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首页> 美国政府科技报告 >Photochemical Systematic Evolution of Ligands by Exponential Enrichment (Photoselex) Employing the 5-BROMO-2'-Deoxyuridine Chromophore for the Basic Fibroblast Growth Factor Target
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Photochemical Systematic Evolution of Ligands by Exponential Enrichment (Photoselex) Employing the 5-BROMO-2'-Deoxyuridine Chromophore for the Basic Fibroblast Growth Factor Target

机译：指数富集（photoselex）配体的光化学系统演化采用5-BROmO-2'-脱氧尿苷发色团为碱性成纤维细胞生长因子靶

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An in vitro selection methodology (PhotoSELEX) was investigated and used to identify ssDNA aptamers capable of binding with high affinity and capable of covalently cross-linking via a high quantum yield photochemical reaction the human basic Fibroblast Growth Factor (bFGF) protein. Beginning with a library of ssDNA aptamers in which thymidine had been replaced with the chromophore 5-bromo-2'-deoxyuridine, five rounds of affinity-based in vitro selection were completed. The resulting library was then irradiated with 308 nm light in the presence of bFGF. Those sequences capable of forming a covalent bond to the target protein were subsequently partitioned from those not capable of forming such a bond. Enzymatic digestion of the protein component yielded a template suitable for polymerase chain reaction (PCR) amplification permitting additional rounds of such photocross-linking selection. Systematic and exclusive application of rounds of PhotoSELEX on such a library identified ssDNA aptamers with high affinity binding and high quantum yield cross-linking potential for the target. Moreover, it was shown that PhotoSELEX can be manipulated to yield: (1) exceptionally high affinity binding or (2) exceptionally high quantum yield cross-liking aptamers depending on the nature of the pressure applied during the experiment. Based upon these results, a PhotoSELEX experiment was performed in which photochemical and affinity pressure were applied simultaneously to a completely random oligonucleotide library. This experiment showed that PhotoSELEX can converge to unique sequences much more rapidly than affinity- based SELEX. Finally, the application of electrospray mass spectrometry (ESMS) and Maxam-Gilbert sequencing as novel methods to characterize the results of covalent-bond based SELEX protocols were demonstrated.

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