Photochemical Systematic Evolution of Ligands by Exponential Enrichment (Photoselex) Employing the 5-BROMO-2'-Deoxyuridine Chromophore for the Basic Fibroblast Growth Factor Target.
Abstract
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.
Document Details
- Document Type
- Technical Report
- Publication Date
- Aug 20, 1999
- Accession Number
- ADA367833
Entities
People
- Mace C. Golden
Organizations
- Air Force Institute of Technology