Tamaki Endoh

Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape

RNAi-mediated silencing of specific genes is a promising strategy for gene therapy. To utilize RNAi for therapy, an efficient and safe method for delivery of RNA into the cell cytosol is necessary. The plasma membrane is the primary, and most difficult, barrier for RNA to cross, because negatively charged RNA is strongly repulsed by the negatively charged membrane. A variety of cationic polymers can be used as RNA carriers by interacting with RNA and covering its negative charges to form a cell-penetrating complex. Among the emerging candidates for RNA carriers are cationic cell-penetrating peptides (CPPs), which can cross the plasma membrane and internalize into cells together with RNA. This review focuses on CPP-based RNA delivery strategies. In using CPP-based RNA delivery, most of the RNA internalized by the cell is entrapped in endosomes. Strategies for endosomal escape of RNAs are also reviewed.

Cellular siRNA Delivery Mediated by a Cell-Permeant RNA-Binding Protein and Photoinduced RNA Interference

HIV-1 TAT peptide, which is a cell-penetrating peptide (CPP), was fused to the U1A RNA-binding domain (TatU1A) to generate a sequence-specific siRNA delivery system for mammalian cells. The siRNA contained a short 5-extension that is specifically recognized by the U1A RNA-binding domain (U1AsiRNA). Specific binding of TatU1A to the U1AsiRNA was confirmed using a gel mobility shift assay. The U1AsiRNA was internalized by cells only when it was preincubated with TatU1A before addition to the cells. Although most of the internalized siRNA seemed to be entrapped in endocytic compartments, efficient redistribution of the entrapped siRNAs was achieved by photostimulation of a fluorophore attached to TatU1A. Once in the cytoplasm, the siRNA induced RNAi-mediated gene silencing. We refer to this delivery strategy as CLIP-RNAi. CLIP-RNAi is a promising strategy for RNAi experiments and for pinpoint RNAi therapy.

Spatial regulation of specific gene expression through photoactivation of RNAi

In this study we describe the spatial regulation of RNA interference (RNAi) using an RNA-carrier protein labeled with a fluorescent dye and a light source to trigger the RNAi. We demonstrate photo-dependent gene silencing using several dyes with different excitation wavelengths. Additionally, we use light from a halogen lamp and a photomask to produce photopatterned RNAi, and laser light to trigger single-cell RNAi on cell culture plates.

Carrier PNA for shRNA delivery into cells

A peptide nucleic acid (PNA)-cell-penetrating peptide (CPP) conjugate (carrier PNA) was used as bridge-builder to connect a CPP with an shRNA. The carrier PNA successfully formed a hybrid with an shRNA bearing complementary dangling bases and the shRNA was introduced into cells by the carrier PNA, and RNAi was induced by the shRNA.

Detection of bioactive small molecules by fluorescent resonance energy transfer (FRET) in RNA-protein conjugates.

Bioactive small molecules such as metabolites and drugs play important roles in regulating biological functions. A technique for visualizing such small molecules is very useful to understand their molecular mechanisms. In this study, an RNA-protein conjugate, which consists of an RRE-RNA sensor protein (EYFP-Rev-ECFP) and an altered RRE-RNA, was constructed to detect bioactive small molecules by fluorescent resonance energy transfer (FRET). We designed a theophylline-aptamer-inserted RRE-RNA (Theo-RRE) to detect theophylline as a model target molecule. Theo-RRE formed an RNA-protein conjugate with EYFP-Rev-ECFP in the presence of theophylline. As a result, theophylline was specifically detected down to 10 microM by the FRET increase in distinction from theophylline analogue, caffeine, in cell lysates.

