Journal: Journal of Extracellular Vesicles

Article Title: A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery

doi: 10.3402/jev.v5.31027

Figure Lengend Snippet: Evaluation of RNA loading into EVs via the TAMEL platform. (a) This cartoon summarizes the concept of facilitating active loading of cargo RNA into EVs via our TAMEL platform. A TAMEL EV-loading protein comprises an EV-enriched protein (EEP, blue) fused to an RNA-binding domain (RBD, green), which localizes to EVs. Actively loaded RNA (green) contains a motif that binds to the RBD, resulting in enhanced loading into EVs relative to passively loaded RNA (orange). (b) RNA cargo design impacts active loading. The “fold enrichment of cargo mRNA +/−MS2” is defined as the ratio of cargo RNA/GAPDH mRNA in EVs derived from cells expressing Lamp2b–MS2–HA divided by the same RNA ratio in EVs derived from cells expressing Lamp2b–HA. All experiments were performed in biological triplicates. (c) Cartoon illustrating the 3′ RNA fragment analysis technique. Cargo RNA is first reverse transcribed using an oligo dT primer, and amplicons corresponding to the RNA 5′ or 3′ ends (the latter is located ~500 bases upstream of the polyA site) are then quantified by qPCR using the primer pairs indicated. Note that the amplicon near the RNA 3′ end will be present in cDNA derived from both full-length RNA and 3′ RNA fragments. (d) Analysis of 3′ RNA fragment loading into EVs. Cargo RNA levels were quantified as depicted in panel c and normalized to GAPDH. Passive loading: cells transfected with Lamp2b–HA; active loading: cells transfected with Lamp2b–MS2–HA. (e) Full-length RNA and 3′ fragment RNA levels in EVs were quantified following incubation at 37°C; experiments were performed in technical duplicate with a biological replicate shown in Supplementary Fig. 2a. Error bars indicate 1 standard deviation, throughout. MVB, multivesicular body.

Article Snippet: The pMS2-GFP plasmid, which encodes the MS2 bacteriophage coat protein tandem dimer (mutant d1FG and V29I ( , )), was obtained from Addgene (plasmid #27121), deposited by Robert Singer ( ).

Techniques: RNA Binding Assay, Derivative Assay, Expressing, Reverse Transcription, Amplification, Transfection, Incubation, Standard Deviation

Journal: Journal of Extracellular Vesicles

Article Title: A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery

doi: 10.3402/jev.v5.31027

Figure Lengend Snippet: Impact of EEP choice on TAMEL-mediated active RNA loading into vesicles. (a) Effects of EEP choice on cargo RNA loading into EVs. Experiments were performed in biological triplicate. (b) Effects of EEP choice on cargo loading into gesicles. Experiments were performed in biological triplicate. (c) Protein abundance was quantified by densitometry analysis of anti-HA western blots (Supplementary Fig. 3), and each blot was internally normalized by the intensity for VSVG–MS2–HA in gesicles (maximal intensity case). This experiment was performed in biological duplicate. The y-axis is in log scale to enable visualization of all values. Error bars indicate 1 standard deviation, throughout.

Article Snippet: The pMS2-GFP plasmid, which encodes the MS2 bacteriophage coat protein tandem dimer (mutant d1FG and V29I ( , )), was obtained from Addgene (plasmid #27121), deposited by Robert Singer ( ).

Techniques: Quantitative Proteomics, Western Blot, Standard Deviation

Journal: Journal of Extracellular Vesicles

Article Title: A platform for actively loading cargo RNA to elucidate limiting steps in EV-mediated delivery

doi: 10.3402/jev.v5.31027

Figure Lengend Snippet: Comparative analysis of dTomato delivery by actively or passively loaded vesicles. For panels a–c, the cartoons at left summarize the experimental designs, and in the panels at right, each data point represents the average of duplicate wells of cells treated with the same type of vesicle. Error bars indicate 1 standard deviation. “Normalized fluorescence” is defined as the mean fluorescence of cells receiving vesicles divided by the mean fluorescence of cells receiving a medium change only. (a) Time course of EV delivery to cells. Grey squares: CD63–HA EVs; orange circles: CD63–MS2–HA EVs. The solid arrow represents cells that received a medium change after 4 h of EV treatment. The dashed arrow indicates that cells did not receive a medium change. Statistically significant differences (p<0.05, not shown for clarity): CD63–MS2–HA +/− medium change. An independent repeat of this experiment is shown in Supplementary Fig. 3a. (b) Time course of gesicle delivery to cells. Purple squares: VSVG–HA gesicles; green circles: VSVG–MS2–HA gesicles. Solid and dashed arrows carry the same meaning as in panel (a). Statistically significant differences (p < 0.05, not shown for clarity): VSVG–HA versus VSVG–MS2–HA at 4 and 16 h (comparisons were made for each time point), VSVG–HA +/− medium change, and VSVG–MS2–HA +/− medium change. An independent repeat of this experiment is shown in Supplementary Fig. 3c. (c) Comparison of delivery by gesicles from cells transfected with VSVG–HA (purple), VSVG–MS2–HA (green) or a 50:50 mix of VSVG–HA and VSVG–MS2–HA (hybrid gesicles, magenta). (d) dTomato RNA levels (normalized to GAPDH) in VSVG–HA gesicles (purple), VSVG–MS2–HA gesicles (green) or hybrid gesicles (magenta). Error bars indicate 1 standard deviation of technical duplicate samples. *Significant difference was evaluated with a Student's t -test using a cut-off of p < 0.05.

Article Snippet: The pMS2-GFP plasmid, which encodes the MS2 bacteriophage coat protein tandem dimer (mutant d1FG and V29I ( , )), was obtained from Addgene (plasmid #27121), deposited by Robert Singer ( ).

