Autonomous Generation and Loading of DNA Guides by Bacterial Argonaute.

Several prokaryotic Argonaute proteins (pAgos) utilize small DNA guides to mediate host defense by targeting invading DNA complementary to the DNA guide. It is unknown how these DNA guides are being generated and loaded onto pAgo. Here, we demonstrate that guide-free Argonaute from Thermus thermophilus (TtAgo) can degrade double-stranded DNA (dsDNA), thereby generating small dsDNA fragments that subsequently are loaded onto TtAgo. Combining single-molecule fluorescence, molecular dynamic simulations, and structural studies, we show that TtAgo loads dsDNA molecules with a preference toward a deoxyguanosine on the passenger strand at the position opposite to the 5' end of the guide strand. This explains why in vivo TtAgo is preferentially loaded with guides with a 5' end deoxycytidine. Our data demonstrate that TtAgo can independently generate and selectively load functional DNA guides.

Synthesis and application of stationary phase for DNA-affinity chromatographic analysis of unmodified and antisense oligonucleotide.

The goal of the research was the synthesis and application of an oligonucleotide immobilized stationary phase for the analysis of unmodified and antisense oligonucleotides. The method for attaching these molecules to aminopropyl silica modified with pentanedioic acid was developed. Each step of the synthesis was carefully controlled with the application of spectroscopic, elemental, and chromatographic analyses. The oligonucleotide-based stationary phase was applied for the retention studies. Unmodified oligonucleotides of different complementarity to the molecule attached as a stationary phase, as well as antisense oligonucleotides, were tested. The comparative study upon complex optimization of oligonucleotide analysis in different liquid chromatography modes was performed. Results have shown that this stationary phase may be applied for oligonucleotide analysis in hydrophilic interaction liquid chromatography and ion exchange chromatography, but no unique sequence-based selectivity was obtained. Contrary results were observed for affinity chromatography, which allowed for specific separation of the complementary strands based on hydrogen bonding and stacking interactions, where the temperature was the main factor influencing the selectivity of the separation. Furthermore, the oligonucleotide-based stationary phase may be applied for comparative antisense oligonucleotide hybridization studies to a specific RNA sequence. All of the results have shown that affinity chromatography with oligonucleotide-based stationary phases is a powerful technique for the specific base recognition of polynucleotides.

Enhanced Performance of DNA Methylation Markers by Simultaneous Measurement of Sense and Antisense DNA Strands after Cytosine Conversion.

BACKGROUND: Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. METHODS: Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)-specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. RESULTS: Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. CONCLUSION: Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.

RNA polymerase II collision interrupts convergent transcription.

Antisense noncoding transcripts, genes-within-genes, and convergent gene pairs are prevalent among eukaryotes. The existence of such transcription units raises the question of what happens when RNA polymerase II (RNAPII) molecules collide head-to-head. Here we use a combination of biochemical and genetic approaches in yeast to show that polymerases transcribing opposite DNA strands cannot bypass each other. RNAPII stops but does not dissociate upon head-to-head collision in vitro, suggesting that opposing polymerases represent insurmountable obstacles for each other. Head-to-head collision in vivo also results in RNAPII stopping, and removal of collided RNAPII from the DNA template can be achieved via ubiquitylation-directed proteolysis. Indeed, in cells lacking efficient RNAPII polyubiquitylation, the half-life of collided polymerases increases, so that they can be detected between convergent genes. These results provide insight into fundamental mechanisms of gene traffic control and point to an unexplored effect of antisense transcription on gene regulation via polymerase collision.

Intracellular Delivery of Antisense DNA and siRNA with Amino Groups Masked with Disulfide Units.