RNA detection using peptide-inserted Renilla luciferase

A novel complementation system with short peptide-inserted-Renilla luciferase (PI-Rluc) and split-RNA probes was constructed for noninvasive RNA detection. The RNA binding peptides HIV-1 Rev and BIV Tat were used as inserted peptides. They display induced fit conformational changes upon binding to specific RNAs and trigger complementation or discomplementation of Rluc. Split-RNA probes were designed to reform the peptide binding site upon hybridization with arbitrarily selected target RNA. This set of recombinant protein and split-RNA probes enabled a high degree of sensitivity in RNA detection. In this study, we show that the Rluc system is comparable to Fluc, but that its detection limit for arbitrarily selected RNA (at least 100xa0pM) exceeds that of Fluc by approximately two orders of magnitude.

Gene Regulation System with an Artificial RNA Switch Operating in Human Cells

Characteristic gene-regulation systems termed riboswitches have been discovered in various organisms. 2] Riboswitches are located primarily in the 5’-untranslated regions (UTRs) of mRNAs and control gene expression through RNA conformational transitions in response to specific binding of target metabolites to riboswitches. Riboswitches could therefore be regarded as gene regulation systems in which only oligonucleotides (RNAs) and target metabolites operate, without any contributions from proteins. These simple systems are suitable for construction of artificial gene regulation systems (artificial riboswitches) in which gene expression is controlled by artificial trigger molecules. Various artificial riboswitches have been constructed by utilization of artificially designed and selected RNAs such as aptamers and allosteric ribozymes or by modification of natural riboswitch systems. 6] However, most reported artificial riboswitches are functional in prokaryotes, fungi and plants, with only a few examples in mammalian cells. In addition, all systems in mammalian cells operate at the post-transcriptional level through mRNA splicing or translation. 8] The mRNA transcription mechanisms in higher eukaryotes, including mammals, are more complex than those in prokaryotes, being controlled by contributions of many functional proteins at the initiation and elongation steps. Because riboswitches function without any contribution from any proteins, it has been considered that they are too simple to control mRNA transcription, especially in mammalian cells. Indeed, the only riboswitch so far found in eukaryotes is a thiamine-pyrophosphate-specific riboswitch that controls mRNA splicing. In addition, there are no examples of natural riboswitches in mammalian cells, whereas various target metabolites and their control operations such as transcription, translation and mRNA editing have been discovered in investigations of prokaryote riboswitches. 3] However, control of mRNA transcription in mammalian cells as a response to small target molecules could contribute to an efficient and useful gene regulation system, because transcription is the first step in gene expression. In this study we demonstrate an artificial gene regulation system capable of controlling gene expression at the transcriptional level in human cells. The mechanism is similar to that of natural riboswitches, in which control of gene expression is triggered by binding of a trigger molecule to a specific RNA sequence. One difference from natural riboswitches is that the system constructed in this study benefits from a cooperative function of a protein to induce transactivation of mRNA transcription. Because proteins display multiple functions in contrast to RNAs, assignation of functions of molecular recognition and signal output to an RNA and a protein, respectively, and association of these functions through an allosteric RNA–protein interaction in response to target molecules facilitates complex output signals. Gene expression under the control of the long terminal repeat (LTR) promoter of human immunodeficiency virus type 1 (HIV-1) is activated by a viral protein: trans-activator of transcription (Tat). Tat binds to trans-activation-responsive RNA (TAR-RNA) located at the 5’ terminus of nascent viral mRNA through its C terminus RNA binding domain (RBD), designated Tat-peptide. The N terminus of Tat contains an activation domain (AD) that interacts with a complex of cyclin T1 and cyclin-dependent kinase 9 (cdk-9) in host cells. Binding of Tat to TAR–RNA recruits the cyclin T1/cdk-9 complex to nascent mRNA and transactivates elongation of transcription through phosphorylation of a C-terminal domain of RNA polymerase II (Figure 1 A). 18] Previous reports demonstrated that this transactivation occurs when RNA–peptide (TAR–RNA and Tat–peptide) interaction is replaced with that of other immunodeficiency viruses or other RNA–protein interactions. 19] In addition, RNA oligonucleotides, which interact with TAR–RNA, through a loop–loop interaction inhibit the interaction between TAR– RNA and Tat-peptide in vitro and suppress gene expression from the HIV-1 LTR promoter in vivo. These observations indicate flexible and designable characteristics of transactivation mechanisms as well as the possibility that Tat-mediated transactivation of transcriptional elongation could be an operating procedure for artificial gene regulation systems. Here we have designed a modified TAR–RNA (thTAR–RNA) containing an RNA sequence of a theophylline-specific aptamer at the loop region of TAR–RNA derived from bovine immunodeficiency virus (BIV; Figure 1 B). The theophylline-specific aptamer is an RNA aptamer selected in vitro that specifically binds to the small bronchodilator theophylline. NMR structural studies revealed that the theophylline-specific aptamer assumes an intricate and compact structure to form a theophylline binding pocket, which enables extensive stacking and hydrogen-bonding interactions with theophylline. In cell studies, insertion of additional RNA sequences into the loop region of BIV TAR–RNA permitted transactivation of mRNA [a] Prof. Dr. N. Sugimoto Faculty of Frontiers of Innovative Research in Science and Technology (FIRST) Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University 7-1-20 Minatojima-minamimachi, Kobe 650-0047 (Japan) Fax: (+ 81) 78-303-1495 E-mail : sugimoto@konan-u.ac.jp [b] Dr. T. Endoh Frontier Institute for Biomolecular Engineering Research (FIBER) Konan University 7-1-20 Minatojima-minamimachi, Kobe 650-0047 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100093.

