Active Conformation of DNA Aptamer Against Recombinant Human Erythropoietin-α†

doi:10.7503/cjcu20170312

Abstract

Abstract:

A series of spectroscopic techniques such as surface plasmon resonance, circular dichroism and fluorescence was employed to illustrate the relationship between the binding activity and conformation of the shortest aptamer In27 against recombinant human erythropoietin-α(EPO-α). It was found that the presence and concentration of sodium cation were the key factors that affected the specific affinity recognition of aptamer In27. Sodium cation induced aptamer In27 to form a parallel/antiparallel hybrid G-quadruplex(GQ) conformation. GQ-specific fluorescent probe thysmenonine T(ThT) specifically bound to the hybrid GQ conformation of aptamer In27. The addition of EPO-α could compete with ThT to facilitate to create a label-free fluorescent “turn-off” probe for EPO-α. This research provides an effective combination of techniques for elucidating the active conformation of novel aptamers, and provides a kind of aptameric modules with clear interaction features for analytical, sensing and diagnostic applications.

Key words: Surface plasmon resonance, Circular dichroism, Fluorescent probes, G-Quadruplex, Sodium cation

CLC Number:

O657.3

TrendMD:

HE Xiaoqin, HE Junlin, XU Hua, GUO Lei, XIE Jianwei. Active Conformation of DNA Aptamer Against Recombinant Human Erythropoietin-α^†[J]. Chem. J. Chinese Universities, 2018, 39(1): 48.

Figures/Tables 6

Fig.1 Influences of cation kind and concentration on the binding response between aptamer and EPO-α as observed in SPR a. In27; b. 39nt. Buffer: Tris-S.

Fig.2 Multiple cycle kinetics of In27 towards immobilized EPO-α in Tris-S(A) or Tris-140Na buffer(B)(A) Ka=2.85×104 L·mol-1·s-1, Kd=1.52×10-3 s-1, KD=53.3 nmol/L, Chi2=0.197, U-Value=1; (B) Ka=3.84×104 L·mol-1·s-1, Kd=2.02×10-3 s-1, KD=52.7 nmol/L, Chi2=0.779, U-Value=1.

Fig.3 CD spectra of aptamer In27 or 39nt at 5 μmol/L in Tris-S buffer(A), In27 in Tris-Na buffer(B) and In27 in Tris-K buffer(C)

Fig.4 Fluorescence spectra of ThT(5 μmol/L) with In27(4 μmol/L) in Tris-Na buffer(A) and

Fig.5 Fluorescence spectra of 5 μmol/L ThT upon titration with In27 from 0 to 10 μmol/L(A) and Job’s experiments on binding ratio of In27 and ThT(B)(A) Concentration of In27 from top to bottom/(μmol·L-1): 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05, 0; (B) the concentration of In27 or ThT was varied from 0 to 5 μmol/L to compose the molar ratio of In27 to In27+ThT between 0 to 1, both slit widths of emission and excitation were 5 nm.

Fig.6 Fluorescence spectra(A) and titration curve(B) of ThT in the presence of In27 upon addition of EPO-αBoth concentrations of In27 and ThT were 2 μmol/L. Concentration of EPO-α, from top to bottom/(μmol·L-1): 0.1, 0.25, 0.5, 2.5, 5, 10. Both slit widths of emission and excitation were 10 nm. Inset of (B) shows the linearity curve between fluorescence intensity and the concentration of EPO-α.

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