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Isolation and Characterization of RNA Aptamers against Glutamate Ion Channel Receptors
Başlık:
Isolation and Characterization of RNA Aptamers against Glutamate Ion Channel Receptors
Yazar:
Jaremko, William J., author.
ISBN:
9780438045736
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (235 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: Li Niu Committee members: Alan Chen; Jayanti Pande; Alexander Shekhtman; Jia Sheng; Hua Shi.
Özet:
Ionotropic glutamate receptors (iGluRs) are ligand-gated transmembrane ion channels, and they mediate the majority of excitatory synaptic transmission in the central nervous system (CNS). iGluRs are divided into three subtypes, namely alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) receptors. These receptors are essential for neuronal development and brain function. Over-activation or excessive activity of these receptors, however, has been linked to various neurological diseases, such as stroke, epilepsy and neuropathic pain. Inhibitors of these receptors are potential drug candidates for the treatment of these neurological diseases. Therefore, developing potent and selective inhibitors against these receptors is significant. Specifically, my thesis work has focused on developing ribonucleic acid (RNA) aptamers against AMPA and kainate receptors.
Previously, small-molecule compounds with various chemical structures have been synthesized as AMPA and kainate receptor antagonists. In general, however, these chemical inhibitors have low potency and selectivity towards various subunits of these receptors as well as poor water solubility. Not surprisingly, these compounds have not shown significant therapeutic efficacy in clinical trials. One of the non-traditional ways to develop potent and selective inhibitors of AMPA or kainate receptors is through an in vitro evolutionary approach, known as systematic evolution of ligands by exponential enrichment (SELEX), to isolate novel RNA molecules as potential antagonists (or RNA aptamers) from a large RNA library (~1014 sequences).
As described in Chapter 1, I have used SELEX, and thus far identified RNA aptamers capable of inhibiting AMPA and/or kainate receptors. For instance, one aptamer, which I termed as AB9, was found capable of inhibiting both AMPA and kainate receptors, though with a strong preference towards AMPA receptor subunits. By sequence truncation and mutation, I have revealed that AB9s, i.e., a 55-nucleotide (nt) RNA as compared with the original 101-nt sequence RNA, contains two critical sequence segments, which I termed as "blue or b" and "red or r" sequences. This truncated aptamer retained the inhibition toward AMPA receptor subunits, but became significantly more potent against the kainate receptors subunits of GluK1 and GluK2. Modification of aptamer AB9s showed that the inhibition toward AMPA or kainate receptors could be decoupled. Testing revealed that AB9s-b has selective activity against only the kainate receptors. The aptamer that contains only the red sequence or AB9s-r is AMPA receptor selective. Both aptamers showed no activity on NMDA receptors. To make these aptamers practically useful, I have also chemically modified them. This is because unmodified, regular RNAs with canonical nucleotides are quickly digested in vivo, catalyzed by a variety of ribonuclease enzymes. In my experiments, I have created 2'-F aptamers, i.e., replacing the 2'-OH group in all As, Cs, and Us in the corresponding RNA sequences with 2'-F modified nucleotides. These 2'-F modified aptamers, i.e., FB9s, FB9s-b and FB9s-r, have shown similar inhibition potency and selectivity profiles, as compared with the unmodified aptamers. These 2'-F modified RNA aptamers are now significantly stable, as tested in both serum-containing medium and mouse cerebrospinal fluid (CSF), with half-lives of days as compared with mins for regular, unmodified RNAs. Using homologous binding and whole-cell recording assays, I have found that the aptamer most likely binds to the receptor's regulatory site and inhibits it noncompetitively. My results further suggest the potential of using a single receptor target (I used GluA1/2R complex channel in my SELEX) to develop RNA aptamers with dual activity for effectively blocking both AMPA and kainate receptors. Most importantly, I have revealed that the blue and red sequences in the AB9 or AB9s RNA sequences code distinct kainate and AMPA receptor activities respectively. The separation of the dual-activity observed for aptamer AB9s suggests a potential to evolve unique RNA motifs and the possibility of grafting these motifs for a tailor-designed selectivity.
In Chapter 2, I have carried out two additional SELEX experiments against GluA1 and GluA3, two AMPA receptor targets. Both of these targets are important due to their roles in neurological diseases, and compounds capable of inhibiting them would provide further information on their role in the central nervous system. The main difference in the work I have done in Chapter 2 as compared with that in Chapter 1 is the use of next generation sequencing (NGS) to analyze DNA sequences from SELEX; whereas in Chapter 1, the traditional Sanger sequencing method was used. This new method allows me to reveal millions of sequence reads, as opposed to only a few hundred reads as shown by Sanger sequencing at the end of the SELEX round. The goal is to identify more DNA sequences (or RNA aptamer candidates) and do so in earlier rounds of SELEX experiments where the libraries are still more diversified. (Abstract shortened by ProQuest.).
Notlar:
School code: 0668
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(681926.1) | 681926-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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