•TNRC6 is the central organizing factor for nuclear RNAi•Interactions with mediator, CCR4-NOT, anaphase promoting complex, and histone modifiers•TNRC6, MED14, NAT10, and WDR5 are essential for RNA-mediated gene activation•Biochemical connection between transcriptional control and nuclear RNAiIn the cytoplasm, small RNAs can control mammalian translation by regulating the stability of mRNA. In the nucleus, small RNAs can also control transcription and splicing. The mechanisms for RNA-mediated nuclear regulation are not understood and remain controversial, hindering the effective application of nuclear RNAi and investigation of its natural regulatory roles. Here, we reveal that the human GW182 paralogs TNRC6A/B/C are central organizing factors critical to RNA-mediated transcriptional activation. Mass spectrometry of purified nuclear lysates followed by experimental validation demonstrates that TNRC6A interacts with proteins involved in protein degradation, RNAi, the CCR4-NOT complex, the mediator complex, and histone-modifying complexes. Functional analysis implicates TNRC6A, NAT10, MED14, and WDR5 in RNA-mediated transcriptional activation. These findings describe protein complexes capable of bridging RNA-mediated sequence-specific recognition of noncoding RNA transcripts with the regulation of gene transcription.Download high-res image (206KB)Download full-size image

•Quantitative biochemical link between RNA numbers and disease•Less than four mutant c9orf72 molecules per cell on average•∼1:1 correspondence between c9orf72 foci and mutant intronic RNA•Small numbers of disease RNA molecules can have major consequencesThe chromosome 9 open reading frame 72 (c9orf72) gene contains a hexanucleotide (GGGGCC) repeat expansion responsible for many cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mutant intronic RNA forms “foci” within nuclei, but the connection between transcript expression, foci, and biochemical disease mechanisms is unclear. Knowing the absolute numbers of cellular RNAs, in any system, is important for understanding the molecular mechanisms of natural physiology, disease, and drug action. Absolute numbers, however, are rarely determined, and this absence is a major impediment to understanding complex systems. Using quantitative methods, we demonstrate that foci are single RNA molecules. Most cells have no foci while 1%–2% have more than ten. Knowing the number of disease-causing molecules may contribute to understanding ALS and FTD pathology and successful drug discovery. More broadly, our data suggest that small numbers of RNA molecules may have a sizable impact on disease.

•Duplex RNAs can be engineered to recognize RNA targets composed entirely of C/G•Duplex RNAs inhibit sense and antisense foci at C9orf72 locus•RNAi is active at suppressing foci in mammalian cell nucleiA GGGGCC expansion within an intronic region of the C9orf72 gene forms RNA foci that are associated with one-third of familial amyotrophic lateral sclerosis and one-quarter of frontotemporal dementia. The C9orf72 locus also expresses an antisense transcript with a CCCCGG expansion that forms foci and may contribute to disease. Synthetic agents that bind these hexanucleotide repeats and block foci would be leads for therapeutic discovery. We have engineered duplex RNAs to enable them to recognize difficult C/G targets. Recognition inhibits foci formed by both GGGGCC and CCCCGG RNA. Our findings show that a single duplex RNA can be used to recognize both disease-related C9orf72 transcripts. More broadly, we extend RNAi to previously inaccessible C/G sequences and provide another example of target recognition in human cells by nuclear RNAi.Figure optionsDownload full-size imageDownload high-quality image (173 K)Download as PowerPoint slide

Dentatorubral-pallidoluysian atrophy (DRPLA) is a progressive neurodegenerative disorder that currently has no curative treatments. DRPLA is caused by an expansion of a CAG trinucleotide repeat region within the protein-encoding sequence of the atrophin-1 (ATN-1) gene. Inhibition of mutant ATN-1 protein expression is one strategy for treating DRPLA, and allele-selective gene silencing agents that block mutant expression over wild-type expression would be lead compounds for therapeutic development. Here we develop an assay for distinguishing mutant from wild-type ATN-1 protein by gel electrophoresis. We use this assay to evaluate duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) for allele-selective inhibition of ATN-1 protein expression. We observed potent and allele-selective inhibition by RNA duplexes that contain mismatched bases relative to the CAG target and have the potential to form miRNA-like complexes. ss-siRNAs that contained mismatches were as selective as mismatch-containing duplexes. We also report allele-selective inhibition by duplex RNAs containing unlocked nucleic acids or abasic substitutions, although selectivities are not as high. Five compounds that showed >8-fold allele selectivity for mutant ATN-1 were also selective for inhibiting the expression of two other trinucleotide repeat disease genes, ataxin-3 (ATXN-3) and huntingtin (HTT). These data demonstrate that the expanded trinucleotide repeat within ATN-1 mRNA is a potential target for compounds designed to achieve allele-selective inhibition of ATN-1 protein, and one agent may allow the targeting of multiple disease genes.

