HIV-1 RNA structure and heterogeneity analysis

The goal of this set of experiments is to determine the structure of HIV-1 NL4-3 and NHG RNA structure using a novel algorithm that allows for the identification of multiple RNA folding conformations. The input data used for this algorithm is DMS-MaPseq data. We focused on two specific areas of the HIV-1 genome, the Rev Response Element (RRE) and A3 splice acceptor site, as well as probing the whole HIV-1 genome. RNA is DMS-modified in vitro and in vivo as indicated. The goal of this data set was to test the novel alternative RNA structure detecting algorithm (DREEM) using RNA molecules with known structures. Overall design: DMS-Modification and targeted sequencing of the HIV-1 A3 splice acceptor site in CD4+ T-cells or transfected HEK293t cells. 1 sample in CD4 T cells, 3 in HEK293t. DMS-modification and targeted sequencing of the HIV-1 A3 splice acceptor site in transfected HEK293t cells using two designed mutant viruses. 1 sample per mutant. HEK293t cells were transfected with a plasmid containing HIV-1 NHG. The cells were DMS-modified and RNA was extracted and sequenced. RNA strucural models were generated using a novel algorithm for regions of interest across the HIV-1 genome. Two HIV-1 RRE mutants were transfected in a HIV-1NHG plasmid into HEK293. Structure of both mutants were analyzed and mixed computationally in order to validate the DREEM pipeline. HIV-1 RNA structure of RRE was analyzed after DMS modification in vitro, in vivo and in the virion. For in vitro samples, HIV-1 RRE was in vitro transcribed. For in vivo and in virion, isolated CD4+ T-cells were infected with HIV-1 NL4-3. U4-6 core-domain was in vitro transcribed, refolded and DMS modified. The RNA was then prepared for sequencing following the DMS-MaPseq library generation protocol and clustered by DREEM. Adenine Riboswitch was in vitro transcribed, refolded and DMS modified with and without 5 mM adenine. The RNA was then prepared for sequencing following the DMS-MaPseq library generation protocol and clustered by DREEM. Samples StemA_C_mix_1-5: The goal of this set of experiments is to validate a novel RNA structure clustering algorithm. Two closely related structured RNAs were in vitro transcribed and mixed at different proportions. The RNA was DMS-modified and used for DMS-MaPseq and clustering by the algorithm DREEM. Two RNAs the form different structures within the human gene MRPS21 we in vitro transcribed, mixed at different proportions and DMS-modified. The RNA structure was determined by DMS-MaPseq and clustering with the DREEM algorithm.