Biochemical and Biophysical Research Communications
Probing the biophysical interaction between Neocarzinostatin toxin and EpCAM RNA aptamer
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′ 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 β strands (Inner and Outer as depicted in Fig. 1) and a loop region [7]. The arrangement of β strands are that outer β-sheet contains strands A (residues 5–8), B (residues 17–24), and E (residues 62–69), while the inner β-sheet contains strand C (residues 31–39), strand D (residues 45–56), Strand H (residues 93–98) and strand I (residues 107–109). Strand D is separated into two sub-strands D1 and D2 by a short loop (Loop 47–53) [8], [9]. The apoNCS is bound to enediyne chromophore (Affinity Kd = 0.1 nM) and thereby, protects the chromophore's functional 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 β 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 (MD) study, it is inferred that the β sheets possess restricted movement while the loop region (residue 99–104) has more flexibility and movement by which it release the chromophore. The cleft formation and chromophore binding is formed by three loop region such as residues 40–44, 76–83, and 99–104 [8]. In the present study, 2F Modified EpCAM RNA aptamer interacted with NCS and the interaction pattern was analyzed using Circular Dichroism and Infra-Red spectroscopy. Furthermore, in-silico Molecular modeling was performed for native aptamer followed by docking and dynamics with NCS. The result of modeling and docking was in agreement with CD spectra analysis that β sheets of NCS peptide are engaged in the interaction with aptamer residues. Given the high potency of the toxin, it may be useful to exploit this property of NCS and find out the action on EpCAM positive cells by targeting the cells using EpCAM aptamers. In this scenario, the interaction of NCS with 2F Modified EpCAM aptamer need to be investigated which is being reported in this study.
Section snippets
Circular Dichroism (CD) spectroscopy
Equimolar concentrations of Neocarzinostatin (20uM) (NCS, Sigma–Aldrich) and 2 F Modified EpCAM RNA aptamer (IKA gene technologies) were mixed in the 1.5 ml tube at RT. The interactions were studied in different physiological buffers such as Tris Acetate EDTA (TAE) buffer (pH 7.6), PBS (pH 7.6) and MES buffer (pH 5.5). Due to buffer interference in the region <200 nm, PBS (pH 7.6) was used for further experiments. The CD spectra were measured using a spectropolarimeter, model J-710 (Jasco,
Circular Dichroism spectra of interaction between 2F Modified EpCAM aptamer and NCS
Ellipticity changes were observed in the region between 200 nm and 300 nm for the native NCS and 2F Modified EpCAM aptamer (Fig. 2A4). The Microweb analysis of CD spectra for native NCS showed peaks such as +224 nm (helix π–π*), −216 nm, and −210 nm (Fig. 2A1). The native 2F Modified EpCAM aptamer revealed peak at −210 nm reflecting 60% GC (Fig. 2A3) content from the sequence [28]. The physical mix of 2-Fluro Modified EpCAM aptamer with NCS showed the perturbation of β sheets in NCS (Fig. 2A2).
Discussion
The CD Spectra of NCS has signature peaks at −210, −190 and +225 nm, which is in agreement with an earlier report [30]. The hydrophobic cluster of the inner sheet was found to participate when the chromophore of NCS interacts with 2F Modified EpCAM aptamer residues. The chromophore binding site on Apo NCS consists of three pockets, a hydrophilic, medium and large hydrophobic cavity [31]. CD spectra reveals the appearance of a peak at 205 nm for helix structure induction in NCS by 2F Modified
Acknowledgment
JN, KK, and PK thank Department of Biotechnology, Government of India for funding the project: (NO.BT/PR2285/MED/31/128/2011, 29/12/2011). PK thank DBT for the fellowship. Authors thank Dr. Ramarajaram, VRF for critical suggestions in the manuscript and Bioinformatics Facility supported by DBT under RGYI scheme [BT/PR6476/GBD/27/496/2013].
Authors thank Dr. Nishad Fathima, CLRI, Chennai for helping in CD experiments.
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Equal contribution for the manuscript.