Chemical Mapping 7SK snRNA

Jacob Sorensen Author

04/05/2021 Added

37 Plays

Description

Enjoy my research on chemical mapping 7SK!

Searchable Transcript

Search:

[00:00:01.050]Hi, my name is Jacob Sorenson,
[00:00:02.850]and I'm going to be talking to you about my research on chemical mapping,
[00:00:05.970]the secondary structures of seven S K S and RNA
[00:00:10.350]organisms contain coding RNA, which is also known as M RNA,
[00:00:14.340]which can be translated into proteins as well as non-coding RNA,
[00:00:17.850]which can not be translated.
[00:00:20.040]Non-coding RNA is a diverse class of RNA with many different functions.
[00:00:25.080]An example of non-coding RNA is small nuclear RNA,
[00:00:28.260]which function and the regulation of gene expression,
[00:00:31.380]seven S K S and RNA makes up the core of the seven S K ribonucleoprotein
[00:00:36.270]complex and is abundant in the human body.
[00:00:39.330]Seven SK functions to regulate the activity of positive transcription,
[00:00:43.770]elongation factor B or P T E F B,
[00:00:47.160]which in turn regulates RNA polymerase two,
[00:00:49.890]which promotes the efficiency of M RNA in the cell.
[00:00:54.420]A big part of my research prior to conducting experiments on my own was
[00:00:58.290]analyzing literature pertaining to 700 K probing.
[00:01:01.680]I did a lot of research on methods to get a good understanding on the how and
[00:01:05.790]why of chemical mapping as well as obtain raw data that I could
[00:01:10.470]input into spreadsheets for later. Comparison,
[00:01:14.040]figure one on the right is a structure from a paper published by the Howard
[00:01:17.550]Chang lab.
[00:01:18.540]Here I see shape was used to probe seven SK when bound to different proteins.
[00:01:23.040]Hexham one in bath to see how it affected the structure.
[00:01:27.090]This paper was one of the biggest inspirations for my project and gave me a lot
[00:01:30.720]of ideas on the direction I want my project to go.
[00:01:34.770]The purpose of my study is to use chemical mapping methods and computational
[00:01:38.490]modeling to determine the seven SK RNA secondary structure of STEM loop three
[00:01:42.900]specifically in various buffer conditions by determining the structure of seven
[00:01:47.160]SK in various conditions,
[00:01:49.320]the function can later be hypothesized by understanding how the RNA interacts in
[00:01:53.430]certain circumstances.
[00:01:55.410]Further structural analysis can help determine how PTE F B binds and releases in
[00:02:00.360]the seven S K R and P complex.
[00:02:03.780]Here's a little overview on chemical mapping.
[00:02:06.390]It works by modifying bases that are solvent exposed or accessible to the
[00:02:10.200]probing agent. Also known as single stranded regions, depending on the probe.
[00:02:14.910]Different positions on different bases can be modified DMS.
[00:02:19.140]Methylates the end seven position of adenine and N three position of cytosine
[00:02:24.210]shape isolates the two primary Doxil group of solvent exposed spaces
[00:02:29.310]because of this,
[00:02:30.240]it is not based specific and can modify all four bases.
[00:02:35.490]CMCT is a carbo Diomede,
[00:02:38.010]which covalently a ducks gone in and [inaudible].
[00:02:41.130]And finally [inaudible] forms five member rings on single-stranded guanine,
[00:02:45.960]different chemical probes provide complimentary data on seven S K secondary
[00:02:50.640]structure and can be used together to form a more comprehensive structure.
[00:02:55.620]Finally, magnesium is a crucial part to this process because it allows for the
[00:03:00.130]stabilization of the secondary structures in buffer additions,
[00:03:04.620]before chemical mapping,
[00:03:06.150]I had to first optimize my transcription reactions of STEM loop three.
[00:03:11.520]The reactions were run for three to four hours at 37 degrees Celsius.
[00:03:15.540]And as you can see in table one, the conditions that were optimized,
[00:03:19.470]where the Ribo nucleoside triphosphate levels, the magnesium concentration,
[00:03:24.330]the DNA template concentration, as well as the enzyme conscience concentration.
[00:03:29.460]After the transcription reactions,
[00:03:31.650]the yield was analyzed on Agros gels
[00:03:36.600]in table two,
[00:03:37.530]you can see the final optimized values for seven S K STEM loop through
[00:03:40.950]transcription, which I used in my case, chemical mapping.
[00:03:44.940]The first step in chemical mapping is to fold the RNA.
[00:03:48.870]You need to heat and cool it to destabilize any preexisting secondary
