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A combined atomic force/fluorescence microscopy technique to select aptamers in a single cycle from a small pool of random oligonucleotides
Author(s) -
Peng Lu,
Stephens Bryan J.,
Bonin Keith,
Cubicciotti Roger,
Guthold Martin
Publication year - 2007
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20421
Subject(s) - aptamer , oligonucleotide , fluorescence microscope , fluorescence , biophysics , chemistry , microscopy , nanotechnology , combinatorial chemistry , materials science , dna , biology , microbiology and biotechnology , biochemistry , optics , physics
We develop a method, which utilizes a combined atomic force microscope (AFM)/fluorescence microscope and small copy number polymerase chain reaction (PCR), to affinity‐select individual aptamer species in a single cycle from a small pool of random‐sequence oligonucleotides (oligos). In this method, a library of small beads, each of which is functionalized with fluorescent oligos of different sequences, is created. This library of oligo‐functionalized beads is flowed over immobilized target molecules on a glass cover slip. High‐affinity target‐specific aptamers bind tightly to the target for prolonged periods and resist subsequent washes, resulting in a strong fluorescence signal on the substrate surface. This signal is observed from underneath the sample via fluorescence microscopy. The AFM tip, situated above the sample, is then directed to the coordinates of the fluorescence signal and is used to capture a three‐dimensional high‐resolution image of the surface‐bound bead and to extract the bead (plus attached oligo). The extracted oligo is PCR‐amplified, sequenced, and may then be subjected to further biochemical analysis. Here, we describe the underlying principles of this method, the required microscopy instrumentation, and the results of proof‐of‐principle experiments. In these experiments, we selected aptamers in eight trials from a binary pool containing a 1:1 mixture of thrombin aptamer oligo and a nonsense oligo. In each of the eight trials, the positive control aptamer was successfully detected, imaged, extracted, and characterized by PCR amplification and sequencing. In no case was the nonsense oligo selected, indicating good selectivity at this early stage of technology development. Microsc. Res. Tech., 2007. © 2007 Wiley‐Liss, Inc.

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