DNA Origami Nanopores: An Emerging Tool in Biomedicine

Keywords::

• biosensor
• DNA nanotechnology
• DNA origami
• protein nanopore
• resistive pulse technique
• solid-state nanopore

Natural systems have developed a precise and intricate machinery to preserve life. The complex mechanisms that living organisms employ to control the transport of ions and molecules across their lipid membranes represent a remarkable example. Membrane transport is of indisputable importance, as it is ultimately implicated in such important functions as energy production and protein synthesis. Many transport pathways are mediated by the presence of small holes (known as nanopores in biotechnology) in the lipid membranes based on active or passive proteins. Since living organisms so efficiently utilize these protein-based holes in their membranes to control the passage of biomolecules, why not employ them to obtain devices with potential interest in biotechnology and biomedicine?

A key achievement in biotechnology was the detection of single molecules utilizing nanopores. Two decades ago, Kasianowicz et al.[1] were able to detect single DNA and RNA molecules for the first time using a natural nanopore (α-hemolysin) inserted into an artificial lipid membrane by means of the resistive pulse technique [2]. The idea is easy and elegant: individual molecules pass through a nanopore leading to changes in the ionic current characteristic of the translocating molecule.

α-hemolysin (a Staphylococcus aureus pore-forming protein) has been by far the most employed biological pore due to its dimensions, stability and commercial availability. The main advantage of using these biological pores is their perfectly defined structure, which guarantees reproducibility of measurements and the possibility of introducing different chemical groups by mutating the DNA sequence of the protein pore. This can be used to increase specificity for the identification of molecules. However, these biological nanopores depend on a lipid bilayer, which can present challenges when the measurement requires extreme external parameters. Some of the limitations of biological nanopores have been overcome by the use of solid-state nanopores, first demonstrated by Li et al.[3]. They can be made using different materials such as silicon nitride, graphene or organic polymer membranes by drilling a hole with focused electron or ion beams, or by chemical etching [4]. A simpler and cheaper alternative is to use glass nanopores obtained by pulling glass capillaries [5]. In addition to their robustness, solid-state nanopores can be prepared to have the desired diameter, which is very difficult to achieve with biological nanopores.

Biological and solid-state nanopores have been used to detect a broad range of biomolecules via the resistive pulse technique [6]; DNA, RNA and proteins have all been detected at the single-molecule level using these nanopores. Furthermore, interesting physical and chemical features of these biomolecules have been revealed with this technology.

A current challenge in the field is the construction of more sophisticated nanopores that possess both precise sizes and adaptability for chemical modification similar to biological pores, together with the versatility in pore size and robustness provided by solid-state nanopores. These next-generation nanopores will expand the range of detectable molecules and even facilitate DNA sequencing, reducing the time and cost of current methods.

One possibility is to make solid-state nanopores more similar to natural pores. To this end, and inspired by natural systems, the surfaces of solid-state nanopores were coated with lipid bilayers, which maximized sensitivity in protein detection and improved the yield of DNA detection [7,8].

Another approach is to combine protein pores with solid-state nanopores in order to exploit the advantages of both kinds of pores. The Bayley and Dekker groups demonstrated the first hybrid nanopore based on a single α-hemolysin protein inserted into a silicon nitride pore. They engineered the natural protein by attaching a long dsDNA molecule that acted as a guiding strand to drive its insertion by electrophoretic force into the solid-state nanopore [9]. Since this has only been achieved with modified α-hemolysin (1.4 nm inner diameter), only ssDNA can be detected. Therefore, new strategies are required to expand the functions of these hybrid pores.

Inspired by this work, our group constructed a totally artificial hybrid nanopore utilizing DNA nanotechnology. DNA is an ideal molecular building material as its intrinsic properties enable custom-made designs with tailored shapes, sizes and functionalities, as well as very good stability. Indeed, nanotechnology has exploited the structural stability and programmability of sequences, as well as the predictability of the self-assembly of DNA to create complex entities by means of scaffolded DNA origami [10,11]. This method uses a long ssDNA scaffold that is folded with a number of short ssDNA staples into 2D and 3D structures predesigned using the open-source DNA origami software caDNAno [12].

