Poster abstracts

Poster number 64 submitted by Yi Shu

Chemical modification of ribonucleotides to produce ribonuclease resistant RNA in nanotechnology and nanomedicine

Jing Liu (Department of Biomedical Engineering, University of Cincinnati; Department of Immunology, Huazhong University of Science and Technology, China), Yi Shu (Department of Biomedical Engineering, University of Cincinnati), Peixuan Guo (Department of Biomedical Engineering, University of Cincinnati)

Abstract:
The pRNA (packaging RNA) of bacteriophage phi29 has been demonstrated having a novel role in gearing the phi29 DNA packaging motor and in delivering therapeutic RNAs for the treatment of cancers, viral infections, and genetics diseases. Since RNA is vulnerable to RNase and can be easily degraded in vitro and in vivo, approaches with chemical modification have been extensively explored in the quest of reliable methods to produced stable RNA. One critical question remains to be answered is whether the RNA maintain its correct folding and biological activity after chemical modification. Here we evaluate the property of 2¡¯-Fluorine (2¡¯-F), 2¡¯-Amino (2¡¯-NH2), 2¡¯-O-Methyl (2¡¯-O-Me), and modified RNA using the highly sensitive phi29 DNA virion assembly system. It was found that with the 2¡¯ modification on only pyrimidines but not purines, the resulting RNA was completely resistant to RNase digestion and remains intact even after 19 hours of incubation with fetal bovine serum. Moreover, pRNA with 2¡¯F modification retained dimer formation potential via loop-loop interaction. More interestingly, the 2¡¯F modified pRNA retained its activity in binding to the procapsid, gearing the motor to package the genomic DNA, and retained its property in the process of assembling infections phi29 virion. 2¡¯-NH2 modification on pyrimidines also resulted in RNase resistant pRNA, however, the resulted pRNA could not form dimers, and was incompetent in viral assembly. For the 2¡¯-O-Me modification, pRNAs could be synthesized through in vitro transcription with 2¡¯-O-Me modified cytidines (2¡¯-O-Me-C) as substrate, while synthesis was failed using 2¡¯-O-Me modified uridines as substrate. pRNA with 2¡¯-O-Me-C modification was competent in pRNA dimer formation and procapsid binding. However, the modified pRNA were inactive in phi29 virion assembly. The pRNA containing 2¡¯-O-Me-C was not as stable as pRNA with 2¡¯-F and 2¡¯-Amino pyrimidines modification. Taken together, our data suggested that 2¡¯-F modification did not affect pRNA folding or hinder its biological activity. However, 2¡¯-O-Me and 2¡¯-NH2 modification is seriously detrimental to RNA folding, structure and function.

Keywords: Bacteriophage phi29, RNA modification, pRNA