Assessment of Improved Aptamers by RNA Rational Design
Aptamers are single-stranded nucleic acids, DNAs, or RNAs, which recognize a wide variety of molecules. In biotechnology research, their ability to universe specifically and with great affinity to a specific molecule allows them to be used in separation systems. This project It is related to the improvement of these aptamers by putting them in the tetraloop-receptor scaffold (TTR) in chemical mapping experiment and teaches how Spinach and ATP aptamers bind to the desired ligand at different conditions or concentrations so that it has better use over time.
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[00:00:00.080]Hello, my name is Stephanie Orraca,
[00:00:01.480]and today I will be showing you my results
[00:00:03.250]towards the research summer program with the topic of
[00:00:06.680]Assessment of Improved Aptamers by RNA Rational Design.
[00:00:10.440]Aptamers are single-strand nuclear acid DNAs or RNA
[00:00:14.800]which recognize a wide variety of molecules.
[00:00:17.450]Each aptamer has a particular two-dimensional structure
[00:00:20.260]that allows it to bind with high affinity
[00:00:22.420]and specificity to the target molecule.
[00:00:24.500]They can be used as recognition molecules
[00:00:26.840]in different bio-social system.
[00:00:28.630]In biotechnology research,
[00:00:30.110]their ability to universe a specifically,
[00:00:32.500]and with high affinity to a specific molecule
[00:00:35.990]allows them to be used in separation system.
[00:00:38.580]And these are created by a biological technique
[00:00:42.230]But aptamers with high affinity are not always isolated
[00:00:45.331]treated by conventional SELEX.
[00:00:46.900]So this is because a one is the limitation
[00:00:49.390]of moleculars diversity is in the initial library
[00:00:52.150]and the other is the loss of potential high affinity
[00:00:54.920]aptamers during the polymerase chain reaction.
[00:00:57.660]This project is related to the improvement of these aptamers
[00:01:01.290]by putting them in the tetraloop-receptor scaffold
[00:01:04.580]in chemical mapping experiment and teaches
[00:01:06.760]how spinach and ATP aptamers bind to the desired ligand
[00:01:10.150]at different concentrations so that it has
[00:01:13.430]better use over time.
[00:01:14.810]This is a method to observe binding
[00:01:18.650]of an aptamer to its ligand.
[00:01:21.360]As can be seen from it's two-dimensional structure,
[00:01:24.260]this is what happened throughout the experiments.
[00:01:26.380]So the ligand will be in the RNA.
[00:01:29.290]And a motive is created, complete the path of the complex
[00:01:33.730]and the aptamer is positioning to bind the ligand.
[00:01:37.860]So when the aptamer binds its target it
[00:01:41.290]undergo a conformational change,
[00:01:43.293]example sample goes from one structure from another,
[00:01:47.500]and by building a scaffold around the aptamer
[00:01:50.120]it locks the RNA into the bound like conformation
[00:01:53.410]thus improving the aptamer.
[00:01:56.030]For the spinach one is the same process
[00:01:58.670]at the ATP aptamer.
[00:02:01.630]These aptamers change in chemical reactivity
[00:02:04.550]thanks to the fact that dimethyl sulfide
[00:02:07.460]was used within the chemical mapping.
[00:02:09.770]Here's as shown in the reactions simply
[00:02:12.804]a methyl group is added to it when it reacts with DMS.
[00:02:18.530]The SELEX process which stands for
[00:02:20.990]Systematic Evolution of Ligand with Exponential enrichment,
[00:02:25.060]also known as in vitro selection,
[00:02:28.580]is the technique used for the selection
[00:02:32.080]and installation of aptamers.
[00:02:34.290]This method use combinational chemistry
[00:02:37.310]for the selection of synthetic nucleic acids
[00:02:40.280]with high affinity for the target molecule.
[00:02:43.740]So the immunization consists of the chemical synthesis
[00:02:47.400]for the library.
[00:02:48.700]Then the library and the target molecule come into contact
[00:02:53.760]and all the diphenyl members remain attached.
[00:02:57.500]Binding library members are separated and sequences
[00:03:01.690]in the DNA libraries or by reverse transcription and PCR,
[00:03:07.350]which is polymerase chain reaction,
[00:03:10.160]in this case of libraries.
[00:03:13.410]A new cycle begin which will be repeated
[00:03:17.280]until libraries that contain an intermediate group
[00:03:21.470]of sequences that bind to the target molecule are found
[00:03:24.790]and sequencing of the last cycle to determine
[00:03:27.799]of individual sequences with high affinity.
