Single-cell coating with biomimetic extracellular nanofiber matrices.

Cell therapies offer great promise in the treatment of diseases and tissue regeneration, but their clinical use has many challenges including survival, optimal performance in their intended function, or localization at sites where they are needed for effective outcomes. We report here on a method to coat a biodegradable matrix of biomimetic nanofibers on single cells that could have specific functions ranging from cell signaling to targeting and helping cells survive when used for therapies. The fibers are composed of peptide amphiphile (PA) molecules that self-assemble into supramolecular nanoscale filaments. The PA nanofibers were able to create a mesh-like coating for a wide range of cell lineages with nearly 100 % efficiency, without interrupting the natural cellular phenotype or functions. The targeting abilities of this system were assessed in vitro using human primary regulatory T (hTreg) cells coated with PAs displaying a vascular cell adhesion protein 1 (VCAM-1) targeting motif. This approach provides a biocompatible method for single-cell coating that does not negatively alter cellular phenotype, binding capacity, or immunosuppressive functionality, with potential utility across a broad spectrum of cell therapies. STATEMENT OF SIGNIFICANCE: Cell therapies hold great promise in the treatment of diseases and tissue regeneration, but their clinical use has been limited by cell survival, targeting, and function. We report here a method to coat single cells with a biodegradable matrix of biomimetic nanofibers composed of peptide amphiphile (PA) molecules. The nanofibers were able to coat cells, such as human primary regulatory T cells, with nearly 100 % efficiency, without interrupting the natural cellular phenotype or functions. The approach provides a biocompatible method for single-cell coating that does not negatively alter cellular phenotype, binding capacity, or immunosuppressive functionality, with potential utility across a broad spectrum of cell therapies.

Microfluidic Technology for Nucleic Acid Aptamer Evolution and Application.

The intersection of microfluidics and aptamer technologies holds particular promise for rapid progress in a plethora of applications across biomedical science and other areas. Here, the influence of microfluidics on the field of aptamers, from traditional capillary electrophoresis approaches through innovative modern-day approaches using micromagnetic beads and emulsion droplets, is reviewed. Miniaturizing aptamer-based bioassays through microfluidics has the potential to transform diagnostics and embedded biosensing in the coming years.

Aptamer Display on Diverse DNA Polyhedron Supports.

DNA aptamers are important tools for molecular recognition, particularly for a new generation of tools for biomedicine based on nucleic acid nanostructures. Here, we investigated the relative abilities of different shapes and sizes of DNA polyhedra to display an aptamer which binds to the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH). The aptamer was shown to perform an Aptamer-Tethered Enzyme Capture (APTEC) assay with the hypothesis that the display of the aptamer above the surface through the use of a polyhedron may lead to better sensitivity than use of the aptamer alone. We compared different numbers of points of contact, different shapes, including tetrahedron, square, and pentagon-based pyramids, as well as prisms. We also investigated the optimal height of display of the structure. Our results demonstrated that the display of an aptamer on an optimized nanostructure improved sensitivity up to 6-fold relative to the aptamer alone in the APTEC assay. Other important factors included multiple basal points of contact with the surface, a tetrahedron proved superior to the more complex shaped structures, and height above the surface only made minor differences to efficacy. The display of an aptamer on a nanostructure may be beneficial for higher sensitivity aptamer-mediated malaria diagnosis. Aptamer displays using DNA nanostructure polyhedron supports could be a useful approach in a variety of applications.

Selection and Characterization of a DNA Aptamer Specifically Targeting Human HECT Ubiquitin Ligase WWP1.

