A genome-inspired DNA ligand for the affinity capture of insulin and insulin-like growth factor-2.
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 MS 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 K(D) approximately 10(-13) M and K(D) approximately 10(-7) M, which was not observed for insulin with variant h (K(D) approximately 10(-8) M) or IGF-2 with either variant (K(D)s approximately 10(-9) M). The results provide a basis for the 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.
Xiao
Junfeng
J
Department of Chemistry and Chemical Biology, 118 Cogswell Laboratory, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Carter
Jennifer A
JA
Frederick
Kimberley A
KA
McGown
Linda B
LB
D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry* / Q000235:genetics; D054856:G-Quadruplexes; D006801:Humans; D007328:Insulin / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry* / Q000235:genetics; D011401:Promoter Regions, Genetic; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
D007328:Insulin; D008024:Ligands; D009841:Oligonucleotides; D007335:Insulin-Like Growth Factor II; D004247:DNA