Techniques: Standard Deviation, Fluorescence, Comparison, Transfection

Journal: Nature biotechnology

Article Title: Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

doi: 10.1038/nbt.2880

Figure Lengend Snippet: (a) Steps for generating RNA tethered to DNA clusters on a high-throughput DNA sequencing flow cell. (b) Structure of the MS2 coat protein homodimer bound to the 19 nt hairpin RNA (PDB ID: 2BU1) . (c) Images of fluorescently labeled MS2 bound to RNA clusters at increasing concentrations of protein and at time points following perfusion of unlabeled MS2 competitor. Below, fitted sum of Gaussians used to assign fluorescence to clusters. Scale bars (white) represent 2.5 μ m . (d) Fluorescence decay of MS2 dissociating from clusters containing the consensus sequence (-5C) ( t 1/2 =8.39 minutes). (e) Fit binding curves to clusters labeled in panel ( c ). (f) The probability distribution of binding energies from all clusters with labeled variants; mean K d = 2.57 nM, 36.8 nM, and 415 nM for the -5C, -5U, and -5A variants, respectively. (g) Correlation between binding energies reported in the literature and measured on the RNA array (squares, Carey et al. , circles, Romaniuk et al. ). (Dashed line indicates our affinity measurement cutoff.)

Article Snippet: The MS2-dlFG mutant of the MS2 Coat Protein was cloned into a custom expression vector containing an N -terminal FLAG and SNAPtag (NEB) and a C -terminal 6xHis tag ( https://benchling.com/s/oYAOq4 ).

Techniques: High Throughput Screening Assay, DNA Sequencing, Labeling, Fluorescence, Sequencing, Binding Assay

Journal: Nature biotechnology

Article Title: Quantitative analysis of RNA-protein interactions on a massively parallel array for mapping biophysical and evolutionary landscapes

doi: 10.1038/nbt.2880

Figure Lengend Snippet: (a) Distribution of observed RNA variants by number of mutations. (b) Clusters measured per molecular variant as a function of mutation number. A median of ~11 clusters are observed for sequences with ≥4 mutations. Affinities for the consensus sequence come from N C =909,385 clusters. (c) Average − ΔΔG of point mutations per position. The − ΔΔG of alanine substitutions to the MS2 binding surface are shown in parentheses ( k B T ). Solid and dashed lines represent base and phosphate interactions, respectively. (d) Matrix of − ΔΔG for single and double mutants of the consensus sequence. Inset contains the m atrix of − ΔΔG for single and double mutants of the +1G variant. All energies are calculated relative to the consensus (-5C) sequence (arrow, − ΔΔG =0), and the number of quality-filtered double mutants in each matrix is indicated (M 2 ) . (e) Epistasis matrix derived from (d) allows de novo reconstruction of the hairpin structure.

Article Snippet: The MS2-dlFG mutant of the MS2 Coat Protein was cloned into a custom expression vector containing an N -terminal FLAG and SNAPtag (NEB) and a C -terminal 6xHis tag ( https://benchling.com/s/oYAOq4 ).

Techniques: Variant Assay, Mutagenesis, Sequencing, Binding Assay, Derivative Assay

Journal: Molecular Cancer

Article Title: LncRNA LINRIS stabilizes IGF2BP2 and promotes the aerobic glycolysis in colorectal cancer

doi: 10.1186/s12943-019-1105-0

Figure Lengend Snippet: LINRIS was associated with IGF2BP2 in CRC. a IGF2BP2 was pulled down by biotin-labeled sense LINRIS (S) but not LINRIS anti-sense (AS) RNA in the indicated cells. b RIP assays were applied using anti-IGF2BP2 antibodies with extractions from HCT116 cells. Relative enrichment (mean ± SD) represents the RNA levels associated with the indicated protein relative to an input control from three independent experiments after immunoprecipitation with the anti-IGF2BP2 antibody compared with that with the IgG antibody. MYC mRNA was uesd as the positive control and GAPDH mRNA was used as the negative control. c Expression vectors for FLAG-tagged MCP and MS2-tagged LINRIS were transfected into CRC cells to establish the FLAG-MCP-MS2 system. And IGF2BP2 was then pulled down using the anti-FLAG® M2 affinity gel followed by the Western blot analysis. d Western blot detection of IGF2BP2 binding to LINRIS after FLAG-MCP-MS2 pull-down assays. e In vitro-synthesized full-length (FL), N-terminal (NT) and C-terminal (CT) fragments of LINRIS were incubated with protein lysates from HCT116 cells. RNA pull-down and Western blotting assays were then performed. The data shown represent three independent experiments. f Western blot analysis shows the levels of IGF2BP2 in 11 CRC cell lines with GAPDH as the loading control. g IGF2BP2 expression was positively correlated with LINRIS expression in CRC cells. The r values and P values are from Pearson’s correlation analysis. h Western blot analysis shows the expression of IGF2BP2 with or without knockdown of LINRIS in the indicated cells. i CRC cells transfected with shRNAs specific for LINRIS or a scrambled control. Cell lysates were immunoprecipitated with either an antibody against IGF2BP2 or an IgG control and then analyzed by immunoblotting with a ubiquitin (Ub)-specific antibody

Article Snippet: The expression vectors for 3FLAG-tagged MS2 coat protein (MCP) and MS2-tagged LINRIS were provided by OBiO Technology Co., Ltd. (Shanghai, China), and FLAG-tagged expression vectors for full-length IGF2BP2 and site-directed mutants (K77R and K139R) were provided by Kidan BioTechnology Co., Ltd. (Guangzhou, China).

Techniques: Labeling, Immunoprecipitation, Positive Control, Negative Control, Expressing, Transfection, Western Blot, Binding Assay, In Vitro, Synthesized, Incubation