Development of functional biomaterials using RNA aptamer

Abstract

Event Abstract Back to Event Development of functional biomaterials using RNA aptamer Yusuke Nomura1, Chie Fukui1, Hitomi Toida1, Yuki Morishita1, Shingo Niimi1, Shin Miyakawa2, Ling Jin2, Yoshikazu Nakamura2, 3 and Yuji Haishima1 1 National Institute of Health Sciences, Division of Medical Devices, Japan 2 RIBOMIC Inc., Japan 3 University of Tokyo, Institute of Medical Science, Japan Introduction: Biomaterials used for implant interact with cells and tissues through the protein layer present at the biointerface. These proteins could adversely affect the function and biocompatibility of the materials. Their adsorption behavior is basically dependent on the physicochemical property of the materials, but it is possible to provide favorable functions to the materials by artificially controlling the protein adsorption behavior on the material surface. In this study, we developed a novel and functional biomaterial immobilized the RNA aptamer (RNA-AP) which specifically captures active FGF2. Materials and Methods: Human FGF2, FGF2 receptor and Lysozyme were purchased from Peprotec, R&D systems and SIGMA, respectively. RNA-APs were prepared by in vitro transcription using the DuraScribe T7 Transcription kit. RNA-AP-SH, poly dT-SH (pdT-SH) and PEG (PEG2k-SH and PEG5k-SH) were immobilized on the Au-coated glass plate. The inhibition of FGF2-mediated phosphorylation in NIH 3T3 cells was analyzed by western blotting using phospho-specific antibody (P-FRS2, P-ERK). RNA-AP/protein interaction and cell proliferation were analyzed using BIAcore2000 and BZ-9000, respectively. Results and Discussion: Candidate of RNA-APs against the human FGF2 were selected by surface plasmon resonance (SPR) method, that did not inhibit the binding of FGF2 to FGF2 receptor in SPR analysis and the phosphorylation of FRS2 and ERK in the NIH 3T3 cells. The several SPR sensor chips were prepared and used for evaluating the protein adsorption. The bare gold and pdT-immobilized surfaces did not show a difference between Lysozyme and FGF2 adsorption, indicating that only nonspecific adsorption of the proteins was observed. Although the specific adsorption of FGF2 was observed on the RNA-AP-immobilized surface, the nonspecific adsorption of Lysozyme was still high. On the other hand, the RNA-AP/PEG2k/PEG5k-coimmobilized surfaces showed a remarkable reduction of the nonspecific adsorption due to the excluded volume effect of the PEG chains[1]. In addition, cell proliferation on the RNA-AP/PEG-coated surfaces was significantly increased in comparison with the pdT/PEG-coated surfaces, indicating that RNA-AP/PEG-immobilized surfaces effectively capture and concentrate the active FGF2. Conclusion: In this study, we succeeded in selectively capturing and concentrating the target protein on the biomaterial surface by immobilizing the functional RNA-AP. It is expected that biomaterials combinationally immobilized several RNA-APs may exhibit the potency to control different function such as proliferation and differentiation of various stem cells. Thus, the innovative biomaterials using RNA aptamer are useful for not only the application to regenerative medicine product, but also other medical devices.