In surface-based assays, a critical challenge lies in controlling the structure, orientation, and density of DNA probes at the biosensing interface, which play important roles in efficiency and kinetics of target capture , In this context, DNA tetrahedra allow control over spatial density and orientation of DNA probes, maximizing target accessibility and minimizing non-specific adsorption and lateral interactions between probes The programmability of DNA also opens up some entirely new sensing possibilities based on reconfiguration of DNA objects in response to a target sequence.
A few examples in DNA origami and almost all in DNA devices and assemblies sections take advantage of the programmability of DNA to offer interesting new sensing approaches that help convey the many possibilities of DNA nanostructures for sensing. Using DNA nanostructures in microRNA sensing opens up the possibility of engineered biological sensors with inherent biocompatibility, and also offers the longer-term possibility of integrating such sensors with living biological systems.
Some examples of this are already starting to emerge, where DNA nanotechnology is being used in living biological systems 22 , — Additionally, the programmable nature of DNA nanostructures enables unique and complex functionality. There are several such examples in this review including multiplexing , logic operations , and cascading events Relating back to Table 2 , we draw attention to a few common features in the surveyed DNA nanotechnology approaches that address common disadvantages of current approaches. For most of the surveyed approaches, a key advantage is that the microRNA does not need to be amplified, in contrast to some popular current approaches such as qPCR and next-generation sequencing.
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Relatedly, amplification typically exponential can pose a challenge for absolute and even relative quantification. Some of the DNA nanotechnology approaches discussed in this survey demonstrate direct detection of microRNA, thus eliminating any amplification-based errors. Many of the techniques have also demonstrated multiplexing, which is typically absent from qPCR and Northern blotting. Several also offer advantages in lowering cost or complexity compared to existing methods, sometimes enabling detection with little or no equipment, or with minimal training. Despite these advantages, some challenges still remain in these approaches.
The primary one among these may be formation of the nanostructures themselves, which typically requires thermal annealing of multiple components. While it can be straightforward, some structures and devices can require multiple steps, precise molar ratios, or long times that stretch over days. After structure formation, purification can sometimes be necessary to remove excess reagents or malformed structures.
In part due to the construction issue, some biosensing assays using DNA nanostructures are complex, requiring many different steps in assay preparation and execution. A few even appear to be unnecessarily complex, sometimes resembling a Rube Goldberg apparatus. These sensing assays also need to be integrated into workflows that can be performed by end users, necessarily moving away from AFM imaging that is common for verifying structures.
Since they are formed by base pairing of DNA, thermal stability and susceptibility to nucleases can both pose problems for certain applications. These challenges are solvable, and many of these aspects are currently being addressed by this relatively new field — Sensitivity and specificity tend to be the most widely reported metrics for microRNA detection.
For sensitivity, needs vary by application but some realistic limits can be defined for cellular extracts and biological fluids. This corresponds to sub-pM to sub-nM range concentrations assuming microliter volumes. In biological fluids, microRNAs are diluted in milliliter to liter-scale volumes, resulting in lower concentrations. Published clinical results of microRNAs in body fluids tend to be primarily in the fM range — , with some samples reportedly as low as 20 aM and as high as 20 nM It is useful to note, however, that microRNAs need not be detected at their native concentrations; body fluids typically collected at the milliliter scale can be concentrated to the microliter scales typical for biosensing assays.
Given these considerations, sensitivity is important but only to a degree; extreme sub-fM sensitivity may not be meaningful for microRNA detection. This assertion is further supported by the continued use of Northern blotting in microRNA detection, which has the lowest sensitivity of the common approaches typically pM-nM Cross-talk between microRNAs can be problematic, and due to the relative rarity of individual microRNAs in biological samples, cross talk from other RNAs can increase the background signal and reduce sensitivity.
While the ultimate specificity is to detect single-nucleotide variations, many applications may not require this level of specificity e. It is also worth pointing out that specificity is likely to be a solvable problem for most assays. Nucleic acid analogs such as peptide nucleic acid PNA and locked nucleic acid LNA with different base pairing affinities can also be added to tune or improve specificity , Thus, we contend that specificity differences between many assays are largely due to probe design and experimental conditions and not necessarily inherent to the different approaches.
Aside from sensitivity and specificity, there are several other aspects of sensing that are important for practical use that include cost, time, and complexity. These features can be underappreciated, which makes them both underreported in the literature and difficult to compare between different assays. Still, these features are important and can be critical for establishing an assay with a wide user base.
