Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury

We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

Establishment and Verification of An Efficient Virus-induced Gene Silencing System in Forsythia

To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS(Forsythia phytoene desaturase)using stem infiltration and leaf infiltrationmethods.All the treated plants were pruned below the injection site after 7–15 d infection;the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level.Meanwhile,this system has been successfully tested and verified through virus detection and qRT-PCR analysis.After the optimization,Forsythia magnesium chelatase subunit H(FsChlH)was silenced successfully in Forsythia using this system,resulting in yellow leaveswith decreased chlorophyll content.The system was stable,highly efficient and had greater rapidity and convenience,which made it suitable to study the function of genes related to physiological pathways such as growth and development,and metabolic regulation in Forsythia.

Aquaporin-4 gene silencing protects injured neurons after early cerebral infarction

Aquaporin-4 regulates water molecule channels and is important in tissue regulation and water transportation in the brain. Upregulation of aquaporin-4 expression is closely related to cellular edema after early cerebral infarction. Cellular edema and aquaporin-4 expression can be determined by measuring cerebral infarct area and apparent diffusion coefficient using diffusion-weighted imaging（DWI）. We examined the effects of silencing aquaporin-4 on cerebral infarction. Rat models of cerebral infarction were established by occlusion of the right middle cerebral artery and si RNA-aquaporin-4 was immediately injected via the right basal ganglia. In control animals, the area of high signal intensity and relative apparent diffusion coefficient value on T2-weighted imaging（T2WI） and DWI gradually increased within 0.5–6 hours after cerebral infarction. After aquaporin-4 gene silencing, the area of high signal intensity on T2 WI and DWI reduced, relative apparent diffusion coefficient value was increased, and cellular edema was obviously alleviated. At 6 hours after cerebral infarction, the apparent diffusion coefficient value was similar between treatment and model groups, but angioedema was still obvious in the treatment group. These results indicate that aquaporin-4 gene silencing can effectively relieve cellular edema after early cerebral infarction; and when conducted accurately and on time, the diffusion coefficient value and the area of high signal intensity on T2 WI and DWI can reflect therapeutic effects of aquaporin-4 gene silencing on cellular edema.

Piwi like RNA-mediated gene silencing 1 gene as a possible major player in gastric cancer

AIM To establish a permanent piwi like RNA-mediated genesilencing 1(PIWIL1) gene knockout in AGP01 gastric cancer cell line using CRISPR-Cas9 system and analyze phenotypic modifications as well as gene expression alterations.METHODS CRISPR-Cas9 system used was purchased from Dharmacon GE Life Sciences(Lafayette, CO, United States) and permanent knockout was performed according to manufacturer's recommendations. Woundhealing assay was performed to investigate the effect of PIWIL1 knockout on migration capability of cells and Boyden chamber invasion assay was performed to investigate the effect on invasion capability. For the gene expression analysis, a one-color microarray-based gene expression analysis kit(Agilent Technologies, Santa Clara, CA, United States) was used according to the protocol provided by the manufacturer. RESULTS PIWIL1 gene knockout caused a significant decrease in AGP01 migration capacity as well as a significant decrease in cell invasiveness. Moreover, functional analysis based on grouping of all differentially expressed m RNAs identified a total of 35 genes(5 up-regulated and 30 down-regulated) encoding proteins involved in cellular invasion and migration. According to current literature, 9 of these 35 genes(DOCK2, ZNF503, PDE4 D, ABL1, ABL2, LPAR1, SMAD2, WASF3 and DACH1) are possibly related to the mechanisms used by PIWIL1 to promote carcinogenic effects related to migration and invasion, since their functions are consistent with the changes observed(being up-or down-regulated after knockout). CONCLUSION Taken together, these data reinforce the idea that PIWIL1 plays a crucial role in the signaling pathway of gastric cancer, regulating several genes involved in migration and invasion processes; therefore, its use as a therapeutic target may generate promising results in the treatment of gastric cancer.

