Bifunctional RNA Research Articles

535. In Vivo Study of a Bi-Functional Short-Hairpin RNA (bi-shRNA) Targeting Multiple Single-Nucleotide KRAS Mutations in Pancreatic Cancer

535. In Vivo Study of a Bi-Functional Short-Hairpin RNA (bi-shRNA) Targeting Multiple Single-Nucleotide KRAS Mutations in Pancreatic Cancer

Dual Masking of Specific Negative Splicing Regulatory Elements Resulted in Maximal Exon 7 Inclusion of SMN2 Gene

Spinal muscular atrophy (SMA) is a fatal autosomal recessive disease caused by survival motor neuron (SMN) protein insufficiency due to SMN1 mutations. Boosting SMN2 expression is a potential therapy for SMA. SMN2 has identical coding sequence as SMN1 except for a silent C-to-T transition at the 6th nucleotide of exon 7, converting a splicing enhancer to a silencer motif. Consequently, most SMN2 transcripts lack exon 7. More than ten putative splicing regulatory elements (SREs) were reported to regulate exon 7 splicing. To investigate the relative strength of each negative SRE in inhibiting exon 7 inclusion, antisense oligonucleotides (AONs) were used to mask each element, and the fold increase of full-length SMN transcripts containing exon 7 were compared. The most potent negative SREs are at intron 7 (in descending order): ISS-N1, 3' splice site of exon 8 (ex8 3'ss) and ISS+100. Dual-targeting AONs were subsequently used to mask two nonadjacent SREs simultaneously. Notably, masking of both ISS-N1 and ex8 3'ss induced the highest fold increase of full-length SMN transcripts and proteins. Therefore, efforts should be directed towards the two elements simultaneously for the development of optimal AONs for SMA therapy.

Bifunctional siRNAs for tumor therapy.

Double-stranded RNA molecules carrying a triphosphate moiety represent a molecular structure by which the host recognizes viral infections. Such RNA molecules can be generated synthetically by chemical synthesis or by in vitro transcription (see Chapter 2, Hornung et al.). Similar to viruses, they initiate an antiviral immune response, e.g., by stimulation of the immune system. Short, double-stranded RNA in the cytosol can also trigger the RNA interference mechanism, which also has been considered as an antiviral response. Notably, synthetic RNAs that are designed to be specific for a certain host mRNA inhibit expression of the respective gene, leading to specific gene silencing. Both effects-gene silencing and immunostimulation-are interesting from a therapeutic perspective, e.g., for cancer therapy. Notably, both effects can be activated by a single molecule, an siRNA carrying a triphosphate moiety. This chapter provides information how to design such compounds with respect to the associated signaling pathways and the techniques to evaluate bifunctional RNAs in the context of tumor therapy.

A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications

A Chemical View of Oligonucleotides for Exon Skipping and Related Drug Applications

317. Multiplex Bi-Functional Short-Hairpin RNA (shRNA) Targeting Single-Nucleotide KRAS Mutations in Pancreatic Cancer

317. Multiplex Bi-Functional Short-Hairpin RNA (shRNA) Targeting Single-Nucleotide KRAS Mutations in Pancreatic Cancer

A short bifunctional element operates to positively or negatively regulateESAG9expression in different developmental forms ofTrypanosoma brucei

In their mammalian host trypanosomes generate 'stumpy' forms from proliferative 'slender' forms as an adaptation for transmission to their tsetse fly vector. This transition is characterised by the repression of many genes while quiescent stumpy forms accumulate during each wave of parasitaemia. However, a subset of genes are upregulated either as an adaptation for transmission or to sustain infection chronicity. Among this group are ESAG9 proteins, whose genes were originally identified as a component of some telomeric variant surface glycoprotein gene expression sites, although many members of this diverse family are also transcribed elsewhere in the genome. ESAG9 genes are among the most highly regulated genes in transmissible stumpy forms, encoding a group of secreted proteins of cryptic function. To understand their developmental silencing in slender forms and activation in stumpy forms, the post-transcriptional control signals for a well conserved ESAG9 gene have been mapped. This identified a precise RNA sequence element of 34 nucleotides that contributes to gene expression silencing in slender forms but also acts positively, activating gene expression in stumpy forms. We predict that this bifunctional RNA sequence element is targeted by competing negative and positive regulatory factors in distinct developmental forms of the parasite. Analysis of the 3'UTR regulatory regions flanking the highly diverse ESAG9 family reveals that the linear regulatory sequence is not highly conserved, suggesting that RNA structure is important for interactions with regulatory proteins.

