Novel Cell type-specific aptamer-siRNA delivery system for HIV-1 therapy

The successful use of small interfering RNAs (siRNAs) for therapeutic purposes requires safe and efficient delivery to specific cells and tissues. Here we demonstrate cell type-specific delivery of anti-HIV siRNAs via fusion to an anti-gp120 aptamer. The envelope glycoprotein is expressed on the surface of HIV-1 infected cells, allowing binding and interalization of the aptamer-siRNA chimeric molecules. We demonstrate that the anti-gp120 aptamer-siRNA chimera is specifically taken up by cells expressing HIV-1 gp120, and the appended siRNA is processed by Dicer, releasing an anti-tat/rev siRNA which in turn inhibits HIV replication. We show for the first time a dual functioning aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities and that gp120 expressed on the surface of HIV infected cells can be used for aptamer mediated delivery of anti-HIV siRNAs

Engineering ligand-responsive RNA controllers in yeast through the assembly of RNase III tuning modules

The programming of cellular networks to achieve new biological functions depends on the development of genetic tools that link the presence of a molecular signal to gene-regulatory activity. Recently, a set of engineered RNA controllers was described that enabled predictable tuning of gene expression in the yeast Saccharomyces cerevisiae through directed cleavage of transcripts by an RNase III enzyme, Rnt1p. Here, we describe a strategy for building a new class of RNA sensing-actuation devices based on direct integration of RNA aptamers into a region of the Rnt1p hairpin that modulates Rnt1p cleavage rates. We demonstrate that ligand binding to the integrated aptamer domain is associated with a structural change sufficient to inhibit Rnt1p processing. Three tuning strategies based on the incorporation of different functional modules into the Rnt1p switch platform were demonstrated to optimize switch dynamics and ligand responsiveness. We further demonstrated that these tuning modules can be implemented combinatorially in a predictable manner to further improve the regulatory response properties of the switch. The modularity and tunability of the Rnt1p switch platform will allow for rapid optimization and tailoring of this gene control device, thus providing a useful tool for the design of complex genetic networks in yeast

Model-guided design of ligand-regulated RNAi for programmable control of gene expression

Progress in constructing biological networks will rely on the development of more advanced components that can be predictably modified to yield optimal system performance. We have engineered an RNA-based platform, which we call an shRNA switch, that provides for integrated ligand control of RNA interference (RNAi) by modular coupling of an aptamer, competing strand, and small hairpin (sh) RNA stem into a single component that links ligand concentration and target gene expression levels. A combined experimental and mathematical modelling approach identified multiple tuning strategies and moves towards a predictable framework for the forward design of shRNA switches. The utility of our platform is highlighted by the demonstration of fine-tuning, multi-input control, and model-guided design of shRNA switches with an optimized dynamic range. Thus, shRNA switches can serve as an advanced component for the construction of complex biological systems and offer a controlled means of activating RNAi in disease therapeutics

A novel colorimetric biosensor based on non-aggregated Au@Ag core–shell nanoparticles for methamphetamine and cocaine detection

We report a novel colorimetric biosensor based on non-aggregation Au@Ag core-shell nanoparticles to detect methamphetamine and cocaine. The biosensor consisted of a reporter probe (RP) that is a specific single-stranded DNA (ssDNA) sequence coated on Au@Ag nanoparticles, a capture probe (CP) conjugated with magnetic beads, and an illicit drug-binding DNA aptamer (Apt). Au@Ag nanoparticles were synthesized by seed growth and characterized by scanning electron microscope (SEM), high-resolution transmission electron microscopy (HR-TEM), and UV–vis spectra. Methamphetamine (METH) was used as an example to evaluate the feasibility of the biosensor and to optimize the detection conditions. We demonstrated that this sensing platform was able to detect as low as 0.1 nM (14.9 ng L−1) METH with a negligible interference from other common illicit drugs. Various concentrations of METH were spiked into urines, and the biosensor yielded recoveries more than 83.1%. In addition, the biosensor also showed a high sensitivity to detect cocaine. These results demonstrated that our colorimetric sensor holds promise to be implemented as a visual sensing platform to detect multiple illicit drugs in biological samples and environmental matrices

Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (KD). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions.

Three-dimensional modeling of single stranded DNA hairpins for aptamer-based biosensors.

Aptamers consist of short oligonucleotides that bind specific targets. They provide advantages over antibodies, including robustness, low cost, and reusability. Their chemical structure allows the insertion of reporter molecules and surface-binding agents in specific locations, which have been recently exploited for the development of aptamer-based biosensors and direct detection strategies. Mainstream use of these devices, however, still requires significant improvements in optimization for consistency and reproducibility. DNA aptamers are more stable than their RNA counterparts for biomedical applications but have the disadvantage of lacking the wide array of computational tools for RNA structural prediction. Here, we present the first approach to predict from sequence the three-dimensional structures of single stranded (ss) DNA required for aptamer applications, focusing explicitly on ssDNA hairpins. The approach consists of a pipeline that integrates sequentially building ssDNA secondary structure from sequence, constructing equivalent 3D ssRNA models, transforming the 3D ssRNA models into ssDNA 3D structures, and refining the resulting ssDNA 3D structures. Through this pipeline, our approach faithfully predicts the representative structures available in the Nucleic Acid Database and Protein Data Bank databases. Our results, thus, open up a much-needed avenue for integrating DNA in the computational analysis and design of aptamer-based biosensors

Combined aptamer and transcriptome sequencing of single cells.

The transcriptome and proteome encode distinct information that is important for characterizing heterogeneous biological systems. We demonstrate a method to simultaneously characterize the transcriptomes and proteomes of single cells at high throughput using aptamer probes and droplet-based single cell sequencing. With our method, we differentiate distinct cell types based on aptamer surface binding and gene expression patterns. Aptamers provide advantages over antibodies for single cell protein characterization, including rapid, in vitro, and high-purity generation via SELEX, and the ability to amplify and detect them with PCR and sequencing

Label-Free, Highly Sensitive Electrochemical Aptasensors Using Polymer-Modified Reduced Graphene Oxide for Cardiac Biomarker Detection

Acute myocardial infarction (AMI), also recognized as a ???heart attack,??? is one leading cause of death globally, and cardiac myoglobin (cMb), an important cardiac biomarker, is used for the early assessment of AMI. This paper presents an ultrasensitive, label-free electrochemical aptamer-based sensor (aptasensor) for cMb detection using polyethylenimine (PEI)-functionalized reduced graphene oxide (PEI???rGO) thin films. PEI, a cationic polymer, was used as a reducing agent for graphene oxide (GO), providing highly positive charges on the rGO surface and allowing direct immobilization of negatively charged single-strand DNA aptamers against cMb via electrostatic interaction without any linker or coupling chemistry. The presence of cMb was detected on Mb aptamer-modified electrodes using differential pulse voltammetry via measuring the current change due to the direct electron transfer between the electrodes and cMb proteins (Fe3+/Fe2+). The limits of detection were 0.97 pg mL???1 (phosphate-buffered saline) and 2.1 pg mL???1 (10-fold-diluted human serum), with a linear behavior with logarithmic cMb concentration. The specificity and reproducibility of the aptasensors were also examined. This electrochemical aptasensor using polymer-modified rGO shows potential for the early assessment of cMb in point-of-care testing applications