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Many assays that have been successful in reaching a wide user base build or improve on existing assays in a way that minimally disrupt existing workflows — This suggests that new assays should consider workflow and the likelihood of end users to have the resources and the skills to perform the assay. For clinical use, there can be a compromise on parameters such as sensitivity for rapid detection of biomarkers. For tools that are primarily developed for lab-based research, faster detection times are not as important, but eliminating the need for expensive equipment and creating easy-to-use methodologies are important especially in low resource settings.
This movement has produced interesting scientific tools such as centrifuges , microscopes , and water filtrations systems with ultralow cost. As some examples in this review have shown, DNA nanotechnology approaches can integrate into existing workflows and due to low cost synthesis can be very cost-effective.
The methods described in this article span the range from proof-of-concept explorations to more mature technologies with a history of progressive improvement. The authors appreciate the importance of experimentation with different methods for detecting microRNA, but it is not immediately obvious which of these techniques may become widely adopted for their intended purpose.
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In this section we discuss the relative merits of the different approaches, and provide some speculation on methods that may become more widespread. Most methods described in this article fall within the useful range of sensitivity and specificity for lab-based use, and some meet higher standards that might be needed for clinical use. As we mentioned earlier, improved sensitivity and specificity are attractive features, but we suspect that other features such as ease-of-use, cost, and equipment requirements may play a larger role in facilitating adoption.
So, where do these approaches sit among the landscape of existing tools? The niche of genome-wide screening of microRNAs is well established by next generation sequencing technologies and microarray.
To our view, none of the methods in this report are poised to be competitive in this area; to our knowledge none have described a strategy for scaling up to the genome scale with thousands of microRNAs. Instead, the methods described here focus on the area of targeted detection and quantification of microRNAs, occupying a niche with the more dominant technologies of qPCR and Northern blotting. These two established methods arguably occupy their own niches, with qPCR based on amplification and being relatively expensive and Northern blotting offering direct detection and being relatively inexpensive but cumbersome.
Each of these have their own features and limitations as mentioned in the section on current microRNA detection methods above, so new methods shown here may occupy the same niche or carve out a new one. Moreover, these DNA nanotechnology-based approaches have compared their performances to traditional methods mostly qPCR but a comparative analysis of DNA nanostructure-based assays is lacking.
Such analyses will be important in improving and adapting the techniques mentioned in this article, especially since the outcome of the assay can also be dependent on the methodology used even in traditional methods , The electrochemical approaches have largely demonstrated improved detection using DNA nanostructures to control the surface interface. This is a great benefit of the nanoscale control that comes with DNA nanostructures, though there is some counter-evidence showing that similar results can be obtained with chemical monolayers Overall, however, these DNA nanostructured electrochemical systems which were predominantly tetrahedra in the literature have impressively high sensitivity and specificity and have some potential for further adoption.
The main drawbacks we see are the multiple steps including several washes in the assay as well as the relatively costly electrochemical workstations that are used. However, recent work shows that electronics can be simplified, potentially enabling this type of approach to be used as a general lab or point-of-care approach It is worth noting here that although the electrochemical approaches used tetrahedra, it is possible that other shapes including DNA origami structures may provide similar results but were not reported in our literature search.
Obtaining and analyzing high-resolution images by fluorescence microscopy or AFM is costly in time and money. Furthermore, the resulting detection metrics from these techniques is not compelling enough to justify the effort. Technologies such as DNA-PAINT have found success in the context of adding new features to imaging techniques that are used in biology , but when such imaging is a prerequisite for detecting microRNA or another analyte it becomes less useful. There are, however, some applications where imaging is an important aspect of detection.
For example, some of these methods could be adapted for use when it is important to know the spatial distribution of RNAs within a biological sample such as a cell or a tissue. For many such applications in biology, imaging especially fluorescence imaging may already be part of the workflow and could potentially benefit from some of these DNA nanotechnology approaches.
The approaches that rely on optical changes may be the most likely in general to become widespread, due in part to the existing infrastructure in the life sciences and medicine to measure such changes. Fluorescent plate readers and gel assays are incredibly common in biology, so techniques that take advantage of these workflows have the lowest barrier to overcome in translating the technology. Our own technique of DNA nanoswitches , , falls into this category, with one of the most compelling features being integration with existing workflows.
Other examples that look promising in this category are fluorescence-based approaches such as those involving DNA-nanoparticle complexes , and the CD-based detection of the DNA origami cross Some of these techniques could also benefit from handheld or portable readers, of which there are several examples being developed in the literature In looking at potential widespread adoption of particular technologies, another aspect to consider is evidence of sustained growth of a strategy or its demonstrated use for multiple applications.
