RNase P is an essential ribonucleoprotein complex found in all three domains of life. The RNase P holoenzyme is composed of an RNA subunit and one or more protein subunits. The RNA component is the catalytic moiety of RNase P across all phylogenetic domains, and is responsible for the maturation of 5′ termini of all pre-tRNAs, which account for about 2% of total cellular RNA . A unique feature of RNase P is its recognition of substrate structures and thus is able to hydrolyze different natural substrates . This is of great advantage because recognition of structures rather than sequences may help the fight against variable viruses as single or even double mutations in the target sequence may well be tolerated . It is well known that HCV is highly variable, and it has been proved that HCV genome cannot be defined by a single sequence but by a population of closely related variant sequences . In consideration of the “quasispecies” nature of HCV genome , the RNase P-based M1GS ribozyme is a promising antisense technique in HCV therapeutic studies.
HCV 5′ UTR is the most conserved locus within its genome and thus efforts related to HCV RNA therapeutics have been focused on this locus . Nevertheless, this region has a highly stable RNA structure and is modulated by miRNAs and RNA-binding proteins, which limits the number of accessible sites for ribozyme targeting . Therefore, it is important to select the target regions from the 5′ UTR of HCV RNA that are accessible to M1GS binding. In this study, we first analyzed the sequence of HCV 5′ UTR and found that three sites, i.e. C20-G21, C67-G68 and U76-G77, met the general features for M1GS cleavage activities . All the three sites were located in the front one third of HCV 5′ UTR. Further analysis on the secondary structure of this portion with RNA structure software revealed that the 3′ flanking sequence of the site C67-G68 formed a long single-strand region but the flanking sequence of C20-G21 or U76-G77 folded into a stable stem-loop structure (Figure 2), while the effect of long range annealing would make relevant regions less accessible for targeting [33, 34]. Therefore, the region near C67-G68 may be more accessible for ribozyme binding.
Based on the above putative site (C67-G68), a custom guide sequence was designed, which was covalently linked to the 3′ termini of M1 RNA through an 88 nt-long bridge sequence, and a new targeting enzyme (i.e. M1GS-HCV/C67) for the 5′ UTR of HCV RNA was successfully constructed. In consideration of the influence of bridge sequence on the cleavage activities of M1GS as previously reported , a M1GS without a bridge, i.e. M1GS-HCV/C67*, was also constructed as a control. As shown in Figures 7 and 8, about 85% reduction of the expression level of HCV core protein and >1000-fold reduction of viral growth were observed in supernatant of cultured cells transfected with M1GS-HCV/C67 ribozyme. On the other hand, no obvious reduction of the levels of core gene expression and viral growth was observed in cells transfected with M1GS-HCV/C67*. Because M1GS-HCV/C67* contained an identical guide sequence with M1GS-HCV/C67 and thus had a similar binding affinity to target sequence. Therefore, the overall inhibition of viral gene expression and growth by M1GS-HCV/C67 was mainly due to the targeted cleavage by the ribozyme, as opposed to antisense or other nonspecific effects of the guide sequence.
It has been reported that cholesterol-modified siRNAs can be easily bound to human serum albumin, and thus cholesterol modification has the potential to improve in vivo pharmacokinetic properties of RNA therapeutics and broaden their tissue biodistribution . Therefore, M1GS RNA was modified in this study with cholesterol on the 5′ terminus of M1 RNA within the ribozyme, and the cholesterol-conjugated M1GS ribozyme (i.e. Chol-M1GS-HCV/C67) did not lose its cleavage activity in vitro (Figure 5 Lane 4). Furthermore, similar to the unconjugated M1GS RNAs, Chol-M1GS-HCV/C67 was able not only to efficiently inhibit HCV gene expression in transiently transfected Huh7.5.1 cells (Figure 6 Lane 5 and Figure 7 Lane 4) but also to significantly reduce viral titers in the culture supernatant (Figure 8). Together, our data demonstrate the successful use of an M1GS ribozyme in the inhibition of HCV multiplication and provide an insight into the potential of M1GS-base therapeutics against HCV infection.