Abstract
The potential of photoaptamers as proteomic probes was investigated. Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report, 5-bromo-2'-deoxyuridine. A key question regarding the use of photoaptamer probes is the specificity of the cross-linking reaction. The specificity of three photoaptamers was explored by comparing their reactions with target proteins and non-target proteins. The range of target/non-target specificity varies from 100- to >10(6)-fold with most values >10(4)-fold. The contributions of the initial binding step and the photocross-linking step were evaluated for each reaction. Photocross-linking never degraded specificity and significantly increased aptamer specificity in some cases. The application of photoaptamer technology to proteomics was investigated in microarray format. Immobilized anti-human immunodeficiency virus-gp120 aptamer was able to detect subnanomolar concentrations of target protein in 5% human serum. The levels of sensitivity and specificity displayed by photoaptamers, combined with other advantageous properties of aptamers, should facilitate development of protein chip technology.
Highlights
The potential of photoaptamers as proteomic probes was investigated
Proteomics, the study of protein expression at the scale of cell, tissue, or organism [1, 2], has been defined by a single technology: two-dimensional gel separation followed by mass spectrometric analysis [3, 4]
This technology is mature, powerful, and wonderfully sophisticated, it suffers from evident limitations in speed and sensitivity
Summary
The potential of photoaptamers as proteomic probes was investigated. Photoaptamers are defined as aptamers that bear photocross-linking functionality, in this report, 5-bromo-2-deoxyuridine. The levels of sensitivity and specificity displayed by photoaptamers, combined with other advantageous properties of aptamers, should facilitate development of protein chip technology. Proteomics, the study of protein expression at the scale of cell, tissue, or organism [1, 2], has been defined by a single technology: two-dimensional gel separation followed by mass spectrometric analysis [3, 4]. This technology is mature, powerful, and wonderfully sophisticated, it suffers from evident limitations in speed and sensitivity. Nucleic acids can be selectively amplified to overcome these limitations, but proteins cannot
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