Final_Poster_Presentation_ReenaDosanjh

Reena Dosanjh Author

07/28/2021 Added

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Final research presentation

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[00:00:03.210]Hi, my name is Reena Dosanjh
[00:00:04.940]and today I will be discussing my project
[00:00:07.820]focusing on probing the secondary structure
[00:00:09.920]and kinetics of an RNA methyltransferase enzyme.
[00:00:14.770]Recently, a group of researchers have identified
[00:00:17.160]a novel ribozyme that has the ability
[00:00:19.530]to catalyze the transfer of a methyl group
[00:00:22.020]to the n1 position of an amine in a site-specific manner.
[00:00:26.320]This methyltransferase ribozyme, which is named MTR1,
[00:00:30.130]uses the co-factor O6-Methylguanine
[00:00:33.230]as a co-substrate to transfer a methyl group to the adenine
[00:00:36.940]at the n1 position as shown here.
[00:00:42.240]Methylation of adenine is a type
[00:00:43.710]of post transcriptional modification
[00:00:46.810]and post transcriptional modifications
[00:00:48.460]have been shown to affect the structure of RNA molecules.
[00:00:52.800]One specific effect is that methylation of amines
[00:00:55.720]can prevent shifting between two confirmations
[00:00:58.150]of an RNA molecule when both confirmations
[00:01:00.680]are equally favorable.
[00:01:02.740]Therefore, MTRI can be optimized
[00:01:04.720]as a tool for a site-specific methylation of adenine
[00:01:07.630]to lock an RNA molecule in one conformation.
[00:01:13.120]Our research goals for this project
[00:01:14.740]are to determine the secondary structure of MTR1 constructs
[00:01:18.490]using dimethyl sulfate or DMS
[00:01:21.140]as a probe to assess the kinetics of MRTR1 constructs
[00:01:27.210]using real-time NMR kinetic studies.
[00:01:30.030]And lastly, to analyze the overall effect
[00:01:32.300]of mutations in the stem region of MTR1 constructs
[00:01:35.530]on catalytic activity.
[00:01:39.930]In this project, we're using wild-type MTR1
[00:01:43.110]shown here on the left, along with two immune constructs
[00:01:46.180]that I designed that contain mutations in the stem region.
[00:01:51.030]So we have MTR1 UA which contains two UA-based pairs
[00:01:55.310]in the stem, shown in red
[00:01:57.300]and MTR1 UGA which has a UGA there that places
[00:02:01.090]the GAG-based pairs in MTR1 wildtype.
[00:02:06.860]So which here MRT1 methylates adenines at the n1 position.
[00:02:10.740]In this project, we're using the solvent dimethyl sulfate,
[00:02:14.080]which also methylates adenines at the n1 position
[00:02:16.800]along with cytosines at the n3 position
[00:02:19.700]as a probe in chemical mapping experiments
[00:02:21.880]to determine secondary structure.
[00:02:24.730]We're also conducting real-time NMR kinetic studies
[00:02:27.640]to obtain the kinetic parameters
[00:02:29.220]of the wildtype imino constructs.
[00:02:34.180]To give a brief overview of chemical mapping experiments,
[00:02:38.020]first we have our RNA construct
[00:02:41.980]and then we introduce DMS to the reaction mixture,
[00:02:45.550]which then methylates the unpaired adenines and cytosines.
[00:02:51.130]And then we use a reverse transcriptase
[00:02:53.960]to convert RNA to complimentary DNA
[00:02:57.270]and this reverse transcriptase also produces mutations
[00:03:00.770]that mutates methylated adenines or cytosines to thymines.
[00:03:07.490]And then we amplify the cDNA
[00:03:09.220]using polymerase chain reaction.
[00:03:12.750]And next we input these samples into the sequencer
[00:03:16.990]and then we're able to align the sequences
[00:03:19.430]and determine which adenines and cytosines were methylated
[00:03:24.790]based on which were mutated to thymines.
[00:03:30.050]First, we conducted chemical mapping
[00:03:31.650]with MTR1 wildtype.
[00:03:33.470]And on the left, we had the reactivity values
[00:03:35.380]along with the conditions that we used.
[00:03:38.020]There's an increase in reactivity with the addition of DMS
[00:03:41.826]and the base pairs there in the loop region
[00:03:43.320]are more reactive than the base pairs in the stem.
[00:03:48.840]And now looking at MTR1 wildtype with substrate,
[00:03:51.710]we can see that these base pairs at the ends
[00:03:53.630]are less reactive now that they're base paired
[00:03:55.530]to the substrate.
[00:04:00.010]Next, we conducted chemical mapping
[00:04:01.680]with MTR1 UA, which again,
[00:04:03.960]contains two extra base pairs, UA and AU in the stem region.
[00:04:08.770]Again, we can see that the basis in the stem region
[00:04:11.810]or the bases in the loop region
[00:04:13.480]are more reactive to methylation
[00:04:15.130]than the bases in the stem.
[00:04:19.780]Next, looking at MTR1 UA with substrate.
[00:04:22.620]Again, we observed that the bases there now bound
