Specificity of oligonucleotide gene therapy (OGT) agents.

Oligonucleotide gene therapy (OGT) agents (e. g. antisense, deoxyribozymes, siRNA and CRISPR/Cas) are promising therapeutic tools. Despite extensive efforts, only few OGT drugs have been approved for clinical use. Besides the problem of efficient delivery to targeted cells, hybridization specificity is a potential limitation of OGT agents. To ensure tight binding, a typical OGT agent hybridizes to the stretch of 15-25 nucleotides of a unique targeted sequence. However, hybrids of such lengths tolerate one or more mismatches under physiological conditions, the problem known as the affinity/specificity dilemma. Here, we assess the scale of this problem by analyzing OGT hybridization-dependent off-target effects (HD OTE) in vitro, in animal models and clinical studies. All OGT agents except deoxyribozymes exhibit HD OTE in vitro, with most thorough evidence of poor specificity reported for siRNA and CRISPR/Cas9. Notably, siRNA suppress non-targeted genes due to (1) the partial complementarity to mRNA 3'-untranslated regions (3'-UTR), and (2) the antisense activity of the sense strand. CRISPR/Cas9 system can cause hundreds of non-intended dsDNA breaks due to low specificity of the guide RNA, which can limit therapeutic applications of CRISPR/Cas9 by ex-vivo formats. Contribution of this effects to the observed in vivo toxicity of OGT agents is unclear and requires further investigation. Locked or peptide nucleic acids improve OGT nuclease resistance but not specificity. Approaches that use RNA marker dependent (conditional) activation of OGT agents may improve specificity but require additional validation in cell culture and in vivo.

Binary Antisense Oligonucleotide Agent for Cancer Marker-Dependent Degradation of Targeted RNA.

Antisense oligonucleotide technology is one of the most successful gene therapy (GT) approaches. However, low selectivity of antisense agents limits their application as anticancer drugs. To achieve activation of antisense agent selectively in cancer cells, herein, we propose the concept of binary antisense oligonucleotide (biASO) agent. biASO recognizes an RNA sequence of a gene associated with cancer development (marker) and then activates RNase H-dependent cleavage of a targeted messenger RNA. biASO was optimized to produce only the background cleavage of the targeted RNA in the absence of the activator. The approach lays the foundation for the development of highly selective and efficient GT agents.

RNA-Cleaving DNA Thresholder Controlled by Concentrations of miRNA Cancer Marker.

Oligonucleotide gene therapy (OGT) agents suppress specific mRNAs in cells and thus reduce the expression of targeted genes. The ability to unambiguously distinguish cancer from healthy cells can solve the low selectivity problem of OGT agents. Cancer RNA markers are expressed in both healthy and cancer cells with a higher expression level in cancer cells. We have designed a DNA-based construct, named DNA thresholder (DTh) that cleaves targeted RNA only at high concentrations of cancer marker RNA and demonstrates low cleavage activity at low marker concentrations. The RNA-cleaving activity can be adjusted within one order of magnitude of the cancer marker RNA concentration by simply redesigning DTh. Importantly, DTh recognizes cancer marker RNA, while cleaving targeted RNA; this offers a possibility to suppress vital genes exclusively in cancer cells, thus triggering their death. DTh is a prototype of computation-inspired molecular device for controlling gene expression and cancer treatment.

Binary (Split) Light-up Aptameric Sensors.

This Minireview discusses the design and applications of binary (also known as split) light-up aptameric sensors (BLAS). BLAS consist of two RNA or DNA strands and a fluorogenic organic dye added as a buffer component. When associated, the two strands form a dye-binding site, followed by an increase in fluorescence of the aptamer-bound dye. The design is cost-efficient because it uses short oligonucleotides and does not require conjugation of organic dyes with nucleic acids. In some applications, BLAS design is preferable over monolithic sensors because of simpler assay optimization and improved selectivity. RNA-based BLAS can be expressed in cells and used for the intracellular monitoring of biological molecules. BLAS have been used as reporters of nucleic acid association events in RNA nanotechnology and nucleic-acid-based molecular computation. Other applications of BLAS include the detection of nucleic acids, proteins, and cancer cells, and potentially they can be tailored to report a broad range of biological analytes.

Cut and Paste for Cancer Treatment: A DNA Nanodevice that Cuts Out an RNA Marker Sequence to Activate a Therapeutic Function.

DNA nanotechnology uses oligonucleotide strands to assemble molecular structures capable of performing useful operations. Here, we assembled a multifunctional prototype DNA nanodevice, DOCTR, that recognizes a single nucleotide mutation in a cancer marker RNA. The nanodevice then cuts out a signature sequence and uses it as an activator for a "therapeutic" function, namely, the cleavage of another RNA sequence. The proposed design is a prototype for a gene therapy DNA machine that cleaves a housekeeping gene only in the presence of a cancer-causing point mutation and suppresses cancer cells exclusively with minimal side effects to normal cells.