Effects of Transparent Testa8(TT8) gene and Homeobox12(HB12) gene silencing in alfalfa(Medicago sativa L.) on molecular structure spectral profile in relation to energy,degradation,and fermentation characteristics in ruminant systems

Alfalfa(Medicago sativa L.) is a legume forage that is widely cultivated owing to its high biomass yield and favorable nutrient values. However, alfalfa contains relatively high lignin, which limits its utilization.Downregulation of two transcriptional factors, Transparent Testa8(TT8) and Homeobox12(HB12), has been proposed to reduce lignin content in alfalfa. Therefore, silencing of TT8(TT8i) and HB12(HB12i) in alfalfa was achieved by RNAi technology. The objective of this project was to determine effect of gene modification through silencing of TT8 and HB12 genes in alfalfa plants on lignin and phenolic content,bioenergic value, nutrient supply from rumen degradable and undegradable fractions, and in vitro ammonia production in response to the silencing of TT8 and HB12 genes in alfalfa. All gene silenced alfalfa plants(5 TT8i and 11 HB12i) were grown under greenhouse conditions with wild type as a control.Samples were analyzed for bioactive compounds, degradation fractions, truly digestible nutrients, energetic values and in vitro ammonia productions in ruminant systems. Furthermore, relationships between physiochemical, metabolic and fermentation characteristics and molecular spectral parameters were determined using vibrational molecular spectroscopy. Results showed that the HB12i had higher lignin, while TT8i had higher phenolics. Both silenced genotypes had higher rumen slowly degraded carbohydrate fractions and truly digestible neutral detergent fiber, but lower rumen degradable protein fractions. Moreover, the HB12i had lower truly digestible crude protein, energetic values and ammonia production compared with other silenced genotypes. In addition, in relation to the nutritive values of alfalfa, structural carbohydrate parameters were negatively correlated, whereas alpha/beta ratio in protein structure was positively correlated. Furthermore, good predictions were obtained for degradation of protein and carbohydrate fractions and energy values from molecular spectral parameters. In conclusion, silencing of the TT8 and HB12 genes decreased protein availability and increased fiber availability. Silencing of the HB12 gene also increased lignin and decreased energy and rumen ammonia production. Moreover, nutritional alterations were closely correlated with molecular spectral parameters. Therefore, gene modification through silencing the TT8 and HB12 genes in alfalfa influenced physiochemical, metabolic and fermentation characteristics.

The novel C5 protein from tomato yellow leaf curl virus is a virulence factor and suppressor of gene silencing

Tomato yellow leaf curl virus(TYLCV)is known to encode 6 canonical viral proteins.Our recent study revealed that TYLCV also encodes some additional small proteins with potential virulence functions.The fifth ORF of TYLCV in the complementary sense,which we name C5,is evolutionarily conserved,but little is known about its expression and function during viral infection.Here,we confirmed the expression of the TYLCV C5 by analyzing the promoter activity of its upstream sequences and by detecting the C5 protein in infected cells by using a specific custom-made antibody.Ectopic expression of C5 using a potato virus X(PVX)vector resulted in severe mosaic symptoms and higher virus accumulation levels followed by a burst of reactive oxygen species(ROS)in Nicotiana benthamiana plants.C5 was able to effectively suppress local and systemic post-transcriptional gene silencing(PTGS)induced by single-stranded GFP but not double-stranded GFP,and reversed the transcriptional gene silencing(TGS)of GFP.Furthermore,the mutation of C5 in TYLCV inhibited viral replication and the development of disease symptoms in infected plants.Transgenic overexpression of C5 could complement the virulence of a TYLCV infectious clone encoding a dysfunctional C5.Collectively,this study reveals that TYLCV C5 is a pathogenicity determinant and RNA silencing suppressor,hence expanding our knowledge of the functional repertoire of the TYLCV proteome.

Transgenerational Inheritance and Resetting of Stress, Induced Loss of Epigenetic Gene Silencing in Arabidopsis

Plants, as sessile organisms, need to sense and adapt to heterogeneous environments and have developed sophisticated responses by changing their cellular physiology, gene regulation, and genome stability. Recent work dem- onstrated heritable stress effects on the control of genome stability in plants--a phenomenon that was suggested to be of epigenetic nature. Here, we show that temperature and UV-B stress cause immediate and heritable changes in the epi- genetic control of a silent reporter gene in Arabidopsis. This stress-mediated release of gene silencing correlated with pronounced alterations in histone occupancy and in histone H3 acetylation but did not involve adjustments in DNA meth- ylation. We observed transmission of stress effects on reporter gene silencing to non-stressed progeny, but this effect was restricted to areas consisting of a small number of cells and limited to a few non-stressed progeny generations. Further- more, stress-induced release of gene silencing was antagonized and reset during seed aging. The transient nature of this phenomenon highlights the ability of plants to restrict stress-induced relaxation of epigenetic control mechanisms, which likely contributes to safeguarding genome integrity.