Nucleic acid aptamers hold promise as therapeutic tools for specific, tailored inhibition of protein targets with several advantages when compared to small molecules or antibodies. Nuclear WW domain containing E3 ubiquitin ligase 1 (WWP1) ubiquitin ligase poly-ubiquitinates Runt-related transcription factor 2 (Runx2), a key transcription factor associated with osteoblast differentiation. Since WWP1 and an adapter known as Schnurri-3 are negative regulators of osteoblast function, the disruption of this complex has the potential to increase bone deposition for osteoporosis therapy. Here, we develop new DNA aptamers that bind and inhibit WWP1 then investigate efficacy in an osteoblastic cell culture. DNA aptamers were selected against three different truncations of the HECT domain of WWP1. Aptamers which bind specifically to a C-lobe HECT domain truncation were observed to enrich during the selection procedure. One particular DNA aptamer termed C3A was further evaluated for its ability to bind WWP1 and inhibit its ubiquitination activity. C3A showed a low µM binding affinity to WWP1 and was observed to be a non-competitive inhibitor of WWP1 HECT ubiquitin ligase activity. When SaOS-2 osteoblastic cells were treated with C3A, partial localization to the nucleus was observed. The C3A aptamer was also demonstrated to specifically promote extracellular mineralization in cell culture experiments. The C3A aptamer has potential for further development as a novel osteoporosis therapeutic strategy. Our results demonstrate that aptamer-mediated inhibition of protein ubiquitination can be a novel therapeutic strategy.

An aptamer-enabled DNA nanobox for protein sensing.

DNA nanostructures can show dynamic responses to molecular triggers for a wide variety of applications. While DNA sequence signal triggers are now well-established, there is a critical need for a broader diversity of molecular triggers to drive dynamic responses in DNA nanostructures. DNA aptamers are ideal; they can both seamlessly integrate into DNA nanostructure scaffolds and transduce molecular recognition into functional responses. Here, we report construction and optimization of a DNA origami nanobox locked by a pair of DNA double strands where one strand is a DNA aptamer targeting the malaria biomarker protein Plasmodium falciparum lactate dehydrogenase. The protein acts as the key which enables box opening. We observe highly specific protein-mediated box opening by both transmission electron microscopy and fluorescence. Aptamer-enabled DNA boxes have significant potential for enabling direct responses to proteins and other biomolecules in nanoscale diagnostics, drug delivery and sensing devices.

A portable microfluidic Aptamer-Tethered Enzyme Capture (APTEC) biosensor for malaria diagnosis.

There is a critical need for better biosensors for the detection and diagnosis of malaria. We previously developed a DNA aptamer that recognises the Plasmodium falciparum lactate dehydrogenase (PfLDH) enzyme with high sensitivity and specificity. The aptamer was integrated into an Aptamer-Tethered Enzyme Capture (APTEC) assay as a laboratory-based diagnostic approach. However, a portable equipment-free point-of-care aptamer-mediated biosensor could have a significant impact on malaria diagnosis in endemic regions. Here, we present a new concept for a malaria biosensor whereby aptamers are coated onto magnetic microbeads for magnet-guided capture, wash and detection of the biomarker. A biosensor incorporating three separate microfluidic chambers was designed to enable such magnet-guided equipment-free colorimetric detection of PfLDH. A series of microfluidic biosensor prototypes were optimised to lower rates of inter-chamber diffusion, increase sensitivity, and provide a method for point-of-care sample testing. The biosensor showed high sensitivity and specificity when detecting PfLDH using both in vitro cultured parasite samples and using clinical samples from malaria patients. The high performance of the biosensor provides a proof-of-principle for a portable biosensor that could be adaptable for a variety of aptamer-mediated diagnostic scenarios.

Aptamer Bioinformatics.

Aptamers are short nucleic acid sequences capable of specific, high-affinity molecular binding. They are isolated via SELEX (Systematic Evolution of Ligands by Exponential Enrichment), an evolutionary process that involves iterative rounds of selection and amplification before sequencing and aptamer characterization. As aptamers are genetic in nature, bioinformatic approaches have been used to improve both aptamers and their selection. This review will discuss the advancements made in several enclaves of aptamer bioinformatics, including simulation of aptamer selection, fragment-based aptamer design, patterning of libraries, identification of lead aptamers from high-throughput sequencing (HTS) data and in silico aptamer optimization.

Aptamer-Mediated Protein Molecular Recognition Driving a DNA Tweezer Nanomachine.

