Insulin capture by a G-quadruplex DNA oligonucleotide containing a two-repeat sequence of the insulin-linked polymorphic region (ILPR) of the human insulin gene promoter region is reported. The immobilized oligonucleotide was demonstrated to capture human insulin from standard solutions and from nuclear extracts of pancreatic cells with high selectivity, using affinity MALDI-mass spectrometry and affinity capillary chromatography. Insulin was preferentially captured by the tworepeat ILPR oligonucleotide over another G-quadruplex forming oligonucleotide, the thrombin binding aptamer, as well as over a single repeat of the ILPR sequence that is not capable of forming the G-quadruplex architecture. Binding was shown to involve the beta chain of insulin, most likely through association with the two parallel loops of the G-quadruplex structure. The discovery raises the possibility that insulin may bind to G-quadruplex DNA formed in the ILPR in vivo and thereby play a role in modulation of insulin gene expression, and provides a basis for design of insulin analogs to probe this hypothesis. The availability of a DNA ligand to human insulin has analytical importance as well, offering an alternative to antibodies for in vitro or in vivo detection and sensing of insulin as well as its isolation and purification from biological samples.
Although many chemistry students at small liberal arts colleges participate in undergraduate research projects with faculty members, they do not get much experience framing their own research questions and designing their own projects, which is an important part of science. We have implemented a developmental process to help students design and execute their own research projects in a two-course sequence: seminar in the fall and an integrated laboratory in the January term as a capstone experience in the chemistry curriculum. In seminar, students read scientific literature to generate an unanswered question that becomes the basis for a project proposal. Students then compare and contrast various methods to answer the question and propose a project. In the integrated laboratory, students execute the project where they troubleshoot experiments, collect and interpret data, and draw conclusions. Assessment of final papers and student course evaluations indicated that the students met the above goals. Overall, this educational experience can be implemented at other small liberal arts colleges and elements of this project could be adapted at a larger college or university.
The insulin-linked polymorphic region (ILPR) of the human insulin gene contains tandem repeats of similar G-rich sequences, some of which form intramolecular G-quadruplex structures in vitro. Previous work showed affinity binding of insulin to an intramolecular G-quadruplex formed by ILPR variant a. Here we report on interactions of insulin and the highly homologous insulin-like growth factor 2 (IGF-2) with ILPR variants a, h and i. Circular dichroism indicated intramolecular G-quadruplex formation for variants a and h. Affinity MALDI mass spectrometry and surface plasmon resonance were used to compare protein capture and binding strengths. Insulin and IGF-2 exhibited high binding affinity for variants a and h but not i, indicating the involvement of intramolecular G-quadruplexes. Interaction between insulin and variant a was unique in the appearance of two binding interactions with KD~10−13 M and KD~10−7 M, which was not observed for insulin with variant h (KD~10−8 M) or IGF-2 with either variant (KD’s~10−9 D M). The results provide a basis for design of DNA binding ligands for insulin and IGF-2 and support a new approach to discovery of DNA affinity binding ligands based on genome-inspired sequences rather than the traditional combinatorial selection route to aptamer discovery.
Mitochondrial dysfunction plays a significant role in neurodegenerative disease including ataxias and other movement disorders, particularly those marked by progressive degeneration in the cerebellum. In this study, we investigate the role of mitochondrial oxidative phosphorylation (OXPHOS) deficits in cerebellar tissue of a Purkinje cell-driven spinocerebellar ataxia type 1 (SCA1) mouse. Using RNA sequencing transcriptomics, OXPHOS complex assembly analysis and oxygen consumption assays, we report that in the presence of mutant polyglutamine-expanded ataxin-1, SCA1 mice display deficits in cerebellar OXPHOS complex I (NADH-coenzyme Q oxidoreductase). Complex I genes are upregulated at the time of symptom onset and upregulation persists into late stage disease; yet, functional assembly of complex I macromolecules are diminished and oxygen respiration through complex I is reduced. Acute treatment of postsymptomatic SCA1 mice with succinic acid, a complex II (succinate dehydrogenase) electron donor to bypass complex I dysfunction, ameliorated cerebellar OXPHOS dysfunction, reduced cerebellar pathology and improved motor behavior. Thus, exploration of mitochondrial dysfunction and its role in neurodegenerative ataxias, and warrants further investigation.
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