Streptavidin binding bifunctional aptamers and their interaction with low molecular weight ligands

This paper describes the measurement of the binding affinities of two bifunctional RNA aptamers to their respective ligands. The aptamers comprise either a theophylline or malachite green binding sequence fused to a streptavidin binding sequence. These bifunctional aptamers are shown to bind simultaneously to both the small ligand and to streptavidin whether in free solution or on gold surfaces. Binding isotherms for both interactions were measured by different physiochemical techniques: surface plasmon resonance, fluorescence spectroscopy and dynamic light scattering. Both qualitatively and quantitatively there is little difference in binding affinities between the bifunctional aptamers and their monofunctional components. The respective Kd values for streptavidin binding in the monofunctional aptamer and in the theophylline bifunctional aptamer were 12nM and 65nM, respectively whilst the Kd values for theophylline binding in the monofunctional aptamer and the streptavidin bifunctional aptamer were 300nM and 120nM. These results are consistent with treating each aptamer sequence as a module that can be combined with others without significant loss of function. This allows for the use of streptavidin based immobilization strategies without either the cost of biotinylated dNTPs or the variable yields associated with the chemical biotinylation of RNA.

Unusually High‐Affinity Mg2+ Binding at the AU‐Rich Sequence within the Antiterminator Hairpin of a Mg2+ Riboswitch

Mg(2+)-Responsive riboswitches represent a fascinating example of bifunctional RNAs that sense Mg(2+) ions with high selectivity and autonomously regulate the expression of Mg(2+)-transporter proteins. The mechanism of the mgtA riboswitch is scarcely understood, and a detailed structural analysis is called for to study how this RNA can selectively recognize Mg(2+) and respond by switching between two alternative stem loop structures. In this work, we investigated the structure and Mg(2+)-binding properties of the lower part of the antiterminator loop C from the mgtA riboswitch of Yersinia enterocolitica by solution NMR and report a discrete Mg(2+)-binding site embedded in the AU-rich sequence. At the position of Mg(2+) binding, the helical axis exhibits a distinct kink accompanied by a widening of the major groove, which accommodates the Mg(2+)-binding pocket. An unusually large overlap between two adenine residues on the opposite strands suggests that the bending may be sequence-induced by strong stacking interactions, enabling Mg(2+) to bind at this so-far not described metal-ion binding site.

131. Systematic Identification of Bi-Functional Short-Hairpin RNA (shRNA) Specific for Single-Nucleotide KRAS Mutations

131. Systematic Identification of Bi-Functional Short-Hairpin RNA (shRNA) Specific for Single-Nucleotide KRAS Mutations

Bifunctional RNAs Targeting the Intronic Splicing Silencer N1 Increase SMN Levels and Reduce Disease Severity in an Animal Model of Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients. Although the SMN2 coding sequence has the potential to produce full-length SMN, nearly 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein. SMN2, however, is an excellent therapeutic target. Previously, we developed antisense-based oligonucleotides (bifunctional RNAs) that specifically recruit SR/SR-like splicing factors and target a negative regulator of SMN2 exon-7 inclusion within intron-6. As a means to optimize the antisense sequence of the bifunctional RNAs, we chose to target a potent intronic repressor downstream of SMN2 exon 7, called intronic splicing silencer N1 (ISS-N1). We developed RNAs that specifically target ISS-N1 and concurrently recruit the modular SR proteins SF2/ASF or hTra2β1. RNAs were directly injected in the brains of SMA mice. Bifunctional RNA injections were able to elicit robust induction of SMN protein in the brain and spinal column of neonatal SMA mice. Importantly, hTra2β1-ISS-N1 and SF2/ASF-ISS-N1 bifunctional RNAs significantly extended lifespan and increased weight in the SMNΔ7 mice. This technology has direct implications for SMA therapy and provides similar therapeutic strategies for other diseases caused by aberrant splicing.

