IGEM is based on a combination of viral RNA and viral protein to study HIV-1 RRE RNA and HIV-1 Rev protein. In vitro transcription of RNA and overexpression of Rev proteins can provide a reliable molecular basis and mechanism of action for the development of viral RNA vaccines, nucleic acid integrase inhibitors, and site-targeted drugs. We validated and characterized the interaction after in vitro transcription of HIV-1 RNA and overexpression of Rev protein.
Mechanism of RNA transcription in vitro
Transcription generally refers to the synthesis of RNA from DNA.RNA synthesis is divided into three stages: recognition and initiation, extension and termination.
RNA viruses synthesize double-stranded DNA by reverse transcriptase, and then the DNA is transcribed to synthesize RNA.The essence of transcription here is still DNA to RNA.RNA undergoes a folding mechanism similar to that of proteins in vivo in order to carry out its function during transcription in the cell. Therefore, in order to realize the environment and conditions similar to the cellular transcription process in the in vitro transcription process, we also maintain the normal folding of RNA to perform the function of RNA in the laboratory transcription by figuring out the Mg2+ conditions and reverse transcriptase, etc.[1]
Parts design.
The basic parts (HIV-1RRE and HIV-Rev) were both genome searched through the NCBI[2] database and designed to construct the HIV-1 RRE plasmid and HIV-1 Rev plasmid using the SnapGene software, as well as designing and optimizing the required primers through the Snapgene software. Finally, the whole gene was synthesized through DynaTech Biotechnology Co.ltd.
HIV-1 Rev was constructed from the PET28a expression vector, ligated with T7 polymerase, which is resistant to kanamycin, as shown in Figure 1.
HIV-1 RRE was constructed from the pMV vector (which is a derivative of the pUC19 vector, with the LacZa gene and its polyclonal site removed from the pUC19 vector), shown in Figure 2. The resistance was ampicillin. pMV was used as the forward primer and RRE-Reverse primer as the reverse primer.
1. Thaw reagents
Centrifuge the optimized T7 RNA Polymerase cryovial briefly and place it on ice; thaw 10×Transcription Buffer and 4 kinds of ribonucleotides (ATP, CTP, GTP, UTP), mix well and centrifuge to the bottom of the tube and place it on ice for later use; 10×Transcription Buffer at room temperature.
2. Configure the transcription reaction system at room temperature.
Prepare the reaction system according to the following system (Reaction volume will be 100μL so add 62μL of water).
3.Incubate at 37°C for 1.5h to obtain HIV-1 RRE RNA by transcription.
Mix the above reaction solution evenly, centrifuge briefly to the bottom of the tube, and incubate at 37°C for 1.5 hours. The length of the transcript is 358nt, and the reaction time is crucial to the quality of the transcript.
Note: During this experiment, it is recommended to use RNase-free pipette tips and EP tubes, and wear disposable latex gloves and masks. All reagents are prepared with RNase free H2O to prevent RNase contamination during the experimental operation.
HIV-1 Rev overexpression, isolation and purification
1. Select a single colony in A to 100 ml of sterile LB medium corresponding to the resistance, and culture it overnight at 37°C with a shaker at 220 rpm/min for 16 hours.
2. After the bacterial liquid cultured overnight is measured to have an OD600 of 0.6, take 15ml of the bacterial liquid to 1L and add it to the sterile LB medium of the corresponding resistance. Culture at 37°C with a shaker at 220rpm/min until the OD600 is between 0.6-0.8. Start with 1mM IPTG. Induce the expression of the target protein and continue culturing for 12-16 hours at 16°C with a shaker at 220 rpm/min.
3.Cell disruption: Collect the cells by centrifugation, weigh the wet cells, add lysis solution and protease inhibitors, and conduct ultrasonic disruption to release proteins.
4. Collect the protein solution: Collect the protein solution by high-speed centrifugation and remove cell debris.
5.Purification: Use a Ni column for crude purification of the target protein, and use different imidazole concentrations to gradient elute impurity proteins, and finally use high-concentration imidazole to elute the target protein.
6. Dialysis: dialyze the target protein eluted with high-concentration imidazole buffer overnight to remove imidazole.
7. Molecular sieve purification: The dialyzed protein is further purified through a Superdex75 column, and the buffer is replaced to be consistent with the buffer for HIV-1 RRE RNA storage to prevent RNA degradation or protein aggregation/degradation due to buffer inconsistency.
Collect protein components, measure their concentration, and freeze for later use.
Characterization results
Our team collected data on the basic components of HIV-1 RRE. In vitro transcription yielded a single, compact HIV-1 RRE RNA band (Figure 1), indicating that 6mM Mg2+ concentration can stabilize the folding of HIV-1 RRE RNA.
At the same time, our IGEM team also characterized the basic component HIV-1 Rev protein. Through the overexpression, isolation, purification and identification of HIV-1 Rev protein (Figure 2), it was shown that the purified Rev band was single, which basically meets the requirements for further research on the interaction between Rev and HIV-1 RRE RNA, as well as the exploration of interaction sites.
[1]Wong TN, Pan T. RNA folding during transcription: protocols and studies. Methods Enzymol. 2009; 468:167-9
[2]https://www.ncbi.nlm.nih.gov/nuccore/K02007.1