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 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...

Simultaneous topographic and recognition imaging of epidermal growth factor receptor (EGFR) on single human breast cancer cells

“…monoclonal antibodies) to block the binding 45 of EGFR and its ligands, consequently blocking receptor activation and 46 transduction of post-receptor signals [9,10]. 47 Recent study has shown that EGFR functionality can be dependent 48 on its subcellular location and mis-located EGFR may regulate tumor re- 49 sponse to therapy [11]. Therefore, localization of EGFR on cancer cells is 50 of great importance.…”

“…Applications of TREC technique in biomedical research is certainly an 231 increasingly demanding task. Up to now, TREC imaging method has 232 been applied to visualize quite a few different biomolecule systems, 233 such as biotin-avidin [44,45], ligand-receptor [46,47], and antibody-234 antigen interactions [20,23]. In this study, we utilized TREC imaging to 235 visualize EGFR molecules on complex cell membrane surface, providing 236 a new example of the applications of this methodology in biomedical re-237 search.…”

Atomic Force Microscopy of Chromatin

“…Lin et al tested another system of recognition using DNA aptamers, to reduce cross-reactivity and enhance detection (Lin et al, 2007c). These nucleic acid molecules display similar properties to antibody such as their characteristic folding complementary to the structure of their target epitope (Bunka & Stockley, 2006;Colas, 2008).…”

Preparation and characterization of DNA aptamer based spin column for enrichment and separation of histones

“…13,14 Recently, a number of aptamer-based analytical methods have been developed for protein recognition and detection. [15][16][17][18] However, only a few aptamers have been reported to be potentially useful in histone recognition. 18,19 Recently, Lin et al created an aptamer in vitro by capillary electrophoresis based SELEX (systematic evolution of ligands by exponential enrichment, CE-SELEX) for the histone H4 peptide, and it showed a relatively high affinity towards the histone H4 peptide and could recognize different histone classes in recognition imaging microscopy.…”