensure that the reagents, buffers, tubes, tips and other materials used are RNase-free. Keep the working area as clean as possible to diminish RNase contamination. Use RNaseZAP (Thermo Fisher Scientific) reagent to remove RNase from the apparatuses and working benches if necessary.

2. Perform the RdRp reaction

2.1 Reagent preparation

2.1a Thaw RNase-free water, 10×RdRp reaction buffer B, 5×RNA folding buffer T2, Manganese (II) solution and 0.1 M DTT. Shake the tubes gently and thoroughly, then centrifuge briefly before use. Note: 0.1 M DTT should not be used if any precipitation is present.

2.1b Prepare an adequate volume of mixture containing 10 mM of ATP, UTP and GTP from the NEB NTP solution set. Note: This can be stored at -20°C for at least one month. Avoid frozen-thaw of all NTP solutions.

2.1c Prepare an adequate volume of 4 mM CTP from the NEB NTP solution set. Note: This can be stored at -20°C for at least one month. Avoid frozen-thaw of all NTP solutions.

2.1d Prepare the Biotin-labeling Mix as listed below. Note: We usually use freshly-prepared Biotin-labeling Mix. The quantity of the below recipe is sufficient for 4 reactions.

2.1e Dilute adequate quantities of RNA templates with RNase-free water to a volume of 8 μl, then heat the samples at 95°C for 2 min, followed by snap-cooling on ice. Add two microliters of 5×RNA fold buffer T2 to the diluted samples, then incubate them at 37°C for 20 min to re-fold the RNA samples. In our experiments, the concentration of the 10-μl re-folded RNA is 150 ng/μl. The folded RNA should be stored at 4°C, and used within 24-hours.

2.1f Take out one aliquot of NS5Pol protein from the -80°C refrigerator and put it immediately into a room-temperature (25°C) water-bath, shake the tube gently to thaw the protein solution. Keep the thawed protein on ice to minimize activity lost.

2.2 Set up and perform the reaction

Set up the reactions as listed below, the NS5Pol protein should be added lastly. A reaction containing heat-inactivated NS5Pol can serve as the negative control. Mix the reactions gently and centrifuge briefly. Incubate the reaction tubes at 30°C for 10-20 min if 5′-160 nt RNA is used. For reactions using mini-genome templates, the incubation time can be extended up to 40 min.

3. Clean-up of the RdRp product.

The product should be purified before electrophoresis. We used the cost-effective RNAclean kit from Tiangen Biotech. Perform the clean-up procedure as described in the instructions of the manufacturer. Elute using 14 μl of RNase-free water. Add an equal volume of 2×RNA loading dye, mix well and stored at -20°C before use. The purified product can be stored at -20°C for at least one month.

4. Urea denaturing PAGE

4.1 Treat the comb, glass plates, and the other components of the Bio-rad Mini-PROTEAN 3 electrophoresis apparatus and the electrophoresis working bench with 0.5% SDS solution for 5 min, then wash them thoroughly with deionized water, air-dry the electrophoresis apparatus.

4.2 Cast 6% TBE/PAGE gels containing 8 M Urea as listed below, the polymerization of gel should be complete in about 30 min. This recipe is intended for two 0.75-mm thick mini-gels.

4.3 Assemble the electrophoresis system, pre-run at 80 V for 30 min at room temperature with 1×TBE as the running buffer.

4.4 Heat the purified RdRp products at 70°C for 5 min, then cool them on ice. Carefully wash out buffers containing high concentration of urea in the wells with a 2-ml syringe. Load 8-μl of samples into the wells. A dsRNA sample with the same length as the RdRp product can be used as a marker, if one replica of the gel is to be stained with SYBR Gold nucleic acid gel stain. Perform the run at 80 V for 1-1.5 hrs at room temperature. Note: 1. The equal volume of 1×RNA loading dye can be loaded surrounding the sample lanes to improve running performance. 2. The RdRp product stays in the dsRNA-form under the electrophoresis conditions described herein.

5. Electro-blotting and Cross-linking

5.1 Semi-dry electro-blot method was routinely used in our lab. After the electrophoresis, remove the extra lanes of the gel by using the Gel Releaser tool, and cut off a small piece from one corner of the gel (in order to identify the running direction and loading sequence). Measure the dimension of the gel which is to be blotted, then submerge the sliced gel into 0.5×TBE for 10 min.

5.2 During this period, accurately cut one piece of Hybond N+ Nylon membrane and six pieces of Whatman 3MM Chr Chromatography paper (or other filter papers with similar parameters), all of which should be the same size and dimension as the corresponding gel.

5.3 Wet the filter papers and the membrane with 0.5×TBE, take out the gel to be transferred from 0.5×TBE and put it on a clean PE plastic glove. Assemble the filter paper/membrane/gel sandwich carefully to avoid air bubbles and gel cracking. The stacking sequence of the sandwich should be compatible with the design of the electro-blotting system used. Set the current to 1 mA per square centimeters of gel area for the 5′-160 nt RdRp product, for the mini-genome RdRp product, set the current to 2.5 mA/cm². We usually optimize the current between 0.5 to 4 mA/ cm².

5.4 After transferring for 45-60 min, disassemble the transfer cell and recover the membrane and gel carcass respectively. Label the membrane with a soft pencil for the easily-recognition of running and transfer direction, as well as sample sequence. Wash the membrane with 1×TBE once, and dry it in the air.

5.5 Optional: Stain the gel carcass with SYBR Gold Nucleic Acid Gel Stain working solution for 15-30 min to check the efficiency of transfer.

5.6 Cross-linking the RNA with the membrane by exposing the transferred side to a 302 nm UV transilluminator for 10 min, or perform the cross-linking by using a UV Crosslinker & Hybridizer Oven.

6. Detection of chemiluminescence signals

6.1 Submerge the membrane into a 10% non-fat milk solution, and the blocking is performed at 37°C for 2 hrs, followed by an incubation at 4°C for 16 hrs.

6.2 Wash the membrane with PBST once, then incubate the membrane with 20 ml of 1:2500 (mini-genome RNA template) or 1:4000 (5′-160 nt RNA) diluted streptavidin-HRP (in PBST) on an orbital shaker for 2 hrs at room temperature. 

6.3 Wash the membrane with PBST for three times, each wash is performed on an orbital shaker for 20 min.

6.4 Detection of chemiluminescence signal using the Pro-Light HRP chemiluminescence kit and Smartchemi II imaging system, or other chemiluminescence imaging systems, as instructed. The typical detection procedure using the Smarchemi II is to capture the signal every 6 seconds for 6 min.

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