Bifunctional RNA-Targeting Deoxyribozyme Nanodevice as a Potential Theranostic Agent.

Theranostic approaches rely on simultaneous diagnostic of a disease and its therapy. Here, we designed a DNA nanodevice, which can simultaneously report the presence of a specific RNA target through an increase in fluorescence and cleave it. High selectivity of RNA target recognition under near physiological conditions was achieved. The proposed approach can become a basis for the design of DNA nanomachines and robots for diagnostics and therapy of viral infections, cancer, and genetic disorders.

Deoxyribozyme-Based DNA Machines for Cancer Therapy.

Soon after their discovery, RNA-cleaving deoxyribozymes (RCDZ) were explored as anticancer gene therapy agents. Despite low toxicity found in clinical trials, there is no clinically significant anticancer RCDZ-based therapy. Some of the reported disadvantages of RCDZ agents include poor accessibility to folded nucleic acids, low catalytic efficiency inside cells, and problems of intracellular delivery. On the other hand, structural DNA nanotechnology provides an opportunity to build multifunctional nano-associations that can address some of these problems. Herein we discuss the possibility of building RCDZ-based multifunctional DNA nanomachines equipped with RNA unwinding, cancer marker recognition, and RCDZ-based RNA-cleavage functions. An important advantage of such "nanomachines" is the possibility to cleave a housekeeping gene mRNA in a cancer-cell-specific manner. The proposed design could become a starting point for building sophisticated DNA-based nanodevices for cancer treatment.

Towards DNA Nanomachines for Cancer Treatment: Achieving Selective and Efficient Cleavage of Folded RNA.

Despite decades of effort, gene therapy (GT) has failed to deliver clinically significant anticancer treatment, owing in part to low selectivity, low efficiency, and poor accessibility of folded RNA targets. Herein, we propose to solve these common problems of GT agents by using a DNA nanotechnology approach. We designed a deoxyribozyme-based DNA machine that can i) recognize the sequence of a cancer biomarker with high selectivity, ii) tightly bind a structured fragment of a housekeeping gene mRNA, and iii) cleave it with efficiency greater than that of a traditional DZ-based cleaving agent. An important advantage of the DNA nanomachine over other gene therapy approaches (antisense, siRNA, and CRISPR/cas) is its ability to cleave a housekeeping gene mRNA after being activated by a cancer marker RNA, which can potentially increase the efficiency of anticancer gene therapy. The DNA machine could become a prototype platform for a new type of anticancer GT agent.

A universal and label-free impedimetric biosensing platform for discrimination of single nucleotide substitutions in long nucleic acid strands.

We report a label-free universal biosensing platform for highly selective detection of long nucleic acid strands. The sensor consists of an electrode-immobilized universal stem-loop (USL) probe and two adaptor strands that form a 4J structure in the presence of a specific DNA/RNA analyte. The sensor was characterized by electrochemical impedance spectroscopy (EIS) using K3[Fe(CN)6]/K4[Fe(CN)6] redox couple in solution. An increase in charge transfer resistance (RCT) was observed upon 4J structure formation, the value of which depends on the analyte length. Cyclic voltammetry (CV) was used to further characterize the sensor and monitor the electrochemical reaction in conjunction with thickness measurements of the mixed DNA monolayer obtained using spectroscopic ellipsometry. In addition, the electron transfer was calculated at the electrode/electrolyte interface using a rotating disk electrode. Limits of detection in the femtomolar range were achieved for nucleic acid targets of different lengths (22 nt, 60 nt, 200 nt). The sensor produced only a background signal in the presence of single base mismatched analytes, even in hundred times excess in concentration. This label-free and highly selective biosensing platform is versatile and can be used for universal detection of nucleic acids of varied lengths which could revolutionize point of care diagnostics for applications such as bacterial or cancer screening.

Nanoreactors based on DNAzyme-functionalized magnetic nanoparticles activated by magnetic field.

A new biomimetic nanoreactor design, MaBiDz, is presented based on a copolymer brush in combination with superparamagnetic nanoparticles. This cellular nanoreactor features two species of magnetic particles, each functionalized with two components of a binary deoxyribozyme system. In the presence of a target mRNA analyte and a magnetic field, the nanoreactor is assembled to form a biocompartment enclosed by the polymeric brush that enables catalytic function of the binary deoxyribozyme with enhanced kinetics. MaBiDz was demonstrated here as a cellular sensor for rapid detection and imaging of a target mRNA biomarker for metastatic breast cancer, and its function shows potential to be expanded as a biomimetic organelle that can downregulate the activity of a target mRNA biomarker.

Magnetic Field-Activated Sensing of mRNA in Living Cells.