Nucleic acid-mediated nanomachines have significant potential in biomedical applications but new approaches that link molecular recognition of proteins to change in nucleic acid structure and function are required. Here, a split DNA aptamer is integrated into G-quadruplex tweezers, which close in the presence of the malaria biomarker protein Plasmodium falciparum lactate dehydrogenase (PfLDH). Closing of the tweezers enables G-quadruplex hemin mediated peroxidase activity, which can be observed colorimetrically. The PfLDH aptamer is split within an asymmetric internal loop and incorporated into the tweezers maintaining aptamer binding capability. Spacing between the G-quadruplex structure and split aptamer, together with extent of complementarity, is found to be critical for optimization to enhance catalytic performance. The integrated split aptamer is observed to maintain the high specificity to Plasmodium falciparum lactate dehydrogenase of the parent aptamer. Split aptamer approaches have significant potential to functionalize nucleic acid nanostructures for protein molecular recognition.

Aptamer Affinity Maturation by Resampling and Microarray Selection.

Aptamers have significant potential as affinity reagents, but better approaches are critically needed to discover higher affinity nucleic acids to widen the scope for their diagnostic, therapeutic, and proteomic application. Here, we report aptamer affinity maturation, a novel aptamer enhancement technique, which combines bioinformatic resampling of aptamer sequence data and microarray selection to navigate the combinatorial chemistry binding landscape. Aptamer affinity maturation is shown to improve aptamer affinity by an order of magnitude in a single round. The novel aptamers exhibited significant adaptation, the complexity of which precludes discovery by other microarray based methods. Honing aptamer sequences using aptamer affinity maturation could help optimize a next generation of nucleic acid affinity reagents.

Oligonucleotide Functionalised Microbeads: Indispensable Tools for High-Throughput Aptamer Selection.

The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR) and Fluorescence Activated Cell Sorting (FACS) to high-throughput selection techniques. Within these systems, monoclonal aptamer microbeads can be individually generated and assayed to assess aptamer candidate fitness thereby helping eliminate stochastic effects which are common to classical SELEX techniques. Such techniques have given rise to aptamers with 1000 times greater binding affinities when compared to traditional SELEX. Another emerging technique is Fluorescence Activated Droplet Sorting (FADS) whereby selection does not rely on binding capture allowing evolution of a greater diversity of aptamer properties such as fluorescence or enzymatic activity. Within this review we explore examples and applications of oligonucleotide functionalised microbeads in aptamer selection and reflect upon new opportunities arising for aptamer science.

The hydrogen-peroxide-induced radical behaviour in human cytochrome c-phospholipid complexes: implications for the enhanced pro-apoptotic activity of the G41S mutant.

We have investigated whether the pro-apoptotic properties of the G41S mutant of human cytochrome c can be explained by a higher than wild-type peroxidase activity triggered by phospholipid binding. A key complex in mitochondrial apoptosis involves cytochrome c and the phospholipid cardiolipin. In this complex cytochrome c has its native axial Met(80) ligand dissociated from the haem-iron, considerably augmenting the peroxidase capability of the haem group upon H2O2 binding. By EPR spectroscopy we reveal that the magnitude of changes in the paramagnetic haem states, as well as the yield of protein-bound free radical, is dependent on the phospholipid used and is considerably greater in the G41S mutant. A high-resolution X-ray crystal structure of human cytochrome c was determined and, in combination with the radical EPR signal analysis, two tyrosine residues, Tyr(46) and Tyr(48), have been rationalized to be putative radical sites. Subsequent single and double tyrosine-to-phenylalanine mutations revealed that the EPR signal of the radical, found to be similar in all variants, including G41S and wild-type, originates not from a single tyrosine residue, but is instead a superimposition of multiple EPR signals from different radical sites. We propose a mechanism of multiple radical formations in the cytochrome c-phospholipid complexes under H2O2 treatment, consistent with the stabilization of the radical in the G41S mutant, which elicits a greater peroxidase activity from cytochrome c and thus has implications in mitochondrial apoptosis.