Assembly of Therapeutic pRNA-siRNA Nanoparticles Using Bipartite Approach

Abstract

The 117-nucleotide (nt) RNA, called the packaging RNA (pRNA) of bacteriophage phi29 DNA packaging motor, has been shown to be an efficient vector for the construction of RNA nanoparticles for the delivery of small interfering RNA (siRNA) into specific cancer or viral-infected cells. Currently, chemical synthesis of 117-nt RNA is not feasible commercially. In addition, labeling at specific locations on pRNA requires the understanding of its modular organization. Here, we report multiple approaches for the construction of a functional 117-base pRNA using two synthetic RNA fragments with variable modifications. The resulting bipartite pRNA was fully competent in associating with other interacting pRNAs to form dimers, as demonstrated by the packaging of DNA via the nanomotor and the assembly of phi29 viruses in vitro. The pRNA subunit assembled from bipartite fragments harboring siRNA or receptor-binding ligands were equally competent in assembling into dimers. The subunits carrying different functionalities were able to bind cancer cells specifically, enter the cell, and silence specific genes of interest. The pRNA nanoparticles were subsequently processed by Dicer to release the siRNA embedded within the nanoparticles. The results will pave the way toward the treatment of diseases using synthetic pRNA/siRNA chimeric nanoparticles.

Fabrication of 14 different RNA nanoparticles for specific tumor targeting without accumulation in normal organs

“…Following standard pRNA nomenclature (Shu et al 2011b), the right-hand (R-loop) sequence was assigned an uppercase letter (i.e., A, B, …); the left-hand (L-loop) sequence, a lowercase letter with a prime (i.e., a ′ , b ′ , …). A pair of the same uppercase and lowercase letters (e.g., Aa ′ ) designated a complementary sequence in the R/L interlocking loop, while different letters indicate a lack of complementarity in the interlocking sequence.…”

“…A dual-luciferase reporter assay system was used to measure the gene silencing effects of pRNA tetrameric constructs harboring four different siRNAs targeted toward the firefly luciferase gene (Shu et al 2011b). The relative luciferase activity is proportional to the expression level of the firefly luciferase gene when normalized with the activity of the internal control, Renilla luciferase, which remains unaffected against siRNA.…”

Uniqueness, Advantages, Challenges, Solutions, and Perspectives in Therapeutics Applying RNA Nanotechnology

“…1A-C, 2B-C, 3) (Shu et al, 2004). In the succeeding years, through a series of papers, they showed that pRNA molecules could be conjugated with various therapeutic functionalities including aptamers, small interfering RNA (siRNA), ribozymes, and microRNA (miRNA) Guo et al, 2005;Khaled et al, 2005;Guo et al, 2006;Shu et al, 2009;Abdelmawla et al, 2011;Ye et al, 2011;Shu et al, 2011a;Shu et al, 2011b;Shu et al, 2011c;Zhang et al, 2009) 4). These findings have paved the way for RNA nanotechnology to develop into a novel area of therapeutics for the treatment of various diseases such as cancer, viral infections, and genetic diseases.…”

“…The production of oligonucleotides with functional moieties then becomes a scalable process (chemically) and with a modular design the complex can be self-assembled from the basic building blocks. Using this methodology, thermodynamically and chemically stable pRNA-based nanoparticles with functional modules were successfully fabricated using a bipartite, tripartite, and tetrapartite approach with various modifications (Shu et al, 2011a;Shu et al, 2011b).…”

“…Same letter pair (e.g., Aa¢) indicates complementarity interlocking loops; different letter (e.g. Ab¢) indicates noncoomplementary loops Guo et al, 2005;Khaled et al, 2005;Guo et al, 2006;Shu et al, 2009;Abdelmawla et al, 2011;Shu et al, 2011a;Shu et al, 2011b;Shu et al, 2011c;Ye et al, 2012). (A) Construction of pRNA monomers bearing either siRNA, a ribozyme, a receptor-binding aptamer, a targeting ligand, or a detection molecule; scale bar = 15 nm.…”