| In this thesis, we are introducing a new method to select individual aptamer species from a pool of random molecules based on the aptamer's affinity to a target molecule. We refer to this technique as "NanoSelection(TM) technology," as it relies on nanoscale imaging and isolation of target-bound aptamer molecules. The advantage of the NanoSelection(TM) method is that it requires only one selection cycle per molecule, whereas the current method, SELEX (Ellington and Szostak 1990; Tuerk and Gold 1990; Ellington and Szostak 1992), requires many iterative cycles. The key idea of our method is that by being able to both observe single binding events and subsequently extract the binding molecule, we circumvent the need for iterative binding, washing, elution and amplification steps. In this thesis, we describe the method, the required instrumentation and proof-of-principle experiments performed with the known thrombin-binding aptamer GGTTGGTGTGGTTGG (Bock, Griffin et al. 1992; Tsiang, Gibbs et al. 1995).; At the core of this method is instrumentation that combines an atomic force microscope and an inverted optical fluorescence microscope. In our method, target molecules are attached to a cover slip. A library of fluorescent oligos attached to a small bead (for AFM detection and extraction) is then dispensed to the target area. High-affinity, target-specific aptamers will bind tightly to the target for prolonged periods resulting in a strong fluorescence signal (and potentially fluorescence resonance energy transfer (FRET) with donor-labeled oligos and acceptor-labeled targets) and making them resistant to washes. The AFM is then directed to the coordinates of the fluorescence signal, and a high resolution image of the area is obtained. Subsequently, the AFM tip is used to extract the bead plus the attached oligo. The extracted oligo is amplified by small-copy-number PCR, followed by a biochemical analysis. |
