The Evolution and Application of a Novel DNA Aptamer Targeting Bone Morphogenetic Protein 2 for Bone Regeneration.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.

A non-enzymatic test for SARS-CoV-2 RNA using DNA nanoswitches.

The emergence of a highly contagious novel coronavirus in 2019 led to an unprecedented need for large scale diagnostic testing. The associated challenges including reagent shortages, cost, deployment delays, and turnaround time have all highlighted the need for an alternative suite of low-cost tests. Here, we demonstrate a diagnostic test for SARS-CoV-2 RNA that provides direct detection of viral RNA and eliminates the need for costly enzymes. We employ DNA nanoswitches that respond to segments of the viral RNA by a change in shape that is readable by gel electrophoresis. A new multi-targeting approach samples 120 different viral regions to improve the limit of detection and provide robust detection of viral variants. We apply our approach to a cohort of clinical samples, positively identifying a subset of samples with high viral loads. Since our method directly detects multiple regions of viral RNA without amplification, it eliminates the risk of amplicon contamination and renders the method less susceptible to false positives. This new tool can benefit the COVID-19 pandemic and future emerging outbreaks, providing a third option between amplification-based RNA detection and protein antigen detection. Ultimately, we believe this tool can be adapted both for low-resource onsite testing as well as for monitoring viral loads in recovering patients.

Aptamers as Functional Modules for DNA Nanostructures.

Watson-Crick base-pairing of DNA allows the nanoscale fabrication of biocompatible synthetic nanostructures for diagnostic and therapeutic biomedical purposes. DNA nanostructure design elicits exquisite control of shape and conformation compared to other nanoparticles. Furthermore, nucleic acid aptamers can be coupled to DNA nanostructures to allow interaction and response to a plethora of biomolecules beyond nucleic acids. When compared to the better-known approach of using protein antibodies for molecular recognition, nucleic acid aptamers are bespoke with the underlying DNA nanostructure backbone and have various other stability, synthesis, and cost advantages. Here, we provide detailed methodologies to synthesize and characterize aptamer-enabled DNA nanostructures. The methods described can be generally applied to various designs of aptamer-enabled DNA nanostructures with a wide range of applications both within and beyond biomedical nanotechnology.

Self-Assembly of DNA Tiles with G-Quadruplex DNAzyme Catalytic Activity for Sensing Applications.

DNA tiles form through self-assembly of a small number of DNA strands that interact through basic repeated interactions, allowing the growth of nanoscale structures seeded by molecular inputs. If an approach for catalytic signal amplification can be integrated into the resultant nanostructure, then one can anticipate biosensing or diagnostic applications mediated by DNA tile self-assembly. Here, two-dimensional DNA tiles with split quadruplexes were designed as diagnostic tools for nucleic acid sensing without the use of protein enzymes. The presence of a target sequence leads to formation of extended microscale structures with arrayed multiple G-quadruplexes across the tile plane, with catalytic activity coupled to a colorimetric reporter. Such a mechanism has potential for low-cost signal amplification using unmodified DNA without the use of protein enzymes for biosensing.

Aptamer-Enabled Nanomaterials for Therapeutics, Drug Targeting and Imaging.

A wide variety of nanomaterials have emerged in recent years with advantageous properties for a plethora of therapeutic and diagnostic applications. Such applications include drug delivery, imaging, anti-cancer therapy and radiotherapy. There is a critical need for further components which can facilitate therapeutic targeting, augment their physicochemical properties, or broaden their theranostic applications. Aptamers are single-stranded nucleic acids which have been selected or evolved to bind specifically to molecules, surfaces, or cells. Aptamers can also act as direct biologic therapeutics, or in imaging and diagnostics. There is a rich field of discovery at the interdisciplinary interface between nanomaterials and aptamer science that has significant potential across biomedicine. Herein, we review recent progress in aptamer-enabled materials and discuss pending challenges for their future biomedical application.

FRET-Mediated Observation of Protein-Triggered Conformational Changes in DNA Nanostructures.

DNA origami is a powerful technique, which allows virtually limitless 2D or 3D nanostructure designs to be constructed from DNA strands. Such nanostructures can even include programmable nanorobots, which are able to respond to the environment in predetermined ways. DNA aptamers hold particular promise as interfaces, which can enable proteins, peptides, and other non-nucleic acid biomolecules to trigger conformational changes in DNA nanostructures for diagnostic, biosensing, or therapeutic applications. Here, we provide the methodology for FRET-mediated observation of aptamer-triggered conformational change in a DNA origami box nanostructure. The method described can, in principle, be adapted to a wide variety of experimental circumstances where the DNA nanostructure conformational change is mediated by molecular or environmental cues.

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.

DNA Aptamers for the Functionalisation of DNA Origami Nanostructures.

DNA origami has emerged in recent years as a powerful technique for designing and building 2D and 3D nanostructures. While the breadth of structures that have been produced is impressive, one of the remaining challenges, especially for DNA origami structures that are intended to carry out useful biomedical tasks in vivo, is to endow them with the ability to detect and respond to molecules of interest. Target molecules may be disease indicators or cell surface receptors, and the responses may include conformational changes leading to the release of therapeutically relevant cargo. Nucleic acid aptamers are ideally suited to this task and are beginning to be used in DNA origami designs. In this review, we consider examples of uses of DNA aptamers in DNA origami structures and summarise what is currently understood regarding aptamer-origami integration. We review three major roles for aptamers in such applications: protein immobilisation, triggering of structural transformation, and cell targeting. Finally, we consider future perspectives for DNA aptamer integration with DNA origami.

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.

The Three S's for Aptamer-Mediated Control of DNA Nanostructure Dynamics: Shape, Self-Complementarity, and Spatial Flexibility.

DNA aptamers are ideal tools to enable modular control of the dynamics of DNA nanostructures. For molecular recognition, they have a particular advantage over antibodies in that they can be integrated into DNA nanostructures in a bespoke manner by base pairing or nucleotide extension without any complex bioconjugation strategy. Such simplicity will be critical upon considering advanced therapeutic and diagnostic applications of DNA nanostructures. However, optimizing DNA aptamers for functional control of the dynamics of DNA nanostructure can be challenging. Herein, we present three considerations-shape, self-complementarity, and spatial flexibility-that should be paramount upon optimizing aptamer functionality. These lessons, learnt from the growing number of aptamer-nanostructure reports thus far, will be helpful for future studies in which aptamers are used to control the dynamics of nucleic acid nanostructures.

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.