Detection of specific mRNA in living cells has attracted significant attention in the past decade. Probes that can be easily delivered into cells and activated at the desired time can contribute to understanding translation, trafficking and degradation of mRNA. Here we report a new strategy termed magnetic field-activated binary deoxyribozyme (MaBiDZ) sensor that enables both efficient delivery and temporal control of mRNA sensing by magnetic field. MaBiDZ uses two species of magnetic beads conjugated with different components of a multicomponent deoxyribozyme (DZ) sensor. The DZ sensor is activated only in the presence of a specific target mRNA and when a magnetic field is applied. Here we demonstrate that MaBiDZ sensor can be internalized in live MCF-7 breast cancer cells and activated by a magnetic field to fluorescently report the presence of specific mRNA, which are cancer biomarkers.

Enzyme-assisted target recycling (EATR) for nucleic acid detection.

Fast, reliable and sensitive methods for nucleic acid detection are of growing practical interest with respect to molecular diagnostics of cancer, infectious and genetic diseases. Currently, PCR-based and other target amplification strategies are most extensively used in practice. At the same time, such assays have limitations that can be overcome by alternative approaches. There is a recent explosion in the design of methods that amplify the signal produced by a nucleic acid target, without changing its copy number. This review aims at systematization and critical analysis of the enzyme-assisted target recycling (EATR) signal amplification technique. The approach uses nucleases to recognize and cleave the probe-target complex. Cleavage reactions produce a detectable signal. The advantages of such techniques are potentially low sensitivity to contamination and lack of the requirement of a thermal cycler. Nucleases used for EATR include sequence-dependent restriction or nicking endonucleases or sequence independent exonuclease III, lambda exonuclease, RNase H, RNase HII, AP endonuclease, duplex-specific nuclease, DNase I, or T7 exonuclease. EATR-based assays are potentially useful for point-of-care diagnostics, single nucleotide polymorphisms genotyping and microRNA analysis. Specificity, limit of detection and the potential impact of EATR strategies on molecular diagnostics are discussed.

Four-way junction formation promoting ultrasensitive electrochemical detection of microRNA.

MicroRNAs (miRNAs) represent a class of biomarkers that are frequently deregulated in cancer cells and have shown a great promise for cancer classification and prognosis. Here, we endeavored to develop a DNA four-way junction based electrochemical sensor (4J-SENS) for ultrasensitive miRNA analysis. The developed sensor can be operated within the dynamic range from 10 aM to 1 fM and detect as low as 2 aM of miR-122 (∼36 molecules per sample), without PCR amplification. Furthermore, the 4J-SENS was employed to profile endogenouse hsa-miR-122 in healthy human and chronic lymphocyitc leukemia (CLL) patient serum, and the results were validated by qPCR analysis.

Modular aptameric sensors.

We report the first examples of modular aptameric sensors, which transduce recognition events into fluorescence changes through allosteric regulation of noncovalent interactions with a fluorophore. These sensors consist of: (a) a reporting domain, which signals the binding event of an analyte through binding to a fluorophore; (b) a recognition domain, which binds the analyte; and (c) a communication module, which serves as a conduit between recognition and signaling domains. We tested recognition regions specific for ATP, FMN, and theophylline in combinations with malachite green binding aptamer as a signaling domain. In each case, we were able to obtain a functional sensor capable of responding to an increase in analyte concentration with an increase in fluorescence. Similar constructs that consist only of natural RNA could be expressed in cells and used as sensors for intracellular imaging.

Structure-function relationship of the influenza virus RNA polymerase: primer-binding site on the PB1 subunit.

Influenza virus RNA polymerase is composed of three viral P proteins (PB1, PB2, and PA) and involved in both transcription and replication of the viral RNA genome. The catalytic site for RNA polymerization is located on the PB1 subunit. To identify the primer ATP-binding site, we have employed a highly selective cross-linking technique: three structurally diverse ATP analogues with reactive groups on their phosphate moieties were first cross-linked to the viral RNA polymerase, and the cross-linked nucleotides were then elongated to dinucleotides by adding the second substrate [alpha-(32)P]GTP. Only the ATP analogues tethered to the primer-binding site could be elongated to radioactive AG dinucleotides. Using this catalytically competent cross-linking procedure, the PB1 subunit was found to be the only labeled subunit. Limited proteolysis of the labeled PB1 by V8 protease revealed the segment between amino acids 179 and 297 as the only bearer of the radioactive label. Thus, we concluded that this region of PB1 faces the 5' end of the primer nucleotide. In support of this prediction, the cross-linked dinucleotides were further elongated up to 8-10 nucleotides in length upon addition of all four substrates. This result suggests that the substantial mass of RNA can be accommodated between the binding site for the primer (and nascent RNA) and the catalytic center of RNA polymerization.

Superselective labelling of proteins: approaches and techniques.

Affinity labelling is a popular method used for the study of macromolecules and their interactions with ligands. The method is based on the targeted delivery of a chemically cross-linkable group, attached to a reactive molecule with affinity for a particular site in the biopolymer of interest. In complex multicomponent systems, the applications of affinity labelling are restricted by the tendency of the reagents to randomly label nontargetted molecules. This review highlights techniques developed to minimize non-specific cross-linking and to achieve high selectivity for the labelling of target protein. Such techniques might be termed 'superselective labelling', as opposed to traditional, less selective approaches.