Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

Abstract

The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5 0 /3 0 ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems -exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5 0 /3 0 ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

“…16,21,22 In this study, a chimeric pRNA/siRNA targeting firefly or renilla luciferase was constructed, and the silencing efficiency was tested by transient transfection. The chimeric siRNA construct suppressed its target gene specifically and efficiently as demonstrated by a Dual reporter assay, in which the expression To determine whether the knockdown effects shown above were siRNA-specific, chimeric pRNA/siRNA monomers or dimers were treated with cell lysate or recombinant purified Dicer ( Figure 5), which is known for its unique function in processing long doublestranded RNA into 22-bp siRNA.…”

“…14 Therefore, the 5 0 /3 0 proximate double-stranded helical region 15 of pRNA can be redesigned to carry additional sequences without altering its secondary structure or intermolecular interactions. 16,17 Being a poorly differentiated carcinoma of the human upper respiratory tract, nasopharyngeal carcinoma has human folate receptors (hFR) that are highly expressed in epidermal carcinoma (KB) cells. Endocytosis of the ligand/receptor complex mediated by the binding of folate to hFR has been well studied, and macromolecules conjugated to folate have been successfully recognized by folate receptors and internalized into cells.…”

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

“…The unique features uncovered from the studies of phi29 pRNA include (1) independent folding of the ATPase gp16 interaction domain (Lee and Guo 2006) and the motor binding domain (Reid et al 1994a,b,c;Zhang et al 1994Zhang et al , 1995b and (2) the capacity to harbor and escort therapeutic functional modules without causing misfolding or loss of function of the incorporated functionalities. These special properties of phi29 pRNA have led to the development of this RNA as a novel vehicle for applications in nanotechnology and medicine (Hoeprich et al 2003;Guo et al 2005aGuo et al , 2006Guo et al , 2012aKhaled et al 2005;Guo 2010;Abdelmawla et al 2011;Shu et al 2011a,b,c;Shukla et al 2011;Zhou et al 2011). Recently, it was found that the core structure of phi29 pRNA, a 54-nt three-way junction (3WJ) (Fig.…”

Controllable Self-Assembly of Nanoparticles for Specific Delivery of Multiple Therapeutic Molecules to Cancer Cells Using RNA Nanotechnology

“…The 71/75 end has been shown to be located in a tightly folded area, 16 which buries and protects the ends from exonuclease degradation. 7 The chimeric pRNA building block with this aptamer also contained the appropriate right and left loops required for the engineering and fabrication of the trimeric complex.…”

“…Additional functional RNA, such as a hammerhead ribozyme, have been conjugated to the double-stranded domain, resulting in enhanced cleavage efficiency of ribozyme. 7 Extensive studies reveal that siRNA is a double-stranded RNA helix.2 -5 ,28 To test whether it is possible to replace the pRNA helical region with double-stranded siRNA and to determine which construct has the optimal function in gene silencing and trimer formation, we constructed several chimeric pRNA/siRNA and their ability in gene silencing was tested. A 29-bp siRNA was connected to nucleotides 29/91 or 18/99 of pRNA, resulting in pRNA/siRNA(GFP)-29/91 and pRNA/siRNA(GFP)21/99, respectively.…”