Probing the biophysical interaction between Neocarzinostatin toxin and EpCAM RNA aptamer Prasanna Kumar Athyala a, b, d, 1 , Jagat Rakesh Kanwar b , Mohamed Alameen c, 1 , Rupinder Kaur Kanwar b , Subramanian Krishnakumar a , Jon Watson b , Umashankar Vetrivel c, 1 , Janakiraman Narayanan a, * a Department of Nanobiotechnology, Vision Research Foundation, Chennai, India b Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine (SoM), Centre for Molecular and Medical Research (C-MMR), Faculty of Health, Deakin University, Geelong, Pigdons Road, Waurn Ponds, Geelong, Victoria 3217, Australia c Bioinformatics Centre, Vision Research Foundation, Chennai, India d Graduate Student Deakin University (ID: 213510168), Nanomedicine-Laboratory of Immunology and Molecular Biomedical Research (NLIMBR), School of Medicine, Deakin University, Australia article info Article history: Received 20 November 2015 Accepted 24 November 2015 Available online 28 November 2015 Keywords: Neocarzinostatin EpCAM aptamer CD spectroscopy Insilico modeling abstract Neocarzinostatin (NCS) a potent DNA-damaging, anti-tumor toxin extracted from Streptomyces carzi- nostaticus that recognizes double-stranded DNA bulge and induces DNA damage. 2 Fluoro (2F) Modified EpCAM RNA aptamer is a 23-mer that targets EpCAM protein, expressed on the surface of epithelial tumor cells. Understanding the interaction between NCS and the ligand is important for carrying out the targeted tumor therapy. In this study, we have investigated the biophysical interactions between NCS and 2-fluro Modified EpCAM RNA aptamer using Circular Dichroism (CD) and Infra-Red (IR) spectroscopy. The aromatic amino acid residues spanning the b sheets of NCS are found to participate in intermolecular interactions with 2 F Modified EpCAM RNA aptamer. In-silico modeling and simulation studies corrob- orate with CD spectra data. Furthermore, it reinforces the involvement of C and D1 strand of NCS in intermolecular interactions with EpCAM RNA aptamer. This the first report on interactions involved in the stabilization of NCS-EpCAM aptamer complex and will aid in the development of therapeutic mo- dalities towards targeted cancer therapy. © 2015 Elsevier Inc. All rights reserved. 1. Introduction Epithelial cell adhesion molecule (EpCAM) is highly expressed in the epithelial tumors [1]. Aptamers are short oligonucleotides derived from the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technology against a pure protein of interest. Here, we use aptamers that are specific to the EpCAM protein expressed in cell surface [2]. By modification of nucleotide bases with 2 0 fluoro group or locked nucleic acids [3] the aptamers can be stabilized against nuclease degradation. The aptamers are being used to deliver siRNA [4], and drug molecules [5] to the target cells. The drug NCS is primarily composed of a chromophore and a protein component (apoNCS) and exhibit anti-tumor properties against various cancers [6]. This protein is made up of 113 amino acids comprising seven b strands (Inner and Outer as depicted in Fig. 1) and a loop region [7]. The arrangement of b strands are that outer b-sheet contains strands A (residues 5e8), B (residues 17e24), and E (residues 62e69), while the inner b-sheet contains strand C (residues 31e39), strand D (residues 45e56), Strand H (residues 93e98) and strand I (residues 107e109). Strand D is separated into two sub-strands D1 and D2 by a short loop (Loop 47e53) [8,9]. The apoNCS is bound to enediyne chromophore (Af- finity K d ¼ 0.1 nM) and thereby, protects the chromophore's func- tional activity [10,11]. Recent studies suggest that apoNCS release chromophore into the cell [12]. Apo-ncs and chromophore are tightly bound to protect each other against their damage [13]. The apo-NCS protein is stable [14], by the formation of the hydrophobic cluster by internal b sheets. The dynamics of the NCS lies in the loop region [8], that primarily facilitates the release of chromophore from the protein structure. Based on earlier Molecular Dynamics * Corresponding author. E-mail address: drjrn15@gmail.com (J. Narayanan). 1 Equal contribution for the manuscript. Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc http://dx.doi.org/10.1016/j.bbrc.2015.11.109 0006-291X/© 2015 Elsevier Inc. All rights reserved. Biochemical and Biophysical Research Communications 469 (2016) 257e262