Genome-Wide Identification of Small RNAs in the Opportunistic Pathogen Enterococcus faecalis V583

Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the Gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5′ and 3′ RACE-PCR, and Northern blot analysis. Six sRNAs were specifically expressed at exponential phase, two sRNAs were observed at stationary phase, and three were detected during both phases. Searches of putative functions revealed that three of them (EFA0080_EFA0081 and EFB0062_EFB0063 on pTF1 and pTF2 plasmids, respectively, and EF0408_EF04092 located on the chromosome) are similar to antisense RNA involved in plasmid addiction modules. Moreover, EF1097_EF1098 shares strong homologies with tmRNA (bi-functional RNA acting as both a tRNA and an mRNA) and EF2205_EF2206 appears homologous to 4.5S RNA member of the Signal Recognition Particle (SRP) ribonucleoprotein complex. In addition, proteomic analysis of the ΔEF3314_EF3315 sRNA mutant suggests that it may be involved in the turnover of some abundant proteins. The expression patterns of these transcripts were evaluated by tiling array hybridizations performed with samples from cells grown under eleven different conditions some of which may be encountered during infection. Finally, distribution of these sRNAs among genome sequences of 54 E. faecalis strains was assessed. This is the first experimental genome-wide identification of sRNAs in E. faecalis and provides impetus to the understanding of gene regulation in this important human pathogen.

Identification of potentially new bifunctional RNA based on genome-wide data-mining of alternative splicing events

It is now evident that the transcriptional output of the genome is much more complex than estimates based on the number of protein-coding genes, and that non-coding RNA widely increase the source of regulatory molecules, a role previously ascribed to proteins. Furthermore, the recent characterization of bifunctional RNA, i.e. RNA for which both coding capacity and activity as functional RNA have been reported, adds an additional degree of complexity. Based on the SRA (Steroid Receptor RNA Activator) model, where bifunctionality is regulated by alternative splicing, we hypothesized that similar cases, not yet formally tested experimentally, might exist. Using freely available data from high-throughput sequencing projects, we propose here a bioinformatical identification of mRNA whose ORF are disrupted by alternative splicing events, especially by intron retention, and potentially representing a cognate non-coding RNA. Our data-mining approach revealed that the human genome contains around 300 possibilities of potentially new bifunctional RNA.

Novel factor rescues ribosomes trapped on non-stop mRNAs

Ribosomes are trapped at the 3′ ends of mRNAs that lack a natural stop codon. In bacteria, a reaction called trans-translation recycles ribosomes entrapped at such ‘non-stop’ mRNAs. The main player in trans-translation is tmRNA (SsrA-RNA), a bi-functional RNA that acts as both a tRNA and an mRNA. In the trans-translation reaction, alanine-charged tmRNA loads at the ribosomal A-site and translation shifts to the resume codon in tmRNA. Translation of tmRNA stops at a natural stop codon at the end of the small reading frame of tmRNA. In this way, the reaction simultaneously adds a peptide tag to the end of the nascent, incomplete polypeptide and recycles the stalled ribosomes. The peptide tag is recognized by cellular proteases that rapidly degrade the incomplete, potentially harmful polypeptides. The trans-translation reaction is not essential in most bacteria, raising the possibility that ribosomes stalled at non-stop mRNAs can be rescued by alternative routes. In this issue of Molecular Microbiology, Chadani et al. show that a novel translation factor, ArfA, can recycle a ribosome trapped at the 3′ end of a non-stop mRNA in the absence of trans-translation. AfrA is essential in the absence of tmRNA, showing that the two systems work in parallel to resolve stalled ribosomes.