[00:04:25.080]to the substrate over here are less reactive to methylation.
[00:04:31.350]Lastly, we conducted chemical mapping with MTR1 UGA,
[00:04:34.940]which again, contained UGA-based pairs rather than GAG.
[00:04:39.900]And we again see the same trend of base pairs
[00:04:41.970]in the loop being more reactive to methylation
[00:04:44.720]than the bases in the stem region.
[00:04:49.150]Along with chemical mapping,
[00:04:50.520]we also conducted real-time NMR kinetic studies
[00:04:53.620]to not only confirm our chemical mapping results,
[00:04:56.600]but also to track the reaction progress of a M6G to guanine.
[00:05:02.910]And some advantages of NMR are that it allows us
[00:05:07.020]to assess the local or that NMR assesses
[00:05:10.940]the local electronic environment
[00:05:13.260]and it also reports on structure and dynamics.
[00:05:17.350]And so in these NMR kinetic assays,
[00:05:21.776]we're tracking M6G and looking at the conversion
[00:05:26.170]of M6G to guanine.
[00:05:28.480]And so the simple equation that we're referring to
[00:05:32.200]is enzyme plus substrate plus co-factor.
[00:05:35.540]So in this case, MTR one is enzyme
[00:05:38.310]and the RNA substrate is S
[00:05:41.450]and C would be our co-factor or M6G.
[00:05:45.220]And these are then converted to enzyme product and guanine.
[00:05:53.450]So first we conducted a 1D imino NMR study,
[00:05:57.000]which specifically reports on the imino protein
[00:06:00.980]in these bonds shown here to confirm the presence
[00:06:05.500]of the UUCG tetraloop.
[00:06:10.040]So the G in the UUCG tetraloop has a signature
[00:06:14.270]kind of goal shift at around 10 PPM as shown here.
[00:06:19.130]And this was also a way to confirm
[00:06:21.250]that we see no peak with substrate alone,
[00:06:24.100]because peaks only appear when bonds form between bases.
[00:06:27.600]And we also wanted to confirm that the peaks are broader
[00:06:32.540]and MTR1 wildtype plus substrate,
[00:06:35.710]because with a bigger biomolecule,
[00:06:39.140]this results in broader peaks,
[00:06:40.660]whereas we see smaller peaks with MTR1 wildtype
[00:06:44.340]by itself because it's a smaller complex.
[00:06:50.670]And then next we connected our real-time NMR kinetic assays
[00:06:54.640]with MTR1 wildtype.
[00:07:00.010]NMR of enzymes correspond to the molecule size
[00:07:02.270]in tumbling and solution
[00:07:04.210]so small molecules like MG6 have a sharp peak,
[00:07:08.040]while biomolecules such as MRT1 plus substrate complex
[00:07:11.580]have broader peaks, which we'd see around here.
[00:07:15.390]And so here we can see there M6G control
[00:07:18.060]has a peak at around 8.4PPM,
[00:07:22.030]but this pitches to 8PPM,
[00:07:24.130]because it's now bond to enzyme plus substrate,
[00:07:27.560]which is a result to a change
[00:07:29.720]in the local electronic environment.
[00:07:32.350]But after T equals zero, the peak stayed in the same spot,
[00:07:36.620]which then indicates that is slow exchange
[00:07:38.690]between MTR1 wildtype and substrate.
[00:07:42.210]And now looking at a plot of peak height versus time,
[00:07:45.600]we can see that the peak slowly increases
[00:07:49.160]as time goes on.
[00:07:54.190]To conclude our findings,
[00:07:56.250]the loop region of all three constructs without substrate
[00:07:59.130]we're more reacted to methylation than the stem region.
[00:08:02.630]We also found that the loop region of MTR1 UGA
[00:08:07.050]seemed to be the least reactive
[00:08:08.630]of all three constructs without substrate.
[00:08:11.980]We also found that MTR1 wildtype undergoes slow exchange
[00:08:14.820]with substrates in the presence of M6G
[00:08:18.680]and so our future directions
[00:08:20.430]include completing chemical mapping experiments
[00:08:23.480]with MTR1 UA plus substrate plus M6G and MTR1 UGA
[00:08:28.210]plus substrate plus M6G.
[00:08:31.280]The next step for NMR kinetic studies
[00:08:33.180]is to conduct a time-point NMR assay
[00:08:35.740]of the varying substrate concentrations
[00:08:37.560]to obtain the Km along with conducting
[00:08:40.470]NMR kinetic time-point assays of MTR1 UA plus substrate
[00:08:44.480]plus M6G and doing the same for MTR1 UGA.
[00:08:51.100]Lastly, I'd like to thank the Eichhom research group
[00:08:53.390]for providing this research opportunity.
[00:08:56.500]And I'd also like to thank the Yesselman research group
[00:08:59.230]for providing reagents and assisting
[00:09:01.410]during chemical mapping experiments.
[00:09:04.050]This work was also supported in part
[00:09:05.780]by National Science Foundation.
[00:09:08.410]Thanks for listening.

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Final_Poster_Presentation_ReenaDosanjh

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