[00:03:52.770]structures. After that, you can prepare the folding buffers.
[00:03:56.460]The three I use were number one, sodium could catalase magnesium,
[00:04:01.260]number two, sodium Kolkatta late, only in number three,
[00:04:05.010]magnesium and heaps. Yeah.
[00:04:07.410]Folding buffers were then added to my RNA samples for 30 minutes to induce RNA
[00:04:11.970]folding into form secondary structures.
[00:04:15.240]After folding modification took place.
[00:04:18.660]Michael probe I chose to use was DMS.
[00:04:21.390]The DMS was added each RNA tube. In addition,
[00:04:26.250]water was added to separate tubes to serve as a background subtraction later on
[00:04:31.110]in the process,
[00:04:32.730]the modification reaction occurred for exactly six minutes when it was quenched
[00:04:36.630]the BME. And after that it was purified
[00:04:42.150]and the concentration was taken on a cubit, right? Figure three.
[00:04:46.470]You can see the sites on adenine inside ezine that DMS modifies
[00:04:52.050]after modification reverse transcription took place to the modified RNA.
[00:04:56.490]I added Tieger,
[00:04:57.510]which shows the reverse transcriptase enzyme as well as a barcode slash reverse
[00:05:01.800]transcription primer. After the reverse transcription,
[00:05:05.610]the reaction was quenched with quench acid to stop the enzymatic activity.
[00:05:11.340]The newly synthesized cDNA DNA fragments were then purified.
[00:05:15.720]Lastly,
[00:05:16.680]the samples were prepped for sequencing [inaudible] master mix was added to the
[00:05:20.610]cDNA along with forward and reverse primers for PCs.
[00:05:24.870]After the PCs,
[00:05:27.060]the nude double stranded DNA was run on 2% precast Agros
[00:05:31.830]Eagles for 10 minutes,
[00:05:33.510]where it was extracted purified in quantified on a cubit.
[00:05:38.190]Again, lastly, it was input yeah, into the sequence.
[00:05:42.330]The sequencer spit out data,
[00:05:44.220]and that data was input into Varna to predict secondary structures
[00:05:49.350]now onto my data and results. Seven S K STEM loop,
[00:05:52.560]three can adopt two different secondary structures. Number one on the left.
[00:05:56.340]And number two, the right here are the results for buffer condition.
[00:06:00.740]Number one,
[00:06:02.050]as you can see a lot of the solvent exposed a and C basis have
[00:06:06.520]reactivity values.
[00:06:09.250]The scale is set so that blue represents zero or lower activity in
[00:06:14.050]anything above 0.4 is red,
[00:06:17.590]which signifies high reactivity values. As you can see,
[00:06:21.430]there are some reactive double stranded base pairs in
[00:06:26.020]both confirmations,
[00:06:28.780]which suggests that both structures are formed buffer
[00:06:33.460]condition, number two, produced pretty similar results. However,
[00:06:38.350]the basis that our solvent exposed seem to have lower reactivity values.
[00:06:43.210]This could be due to the absence of magnesium,
[00:06:47.050]not stabilizing the structure. And lastly, buffer condition. Number three,
[00:06:51.970]produced really similar results to buffer condition. Number two,
[00:06:56.110]in conclusion,
[00:06:57.250]both secondary structure variations have data that suggests they form.
[00:07:01.480]This is very preliminary data,
[00:07:03.070]but I'm really excited to be making progress during a pandemic as observed
[00:07:07.990]in literature and through data analysis,
[00:07:09.670]both [inaudible] secondary structures seem to form an all three conditions.
[00:07:14.050]The percentage is still to be determined in the future.
[00:07:17.320]I hope to incorporate protein into my buffers to determine the
[00:07:21.910]structure of the Seminis K RNP. In addition,
[00:07:25.510]I would like to use other probes to get a more comprehensive structure.
[00:07:29.890]Ultimately, my goal is to answer the question,
[00:07:32.260]how do RNA protein interactions affect secondary structures?
[00:07:36.700]Finally, I want to say a big thank you to Dr.
[00:07:39.190]Eichorn as well as the rest of the Icahn research group for their contributions
[00:07:42.670]to my project. In addition, I want to thank Dr.
[00:07:45.490]Yes-man for his assistance in the procedure,
[00:07:48.340]data processing and data interpretation.
[00:07:51.310]I would also like to thank the entire UCare fellowship program for giving me
[00:07:55.090]this opportunity.
[00:07:56.530]Stay tuned for more exciting seven SK chemical mapping research.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

Chemical Mapping 7SK snRNA

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments