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[摘要]：While many diagnostic assay platforms enable the measurement of analytes with high sensitivity, most of them result in a disruption of the analyte's native structure and, thus, in loss of function. Consequently, the analyte can be used neither for further analytical assessment nor functional analysis. Herein we report the use of caged aptamers as templates during apta-PCR analysis of targets. Aptamers are short nucleic acids that fold into a well-defined three-dimensional structure in which they interact with target molecules with high affinity and specificity. Nucleic acid aptamers can also serve as templates for qPCR approaches and, thus, have been used as high affinity ligands to bind to target molecules and subsequently for quantification by qPCR, an assay format coined apta-PCR. Caged aptamers in turn refer to variants that bear one or more photolabile groups at strategic positions. The activity of caged aptamers can thus be turned on or off by light irradiation. The latter allows the mild elution of target-bound aptamers while the target's native structure and function remain intact. We demonstrate that this approach allows the quantitative and subsequently the functional assessment of analytes. Since caged aptamers can be generated emanating from virtually every available aptamer, the described approach can be generalized and adopted to any target-aptamer pair and, thus, have a broad applicability in proteomics and clinical diagnostics. |
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