There are a few clear examples of this including use of the DNA tetrahedron to improve electrochemical sensing techniques 86 , and the DNA nanoswitches from our lab , , These techniques have also extended beyond microRNA detection and are used in detection of small molecules and proteins.
There are also a few examples where the opposite seems to be true, and individual labs have demonstrated several different approaches that are largely unrelated , , , This might illustrate more exploratory developments where leading candidates for further development have not yet emerged. A few methods have interesting features that could be especially useful for certain applications. One example is colorimetric detection, where the presence of certain microRNAs can be detected by eye This could be a useful way to quickly and efficiently validate biological samples, and could prove useful for point-of-care detection if the sensitivity can be improved or if it can find application where disease-related microRNAs are abundant.
Another example is live cell microRNA detection , , , which could provide new biological insights about microRNA activity inside living systems. Several of the methods also utilize the programmable nature of DNA to incorporate features such as multiplexed detection , , or logic operations , which are all steps toward microRNA biosensors with more complex functionality. With many of these strategies testing efficiency in biofluids such as serum and urine, the potential of DNA nanostructures for diagnosis and disease monitoring does not seem not far off.
Some readers may be interested to know about the commercial potential for the technologies described above. While DNA nanotechnology-based microRNA techniques have not yet been commercialized to our knowledge, the DNA nanotechnology field has progressed in the last decade to spin out related companies. Most related to biosensing is a start-up company called Esya which uses a DNA device to scan cells for lysosomal disorders.
The DNA nanoswitch from our group was briefly adopted by a now defunct start-up Confer Health for use in home-based fertility diagnostics. This feature also allows monitoring RNA profiles in cells for their spatial distribution. Recently, the tools to create DNA origami structures were also commercialized by Tilibit Nanosystems which provides custom-made DNA scaffold and staple strands depending on customer requirements.
This emerging commercial landscape suggests that these DNA nanotechnology approaches are starting to gain traction for certain applications. Nucleic acids are the natural biological sensors for microRNA, and in fact all of the major established methods for microRNA detection rely on nucleic acid probes hybridizing to all or part of their target microRNA.
Given recent advances in structural DNA nanotechnology that enable precise building and reconfiguration, it is not a huge conceptual leap to suggest that DNA-based biosensors may be combined with molecular devices to impart complex functions that were once purely science fiction. These aspirational goals would include sense-respond devices or sense-compute-respond devices.
Even setting these grand notions aside, we have shown here that there already exists a suite of tools enabled by DNA nanotechnology that offer some important advantages for microRNA detection. While we are not clairvoyant enough or brave enough to predict specific winners and losers among microRNA detection technologies, we can reasonably speculate that DNA nanotechnology approaches will play a role in the future of microRNA detection. Already these approaches are increasing the diversity of tools for microRNA detection, as well as demonstrating the power of DNA nanotechnology when form follows function.
The content is solely the responsibility of the authors and does not necessarily represent the official views of the American Heart Association or the NIGMS. Conflict of interest statement. Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide.
Sign In or Create an Account. Sign In. Advanced Search. Article Navigation. Close mobile search navigation Article Navigation. Article Contents. To whom correspondence should be addressed. Oxford Academic. Google Scholar. Jibin Abraham Punnoose. Lifeng Zhou. Paromita Dey. Bijan K Dey. Correspondence may also be addressed to Bijan K. Ken Halvorsen. Correspondence may also be addressed to Ken Halvorsen.
Cite Citation. Permissions Icon Permissions. Abstract MicroRNAs are involved in the crucial processes of development and diseases and have emerged as a new class of biomarkers. Open in new tab Download slide. Table 1. Open in new tab. Table 2. Advantages and disadvantages of the current methods used to detect microRNAs.
Lack of multiplexing and genome-wide coverage, biases and errors due to exponential amplification. Requirement of large amount of starting materials, radioactivity, less sensitive, time consuming, labor-intensive. Laborious, requires specialized skills and instruments, time consuming, non-specific. Requires specific probes and specialized equipment, data normalization is difficult and lacks reproducibility among various platforms.
Requires specialized equipment, skilled bioinformatician, complicated data analysis. Requires multiple enzymes including a nicking enzyme and probe design is complicated. Table 3. HRP: horseradish peroxidase. Search ADS.
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Our goal is to engineer the CAGE into a modular and programmable diagnostic system that for therapeutic means, regulates gene expression by tuning RNA polymerase activity.
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