RNAi using single-strand RNA would provide new options for therapeutic development and for investigating critical questions of mechanism. Using chemically modified single-strands, we test the hypothesis that single-stranded RNAs can engage the RNAi pathway and silence gene transcription. We find that a chemically modified single-stranded silencing RNA (ss-siRNA) designed to be complementary to a long noncoding RNA (lncRNA) requires argonaute protein, functions through the RNAi pathway, and inhibits gene transcription. These data expand the use of single-stranded RNA to cell nuclei.

Unlocked nucleic acid (UNA) is an acyclic analogue of RNA that can be introduced into RNA or DNA oligonucleotides. The increased flexibility conferred by the acyclic structure fundamentally affects the strength of base pairing, creating opportunities for improved applications and new insights into molecular recognition. Here we test how UNA substitutions affect allele-selective inhibition of expression of trinucleotide repeat genes Huntingtin (HTT) and Ataxin-3 (ATX-3). We find that the either the combination of mismatched bases and UNA substitutions or UNA substitutions alone can improve potency and selectivity. Inhibition is potent, and selectivities of >40-fold for inhibiting mutant versus wild-type expression can be achieved. Surprisingly, even though UNA preserves the potential for complete base pairing, the introduction of UNA substitutions at central positions within fully complementary duplexes leads to >19-fold selectivity. Like mismatched bases, the introduction of central UNA bases disrupts the potential for cleavage of substrate by argonaute 2 (AGO2) during gene silencing. UNA-substituted duplexes are as effective as other strategies for allele-selective silencing of trinucleotide repeat disease genes. Modulation of AGO2 activity by the introduction of UNA substitutions demonstrates that backbone flexibility is as important as base pairing for catalysis of fully complementary duplex substrates. UNA can be used to tailor RNA silencing for optimal properties and allele-selective action.

Oligonucleotides and their derivatives are a proven chemical strategy for modulating gene expression. However, their negative charge remains a challenge for delivery and target recognition inside cells. Here we show that oligonucleotide–oligospermine conjugates (Zip nucleic acids or ZNAs) can help overcome these shortcomings by serving as effective antisense and antigene agents. Conjugates containing DNA and locked nucleic acid (LNA) oligonucleotides are active, and oligospermine conjugation facilitates carrier-free cell uptake at nanomolar concentrations. Conjugates targeting the CAG triplet repeat within huntingtin (HTT) mRNA selectively inhibit expression of the mutant huntingtin protein. Conjugates targeting the promoter of the progesterone receptor (PR) function as antigene agents to block PR expression. These observations support further investigation of ZNA conjugates as gene silencing agents.

Transcriptome studies have revealed that protein-coding loci within the human genome are overlapped at their 3′-termini by noncoding RNA (ncRNA) transcripts. Small duplex RNAs designed to be fully complementary to these 3′ ncRNAs can modulate transcription of the upstream gene. Robust regulation by designed RNAs suggests that endogenous small RNAs might also recognize 3′ ncRNAs and regulate gene expression. A genome-wide evaluation revealed that sequences immediately downstream of protein-coding genes are enriched with miRNA target sites. We experimentally tested miRNA mimics complementary to the well-characterized 3′-terminus of the human progesterone receptor (PR) gene and observed inhibition of PR transcription. These results suggest that recognition of ncRNA transcripts that overlap gene termini may be a natural function of endogenous small RNAs.

Rett Syndrome is an X-linked progressive neurological disorder caused by inactivation of one allele of the MECP2 gene. There are no curative treatments, and activation of wild-type MECP2 expression is one strategy for stabilizing or reversing the disease. We isolated fibroblast clones that express exclusively either the wild-type or a 32-bp-deletion mutant form of MECP2. We developed a sensitive assay for measuring wild-type MECP2 mRNA levels and tested small molecule epigenetic activators for their ability to activate gene expression. Although our pilot screen did not identify activators of MECP2 expression, it established the value of using clonal cells and defined challenges that must be overcome.