Graft-accelerated virus-induced gene silencing facilitates functional genomics in rose flowers

Rose has emerged as a model ornamental plant for studies of flower development, senescence, and morphology, as well as the metabolism of floral fragrances and colors.Virus-induced gene silencing(VIGS) has long been used in functional genomics studies of rose by vacuum infiltration of cuttings or seedlings with an Agrobacterium suspension carrying TRV-derived vectors. However, VIGS in rose flowers remains a challenge because of its low efficiency and long time to establish silencing. Here we present a novel and rapid VIGS method that can be used to analyze gene function in rose,called ‘graft-accelerated VIGS’, where axil ary sprouts are cut from the rose plant and vacuum infiltrated with Agrobacterium. The inoculated scions are then grafted back onto the plants to flower and silencing phenotypes can be observed within 5 weeks, post-infiltration. Using this new method, we successfully silenced expression of the RhDFR, RhA G, and RhNUDXin rose flowers, and affected their color, petal number, as well as fragrance, respectively. This grafting method will facilitate high-throughput functional analysis of genes in rose flowers. Importantly, it may also be applied to other woody species that are not currently amenable to VIGS by conventional leaf or plantlet/seedling infiltration methods.

Virus-induced Gene Silencing in Eggplant(Solanum melongena)

Eggplant （Solanum melongena） is an economically important vegetable requiring investigation into its various genomic functions. The current limitation in the investigation of genomic function in eggplant is the lack of effective tools available for conducting functional assays. Virus-induced gene silencing （VIGS） has played a critical role in the functional genetic analyses. In this paper, TRV-mediated VIGS was successfully elicited in eggplant. We first cloned the CDS sequence of PDS （PHYTOENE DESATURASE） in eggplant and then silenced the PDS gene. Photo-bleaching was shown on the newly-developed leaves four weeks after agroinoculation, indicating that VIGS can be used to silence genes in eggplant. To further illustrate the reliability of VIGS in eggplant, we selected Chl H, Su and CLA1 as reporters to elicit VIGS using the high-pressure spray method. Suppression of Chl H and Su led to yellow leaves, while the depletion of CLA1 resulted in albino. In conclusion, four genes, PDS, Chl H, Su （Sulfur）, CLA1, were down-regulated significantly by VIGS, indicating that the VIGS system can be successfully applied in eggplant and is a reliable tool for the study of gene function.

Efficiency for Gene Silencing Induction in Nicotiana Species by a Viral Satellite DNA Vector

Virus-induced gene silencing （VIGS） is a useful technique for rapid plant gene function analysis. We recently reported a new VIGS vector modified from Tomato yellow leaf curl China virus （TYLCCNV） DNAβ （DNAm β）. In this study we compared in detail DNAmβ-induced gene silencing in four Nicotiana species including N. benthamiana, N. glutinosa, N. tabacum and N. paniculata. We found that DNAmβ-induced gene silencing in the four species was distinct in developing dynamics, tissue specificity, efficiency, and constancy in the plant life span. It was most efficient in N. benthamiana, where development of VIGS was most rapid, without tissue specificity and nearly 100% efficient. DNAmβ-induced gene silencing in N. glutinosa was also efficient despite being slightly less than in N. benthamiana. It initially occurred in veins, later was scattered to mesophyll, finally led to complete silencing in whole leaves. In both species, VIGS constantly expressed until the plants died. However, DNAmβ-mediated VIGS in the other two Nicotiana species, N. tabacum and N. paniculata, was significantly less efficient. It was strictly limited within the veins of the silenced leaves, and constantly occurred only over 3-4 weeks. The upper leaves that emerged later stopped showing the silencing phenotype. DNAmβ-induced gene silencing in N. benthamiana and N. glutinosa was not significantly influenced by the growth stage when the plants were agro-inoculated, and was not sensitive to high growth temperature up to 32℃. Our results indicate that this system has great potential as a versatile VIGS system for routine functional analysis of genes in some Nicotiana species.

