Thinking Outside the DNA Box

The most advanced machines today pale in comparison to the promises and possibilities of modern science and engineering. While large nuclear reactors, ion powered rockets, and self-driving cars amaze all of us, science is just beginning to marvel at the wonders of the microscopic world. Hearing the words ‘microscopic world’ it is only natural to imagine nasty bugs and bacteria. While these creatures are remarkable in their own right, if we go even lower to individual molecules, we see stuff that is nothing short of magic. In fact, the up and coming field of nanobiotechnology harnesses this ‘magic’ to develop solutions for clean energy generation, pollutant remediation, rapid disease diagnostics and effective treatments for diseases that were thought to be incurable. Let’s zoom in on to the poster child of nanobiotechnology, DNA origami.

 

 

What is DNA Origami?

 

DNA origami might sound funny to people familiar with either of the terms, let alone a complete outsider. It describes an approach to nanotechnology that uses DNA as the primary building block. While most of us might be familiar with DNA as the molecule at the centre of genetics and heredity, DNA also has several attractive physical properties that make it an ideal building material for nanoscale applications.

 

DNA or deoxyribonucleic acid molecules are double helices, i.e. they consist of two single strands of DNA polymers that wound around each other and are held together with hydrogen bonds. These DNA polymers are made up of individual units called nucleotides joined by phosphodiester bonds. Further, each nucleotide can be decomposed into a sugar, a nitrogenous base and a phosphate group. The sugars form the core of the nucleotide; nitrogenous bases are the famous ATGC letters (often seen in pop culture), which contribute the hydrogen bonds between complementary bases (A with T, G with C); and the phosphate groups give DNA a net negative charge. These bonds and charges keep the DNA molecule in a highly dynamic state – that is it can change its 3D shape to attain a state with the lowest possible energy, a postulation from thermodynamics. When this phenomenon is used in the creation of novel nanoscale structures, it is known as self-assembly. Scientists simply have to manipulate physical conditions around the DNA molecule and it will respond by changing its shape all by itself!

 

 

fig.1 The structure of deoxyribonucleic acid (DNA)

 

 

However, the structures you can create using this approach are limited. Think of a DNA molecule as a string. You can bend it to form several 2D shapes. Still, in 3D, many of the shapes lack stability, making it highly undesirable for any application where physical conditions are often far from ideal. A more advanced construction technique draws upon the ancient Japanese art of paper folding called origami.

 

 

Construction using DNA origami

 

The art in origami lies in folding a flat sheet of paper into a finished sculpture without cutting, using glue or leaving any extraneous marks on the paper. Origami practitioners use a small number of basic folds to combine them in a variety of ways. Thus, DNA origami refers to an assembly technique that folds one strand or single-stranded DNA (ssDNA) molecules into target structures. A single strand is a natural choice because it is more flexible and because a single strand means that the bases on that single strand are free to form hydrogen bonds with staple strands. These staple strands can help create complex shapes from the single linear strand of DNA, by pinning together parts of the same strand of DNA and stabilizing any folding. Since these staple strands are single-stranded DNA themselves, they can bear specific sequence motifs that allow them to clamp or pin down only desired sections of the base DNA molecule used for construction. In layman’s terms, think of these staples strands as a hypothetical hybrid between guided missiles and staple pins.

 

Let’s take a look at the stapling process. Simple hydrogen bonding between appropriate sequences doesn’t provide the desired kind of structural rigidity. This is where Holliday junctions come in. Holliday junctions, named after their discoverer Robin Holliday, form naturally when DNA in cells needs to be repaired. These junctions are highly dynamic, but at the same time are incredibly stable. DNA origami harnesses both these properties – the staple strands force the base DNA molecule to adopt the desired 2D or 3D structure by folding it and forming Holliday junctions at each of the fold sites. The figure below illustrates a Smiley Face made using this technique. Since the staples are internal, this technique helps achieve a clean exterior, much like the way no paper folds are visible in origami.

 

 

fig.2 A Holliday Junction

 

fig.3 The stapling process using Holliday Junctions

 

 

In their natural setting, Holliday junctions are subject to enzyme-induced cutting and rejoining as part of the DNA repair process. This lends itself well in the upcoming field of DNA origami to dynamic structures which can change shape due to movements in these Holliday junctions upon adding specific enzymes. In fact, current research in the field is focused on developing programmable nanorobots made entirely out of DNA!

 

A common strategy, used widely in the construction of these nanorobots, is called strand displacement. Strand displacement refers to the phenomenon where an external strand, with a greater degree of complementarity (or matching) to a particular strand of a DNA molecule, can knock out the other strand that this DNA strand is currently bound to. This phenomenon has been harnessed to create a nanoscale supply route – where the external strand carries a molecule of interest, and it hops along a predetermined path made up of other DNA. These DNA in the path are designed such that each successive DNA strand in the path bears greater complementarity to the external carrier DNA. Thus, the carrier hops from DNA to DNA and finally delivers the molecule of interest to its target. Strand displacement has also been used to create locked DNA boxes that can open or close upon providing a DNA-key. Let’s take a look at these fascinating DNA boxes below.

