Functional RNAs: combined assembly and packaging in VLPs.

We describe here a one pot RNA production, packaging and delivery system based on bacteriophage Qß. We demonstrate a method for production of a novel RNAi scaffold, packaged within Qß virus-like particles (VLPs). The RNAi scaffold is a general utility chimera that contains a functional RNA duplex with paired silencing and carrier sequences stabilized by a miR-30 stem-loop. The Qß hairpin on the 5΄ end confers affinity for the Qß coat protein (CP). Silencing sequences can include mature miRNAs and siRNAs, and can target essentially any desired mRNA. The VLP-RNAi assembles upon co-expression of CP and the RNAi scaffold in E. coli. The annealing of the scaffold to form functional RNAs is intramolecular and is therefore robust and concentration independent. We demonstrate dose- and time-dependent inhibition of GFP expression in human cells with VLP-RNAi. In addition, we target the 3΄UTR of oncogenic Ras mRNA and suppress Pan-Ras expression, which attenuates cell proliferation and promotes mortality of brain tumor cells. This combination of RNAi scaffold design with Qß VLP packaging is demonstrated to be target-specific and efficient.

Eukaryotic Ribosomal Expansion Segments as Antimicrobial Targets.

Diversity in eukaryotic rRNA structure and function offers possibilities of therapeutic targets. Unlike ribosomes of prokaryotes, eukaryotic ribosomes contain species-specific rRNA expansion segments (ESs) with idiosyncratic structures and functions that are essential and specific to some organisms. Here we investigate expansion segment 7 (ES7), one of the largest and most variable expansions of the eukaryotic ribosome. We hypothesize that ES7 of the pathogenic fungi Candida albicans (ES7CA) could be a prototypic drug target. We show that isolated ES7CA folds reversibly to a native-like state. We developed a fluorescence displacement assay using an RNA binding fluorescent probe, F-neo. F-neo binds tightly to ES7CA with a Kd of 2.5 × 10-9 M but binds weakly to ES7 of humans (ES7HS) with a Kd estimated to be greater than 7 µM. The fluorescence displacement assay was used to investigate the affinities of a library of peptidic aminosugar conjugates (PAs) for ES7CA. For conjugates with highest affinities for ES7CA (NeoRH, NeoFH, and NeoYH), the lowest dose needed to induce mortality in C. albicans (minimum inhibitory concentration, MIC) was determined. PAs with the lowest MIC values were tested for cytotoxicity in HEK293T cells. Molecules with high affinity for ES7CA in vitro induce mortality in C. albicans but not in HEK293T cells. The results are consistent with the hypothesis that ESs represent useful targets for chemotherapeutics directed against eukaryotic pathogens.

Engineering towards nitroreductase functionality in ene-reductase scaffolds.

Nitroreductases (NRs) and ene-reductases (ERs) both utilize flavin mononucleotide cofactors but catalyze distinct reactions. NRs reduce nitroaromatics, whereas ERs reduce unsaturated C=C double bonds, and these functionalities are known to somewhat overlap. Recent studies on the ER xenobiotic reductase A (XenA) from Pseudomonas putida demonstrated the possibility of increasing NR activity with active site modifications. Structural comparison between NRs and ERs led us to hypothesize that active site cavity size plays an important role in determining enzyme functionality. Residues of ER KYE1 from Kluyveromyces lactis were selected to increase the binding pocket size, compensate for hydrogen bonding pattern changes, and eliminate ER activity. Single variants were screened, and promising mutations were combined. Variant F296A/Y275A showed a 100-fold improvement in NR specific activity over wild-type, and variant H191A/F296A/Y375A exhibited complete conversion to a NR.

RNA: packaged and protected by VLPs.

Virus Like Particles (VLPs) are devices for RNA packaging, protection and delivery, with utility in fundamental research, drug discovery, and disease treatment. Using E. coli for combined expression and packaging of non-viral RNAs into Qß VLPs, we investigated the extent of chemical protection conferred by packaging of RNA in VLPs. We also probed relationships between packaging efficiency and RNA size, sequence and intrinsic compaction. We observe that VLP packaging protects RNA against assault by small diffusible damaging agents such as hydroxyl radicals and divalent cations. By contrast, the extent of unmediated cleavage, in the absence of reactive species, is the same for RNA that is free or packaged within VLPs, and is very slow. In vivo packaging of RNA within VLPs appears to be more efficient for intrinsically compact RNAs, such as rRNA, and less efficient for unstructured, elongated RNA such as mRNA. Packaging efficiency is reduced by addition of the ribosome binding site to a target RNA. The Qß hairpin is necessary but not sufficient for efficient packaging.

Yeast rRNA Expansion Segments: Folding and Function.

Divergence between prokaryotic and eukaryotic ribosomal RNA (rRNA) and among eukaryotic ribosomal RNAs is focused in expansion segments (ESs). Eukaryotic ribosomes are significantly larger than prokaryotic ribosomes partly because of their ESs. We hypothesize that larger rRNAs of complex organisms could confer increased functionality to the ribosome. Here, we characterize the binding partners of Saccharomyces cerevisiae expansion segment 7 (ES7), which is the largest and most variable ES of the eukaryotic large ribosomal subunit and is located at the surface of the ribosome. In vitro RNA-protein pull-down experiments using ES7 as a bait indicate that ES7 is a binding hub for a variety of non-ribosomal proteins essential to ribosomal function in eukaryotes. ES7-associated proteins observed here cluster into four groups based on biological process, (i) response to abiotic stimulus (e.g., response to external changes in temperature, pH, oxygen level, etc.), (ii) ribosomal large subunit biogenesis, (iii) protein transport and localization, and (iv) transcription elongation. Seven synthetases, Ala-, Arg-, Asp-, Asn-, Leu-, Lys- and TyrRS, appear to associate with ES7. Affinities of AspRS, TyrRS and LysRS for ES7 were confirmed by in vitro binding assays. The results suggest that ES7 in S. cerevisiae could play a role analogous to the multi-synthetase complex present in higher order organisms and could be important for the appropriate function of the ribosome. Thermal denaturation studies and footprinting experiments confirm that isolated ES7 is stable and maintains a near-native secondary and tertiary structure.