Combining Nanotechnology and Molecular Recognition to Unravel the World’s Biggest Agriculture and Food Challenges

In this interview paper, Dr. DeRosa describes aptamers and their potential as low cost and robust affinity reagents in smart farming, promising exudate aptamers that have been selected and characterized, and early prototypes of smart-release nutrient coatings, to provide the right measures at the right time.

What was your motivation to work in DNA nanotechnology?
The concept of using DNA nanotechnology to help solve problems in agriculture and food has been around now for a few years. A quote from 1994, from a Nobel Laureate chemist, Roald Hoffmann described “DNA as Clay”; this served as inspiration for this whole field. The concept is that DNA, nucleic acids, have an incredibly optimized chemistry, thereby, utilizing them as a material to sculpt new things, beautiful things, unnatural things, and most importantly, useful things.

The concept of utilizing DNA for its properties, to do some interesting things that have nothing to do with genetics or biology in any way. This is the inspiration for the DNA technology work that we're doing in our lab, and in many other labs around the world.

How is your work digging into DNA nanotechnology?

At Carleton University, we use DNA as a synthetic type of receptor or pocket for target molecules, and these special pieces of DNA are called aptamers. We’re interested in this field, specifically, synthetic nucleic acid, or synthetic DNA, because they can bind target molecules with the specificity and affinity that people recognize out of natural receptors - things like antibodies. We're using the synthetic surrogate, for all the reasons why synthetic things can sometimes be easier to work with.
For example, synthetic things can have a better shelf life, less batch to batch of variation, lower cost to solve some important problems, especially agriculture.

The red ribbon (Figure 1) is a strand of DNA, a single stranded piece of DNA that is folded up into what’s called a “hairpin structure”. At the top, a blue molecule is nestled into a pocket that's folded over on top of it, making a nanoscale shape. This is an example of an aptamer that is binding to a small molecule, in this case, an amino acid. This structure allows them to either interact with the target pocket, such as proteins or to form a receptor structure - a pocket that a molecule can bind to. It’s like a lock-and-key, where the red structure is the aptamer - the lock, and the key is the one blue molecule that fits into that structure. This is the foundation that gives this work so much potential.

Why do you believe this technology is so important to advance?

We believe this technology can solve all sorts of problems in food and agriculture. These structures of DNA are easy to make and they're relatively inexpensive, but they bind with the affinity that you would see in things like antibodies, which are more expensive and harder to produce.

They're also very specific. Having an aptamer that recognizes a certain target molecule, we can make very small changes in the structure of that molecule which can turn off binding. The example that we utilize to communicate about the specificity of aptamers, was discovered at 20 years ago; an aptamer for an asthma drug called theophylline.

In a structure of theophylline (Figure 2), one could take the hydrogen molecule and replace it with a methyl. It’s a small chemical change but it transforms theophylline to caffeine. The aptamer that recognizes theophylline, is basically shut off from binding to caffeine even though they're almost identical in structure. It binds 10,000-fold less tightly to caffeine even though most of the structure is basically the same. This level of specificity proves very important when you're trying to make a sensor, like for the detection of theophylline and don’t want to detect caffeine. We don’t want to be tricked into thinking that caffeine is theophylline, so this can be used as a receptor, as the basis for my sensor, to make sure we have the specificity we are looking for. This is just one example of a real-world use of this concept, which highlights the importance of specificity and demonstrates our belief in tiny changes having big impacts.

So where do you start in developing technology towards precision agriculture?
We are doing a lot of discovery work in food and agriculture, like looking at toxin molecules towards food safety and we are finding promising aptamers in that space. The discovery process so important to this work, because unfortunately, we can't design aptamers yet, so we need to screen for them and find them. The next step of our pipeline is to characterize the aptamers and we compared a suite of techniques to find the best workflow, to gain the best understandings of how aptamers work.

We are not just talking about improving point in time agriculture outcomes, we are working towards ongoing precision agriculture and “Smart Farms” (Figure 3). We start by taking measurements of soil to inform us on salinity, pH temperature, and we transmit that information back to a central Hub. That information will enable targeted decisions and changes, with aims to best support the needs of the crop and optimize the output of the farm.

We believe our technology fits in by utilizing that data, that knowledge to take modern agriculture and smart farms to the next level. Once we have a sense of some basic factors of the soil like pH, the temperature, the salinity, or the basic information that was acquired, we can do much more than control basic factors or conditions of the soil.