Immunotherapy of pancreatic carcinoma with siRNA targeting TGF-beta

Increasing evidence suggests that immune responses are important in the control of tumors. Pancreatic cancers are often infiltrated with lymphocytes, which correlates with a better prognosis. Continued tumor growth in the presence of a lymphocytic infiltration indicates a failure of immune control. TGF-beta is a highly active immunosuppressive cytokine and high serum levels correlate with a poor prognosis in pancreatic cancer, making it an interesting target for immunotherapy. The ubiquitously expressed cytosolic helicase retinoic acid-inducible protein I (RIG-I) is an immunoreceptor that signals the presence of viral RNA and activates the immune system via type I IFN. RIG-I detects RNA with a triphosphate group at the 5' end. Modification of siRNA as 5'-triphosphat-RNA (3p-siRNA) allows combining gene silencing and RIG-I activation. Here, we describe immunotherapy of murine pancreatic cancer with 3p-siRNA targeting TGF-beta. The 3p-siRNA reduced TGF-beta production by pancreatic carcinoma cells. In addition, RIG-I activation in tumor cells resulted in up-regulation of MHC-I expression, secretion of type I IFN and IP-10. As a consequence, tumor cells were sensitized to T cell-mediated killing. Moreover, 3p-siRNA induced tumor cell apoptosis via the intrinsic pathway. In vivo, 3p-siRNA against TGF-beta induced a Th1-type cytokine profile, with high levels of IL-12 and type I IFN. Systemic therapy slowed down tumor growth and prolonged survival in mice with orthotopic pancreatic tumors. Bi-functional RNA molecules that combine siRNA activity and activation of the innate immune system are promising candidates for the treatment of pancreatic cancer.

Delivery of bifunctional RNAs that target an intronic repressor and increase SMN levels in an animal model of spinal muscular atrophy

Spinal muscular atrophy (SMA) is a motor neuron disease caused by the loss of survival motor neuron-1 (SMN1). A nearly identical copy gene, SMN2, is present in all SMA patients, which produces low levels of functional protein. Although the SMN2 coding sequence has the potential to produce normal, full-length SMN, approximately 90% of SMN2-derived transcripts are alternatively spliced and encode a truncated protein lacking the final coding exon (exon 7). SMN2, however, is an excellent therapeutic target. Previously, we developed bifunctional RNAs that bound SMN exon 7 and modulated SMN2 splicing. To optimize the efficiency of the bifunctional RNAs, a different antisense target was required. To this end, we genetically verified the identity of a putative intronic repressor and developed bifunctional RNAs that target this sequence. Consequently, there is a 2-fold mechanism of SMN induction: inhibition of the intronic repressor and recruitment of SR proteins via the SR recruitment sequence of the bifunctional RNA. The bifunctional RNAs effectively increased SMN in human primary SMA fibroblasts. Lead candidates were synthesized as 2'-O-methyl RNAs and were directly injected in the central nervous system of SMA mice. Single-RNA injections were able to illicit a robust induction of SMN protein in the brain and throughout the spinal column of neonatal SMA mice. In a severe model of SMA, mean life span was extended following the delivery of bifunctional RNAs. This technology has direct implications for the development of an SMA therapy, but also lends itself to a multitude of diseases caused by aberrant pre-mRNA splicing.

A Negatively Acting Bifunctional RNA Increases Survival Motor Neuron Both In Vitro and In Vivo

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present called SMN2, but this gene cannot compensate for the loss of SMN1 because of a single silent nucleotide difference in SMN2 exon 7. This single-nucleotide difference attenuates an exonic splice enhancer, resulting in the production of an alternatively spliced isoform lacking exon 7, which is essential for protein function. SMN2, however, is a critical disease modifier and is an outstanding target for therapeutic intervention because all SMA patients retain SMN2 and SMN2 maintains the same coding sequence as SMN1. Therefore, compounds or molecules that increase SMN2 exon 7 inclusion hold great promise for SMA therapeutics. Bifunctional RNAs have been previously used to increase SMN protein levels and derive their name from the presence of two domains: an antisense RNA sequence specific to the target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. This study was designed to develop negatively acting bifunctional RNAs that recruit hnRNPA1 to exon 8 and block the general splicing machinery from the exon 8. By blocking the downstream splice site, this could competitively favor the inclusion of SMN exon 7 and therefore increase full-length SMN production. Here we identify a bifunctional RNA that stimulated full-length SMN expression in a variety of cell-based assays including SMA patient fibroblasts. Importantly, this molecule was also able to induce SMN expression in a previously described mouse model of SMA and demonstrates a novel therapeutic approach for SMA as well as a variety of diseases caused by a defect in splicing.