Efficient methods for delivery of antisense DNA or small interfering RNA (siRNA) are highly needed. Cationic materials, which are conventionally used for anionic oligonucleotide delivery, have several drawbacks, including aggregate formation, cytotoxicity and a low endosome escape efficiency. In this report a bio-reactive mask (i.e., disulfide unit) for cationic amino groups was introduced, and the mask was designed such that it was removed at the target cell surface. Insolubility and severe cellular toxicity caused by exposed cationic groups are avoided when using the mask. Moreover, the disulfide unit used to mask the cationic group enabled direct delivery of oligonucleotides to the cell cytosol. The molecular design reported is a promising approach for therapeutic applications.

Inhibiting Methicillin-Resistant Staphylococcus aureus by Tetrahedral DNA Nanostructure-Enabled Antisense Peptide Nucleic Acid Delivery.

One of the biggest obstacles for the use of antisense oligonucleotides as antibacterial therapeutics is their limited uptake by bacterial cells without a suitable carrier, especially in multi-drug-resistant bacteria with a drug efflux mechanism. Existing vectors, such as cell-penetrating peptides, are inefficient and nontargeting, and accordingly are not ideal carriers. A noncytotoxic tetrahedral DNA nanostructure (TDN) with a controllable conformation has been developed as a delivery vehicle for antisense oligonucleotides. In this study, antisense peptide nucleic acids (asPNAs) targeting a specific gene ( ftsZ) were efficiently transported into methicillin-resistant Staphylococcus aureus cells by TDNs, and the expression of ftsZ was successfully inhibited in an asPNA-concentration-dependent manner. The delivery system specifically targeted the intended gene. This novel delivery system provides a better platform for future applications of antisense antibacterial therapeutics and provides a basis for the development of a new type of antibacterial drug for multi-drug-resistant bacterial infections.

Real-time imaging of VCAM-1 mRNA in TNF-α activated retinal microvascular endothelial cells using antisense hairpin-DNA functionalized gold nanoparticles.

Vascular cell adhesion molecule 1 (VCAM-1) is an important inflammatory biomarker correlating with retinal disease progression. Thus, detection of VCAM-1 mRNA expression levels at an early disease stage could be an important predictive biomarker to assess the risk of disease progression and monitoring treatment response. We have developed VCAM-1 targeted antisense hairpin DNA-functionalized gold nanoparticles (AS-VCAM-1 hAuNP) for the real time detection of VCAM-1 mRNA expression levels in retinal endothelial cells. The AS-VCAM-1 hAuNP fluorescence enhancement clearly visualized the TNF-α induced cellular VCAM-1 mRNA levels with high signal to noise ratios compared to normal serum treated cells. The scrambled hAuNP probes were minimally detectable under same image acquisition conditions. Intracellular hAuNPs were detected using transmission electron microscopy (TEM) analysis of the intact cells. In addition, the AS-VCAM-1 hAuNP probes exhibited no acute toxicity to the retinal microvascular endothelial cells as measured by live-dead assay.

Facile functionalization of peptide nucleic acids (PNAs) for antisense and single nucleotide polymorphism detection.

In this report, we show how a convenient on-resin copper-click functionalization of azido-functionalized peptide nucleic acids (PNAs) allows various PNA-based detection strategies. Firstly, a thiazole orange (TO) clicked PNA probe facilitates a binary readout when combined with F/Q labeled DNA, giving increased sensitivity for antisense detection. Secondly, our TO-PNA conjugate also allows single nucleotide polymorphism detection. Since antisense detection is also possible in the absence of the TO label, our sensing platform based on azido-d-ornithine containing PNA even allows for additional and more advanced functionalization and sensing strategies.

NanoFlares for the detection, isolation, and culture of live tumor cells from human blood.

Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood­so-called circulating tumor cells (CTCs)­may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.

Effective Antisense Gene Regulation via Noncationic, Polyethylene Glycol Brushes.

Negatively charged nucleic acids are often complexed with polycationic transfection agents before delivery. Herein, we demonstrate that a noncationic, biocompatible polymer, polyethylene glycol, can be used as a transfection vector by forming a brush polymer-DNA conjugate. The brush architecture provides embedded DNA strands with enhanced nuclease stability and improved cell uptake. Because of the biologically benign nature of the polymer component, no cytotoxicity was observed. This approach has the potential to address several long-lasting challenges in oligonucleotide therapeutics.