 

 

DNA Boxes

DNA boxes are like molecular safehouses designed to open and close upon introducing a variety of DNA and RNA keys. Large enough to fit microscopic virus particles, enzymes and even macromolecules, these boxes act as sensors and drug delivery systems at the molecular level. Among rest, a particularly exciting application of DNA boxes comes in the form of Logic gates, which work on Boolean algebra. These novel boxes can, in turn, be used to create large scale biological computers performing calculations inside a live cell.

 

 

fig.4 Process of forming a DNA Box from individual DNA Molecules

 

 

What are logic gates, you ask? A logic gate is a device that uses logic to solve a problem using a Boolean algebra system, which is based on two values – TRUE and FALSE or the binary digits 1 and 0, respectively. A Boolean logic gate takes one or two binary inputs and converts them into a single binary output. For example:

 

 

fig.5 Truth table for basic Logic Gates.

 

 

DNA logic gates are created using a combination of a novel fluorescent probe, DNA molecules and specific enzymes at the genetic levels. In the basic DNA Logic Gate design, the gate selectively detects a specific DNA structure found in telomeres that protect the end of a human chromosome. On binding to the DNA, the gate exhibits distinct light-emitting behaviour in the presence of nucleases, enzymes that degrade DNA. Such light-emitting properties are exploited to design novel logic gates that eventually combine to form the complex logic system. These systems use this light-emission (determined by photometric analysis) as a means of output.

 

 

fig.6 Truth table for an XOR Gate constructed using DNA strands

 

Danish researchers have now successfully constructed a universal DNA box combined with these DNA gates that can perform six out of seven types of Boolean operations – AND, NAND, OR, NOR, XOR, and XNOR. The designed gates can be incorporated into larger DNA structures by implementing chosen logic gates into a DNA origami box structure with sizes 18 x 18 x 24 nm3. The gates can control the box’s opening lid in a programmable way using Boolean or fuzzy logic. This system, the team believes, can be used for diagnostics by detecting microRNAs (markers of cancerous cells), and for treatment by including a drug inside the large DNA structure that can be released upon activation of the appropriate gate.

 

Another research group from Denmark has designed and synthesized self-assembled DNA complexes that sense two environmental signals and produce a fluorescent output corresponding to the operation of all six Boolean logic gates AND, NAND, OR, NOR, XOR, and XNOR. Moreover, the team shows that the designed gates can be incorporated into larger DNA structures by implementing the logic gate in the previously reported 3D DNA origami box.

 

In addition to the molecular logic calculations, the above DNA logic gates have been designed to operate with miRNAs as inputs. They can thus act as biosensors for specific cancer type detection. Combination of these cancer detection abilities and incorporation of anti-cancer drugs is possible via Denmark researchers’ DNA origami box. They can imagine the origami boxes containing a therapeutic agent – antibody, toxin, enzyme or nucleic acid – caged in.

 

In such a scenario, the DNA origami box would enter cells and only respond to the cancer cells that are programmed into the biosensor and subsequently deliver and release the drug to kill the tumour cells. This ‘smart’ targeted process would only respond to a cancerous cell and thus avoid the undesired side effects of the anti-cancer drug on healthy cells. Moreover, Israeli scientists have built and tested a gate to reduce the activity of the blood-clotting enzyme, Thrombin, which causes severe brain damage. This logic gate acts as a trigger, activated only when Thrombin is present and remaining bound and inert otherwise. Such a drug could be injected in advance, switching on only when required.

 

While single gates continue to expand the outreach of healthcare systems, scientists are now looking to create sequences of gates in order to perform much more complicated operations, the so-called Cascading gates. These sequential gates would be connected with common output-input DNA strands, i.e., the output of one acting as the input for next. This comes with its own set of hurdles, like the depletion of DNA strands over a long time. For all we know, the small DNA box is filled with infinite possibilities waiting to be opened by the right key.

 

Nanobiotechnology is a marvel of interdisciplinary research which calls on the principles of physics, chemistry and of course biology to develop applications that can be used in a whole host of different fields. Nanobiotechnology has been used in medicine for targeted drug delivery and diagnostics, in conservation science for pollutant remediation, in computer science and mathematics for solving large iterative problems using biological computers, etc. Like any new field, nanobiotechnology is not without its teething problems. One of the most prominent ones is these goals is the inability to scale up the production of these structures both in terms of the number of units as well as dimensional size. Advances in structural biology and biochemistry promise to advance this fascinating field from biokleptic to the biomimetic and even in enhancing biological processes. Research in these directions not only promises greater technologies but also promises to answer some fundamental questions about life itself.

 

 

References

  1. https://www.biosyn.com/faq/What-is-DNA-origami.aspx
  2. https://pubmed.ncbi.nlm.nih.gov/25565140/
  3. https://www.newscientist.com/article/dn18989-dna-logic-gates-herald-injectable-computers/
  4. Hong, F., Zhang, F., Liu, Y., & Yan, H. (2017). DNA origami: scaffolds for creating higher-order structures. Chemical Reviews, 117(20), 12584-12640
  5. Feagin, T. A., Maganzini, N., & Soh, H. T. (2018). Strategies for creating structure-switching aptamers. ACS Sensors, 3(9), 1611-1615.

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