In vitro selection of histone H4 aptamers for recognition imaging microscopy

Abstract

Recognition imaging microscopy is an analytical technique used to map the topography and chemical identity of specific protein molecules present in complex biological samples. The technique relies on the use of antibodies tethered to the cantilever tip of an AFM probe to detect cognate antigens deposited onto a mica surface. Despite the power of this technique to resolve single molecules with nanometer-scale spacing, the recognition step remains limited by the availability of suitable quality antibodies. Here we report the in vitro selection and recognition imaging of anti-histone H4 aptamers. In addition to identifying aptamers to highly basic proteins, these results suggest that aptamers provide an efficient, cost-effective route to highly selective affinity reagents for recognition imaging microscopy.

Figure 1

Aptamer enrichment and specificity to different histone classes. (a) Representative chromatograms from rounds 1 and 4 of the selection. The bound fraction is too small to be seen as a distinct peak in round 1, but is clearly visible after round 4. (b) Binding curves were obtained for aptamer 4.15 with the H4, H3, H2A and H2B peptide tail sequences.

Figure 2

Demonstration of aptamer recognition in recognition imaging microscopy. Topography (a) and recognition images (b) were simultaneously acquired for pure histone H4 protein deposited on a mica surface. Recognition events are shown with circles. In this image-pair, 53 out of 62 histone molecules are recognized by the anti-H4 aptamer 4.13.