Aptamer and Antisense-Mediated Two-Dimensional Isolation of Specific Cancer Cell Subpopulations.

Cancer cells, and in particular those found circulating in blood, can have widely varying phenotypes and molecular profiles despite a common origin. New methods are needed that can deconvolute the heterogeneity of cancer cells and sort small numbers of cells to aid in the characterization of cancer cell subpopulations. Here, we describe a new molecular approach to capturing cancer cells that isolates subpopulations using two-dimensional sorting. Using aptamer-mediated capture and antisense-triggered release, the new strategy sorts cells according to levels of two different markers and thereby separates them into their corresponding subpopulations. Using a phenotypic assay, we demonstrate that the subpopulations isolated have markedly different properties. This system provides an important new tool for identifying circulating tumor cell subtypes.

Fluorescence Characterization of Gold Modified Liposomes with Antisense N-myc DNA Bound to the Magnetisable Particles with Encapsulated Anticancer Drugs (Doxorubicin, Ellipticine and Etoposide).

Liposome-based drug delivery systems hold great potential for cancer therapy. The aim of this study was to design a nanodevice for targeted anchoring of liposomes (with and without cholesterol) with encapsulated anticancer drugs and antisense N-myc gene oligonucleotide attached to its surface. To meet this main aim, liposomes with encapsulated doxorubicin, ellipticine and etoposide were prepared. They were further characterized by measuring their fluorescence intensity, whereas the encapsulation efficiency was estimated to be 16%. The hybridization process of individual oligonucleotides forming the nanoconstruct was investigated spectrophotometrically and electrochemically. The concentrations of ellipticine, doxorubicin and etoposide attached to the nanoconstruct in gold nanoparticle-modified liposomes were found to be 14, 5 and 2 µg·mL(-1), respectively. The study succeeded in demonstrating that liposomes are suitable for the transport of anticancer drugs and the antisense oligonucleotide, which can block the expression of the N-myc gene.

Reciprocal Self-Assembly of Peptide-DNA Conjugates into a Programmable Sub-10-nm Supramolecular Deoxyribonucleoprotein.

To overcome the limitations of molecular assemblies, the development of novel supramolecular building blocks and self-assembly modes is essential to create more sophisticated, complex, and controllable aggregates. The self-assembly of peptide-DNA conjugates (PDCs), in which two orthogonal self-assembly modes, that is, ß-sheet formation and Watson-Crick base pairing, are covalently combined in one supramolecular system, is reported. Despite extensive research, most self-assembly studies have focused on using only one type of building block, which restricts structural and functional diversity compared to multicomponent systems. Multicomponent systems, however, suffer from poor control of self-assembly behaviors. Covalently conjugated PDC building blocks are shown to assemble into well-defined and controllable nanostructures. This controllability likely results from the decrease in entropy associated with the restriction of the molecular degrees of freedom by the covalent constraints. Using this strategy, the possibility to thermodynamically program nano-assemblies to exert gene regulation activity with low cytotoxicity is demonstrated.

Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in Backbone (γ-CF2-apg-PNA) Enhance Cellular Uptake.

Fluorous PNA analogues possessing fluorine as inherent part of aminopropylglycine (apg) backbone (γ-CF2-apg PNA) have been synthesized and evaluated for biophysical and cell penetrating properties. These form duplexes of higher thermal stability with cRNA than cDNA, although destabilized compared to duplexes of standard aeg-PNA. Cellular uptake of the fluorinated γ-CF2-apg PNAs in NIH 3T3 and HeLa cells was 2-3-fold higher compared to that of nonfluorinated apg PNA, with NIH 3T3 cells showing better permeability compared to HeLa cells. The backbone fluorinated PNAs, which are first in this class, when combined with other chemical modifications may have potential for future PNA-based antisense agents.