Low-density lipoprotein receptor (LDLR) is a cell-surface receptor that plays a central role in regulating cholesterol levels. Increased levels of LDLR would lead to reduced cholesterol levels and contribute to strategies designed to treat hypercholesterolemia. We have previously shown that duplex RNAs complementary to transcription start sites can associate with noncoding transcripts and activate gene expression. Here we show that duplex RNAs complementary to the promoter of LDLR activate expression of LDLR and increase the display of LDLR on the surface of liver cells. Activation requires complementarity to the LDLR promoter and can be achieved by chemically modified duplex RNAs. Promoter-targeted duplex RNAs can overcome repression of LDLR expression by 25-hydroxycholesterol and do not interfere with activation of LDLR expression by lovastatin. These data demonstrate that small RNAs can activate LDLR expression and affect LDLR function.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (117 K)Download as PowerPoint slideHighlights► Promoter-associated dsRNAs activate LDLR gene expression ► An antisense transcript is expressed at the LDLR promoter ► Activating agRNAs recruit argonaute proteins to the antisense transcript ► Chemical modification of agRNAs is tolerated for LDLR activation

Inhibiting expression of huntingtin (HTT) protein is a promising strategy for treating Huntington's disease (HD), but indiscriminant inhibition of both wild-type and mutant alleles may lead to toxicity. An ideal silencing agent would block expression of mutant HTT while leaving expression of wild-type HTT intact. We observe that fully complementary duplex RNAs targeting the expanded CAG repeat within HTT mRNA block expression of both alleles. Switching the RNAi mechanism toward that used by miRNAs by introducing one or more mismatched bases into these duplex RNAs leads to potent (<10 nM) and highly selective (>30-fold relative to wild-type HTT) inhibition of mutant HTT expression in patient-derived cells. Potent, allele selective inhibition of HTT by mismatched RNAs provides a new option for developing HD therapeutics.Graphical AbstractFigure optionsDownload full-size imageDownload high-quality image (150 K)Download as PowerPoint slideHighlights► Allele-selective inhibition of human HTT expression ► Switching the mechanism of RNAi enhances selectivity ► Selective duplex RNAs are a promising approach to therapy for HD

Huntington’s disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

Double-stranded RNA has become a ubiquitous tool for inhibition of gene expression in the laboratory. If similar success could be achieved in vivo, duplex RNA might provide a new class of therapeutics capable of treating a broad spectrum of disease. Chemists and biologists developing duplex RNA as a drug have made progress but continue to face challenges. This review presents the current status of duplex RNA in the clinic and comments on future prospects for the approach.siRNA: having an impact in clinical trials.

Telomeres are the ends of linear chromosomes. They cannot be fully replicated by standard polymerases and are maintained by the ribonucleoprotein telomerase. Telomeres and telomerase stand at a junction of critical processes underlying chromosome integrity, cancer, and aging, and their importance was recognized by the 2009 Nobel Prize in Physiology or Medicine to Elizabeth Blackburn, Jack Szostak, and Carol Greider. Where will the field go now? What are the prospects for antitelomerase agents as drugs? Nearly 30 years after Szostak and Blackburn's pioneering manuscript on telomere ends, the challenges of discovery remain.

Synthetic small duplex RNAs that are complementary to gene promoters can activate or inhibit target gene expression. The potency and robustness of gene modulation by these RNAs suggests that natural mechanisms may exist to facilitate recognition of sequences within gene promoters by endogenous small RNAs. Here, we describe computational methods for identifying potential miRNA target sites within gene promoters. These methods will facilitate investigations of whether miRNAs interact with sequences outside of 3′-untranslated regions and suggest new targets for the design of synthetic modulators of gene expression.A method for detecting potential microRNA target sites within gene promoters.

Peptide nucleic acid (PNA) is a successful DNA/RNA mimic. A major challenge for research is to invent chemically modified PNAs that retain the favorable properties of the parent compound while improving biological recognition. Here, we test modified PNAs containing [bis-o-(aminoethoxy)phenyl]pyrrolocytosine bases designed to engage guanine with an additional hydrogen bond. We observe elevated melting temperatures, localization to cellular compartments, and allele-selective inhibition of mutant huntingtin protein expression.Modified PNAs enter cells and inhibit expression of huntingtin.

Sequence-selective recognition of DNA inside cells by oligonucleotides would provide valuable insights into cellular processes and new leads for therapeutics. Recent work, however, has shown that noncoding RNA transcripts overlap chromosomal DNA. These RNAs provide alternate targets for oligonucleotides designed to bind promoter DNA, potentially overturning previous assumptions about mechanism. Here, we show that antigene locked nucleic acids (agLNAs) reduce RNA levels of targeted genes, block RNA polymerase and transcription factor association at gene promoters, and bind to chromosomal DNA. These data suggest that the mechanism of LNAs involves recognition of chromosomal DNA and that LNAs are bona fide antigene molecules.