A Negatively-Acting Bifunctional RNA Increases Survival Motor Neuron in vitro and in vivo

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder and is the leading genetic cause of infant mortality. SMA is caused by the loss of survival motor neuron-1 (SMN1). In humans, a nearly identical copy gene is present called SMN2, but this gene cannot compensate for the loss of SMN1 because of a single silent nucleotide difference in SMN2 exon 7. This single-nucleotide difference attenuates an exonic splice enhancer, resulting in the production of an alternatively spliced isoform lacking exon 7, which is essential for protein function. SMN2, however, is a critical disease modifier and is an outstanding target for therapeutic intervention because all SMA patients retain SMN2 and SMN2 maintains the same coding sequence as SMN1. Therefore, compounds or molecules that increase SMN2 exon 7 inclusion hold great promise for SMA therapeutics. Bifunctional RNAs have been previously used to increase SMN protein levels and derive their name from the presence of two domains: an antisense RNA sequence specific to the target RNA and an untethered RNA segment that serves as a binding platform for splicing factors. This study was designed to develop negatively acting bifunctional RNAs that recruit hnRNPA1 to exon 8 and block the general splicing machinery from the exon 8. By blocking the downstream splice site, this could competitively favor the inclusion of SMN exon 7 and therefore increase full-length SMN production. Here we identify a bifunctional RNA that stimulated full-length SMN expression in a variety of cell-based assays including SMA patient fibroblasts. Importantly, this molecule was also able to induce SMN expression in a previously described mouse model of SMA and demonstrates a novel therapeutic approach for SMA as well as a variety of diseases caused by a defect in splicing.

Systematic Deletion of the Adenovirus-associated RNAI Terminal Stem Reveals a Surprisingly Active RNA Inhibitor of Double-stranded RNA-activated Protein Kinase

Adenoviruses use the short noncoding RNA transcript virus-associated (VA) RNAI to counteract two critical elements of the host cell defense system, innate cellular immunity and RNA interference, mediated by the double-stranded RNA-activated protein kinase (PKR) and Dicer/RNA-induced silencing complex, respectively. We progressively shortened the VA RNAI terminal stem to examine its necessity for inhibition of PKR. Each deletion, up to 15 bp into the terminal stem, resulted in a cumulative decrease in PKR inhibitory activity. Remarkably, however, despite significant apparent destabilization of the RNA structure, the final RNA mutant that lacked the entire terminal stem (TSΔ21 RNA) efficiently bound PKR and exhibited wild-type inhibitory activity. TSΔ21 RNA stability was strongly influenced by solution pH, indicating the involvement of a protonated base within the VA RNAI central domain tertiary structure. Gel filtration chromatography and isothermal titration calorimetry analysis indicated that wild-type VA RNAI and TSΔ21 RNA form similar 1:1 complexes with PKR but that the latter lacks secondary binding site(s) that might be provided by the terminal stem. Although TSΔ21 RNA bound PKR with wild-type Kd, and overall change in free energy (ΔG), the thermodynamics of binding (ΔH and ΔS) were significantly altered. These results demonstrate that the VA RNAI terminal stem is entirely dispensable for inhibition of PKR. Potentially, VA RNAI is therefore a truly bi-functional RNA; Dicer processing of the VA RNAI terminal stem saturates the RNA interference system while generating a “mini-VA RNAI” molecule that remains fully active against PKR.

434. Potential SMA Gene Therapy: Identification of Optimal Targets for Bi-Functional RNAs In Vivo

434. Potential SMA Gene Therapy: Identification of Optimal Targets for Bi-Functional RNAs In Vivo

526. A Negatively-Acting Bifunctional RNA Increases Survival Motor Neuron In Vitro and In Vivo

526. A Negatively-Acting Bifunctional RNA Increases Survival Motor Neuron In Vitro and In Vivo