期刊
JOURNAL OF SEPARATION SCIENCE
卷 32, 期 10, 页码 1654-1664出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/jssc.200900060
关键词
Aptamer; Affinity capillary electrophoresis; Insulin; MALDI-mass spectrometry; Surface plasmon resonance
资金
- NIDDK NIH HHS [R21 DK070762, R21 DK070762-02] Funding Source: Medline
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 similar to 10(-13) M and K-D similar to 10(-7) M, which was not observed for insulin with variant h (K-D similar to 10(-8) M) or IGF-2 with either variant (K(D)s similar to 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 Father than the traditional combinatorial selection route to aptamer discovery.
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