Signals can come from the roots of crops, they’re called exudates and if we could receive those signals and grab on to those signal molecules, we could use that to make sensors that could then transmit back to a central hub or let the farmer know that this crop needs a particular nutrient or condition change.

We could take this data and incorporate it directly into a product like “Smart Fertilizer”. We would develop a coating for the fertilizer that contains an aptamer, the aptamer receives the signal, and now the coating allows for the precision release of the fertilizer or whatever that crop needs at the time. We call this a signaling cascade. It might sound like science fiction, but this is the idea we’re working on, and it’s the backbone of this body of work.

We also have other ideas for how we think we can use aptamer technology to facilitate all kinds of advances in agriculture and food. For example, we could use aptamers as a delivery mechanism for materials like herbicides, pesticide, basically applying whatever material is needed into a plant surface or into a certain species in a targeted way.

We may be able to use aptamers to protect against stresses like heat stress or use aptamers as a part of a sensor or a part of an assay that the farmer could use to inform their decision-making. In an example of toxins, or mycotoxins that can be present from commodities and making a simple lateral flow assay could inform if there's toxin present in a crop or not.

How do we find aptamers?

As much as we would like to clearly see a target molecule and identify a sequence of DNA that would be the best aptamer for that target, we don't know enough about how aptamers work to be able to do that.

Since the 1990s, researchers have been working on a technique called “SELEX” (Figure 4), the -systematic, evolution of ligands by exponential enrichment. SELEX is the process for finding aptamers.

Here’s how it works. We start with an initial library of samples, millions, billions and trillions of random sequences of DNA. Our aim is for one of those sequences to fold up, to make that pocket mentioned earlier; it’s what we need to bind the target we’re interested in. However, most of the sequences do not have this fold, they're not doing the job we want to do. We are basically working to find the needle in the haystack, that one red sequence in the sea of all the other sequences.

To find this aptamer, we incubate that one red sequence sample and that library with the target of interest and partition the very few sequences that stick to that target. We can control the incubation step and the partitioning step and then once we've partitioned, and we grab all the binders that we are able to recognize as the target of interest, we’ve significantly narrowed down the sequences. We do an amplification step which allows us to make lots of copies of that DNA sequence and then that information goes back into our library. We have now enriched our library with binders, and we repeat the process repeatedly, until we think we've found the right aptamer for the job - whatever the job is.

What kinds of controls or programming can we do with synthetic aptamers over natural receptors?
There are some great controls in this process that allow us to maximize the advantages of this technology. Since the library has synthetic receptors, it is not like antibodies where we are working with natural receptors, so we can do chemistry to the library. We can make a whole host of modifications, like adding fluorophores or things that glow, we can put redox active probes, change the design of the library, and add more of a certain DNA base. We can play around with these things to help us have the best possible outcome. It's hard to do this in a natural system.

We can also control the binding step by adding the target to our library of DNA sequences, we keep what binds and we throw away what doesn't bind. We can control it because it's done on the bench. If an aptamer is needed which works at a certain pH, a certain salinity, a certain temperature; we can ensure that the experiment is happening under those conditions.

Another important control is the partitioning step, where the separation happens of binders from non-binders. Controlling this step enables us to get really good binders, moderate binders, or selective binders, all depending on my application.

How have aptamers been used to detect crop health?

Some applications of this technology are seen in examples like dipstick or strip tests, which utilize aptamers to signal that something is present or not. These strip tests can look at mycotoxins, toxins that are derivatives of mold and present in foods like corn and wheat. If the target is present, we will see a change of color at the test line. Some of these tests have turn-off signals, so a dark color on the strip disappears to signal more of the toxin is present. Some have turn-on sensors, so as there are more toxins than the test line, the colors become darker.

We’ve made these strips and we've used them on real samples in collaboration with some great researchers at the University of Guelph Ridgetown and another great researcher David Miller at Carleton University. We can show that a very simple strip test can detect a relevant toxin in real samples down to .5, parts per billion (ppb). Detecting if something is present or not is a pretty basic use of aptamers, however, our really exciting work is developing how aptamers can respond to signals coming from crops, as they grow and require nutrients to grow.

