An aptameric molecular beacon-based "Signal-on" approach for rapid determination of rHuEPO-alpha.
The aim of this work is to describe the first example of aptameric molecular beacon (MB)-based probe for the detection of recombinant human erythropoietin (rHuEPO-alpha) in physiological buffer, using a novel 35 nt ssDNA aptamer (807-35 nt) originally isolated by Systematic Evolution of Ligands by Exponential enrichment (SELEX) technique in our laboratory. Both "Signal-on" and "Signal-off" MB modes were developed, respectively, in which the conformational alteration of aptamer before and after binding to rHuEPO-alpha can be demonstrated in terms of the correspondingly fluorescent changes. Comparing with "Signal-off" mode, "Signal-on" mode provided higher sensitivity, while with the addition of target rHuEPO-alpha, quenching between fluorescent 807-35 nt aptamer (F-Apt) and a short quencher-labeled complementary sequence (QDNA) was disturbed by the specific binding between rHuEPO-alpha and F-Apt. QDNA was thus loosened and released from F-Apt, leading to a consequently full fluorescent restoration. Systematic optimization of parameters in "Signal-on" mode were carried out, the choice of QDNA length, the hybridization site of a small supplementary DNA (SDNA) stabilizer, and the existence of Mg(2+) cation played essential roles for the performance characterization. A convenient and sensitive determination of rHuEPO-alpha with a LOD of 0.4 nM was achieved.
Zhang
Zhaoyang
Z
Beijing Institute of Pharmacology and Toxicology, Hai Dian District, Beijing, China.
Guo
Lei
L
Tang
Jijun
J
Guo
Xingjie
X
Xie
Jianwei
J
D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004921:Erythropoietin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D011994:Recombinant Proteins; D052156:SELEX Aptamer Technique
D052157:Aptamers, Nucleotide; D004277:DNA, Single-Stranded; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004921:Erythropoietin; D008274:Magnesium; D011188:Potassium