Post-transcriptional Gene Silencing Induced by Short Interfering RNAs in Cultured Transgenic Plant Cells

Short interfering RNA (siRNA) is widely used for studyingpost-transcriptional gene silencing and holds great promise as a tool for both identifying functionof novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which weredesigned against different specific areas of coding region of the same target green fluorescentprotein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice(Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Eraser fir [Abies fraseri (Pursh) Poir;AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in thebombarded transgenic cells between two siRNAs, and these results were consistent with theinactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis intested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be thesiRNA specific in different plant species. These results indicate that siRNA is a highly specifictool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could bea reliable approach for large-scale screening of gene function and drug target validation.

Small RNAs,RNAi and the Inheritance of Gene Silencing in Caenorhabditis elegans

Invasive nucleic acids such as transposons and viruses usually exhibit aberrant characteristics,e.g.,unpaired DNA or abnormal doublestranded RNA.Organisms employ a variety of strategies to defend themselves by distinguishing self and nonself substances and disabling these invasive nucleic acids.Furthermore,they have developed ways to remember this exposure to invaders and transmit the experience to their descendants.The mechanism underlying this inheritance has remained elusive.Recent research has shed light on the initiation and maintenance of RNA-mediated inherited gene silencing.Small regulatory RNAs play a variety of crucial roles in organisms,including gene regulation,developmental timing,antiviral defense,and genome integrity,via a process termed as RNA interference（RNAi）.Recent research has revealed that small RNAs and the RNAi machinery are engaged in establishing and promoting transgenerational gene silencing.Small RNAs direct the RNAi and chromatin modification machinery to the cognate nucleic acids to regulate gene expression and epigenetic alterations.Notably,these acquired small RNAs and epigenetic changes persist and are transmitted from parents to offspring for multiple generations.Thus,RNAi is a vital determinant of the inheritance of gene silencing and acts as a driving force of evolution.

Targeted Silencing of Heparanase Gene by Small Interfering RNA Inhibits Invasiveness and Metastasis of Osteosarcoma Cells

The effects of targeted silencing of heparanase gene by small interfering RNA（siRNA） on invasiveness and metastasis of osteosarcoma cells（MG63 cells） were investigated in the present study.Two complementary oligonucleotide strands were synthesized and inserted into pGenesil-1 vector based on the mRNA sequence of heparanase gene.The expression vector containing short hairpin RNA（pGenesil-shRNA） was constructed successfully.MG63 cells were randomly allocated into 3 groups：blank group,empty vector（pGenesil） transfected group and expression vector（pGenesil-shRNA） transfected group.Under the induction of Lipofectamine 2000,the recombinants were transfected into MG63 cells.Heparanase gene expression level was detected by RT-PCR and Western blotting.Cell prolifera-tion was measured by MTT assay.Cell invasiveness and metastasis were examined by cell adhesion and Transwell-ECM assays.HUVECs migration assay was applied for the detection of angiogenesis.As compared with negative controls,the mRNA and protein expression levels of heparanase were down-regulated by 76.1%（P0.01） and 75.3%（P0.01） respectively in the pGenesil-shRNA transfected group.Meanwhile,the proliferation,adhesiveness,invasiveness and angiogenesis properties of MG63 cells were all significantly inhibited.It was suggested that targeted silencing of heparanase gene by siRNA could dramatically inhibit the invasiveness and metastasis of osteosarcoma cells.

Virus-Induced Gene Silencing in the Culinary Ginger （Zingiber officinale）： An Effective Mechanism for Down-Regulating Gene Expression in Tropical Monocots

Virus-induced gene silencing （VIGS） has been shown to be effective for transient knockdown of gene expression in plants to analyze the effects of specific genes in development and stress-related responses. VlGS is well established for studies of model systems and crops within the Solanaceae, Brassicaceae, Leguminaceae, and Poaceae, but only recently has been applied to plants residing outside these families. Here, we have demonstrated that barley stripe mosaic virus （BSMV） can infect two species within the Zingiberaceae, and that BSMV-VIGS can be applied to specifically down-regulate phytoene desaturase in the culinary ginger Zingiber officinale. These results suggest that extension of BSMV-VlGS to monocots other than cereals has the potential for directed genetic analyses of many important temperate and tropical crop species.

