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 Supporting Information Available: Detailed protocols, sequences, mFold structures, and AFM images are available in the supplementary section. This material is available free of charge via the Internet at http://pubs.acs.org. 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. NIH Public AccessRecognition imaging microscopy is an analytical technique used to map the topography and chemical identity of specific protein molecules present in complex biological samples. 1-3 The technique relies on the use of affinity reagents immobilized to the cantilever tip of an atomic force microscope (AFM) to identify the precise location of a single protein in an aqueous environment. Since surface images can be acquired in near real-time, recognition imaging has been used to study many time dependent processes. 1 Despite the ability of this technique to resolve single molecules with nanometer-scale spacing, the recognition step remains limited by the availability of antibodies of suitable quality. In our work on chromatin remodeling we have found that many commercial antibodies developed to recognize DNA binding proteins show batch-to-batch variation in performance and mild to severe cross-reactivity with other proteins. 4 To overcome these limitations we initiated an investigation into alternative affinity reagents as antibody mimics in recognition imaging microscopy. 5 Here we report the in vitro selection and evaluation of DNA aptamers selected to bind histone H4 tails.Aptamers are nucleic acid molecules that exhibit antibody-like properties by adopting structures that are complementary in shape and charge to a selected target. 6 In contrast to antibodies, aptamers are smaller in size, easier to engineer, and can be generated relatively quickly using standard in vitro selection technologies. Although aptamers have been selected to bind a diverse array of targets with high affinity and specificity, 6 some concern remains over their ability to bind highly charged molecules due to the potential for nonspecific binding. 7 Recognizing that histones are highly charged proteins that contain many lysine and arginine residues, we wondered whether aptamers could be selected to recognize different histone classes.To address this question we used an in vitro selection protocol th...

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