Antigene agents that could recognize specific sequences in chromosomal DNA would have many applications, including specific inhibition or activation of gene expression, introduction or correction of mutations, and analysis of chromosome structure and function1. Relative to recognition of mRNA, recognition of chromosomal DNA may be more potent because there are only two copies of the target, rather than thousands.Despite the exciting applications of chromosomal recognition, there have been few reports of potent effects inside cells. Antigene agents face special challenges relative to current approaches that target mRNA. They must enter the nucleus and locate their target sequence, actions that require them to overcome the barriers posed by chromatin structure and the double helix itself.We have shown that agPNAs2 and agRNAs3 can overcome these barriers and block gene expression. RNA and PNA are chemically different and provide options for exploring how genes are regulated and how DNA functions in living cells. Alternative strategies for recognizing chromosomal DNA include triple-helix formation4, 5 and using synthetic pyrimidine polyamides6, 7.PNA is a neutral DNA mimic that possesses an outstanding ability to recognize sequences in duplex DNA by strand invasion1, 8. PNAs have a peptide-like amide backbone, in which the phosphate backbone of DNA is replaced by (2-aminoethyl)glycine units and the nucleobases are attached through methylene carbonyl linkages (Fig. 1a). Here we describe two methods for introducing agPNAs into cultured mammalian cells.In the first, PNAs are complexed with complementary DNA oligonucleotides and cationic lipid2, 9, 10. The lipid promotes DNA uptake and the PNA is carried into cells as cargo. Once inside, the PNA is released from the DNA, enabling it to find its target. This method is efficient (silencing requires 20–100 nM PNA) and is closely related to standard methods used to transfect antisense oligonucleotides and duplex RNAs.In the second, we use PNA-peptide conjugates that can enter cells without the aid of cationic lipid11. The use of PNA-peptides is advantageous because it is simple and it avoids any potential toxicity and off-target effects associated with use of cationic lipid. A disadvantage is that higher concentrations of PNA-peptide conjugate (1–10 M rather than 20–100 nM) are needed to achieve silencing in cells.Several studies have described delivery of PNA-peptide conjugates into cells11, 12, 13, 14, 15, 16, 17. These studies provide useful experimental details that supplement our protocol and they also describe effective peptide sequences. Taken together, these studies do not suggest that any one sequence is optimal, nor do they clearly recommend attachment of peptide to the amino or carboxy termini. They do suggest that the direct addition of cationic peptides to the termini of PNAs will result in cellular uptake and that it is not necessary to link the PNA to the peptide through a disulfide bond. The efficiency of silencing increases as more lysine or arginine residues are added. Development of PNA conjugates and modified PNAs for cellular uptake is an active area of research and investigators interested in using PNAs should monitor the literature before choosing a PNA-peptide design.Duplex RNA can also mediate recognition of promoter sequences and inhibit gene expression in mammalian cells3, 18, 19, 20, 21, 22, 23, 24, 25. Recognition by agRNAs (here we distinguish between agRNAs, which target DNA and inhibit transcription, and siRNAs, which target mRNA and promote RNA degradation) is potent and provides an alternative to recognition of mRNA by siRNAs. The mechanism of RNA-mediated recognition of DNA is not well understood, but the high potency of silencing suggests that natural cellular pathways facilitate the recognition of chromosomal DNA by RNA.The first challenge in an experiment using agPNAs or agRNAs is to choose a sequence in chromosomal DNA (Fig. 1b). How much is known about the location of a transcription start site? Which sequences are critical? For many genes, transcription start sites either have not been characterized or have been poorly characterized. Even if a transcription start site has been well characterized in one cell line, there is no guarantee that it will be the same in the cell line that an experimenter wishes to use. Potential uncertainty regarding the location of transcription start sites requires consideration of whether the target sequence for an 'antigene' oligomer might be in mRNA. For single-stranded agPNAs, this issue is straightforward: one simply targets the template strand (Fig. 1b). For double-stranded agRNAs, the problem is more complex because the duplex has the inherent ability to target both strands (Fig. 1b). The nuclear run-on assay26 measures transcription and is a useful tool for differentiating between the transcriptional and posttranscriptional silencing pathways.Regardless of whether agPNAs or agRNAs are used, efficient delivery into cells is a chief goal in any method. Confocal microscopy with live cells shows that fluorescently labeled PNA-DNA–lipid complexes, PNA-peptide conjugates (Fig. 2), or duplex RNA–lipid complexes (data not shown) yield a punctate perinuclear staining11. This result is consistent with localization to endosomes15, 16. Only a small percentage of PNA or RNA seems to be available to recognize cellular targets, emphasizing the need for properly executed delivery methods.We find that agPNAs must be introduced into cells by two transfections. By contrast, siRNAs, antisense PNAs or agRNAs need to be transfected only once. The requirement of two transfections and longer incubation times for agPNAs probably reflects the existence of physical barriers that prevent the agPNAs from rapidly recognizing their chromosomal target sequences.Here we describe the design of antigene experiments and a specific protocol for introducing agRNAs and agPNAs into cells. This protocol can also be used to silence gene expression with PNAs or duplex RNAs that are complementary to mRNA.Step 7Ai: 1 hSteps 7Aii to 7Avii: 1–2 hSteps 7Aviii to 7Ax: 1 hSteps 7Axi to 7Axv: 6–8 dSteps 7Bi to 7Bx: 2–4 hSteps 7Ci to 7Ciii: 2–6 hSteps 8–13: 1–2 hStep 14: 16–24 hStep 15: 16–24 hStep 15Ai: 3 d, depends on cell growth ratesStep 15Aii: 4–5 d, depends on cell growth ratesStep 8–13: 1–2 hStep 14: 16–24 hStep 15: 16–24 hSteps 16–17: 2–4 d, depending on desired harvest time and cell growth rateTroubleshooting advice can be found in Table 1We routinely achieve efficient (>75%) silencing with agPNA-peptide conjugates (at 5–8 M) (Fig. 3) or agPNAs (at 100 nM) (Fig. 4). Experimenters should expect variability from one experiment to the next. The protocols for delivering PNAs into cells are relatively complex, and experimentation will be needed to observe an initial positive result and subsequently optimize it. These protocols can be also used to deliver antisense PNAs and PNA-peptide conjugates to silence expression by targeting mRNA.We identified agRNAs that achieve 60–100% inhibition of gene expression when present at a concentration of 25 nM (Fig. 5). Some agRNAs yield almost total inhibition of expression, but the efficiency of silencing varies from one gene to the next. The efficiency of silencing also depends on the target sequence. A one-base shift in a target site can transform an active agRNA into one that is completely inactive. This protocol can be also used to achieve silencing with duplex RNAs that target mRNA.Antigene agents are a powerful option for investigating and silencing gene expression. This protocol offers a framework for the design and execution of experiments targeting chromosomal DNA. Work in this area, however, has only just begun. The landscape of accessibility and function of chromosomal DNA is a frontier for research and it is likely that experiments will lead to exciting discoveries.