Molecular engineering of dendrimer nanovectors for siRNA delivery and gene silencing

Small interfering RNA （siRNA） therapeutics hold great promise to treat a variety of diseases, as long as they can be delivered safely and effectively into cells. Dendrimers are appealing vectors for siRNA delivery by virtue of their well-defined molecular architecture and multivalent cooperativity. However, the clinical translation of RNA therapeutics mediated by dendrimer delivery is hampered by the lack of dendrimers that are of high quality to meet good manufacturing practice standard. In this context, we have developed small amphiphilic dendrimers that self-assemble into supramolecular structures, which mimic high-generation dendrimers synthesized with cova- lent construction, yet are easy to produce in large amount and superior quality. Indeed, the concept of supramolecular dendrimers has proved to be very promising, and has opened up a new avenue for dendrimer-mediated siRNA delivery. A series of self-assembling supramolecular dendrimers have consequently been established, some of them out-performing the currently available nonviral vectors in delivering siRNA to various cell types in vitro and in vivo, including human primary cells and stem cells. This short review presents a brief introduction to RNAi therapeutics, the obstacles to their delivery and the advantages of dendrimer delivery vectors as well as our bio-inspired struc^rally flexible dendrimers for siRNA delivery. We then highlight our efforts in creating self- assembling amphiphilic dendrimers to construct supramo- lecular dendrimer nanosystems for effective siRNA delivery as well as the related structural alterations to enhance delivery efficiency. The advent of self-assembling supramolecular dendrimer nanovectors holds great pro- mise and heralds a new era of dendrimer-mediated delivery of RNA therapeutics in biomedical applications.

Silencing hepatitis B virus covalently closed circular DNA: The potential of an epigenetic therapy approach

Global prophylactic vaccination programmes have helped to curb new hepatitis B virus(HBV)infections.However,it is estimated that nearly 300 million people are chronically infected and have a high risk of developing hepatocellular carcinoma.As such,HBV remains a serious health priority and the development of novel curative therapeutics is urgently needed.Chronic HBV infection has been attributed to the persistence of the covalently closed circular DNA(cccDNA)which establishes itself as a minichromosome in the nucleus of hepatocytes.As the viral transcription intermediate,the cccDNA is responsible for producing new virions and perpetuating infection.HBV is dependent on various host factors for cccDNA formation and the minichromosome is amenable to epigenetic modifications.Two HBV proteins,X(HBx)and core(HBc)promote viral replication by modulating the cccDNA epigenome and regulating host cell responses.This includes viral and host gene expression,chromatin remodeling,DNA methylation,the antiviral immune response,apoptosis,and ubiquitination.Elimination of the cccDNA minichromosome would result in a sterilizing cure;however,this may be difficult to achieve.Epigenetic therapies could permanently silence the cccDNA minichromosome and promote a functional cure.This review explores the cccDNA epigenome,how host and viral factors influence transcription,and the recent epigenetic therapies and epigenome engineering approaches that have been described.

Nucleotide bias of DCL and AGO in plant anti-virus gene silencing

Plant Dicer-like(DCL)and Argonaute(AGO)are the key enzymes involved in anti-virus post-transcriptional gene silencing(AV-PTGS).Here we show that AV-PTGS exhibited nucleotide preference by calculating a relative AV-PTGS efficiency on processing viral RNA substrates.In comparison with genome sequences of dicot-infecting Turnip mosaic virus(TuMV)and monocot-infecting Cocksfoot streak virus(CSV),viral-derived small interfering RNAs(vsiRNAs)displayed positive correlations between AV-PTGS efficiency and G+C content(GC%).Further investigations on nucleotide contents revealed that the vsiRNA populations had G-biases.This finding was further supported by our analyses of previously reported vsiRNA populations in diverse plant-virus associations,and AGO associated Arabidopsis endogenous siRNA populations,indicating that plant AGOs operated with G-preference.We further propose a hypothesis that AV-PTGS imposes selection pressure(s)on the evolution of plant viruses.This hypothesis was supported when potyvirus genomes were analysed for evidence of GC elimination,suggesting that plant virus evolution to have low GC%genomes would have a unique function,which is to reduce the host AV-PTGS attack during infections.