How can aptamers respond to crop signals?
Every plant has a root system. As seen in Figure 6, the blue circles are moving in at the roots – they are root exudates. Root exudates are chemical signals which are constantly produced by the plant and are being released into the environment around the roots, known as the rhizosphere. There are many studies into the understanding of root exudates, but the main point we focus on here, are that these signals can help us unlock a better understanding of the needs of the crop.

A Real pioneer in this area is our collaborator Carlos Monreal, scientist at Agriculture and Agrifood Canada (AAFC). He has decoded some of these exudate signals related to nitrogen needs and nitrogen uptake, as this is one of the important nutrients for any crop.

The crop is sending out signals into the environment that it’s in need of nitrogen. If we can understand more of this 2-way communication, we can deliver only the nutrient being requested by the crop in very specific amounts.

Why are we so interested in exactly the right amount?
Using the example of nitrogen, it’s a nutrient, but it is also technically a pollutant when it is not taken up into the crop. When excess nitrogen leaches or gets washed away into waterways, it can cause nitrification or toxicity to the crops and potentially cause environmental damage. Therefore, we want to send only the right amount of nitrogen to the crop that needs it.

Getting this right can mean protecting the environment, it can mean great economic advantages to farmers as they minimize waste, and this approach can mitigate the risk of over fertilizing and harming crops.

To achieve just the right amounts of nutrient to just the crops that need it, we can create a coated fertilizer, or a nano-fertilizer, where the coating contains aptamers. When the nutrients are untouched by any root exudates, the coating protects the nutrients and the nutrients are not released. When the root exudates signal for nutrients, this is recognized by the aptamer and a change in the coating occurs. The coating becomes more permeable, and nutrients start to come out. The bulk of the release happens in a burst, only when the signal is received by the aptamer. It has taken years to work through this concept and make it a reality.

How do analytical testing techniques fit into this concept and process?

In order to make this a reality, we needed to know what the signals are or what we could exploit, decode or respond to, to prompt the nutrient uptake, in this case nitrogen. Our objective was to find a signal for when an exudate is detected, that’s when nitrogen is needed. To find this out, Dr. Carlos Monreal and the research team did a series of extensive experiments on wheat and canola, especially by analyzing the mixture of chemical compound that was present around the roots throughout different stages of the growth cycle of these crops.

This is where we start to unravel some of the relationship between nitrogen and uptake. We utilized analytical technologies like Py-FIMS (pyrolysis-field ionization mass spectrometry), GC-MS (gas chromatography mass spectrometry), ESI-MS (electrospray ionization), and LC-ESI-MS/MS (liquid chromatography electrospray ionization mass spec) to analyze soil solutions weekly in crops like wheat and canola. We characterized for composition, enzyme activities, microbial biomass, and more. We found patterns emerging in the exudates that track with concentrations of nitrogen uptake in the crop roots at different stages of growth. (Figure 7)

What’s next?
We are happy to say that we are continuing this work and Dr. Emily Mastronardi (a graduate from my professorship) is pioneering new work with myself, Carlos Monreal and Kathryn Cyr and has recently published in the Journal of Agricultural Food Chemistry, on biosensing with a serine aptamer titled: Selection of DNA Aptamers for Root Exudate l-Serine Using Multiple Selection Strategies

By combining all this knowledge and the findings of the studies described here, we have developed a signal-triggered smart fertilizer prototype. The prototype is not a nanoparticle, it’s a standard urea pellet with a special aptamer coating applied. In a lab setting, we have demonstrated that the coating on the urea pellets protects the pellet, but when we have a certain concentration of serine, or amino acids in the soil, there is a significant release of the nutrient from the coating. This is exactly what we needed to prove this exciting concept of smart fertilizers and we believe it will help us make great strides in addressing some of the biggest challenges in agriculture and food.

Resources:

1. FIGURE 1: Biochemistry 2000, 39, 5, 946–954, Publication Date: January 13, 2000, https://doi.org/10.1021/bi9915061
2. FIGURE 2: SCIENCE, 4 Feb 2000, Vol 287, Issue 5454, pp. 820-825 • DOI: 10.1126/science.287.5454.820
3. FIGURE 5: Analyst, 2018,143, 4566-4574, https://pubs.rsc.org/en/content/articlelanding/2018/an/c8an00963e
4. FIGURE 7: Carlos Monreal, scientist at Agriculture and Agrifood Canada (AAFC)