In the sense of transcription regulation by G-quadruplexes: asymmetric effects in sense and antisense strands.

G-Quadruplexes occupy important regulatory regions in the genome. DNA G-quadruplexes in the promoter regions and RNA quadruplexes in the UTRs (untranslated regions) have been individually studied and variously implicated at different regulatory levels of gene expression. However, the formation of G-quadruplexes in the sense and antisense strands and their corresponding roles in gene regulation have not been studied in much detail. In the present study, we have elucidated the effect of strand asymmetry in this context. Using biophysical methods, we have demonstrated the formation of stable G-quadruplex structure in vitro using CD and UV melting. Additionally, ITC was employed to demonstrate that a previously reported selective G-quadruplex ligand was able to bind and stabilize the G-quadruplex in the present sequence. Further, we have shown using reporter constructs that although the DNA G-quadruplex in either strand can reduce translation efficiency, transcriptional regulation differs when G-quadruplex is present in the sense or antisense strand. We demonstrate that the G-quadruplex motif in the antisense strand substantially inhibits transcription, while when in the sense strand, it does not affect transcription, although it does ultimately reduce translation. Further, it is also shown that the G-quadruplex stabilizing ligand can enhance this asymmetric transcription regulation as a result of the increased stabilization of the G-quadruplex.

A gene expression restriction network mediated by sense and antisense Alu sequences located on protein-coding messenger RNAs.

BACKGROUND: Alus are primate-specific retrotransposons which account for 10.6% of the human genome. A large number of protein-coding mRNAs are encoded with sense or antisense Alus in the un-translated regions. RESULTS: We postulated that mRNAs carrying Alus in the two opposite directions can generate double stranded RNAs, capable of regulating the levels of other Alu-carrying mRNAs post-transcriptionally. A gene expression profiling assay showed that the levels of antisense and sense Alus-carrying mRNAs were suppressed in a reversible manner by over-expression of exogenous sense and antisense Alus derived from mRNAs (Family-wise error rate P=0.0483 and P<0.0001 respectively). Screening through human mRNAs on the NCBI-RefSeq database, it was found that sense and antisense Alu-carrying transcripts were enriched in distinct cellular functions. Antisense Alu-carrying genes were particularly enriched in neurological and developmental processes, while sense Alu-carrying genes were enriched in immunological functions. CONCLUSIONS: Taken together, we proposed a novel Alu-mediated regulation network capable of stabilizing Alu-carrying mRNA levels in different cell types and restricting the activated expression levels of protein-coding, Alu-carrying mRNAs.

Genome-level analyses of Mycobacterium bovis lineages reveal the role of SNPs and antisense transcription in differential gene expression.

BACKGROUND: Bovine tuberculosis (bTB) is a disease with major implications for animal welfare and productivity, as well as having the potential for zoonotic transmission. In Great Britain (GB) alone, controlling bTB costs in the region of £ 100 million annually, with the current control scheme seemingly unable to stop the inexorable spread of infection. One aspect that may be driving the epidemic is evolution of the causative pathogen, Mycobacterium bovis. To understand the underlying genetic changes that may be responsible for this evolution, we performed a comprehensive genome-level analyses of 4 M. bovis strains that encompass the main molecular types of the pathogen circulating in GB. RESULTS: We have used a combination of genome sequencing, transcriptome analyses, and recombinant DNA technology to define genetic differences across the major M. bovis lineages circulating in GB that may give rise to phenotypic differences of practical importance. The genomes of three M. bovis field isolates were sequenced using Illumina sequencing technology and strain specific differences in gene expression were measured during in vitro growth and in ex vivo bovine alveolar macrophages using a whole genome amplicon microarray and a whole genome tiled oligonucleotide microarray. SNP/small base pair insertion and deletions and gene expression data were overlaid onto the genomic sequence of the fully sequenced strain of M. bovis 2122/97 to link observed strain specific genomic differences with differences in RNA expression. CONCLUSIONS: We show that while these strains show extensive similarities in their genetic make-up and gene expression profiles, they exhibit distinct expression of a subset of genes. We provide genomic, transcriptomic and functional data to show that synonymous point mutations (sSNPs) on the coding strand can lead to the expression of antisense transcripts on the opposing strand, a finding with implications for how we define a 'silent' nucleotide change. Furthermore, we show that transcriptomic data based solely on amplicon arrays can generate spurious results in terms of gene expression profiles due to hybridisation of antisense transcripts. Overall our data suggest that subtle genetic differences, such as sSNPS, may have important consequences for gene expression and subsequent phenotype.