The Splicing Factor PRP31 Is Involved in Transcriptional Gene Silencing and Stress Response in Arabidopsis

Although DNA methylation is known to play an important role in the silencing of transposable elements （TEs） and introduced transgenes, the mechanisms that generate DNA methylation-independent transcrip- tional silencing are poorly understood. Previous studies suggest that RNA-directed DNA methylation （RdDM） is required for the silencing of the RD29A-LUC transgene in the Arabidopsis rosl mutant back- ground with defective DNA demethylase. Loss of function of ARGONAUTE 4 （AGO4） gene, which encodes a core RdDM component, partially released the silencing of RD29A-LUC in the rosl/ago4 double mutant plants. A forward genetic screen was performed to identify the mutants with elevated RD29A-LUC trans- gene expression in the rosl/ago4 mutant background. We identified a mutation in the homologous gene of PRP31, which encodes a conserved pre-mRNA splicing factor that regulates the formation of the U4/ U6.U5 snRNP complex in fungi and animals. We previously demonstrated that the splicing factors ZOP1 and STA1 contribute to transcriptional gene silencing. Here, we reveal that Arabidopsis PRP31 associates with ZOP1, STA1, and several other splicing-related proteins, suggesting that these splicing factors are both physically and functionally connected. We show that Arabidopsis PRP31 participates in transcrip- tional gene silencing. Moreover, we report that PRP31, STA1, and ZOP1 are required for development and stress response. Under cold stress, PRP31 is not only necessary for pre-mRNA splicing but also for regulation of cold-responsive gene expression. Our results suggest that the splicing machinery has multiple functions including pre-mRNA splicing, gene regulation, transcriptional gene silencing, and stress response.

Visualized and cascade-enhanced gene silencing by smart DNAzyme-graphene nanocomplex

BCL-2 gene as well as its products is recognized as a promising target for the molecular targeted therapy of tumors.However,due to certain defense measures of tumor cells,the therapeutic effect based on the gene silencing of BCL-2 is greatly reduced.Here we fabricate a smart response nucleic acid therapeutic that could silence the gene effectively through a dual-targeted and cascade-enhanced strategy.In brief,nano-graphene oxide(GO),working as a nano-carrier,is loaded with a well-designed DNAzyme,which can target and silence the BCL-2 mRNA.Furthermore,upon binding with the BCL-2 mRNA,the enzymatic activity of the DNAzyme can be initiated,cutting a substrate oligonucleotide to produce an anti-nucleolin aptamer AS1411.Nucleolin,a nucleolar phosphoprotein,is known as a stabilizer of BCL-2 mRNA.Via binding and inactivating the nucleolin,AS1411 can destabilize BCL-2 mRNA.By this means of simultaneously targeting mRNA and its stabilizer in an integrated system,effective silencing of the BCL-2 gene of tumor cells is achieved at both the cellular and in vivo levels.After being dosed with this nucleic acid therapeutic and without any chemotherapeutics,apoptosis of tumor cells at the cellular level and apparent shrinkage of tumors in vivo are observed.By labeling a molecular beacon on the substrate of DNAzyme,visualization of the enzymatic activity as well as the tumor in vivo can be also achieved.Our work presents a pure bio-therapeutic strategy that has positive implications for enhancing tumor treatment and avoiding side effects of chemotherapeutics.

Y-Shaped Circular Aptamer–DNAzyme Conjugates for Highly Efficient in Vivo Gene Silencing

Oligonucleotide drugs have been used widely as therapeutic agents for gene therapy,while their instability in biological media and inefficiency for intracellular delivery remain major hurdles for practical in vivo applications.Herein,we report a circular Y-shaped aptamer–DNAzyme conjugate(cYAD)for highly efficient in vivo gene silencing via RNA cleavage,which can been employed in various disease treatments,including cancer,inflammation,as well as viral infections.Systematic studies revealed that cyclization of the DNA structure could improve the stability of oligonucleotide drugs in vivo.Besides,the bivalent aptamer motifs provided a specific and enhanced tumor cell targeting ability for accumulation and retention of the oligonucleotide drugs at the tumor site.As a proof of concept,a widely applicable Na+-dependent fluorescent sensor,NaA43 DNAzyme,was used to inhibit MET gene expression in mice tumor model tissues,which exhibited highly efficient gene silencing performance in vivo,which confirmed our findings with cYAD.This strategy provides a novel approach for the construction of oligonucleotide drugs for practical therapeutic applications.