In 2012, we presented the first hybrid DNA origami nanopore. With a funnel-shaped 3D structure and an internal pore of 7.5 × 7.5 nm, our DNA origami structure was capable of forming hybrid nanopores once inserted into a silicon nitride nanopore in a voltage-driven process [13]. This strategy required solid-state nanopores with diameters smaller than the size of the DNA origami entity in order to keep it inserted and prevent its translocation. The versatility of DNA origami is so great that we can create arbitrary shapes in order to adapt our DNA origami structures to solid-state nanopores with even bigger diameters. To this end, DNA origami objects that are only two layers thick (2D structures) were prepared. These 2D structures efficiently form hybrid nanopores when combined with solid-state nanopores with diameters of several tens of nanometers [14,15]. This allowed us to use glass nanopores made from pulled glass capillaries, which significantly reduces costs and device preparation time [15]. Furthermore, we have recently demonstrated the possibility of performing parallel measurements with a multichannel device in which up to 16 DNA origami nanopores can be formed simultaneously [16]. This achievement shortens data collection time by an order of magnitude, hence improving statistical confidence, and is a promising avenue for commercial development.

So far, these DNA origami nanopores have succeeded as detectors for a variety of biomolecules, including ssDNA [14,15], dsDNA [13–15] and proteins [14]. Moreover, they have some distinctive advantages over conventional nanopores. One can tune their characteristics and expand their applications as single-molecule detectors for the following reasons:

• Stability: DNA origami nanopores resemble protein pores in shape and size, but they are less sensitive to temperature, salt concentration or pH changes. Their robustness is quite convenient for expanding the detection range to harsher media conditions;

• Reversibility: by reversing the potential, ejection of the DNA origami from the solid-state nanopore is achieved [13–15], which allows the functionality of a nanopore sensor to be changed during an experiment. This property can be exploited to use DNA origami as a template for specific recognition in a mixture of analytes, which would also reduce the analyte concentration, increasing the sensitivity of detection;

• Tailored size: tuning the size of the pore is extremely easy with DNA origami. In connection with this, our group showed that a small modification in the size of the pore in the DNA origami structure influenced the degree of folding of linear dsDNA [15]. This opens up the possibility of investigating physical features in biomolecules, including protein folding, unfolding or aggregation detection. The latter is of special importance for investigations of neurodegenerative diseases such as Alzheimer‘s;

• Chemical modification: DNA origami allows for the introduction of active molecules at specific positions. This property has been used, for example, to attach a short ssDNA just at the pore of the origami structure so that it can selectively recognize complementary added ssDNA by the ionic current signal [14,15]. DNA sequencing may be feasible using these DNA origami nanopores by designing ssDNA sites with different sequences. The structures could be decorated with fluorophores or gold nanoparticles, which would enable detection using single-molecule fluorescence techniques [17].

Beyond their use as improved biomolecular detectors, DNA origami nanopores may be suitable for other biomedical applications. For instance, certain peptides or DNA fragments prone to modification by enzymatic reactions could be introduced into the DNA origami nanopore frame, and the enzymatic activity at the single-molecule level could be traced by combining ionic current measurements with optical tweezers [18]. Similarly, DNA origami nanopores can be fitted with a collection of molecular receptors to identify a wide range of matched ligands (e.g., exploring viral sequences). This could also be an innovative way to screen and select molecules for molecular target therapies in cancer research. Thanks to the versatility of the chemical modification, DNA origami structures can be altered to have an external hydrophobic nature that enables its insertion into lipid bilayers. This idea, which has recently been demonstrated for single-molecule detection purposes [19], highlights the compatibility of these DNA origami nanopores with lipid membranes. Thus, DNA origami nanopores could be incorporated in different natural systems such as lipid or polymer-based nanocarriers. Furthermore, as DNA origami nanopores can be designed and decorated ‘à la carte‘, they can incorporate any molecule that makes them stimuli responsive and, therefore, once inserted into the nanocarriers, they could act as remote-controlled pathways for drug release.

The possible responsiveness of DNA origami nanopores could serve to construct artificial systems that mimic complex natural architectures such as membrane proteins. These artificial ‘smart’ nanomachines could help us gain insight into the activity of their natural counterparts. These could even be directly inserted into living organisms to help or alter membrane protein dysfunction, helping to recover their normal functionality.

In conclusion, DNA origami nanopores represent an emerging and powerful tool for biomedical research. It is our task now to exploit the properties that DNA nanotechnology and the flexibility of DNA chemistry offer for the design of novel DNA origami nanopores with adaptable characteristics. Without doubt, the initial demonstration of their functionality as single-molecule biosensors shows that the revolution in basic science and biomedical research has just started and that DNA origami nanopores may enable an extremely large range of exciting applications for the future.

Financial & competing interests disclosure

The authors acknowledge support from an ERC starting grant and from Oxford Nanopore Technologies^® (www.nanoporetech.com). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Additional information

Funding

The authors acknowledge support from an ERC starting grant and from Oxford Nanopore Technologies^® (www.nanoporetech.com). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.