[00:03:31.960]In the chemical mapping process, first,
[00:03:34.330]the RNA is diluted to have a lower concentration
[00:03:37.210]and put on the thermocycler and in a heat and cool run.
[00:03:42.010]Then the folding buffer where the process is,
[00:03:45.320]in which a linear RNA molecule acquires
[00:03:48.140]a secondary structure through
[00:03:50.090]intermolecular interaction is added.
[00:03:52.910]For the two aptamers to add the ligand
[00:03:55.700]at different condition, a one-three dilution was made
[00:03:59.730]with 100 millimolar of ligand.
[00:04:03.540]The ligand that was used for spin-out was DFHBI,
[00:04:10.690]and for the ATP one it was AMP,
[00:04:14.930]which is adenosine monophosphate.
[00:04:19.120]The chemical modifier was added to each sample
[00:04:22.520]and was quenched by BME.
[00:04:24.860]RNA was purified by spin column,
[00:04:27.940]and its concentration was measured
[00:04:30.380]by cubit RNA quantification.
[00:04:33.060]Reverse transcription was made, purifying then the CNA,
[00:04:38.680]the cDNA, and then doing the polymerase chain reaction
[00:04:42.658]or PCR reaction, it is run in the thermocycler
[00:04:46.759]with the Q5 Mix Program for 16 cycles
[00:04:51.470]to then run Egel
[00:04:54.590]and do a DNA extraction.
[00:04:57.830]The DNA was purified by spin column,
[00:05:02.320]then the concentration was measured
[00:05:04.360]by qubit DNA quantification,
[00:05:06.490]and finally the DNA cloning and sequence is carried out
[00:05:10.610]to obtain the necessary data or results
[00:05:12.900]to help us to predict or verify the structure of the DNA.
[00:05:17.330]So these whole process proves the structure of RNA
[00:05:21.550]through the chemical modification
[00:05:23.530]that can be read out through it's sequencing.
[00:05:28.260]For the spinach aptamer a titration was carried out
[00:05:31.570]in which the purpose was to measure
[00:05:33.480]the intensity of relative fluorescence
[00:05:35.360]and make a graph versus the concentration of the ligand.
[00:05:38.550]In total, 15 sample were taken into consideration
[00:05:42.550]making a one-three dilution
[00:05:43.901]with 100 millimolar of the ligand.
[00:05:46.980]From this it was possible to determine the value
[00:05:49.810]of the dissociation constant,
[00:05:52.310]which has the purpose of saying whether
[00:05:54.970]the aptamer-ligand binding was effective.
[00:05:57.690]With the Hill equation, the titration curve was adjusted
[00:06:01.330]and thus the values of the constant m and AKD were obtained,
[00:06:05.860]which were 0.86
[00:06:07.867]and 6.80 times 10 to the positive four respectively.
[00:06:13.270]Having a small value of the dissociation constant
[00:06:16.076]means that the binding of the ligand
[00:06:18.170]to the aptamer was effective.
[00:06:19.860]So it shows that has a good affinity between the molecules.
[00:06:24.090]For the ATP results, the chemical mapping plot is shown,
[00:06:28.520]which let us know the position of the aptamer
[00:06:30.600]in the sequence of interest.
[00:06:32.180]This is where the aptamer is positioned
[00:06:36.010]and this sequence mean that without the binding
[00:06:39.500]of the AMP ligand the first adenines should react,
[00:06:46.520]they should have a higher fraction number.
[00:06:49.340]In this case as the ligand was attached,
[00:06:51.920]this signals are, these two, indicating that
[00:06:55.220]this is evidence that MAP is binding to the ATP aptamer.
[00:07:00.830]Because aptamers with this higher affinity
[00:07:03.170]are not always isolated by SELEX,
[00:07:05.300]referencing of select aptamers are required
[00:07:08.120]to improve binding affinity.
[00:07:09.980]The affinities of aptamers can be improved
[00:07:12.200]through sequence optimization.
[00:07:14.120]And at the end of the investigation,
[00:07:16.610]it was found that the results indicated a good bond
[00:07:19.180]between the aptamers and the ligand for spinach and ATP.
[00:07:24.250]Thanks to Professor Dr. Ysselman, Chris Jurich,
[00:07:27.090]UNL and National Science Foundation
[00:07:29.000]for conducting this research
[00:07:31.960]that will be beneficial along the way,
[00:07:34.940]and thank you for listening.
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