Structure of the AvrBs3-DNA complex provides new insights into the initial thymine-recognition mechanism.

Transcription activator-like effectors contain a DNA-binding domain organized in tandem repeats. The repeats include two adjacent residues known as the repeat variable di-residue, which recognize a single base pair, establishing a direct code between the dipeptides and the target DNA. This feature suggests this scaffold as an excellent candidate to generate new protein-DNA specificities for biotechnological applications. Here, the crystal structure of AvrBs3 (residues 152-895, molecular mass 82 kDa) in complex with its target DNA sequence is presented, revealing a new mode of interaction with the initial thymine of the target sequence, together with an analysis of both the binding specificity and the thermodynamic properties of AvrBs3. This study quantifies the affinity and the specificity between AvrBs3 and its target DNA. Moreover, in vitro and in vivo analyses reveal that AvrBs3 does not show a strict nucleotide-binding preference for the nucleotide at the zero position of the DNA, widening the number of possible sequences that could be targeted by this scaffold.

Binding assay of human Dectin-1 variants for DNA/ ß-glucan complex for active-targeting delivery of antisense DNA: Part II.

A ß-1,3-glucan binding receptor called Dectin-1 is mainly expressed on antigen-presenting immunocytes. Dectin-1 may be a target molecule for receptor-mediated and active-targeting delivery of drugs to regulate or interfere with the immune system. Therapeutic oligonucleotides are one such drug of interest. To this end, we have been studying the complex of schizophyllan (SPG, one of the linear (1,3)-ß-ᴅ-glucan family) with oligonucleotide and its delivery mechanism to the Dectin-1 expressing cells. There are at least six types of human Dectin-1 expressed on the cell surface (designated V-1, V-2, etc.), with V-1 having a complete carbohydrate recognition domain (CRD) and stalk, V-2 having a complete CRD but no stalk, and other variants having an incomplete CRD due to exon skipping. Our previous studies have shown that SPG binds only to V-1 and V-2. By contrast, SPG/oligonucleotide complexes bind both V-1 and V-2 more strongly than SPG itself and show a certain affinity, for other variants. As a continuing work, the present paper discusses the structure and nature of all human Dectin-1 variants expressed on the cellular surface. we found that (1) a new N-linked glycosylation site is present in some variants, (2) the glycosylation of Dectin-1 plays an important role in the fate of Dectin-1 and its localization in the cells, and (3) the glycosylation is related to the amount of ingestion of the complex. The present findings suggest that, in addition to V-1 and V-2, two other variants that are highly expressed at the plasma membrane and stabilized by the glycosylation may also be targets of the complex.

Antibody-linked spherical nucleic acids for cellular targeting.

Spherical nucleic acid (SNA) constructs are promising new single entity gene regulation materials capable of both cellular transfection and gene knockdown, but thus far are promiscuous structures, exhibiting excellent genetic but little cellular selectivity. In this communication, we describe a strategy to impart targeting capabilities to these constructs through noncovalent functionalization with a complementary antibody-DNA conjugate. As a proof-of-concept, we designed HER2-targeting SNAs and demonstrated that such structures exhibit cell type selectivity in terms of their uptake, and significantly greater gene knockdown in cells overexpressing the target antigen as compared to the analogous antibody-free and off-target materials.