Insulin Capture by an Insulin-Linked Polymorphic Region G-Quadruplex DNA Oligonucleotide

Abstract

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.

Affinity Capture and Detection of Immunoglobulin E in Human Serum Using an Aptamer-Modified Surface in Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry

“…DNA-coated spots were covalently attached to fused silica slides as previously described [8,9]. The surface of the glass plate was first cleaned and activated by rinsing with methanol, water, and sodium hydroxide.…”

“…In previous work, we demonstrated proof-of-principle of aptamer surfaces for affinity MALDI-MS using the model system of thrombin capture by the G-quartet DNA thrombinbinding aptamer [8]. The approach was subsequently applied in a non-aptameric system of insulin capture from nuclear extracts of cell lysates by a genomic DNA sequence that forms a G-quadruplex [9].…”

G4 Resolvase 1 Binds Both DNA and RNA Tetramolecular Quadruplex with High Affinity and Is the Major Source of Tetramolecular Quadruplex G4-DNA and G4-RNA Resolving Activity in HeLa Cell Lysates

“…More indirect empiric evidence for the existence of quadruplex structures in control regions is found in a number of specific genetic control elements that readily form quadruplex structures in vitro under physiological salt conditions including: the aforementioned telomeres (15,16), the immunoglobin heavy chain switch region (3), guanine-rich regions of ribosomal DNA (17), the d(pCGG) repeats of the fragile-X mental retardation gene (18), and the promoters of a number of proliferation associated genes such as c-MYC (19,20), PDGF-A (21,22), RET (22), and the diabetes susceptibility locus (23). In addition, a promoter region in the human insulin gene can form a quadruplex structure in vitro capable of specifically binding insulin suggesting a quadruplex based feedback expression loop (24). In RNA the 5Ј untranslated region of a number of proto-oncogenes contain quadruplex forming motifs including NRAS, BCL2, JUN, and FGR (25).…”

Direct detection of aptamer-thrombin binding via surface-enhanced Raman spectroscopy

“…͔; cancer biomarkers; [22][23][24][25] HIVassociated proteins; [26][27][28][29] food-borne pathogens; 30 and other biologically important biomolecules such as insulin, 31,32 immunoglobulin E ͑IgE͒, 33 and thrombin. 34 They exhibit protein binding affinities that are comparable to those of corresponding antibodies.…”