Short interfering RNAs (siRNAs) are valuable tools for analyzing protein function in mammalian cell culture. This success has led to high expectations for in vivo and therapeutic applications. However, the pharmacokinetic properties of siRNA are not known. Here we report the biodistribution of a phosphodiester (PO) siRNA duplex and examine the effect of phosphorothioate (PS) linkages. Our findings indicate that biodistribution of siRNA is similar to that for single-stranded antisense oligonucleotides and offer insights for use of siRNA in vivo.Graphic

MicroRNAs (miRNAs) are small noncoding transcripts that regulate gene expression. Aberrant expression of miRNAs can affect development of cancer and other diseases. Synthetic miRNA mimics can modulate gene expression and offer an approach to therapy. Inside cells, mature miRNAs are produced as double-stranded RNAs and miRNA mimics typically retain both strands. This need for two strands has the potential to complicate drug development. Recently, synthetic chemically modified single-stranded silencing RNAs (ss-siRNA) have been shown to function through the RNAi pathway to induce gene silencing in cell culture and animals. Here, we test the hypothesis that single-stranded miRNA (ss-miRNA) can also mimic the function of miRNAs. We show that ss-miRNAs can act as miRNA mimics to silence the expression of target genes. Gene silencing requires expression of argonaute 2 (AGO2) protein and involves recruitment of AGO2 to the target transcripts. Chemically modified ss-miRNAs function effectively inside cells through endogenous RNAi pathways and broaden the options for miRNA-based oligonucleotide drug development.

Expanded trinucleotide repeats cause Huntington's disease (HD) and many other neurodegenerative disorders. There are no cures for these devastating illnesses and treatments are urgently needed. Each trinucleotide repeat disorder is the result of the mutation of just one gene, and agents that block expression of the mutant gene offer a promising option for treatment. Therapies that block expression of both mutant and wild-type alleles can have adverse effects, challenging researchers to develop strategies to lower levels of mutant protein while leaving adequate wild-type protein levels. Here, we review approaches that use synthetic nucleic acids to inhibit expression of trinucleotide repeat genes.