3D Structural Integrity and Interactions of Single-Stranded Protein- Binding DNA in a Functionalized Nanopore Mohammed Arif I. Mahmood, †,‡,§ Waqas Ali, †,‡,§ Ashfaq Adnan, ∥ and Samir M. Iqbal* ,†,‡,§,⊥,# † Nano-Bio Lab, ‡ Department of Electrical Engineering, § Nanotechnology Research Center, Shimadzu Institute for Research Technologies, ∥ Department of Mechanical and Aerospace Engineering, ⊥ Department of Bioengineering, # Joint Graduate Studies Committee of Bioengineering Program, University of Texas at Arlington and University of Texas Southwestern Medical Center at Dallas, University of Texas at Arlington, Arlington, Texas 76019, United States ABSTRACT: Biomarker-binding nucleotide sequences, like aptamers, have gained recent attention in cancer cell isolation and detection works. Self-assembly and 3D conformation of aptamers enable them to selectively capture and bind diseased cells and related biomarkers. One mode of utilizing such an extraordinary selective property of the aptamers is by grafting these in nanopores. Coating the inside walls of the nanopore with biomarker specific ligands, like DNA, changes the statistics of the dynamic translocation events. When the target protein passes through the nanopore, it interacts with ligand coated inside the nanopore, and the process alters the overall potential energy profile which is essentially specific to the protein detected. The fundamental goal in this process is to ensure that these detection motifs hold their structure and functionality under applied electric field and experimental conditions. We report here all-atom molecular dynamics simulations of the effects of external electric field on the 3D conformation of such DNA structures. The simulations demonstrate how the grafted moieties affect the translocation time, velocity, and detection frequency of the target molecule. We also investigated a novel case of protein translocation, where DNA is prebound to the protein. As model, a thrombin-specific G- quartet and thrombin pair was used for this study. 1. INTRODUCTION Many diseases can be diagnosed using one or multiple biomarkers. These biomarkers may consist of alien entities inside a host body or disease-induced proteins overexpressed or downregulated by the host itself as part of its defense mechanisms. These proteins become available in the circulatory bloodstream at early stages of the disease and work as disease precursors. Detection and identification of these proteins is important for diagnosis and prognostic approaches henceforth. Over the past several years, significant progress has been made on nanopore-based DNA detection technologies. 1,2 Recently, it has been shown that such nanopore-based detection can also be used toward the detection of protein biomarkers. 3 In a nanopore-based system, DNA traveling through functionalized nanopore has been shown to alter due to ligand specificaffinity. 1 We’ve shown here that proteins allowed to pass through a nanopore whose interior wall is functionalized with the protein-specific DNA would exhibit similar discriminatory effects. In other words, proteins would slow down or even chemically bind to the surface of the nanopore depending on the nature of stimuli applied inside the nanopore (e.g., electric field, mechanical forces, etc.). DNA and protein are two intertwined moieties by virtue of their functions in cellular mechanisms. Proteins are synthesized by DNA transcription; on the other hand, certain proteins play significant roles in regulation of such transcriptions. This regulation is accomplished by selectivity between the DNA segments and proteins. This selectivity is a useful property that can be used in vitro for detection of certain proteins. Nanopore is a highly suitable yet simple platform for utilizing such extraordinary property. Compared to the gel electrophoresis, the widely employed protein detection scheme, a nanopore- based method, not only promises easy and quick detection of as few as a single copy of rare biomarkers without need of expert supervision, but it also eliminates the requirement of a strict lab environment. 4 Therefore, it can be perceived why detection and identification of protein or DNA based on the translocation behavior through a nanopore has received recent growing attention. 4 In this method, protein or DNA is allowed to pass through a nanopore of comparable size in an ionic solution and under applied electric bias (Figure 1). The ionic current is measured, and parameters like translocation time, velocity, and current dip are calculated as electronic signatures. When proteins pass through the function- alized nanopores, these create statistically different ionic current dips than those through bare nanopores. Unraveling the mystery of how protein transport takes place in a cellular environment has opened up new windows toward such methodologies. Enzyme assisted protein translocation through nanopores has indicated promise of protein sequencing through nanopores. 5 However, many biophysical phenomena are yet to Received: December 2, 2013 Revised: April 4, 2014 Published: April 8, 2014 Article pubs.acs.org/JPCB © 2014 American Chemical Society 5799 dx.doi.org/10.1021/jp411820w | J. Phys. Chem. B 2014, 118, 5799−5806