Development of an aptamer-conjugated fluorescent nanoprobe for MMP2

Myoung-Eun Han 0 1 Sungmin Baek 0 1 Hyun-Jung Kim 1 Jung Hwan Lee Sung-Ho Ryu Sae-Ock Oh 0 1 0 Medical Research Center for Ischemic Tissue Regeneration, Pusan National University , 49 Busandaehak-ro, Mulgeum-eup, Yangsan 626-870, Republic of Korea 1 Department of Anatomy, School of Medicine, Pusan National University , 49 Busandaehak-ro, Mulgeum-eup, Yangsan 626-870, Republic of Korea Matrix metalloproteinase 2 (MMP2) plays critical roles in various diseases, such as atherosclerosis and cancer, and has been suggested to contribute to the instability of atherosclerotic plaque. To visualize MMP2 in pathologic tissues, we developed an aptamer targeting MMP2 protein by performing eight rounds of modified DNA systematic evolution of ligands by exponential enrichment (SELEX). The aptamer showed high affinity for MMP2 (Kd = 5.59 nM), precipitated MMP2, and detected MMP2 protein in pathological tissues such as atherosclerotic plaque and gastric cancer tissues. Furthermore, a MMP2 aptamer-conjugated fluorescent nanoprobe successfully visualized atherosclerotic plaques in apolipoprotein E (ApoE) knockout mice. These results suggest that the devised MMP2 aptamer could be useful for the development of various diagnostic tools. - Background Matrix metalloproteinases (MMPs) are zinc- and calciumdependent proteolytic enzymes [1,2]. MMPs can digest extracellular matrix proteins, such as collagen and fibronectin, and many other proteins, such as proteinases, growth factors, cytokines, chemokines, and cell receptors and thus regulate their activities. MMP was first identified in 1962 [3], and since then, other MMPs have been identified. Interestingly, whereas many MMPs are secreted by cells, others are anchored on cellular membranes. Members of this family play important roles in various cellular processes, such as migration, differentiation, and proliferation. Furthermore, they have been associated with pathophysiologies of various diseases, such as cancer, atherosclerosis, and arthritis. During the progression of atherosclerosis, inflammatory cells such as monocytes and lymphocytes [4] play critical roles. Monocytes are recruited into atherosclerotic sites and differentiate into macrophages. After excessive lipid uptake, they become foamy cells. Notably, plaque macrophages secrete critical molecules such as MMPs and prothrombotic tissue factor. Then, MMPs destabilize atherosclerotic plaque by degrading extracellular matrix [5,6]. In addition to the roles in atherosclerosis, MMPs can aid the metastasis of cancer cells [2,7]. Information about the stability of atherosclerotic plaque is critical for the stratification and management of patients [8], and unfortunately, anatomical imaging modalities, such as CT or MRI, do not provide this type of information. Because MMPs are associated with the stability of atherosclerotic plaque, their visualization will be helpful in the stratification and management of patients. The above mentioned actions of MMPs encouraged us to develop a molecular imaging modality that specifically targets MMP molecules. As compared with antibodies, aptamers have several beneficial characteristics, such as low immunogenicity, low molecular weight (8 to 15 kDa), high stability, better penetration, high affinity, and ease of production [9]. From these reasons, we decided to develop a MMP2-specific aptamer. By performing modified DNA systematic evolution of ligands by exponential enrichment (SELEX), we successfully developed a MMP2-specific aptamer which had high affinity and specificity and showed the possibility that it can be applied for molecular imaging. Figure 1 Sequence and secondary structure of the MMP2 aptamer. (a) Sequence of the 40-nucleotide random region (N40, shaded) and of the two constant regions flanking the random region. (b) The hairpin-like secondary structure of the aptamer is presented in the lower panel. Methods In vitro selection of MMP2 DNA aptamers To select MMP2-specific aptamers, a modified DNA SELEX procedure was used, as previously described [10]. Briefly, an ssDNA library template consisting of a 40-nucleotide random region (N40) flanked by two constant regions was prepared and immobilized on streptavidin-coated beads (Pierce, Rockland, MA, USA) via its 5OH-end biotin. A primer extension was then performed using the dATP, dCTP, dGTP, and benzyldUTP nucleotides. The modified DNA library was detached from the template under high pH conditions and then incubated with biotin-tagged target, partitioned using Dynabeads MyOne (Invitrogen, Carlsbad, CA, USA) and amplified by conventional PCR using a 5OH terminal biotinylated reverse primer. A primer extension was then performed, and an enriched pool was prepared for the next round. After eight rounds of SELEX, the enriched DNA pool was cloned and sequenced using standard procedures. After each round of SELEX, binding assays were performed to measure the dissociation constant (Kd) value of the aptamer pool to ensure that its Kd value exhibited a decreasing trend. Figure 2 Affinity of the MMP2 aptamer. (a) 32P-labeled aptamers and different MMP2 protein concentrations were used to examine the binding affinity of the MMP2 aptamer. (b) Images of radiolabeled aptamer that interacted with proteins in the binding assay. Aptamer Pellet Sup Antibody Figure 3 Precipitation of MMP2 protein by MMP2 aptamer. MMP2 protein in buffer containing 10% serum was incubated with the aptamer (0.2 g/ml) overnight at 4C. The protein was detected by immunoblotting with anti-MMP2 antibody. Binding assay MMP2 aptamers were assayed for their ability to bind recombinant MMP2 (R&D Systems, Minneapolis, MN, USA). Aptamers were end-labeled with [-32P]ATP and heated at 95C for 3 min and then slowly ramped to 37C at 0.1C/s in buffer (40 mM HEPES (pH 7.5), 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 0.002% tween-20) for aptamer refolding. Aptamers were then incubated with purified MMP-2 at various concentrations for 30 min at 37C. In order to capture MMP-2, the solution was incubated with Zorbax silica beads (Agilent, Santa Clara, CA, USA) for 1 min with shaking. The protein bead complex was then partitioned through nitrocellulose filter plates (Millipore, Billerica, MA, USA), which were then washed in buffer and exposed to photographic film. Amounts of radiolabeled aptamer that interacted with proteins were quantified using a Fuji FLA-5000 Image Analyzer (Tokyo, Japan). Dissociation constants were calculated by plotting bound MMP2 aptamer versus protein concentration using the following equation: Y = BmaxX/(Kd + X), where Bmax is the extrapolated maximal amount of bound aptamer/ protein complex. Precipitation of MMP2 protein by the aptamer and western blotting A protein solution containing MMP2 protein (R&D Systems, Minneapolis, MN, USA) was precleared with streptavidin-coupled beads for 2 h. The solution was then precipitated with biotin-labeled MMP2 or control aptamer at 4C overnight. Beads were washed four times with 1 m (...truncated)