Cellular siRNA Delivery Using TatU1A and Photo-Induced RNA Interference

RNA interference (RNAi)-mediated silencing of specific genes represents a powerful tool for analyzing protein function. It also has profound biotechnological applications for cellular engineering and therapeutics. However, it is necessary to have a method that controls RNAi in response to artificially regulated stimulation. We designed a fluorescently labeled carrier protein to deliver short hairpin RNA (shRNA) with activity that could be regulated via photostimulation. We constructed a cell-permeable RNA-binding protein (RBP) by fusing the U1A RBP and a HIV-1 Tat peptide, which was labeled with an Alexa Fluor 546 fluorophore (TatU1A-Alexa). TatU1A-Alexa bound specifically to shRNA, which contains a U1A-binding sequence. The TatU1A-Alexa/shRNA complex was then internalized into cells via an endocytotic pathway and redistributed from endosomes to the cytosol by photostimulation, which induced RNAi-mediated gene silencing. This successive strategy was termed CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi).

Evaluation of small ligand-protein interactions by using T7 RNA polymerase with DNA-modified ligand.

The interaction between proteins and ligands was evaluated by T7 RNA polymerase transcription with a DNA-modified ligand. The principle of this method is suppression of T7 RNA polymerase transcription by binding of a protein to small ligand modified by conjugation with a T7 RNA polymerase promoter. To demonstrate proof of principle, biotin or antifolate methotrexate was modified by covalent attachment of a T7 RNA promoter. Using these T7 RNA promoter-modified ligands, T7 RNA polymerase transcriptions were performed in the presence or absence of an anti-biotin antibody or recombinant human dihydrofolate reductase, respectively. Transcription was suppressed in the presence of each binding protein plus its modified ligand, but not in the absence of the binding protein.

Improvement of CPP-RBD for siRNA Delivery

Small interfering RNA (siRNA) causes sequence-specific gene silencing. In this study, siRNAs were induced into the cells by using a cell-penetrating peptide (CPP) and RNA-binding protein (RBP) as a safe and effective siRNA carrier (CPP-RBP). The diffusion of the siRNAs into the cytoplasm and RNAi mediated gene silencing were induced by photostimulation of Alexa546, which was connected to the CPP-RBP. Here, we investigated the effects of several CPP-RBPs for effective inducing the RNAi.