While the world of chemistry rejoiced the discovery of new generation of green solvents called ionic liquids, little did biologists know that this would be a blessing in disguise that will help them overcome their long persistent problem of bio-molecule preservation.
Much of chemistry deals with the study of the interactions between different types of matter. Such interactions are easily imaginable between two or more fluids as the molecules of one fluid have the freedom to occupy the empty spaces between the molecules of the other. But what happens when the candidates involved are solids? Due to their physical constraints, two solids do not interact until you provide them a medium that facilitates such interactions. More often than not, these mediums are liquids called solvents, the excess liquid phase in which one or more solids (now called solutes) are dissolved to form a homogeneous solution. With a global market worth billions of dollars, solvents are an essential part of many sectors of the economy, including manufacturing, processing and transportation.
Despite being critical in addressing some of the most important problems in chemistry research as well as other challenges that society is currently facing, solvents also raise many environmental, health and safety issues. Firstly, most solvents come from finite sources, such as petrochemical or fresh-water resources. To add to this, the life-cycle of a solvent, right from its manufacture to its disposal, requires a considerable amount of energy input. Also, most of the traditional non-aqueous solvents, i.e., those other than water, such as benzene and toluene, tend to be toxic and evaporate easily (also called volatile). This makes them difficult to store and transport, and eventually poses threats of atmospheric pollution and accumulation in living organisms. Given these drawbacks, there are several challenges that need to be overcome for the short- and long-term usage of solvents.
In the pursuit of more environmentally friendly solvents, scientists stumbled upon a class of molecules called ionic liquids. Ionic liquids are non-volatile molecules that seem to be able to dissolve everything, and thus have the ability to replace the conventional manufacturing medium. Sounds magical, doesn’t it? But as the great science fiction writer, Arthur C. Clarke pointed out, “Magic is nothing but science that we don’t understand yet.” And this magic is what Prof. Sanjib Senapati’s group at the Computational Biophysics laboratory, Department of Biotechnology, are trying to unravel — figuring out what makes ionic liquids (or ‘green solvents’ as they have come to be known in recent times) a chemical possibility and gives them these special powers.
Ionic liquids have a very unique chemistry. They are composed of ions like all salts, but, unlike conventional salts, such as sodium chloride, which have ions of comparable sizes, in an ionic liquid, the ions differ a great deal in their sizes. The comparable sizes of the ions in a conventional salt lead to strong electrostatic interactions between them, allowing them to pack together uniformly, giving rise to a regular lattice structure. This does not happen in an ionic liquid, and therefore, they remain liquids at room temperature.
So, to put it in simple words, ionic liquids are salts that remain liquids at room temperature. Interestingly, since the ions involved in ionic liquids are large, a part of them is polar, or in other words, charged, whereas the other is non-polar. By rule, polar solvents only dissolve polar solutes, whereas, non-polar solvents only dissolve non-polar solutes. Now since ionic liquids have both kinds of components, they have the advantage of being amphiphilic – they can dissolve just about anything, even cellulose.
Prof. Sanjib Senapati, a theoretical chemist by training, focused on the application of chemistry in the field of biology during his post-doctoral days. Here, he got introduced to the fascinating field of ionic liquids and has since been studying them keenly. His lab, recently declared one of the best performing bioinformatics facility in India by DBT Biotechnology Information System Network, approaches research problems using state-of-the-art molecular dynamics simulations. By using structural parameters (properties of a molecule such as lengths, angles and planes between the atoms) to define the interactions between molecules and computer codes to replicate the conditions of a lab experiment (such as temperature, pH, salt concentration and atmospheric pressure), they are able to simulate what goes on inside a test tube on a computer screen. The advantage of simulations over test tube is quite evident; MD simulations allow one to see exactly what is happening at the molecular level in the course of a reaction.
Debostuti, one of the graduate students working on ionic liquids at Prof. Sanjib’s lab is amused at the naivety of our query when we ask her, “But what have ionic liquids got to do with biology?” She replies, “Well, there is much more to ionic liquids than meets the eye. After ionic liquids became a super hit in the manufacturing industry, the next big step for the industry was quite obvious — to try their hands on ionic liquids in enzymatic catalysis.” Catalysis is the speeding up of a chemical reaction in the presence of an additional participant called the catalyst. When bio-molecules act as such catalysts, the bio-molecules are termed as enzymes and the reaction is termed as enzymatic catalysis. She then continues, “But, to be able to carry out an enzymatic reaction in ionic liquids, it becomes important that the enzymes themselves are stable in ionic liquids first. That is where the stability of bio-molecules in ionic liquids came into the picture.”
Stability of bio-molecules such as DNA and proteins is one of the most critical issues that research in bio-sciences faces every day. These bio-molecules are delicate entities, with very short half-lives when stored at room temperature in water, their natural solvent. This is either due to hydrolysis (explained later) or the action of degrading enzymes, both of which cause their breakdown. Long-term preservation of bio-molecules is carried out by storing them at refrigerated conditions of -20ºC to -80ºC, temperatures which reduce the rate of hydrolysis and render the degrading enzymes inactive. Common sights in all biology labs are rows of refrigerators which house many precious samples. The failure of these refrigerators can lead to very significant losses – sometimes the whole career of a scientist is lost, sometimes irreplaceable samples such as rare cancer tissues are lost and sometimes, arrays of samples for future experiments are lost. The maintenance of samples at such ultra-low temperatures is therefore crucial and adds significant costs and concerns to research. It is in fact ironic that this article was written while guarding a -80ºC freezer which threatened to go off during the Chennai floods power outage.
In such a scenario, the evolution of ionic liquids to the latest generation of bio-compatible ionic liquids ushered in the promise of an economical and hassle-free solution to this age-old problem. Research in this field was pioneered by Dr. Prabhakar Ranganathan, an alumnus of IIT Madras, and his group at the University of Monash, Australia. They found that when DNA is stored in these bio-compatible ionic liquids, the structural features of DNA were maintained even after six months of storage at room temperature as opposed to a few weeks in water!
DNA is one of the three common bio-molecules (DNA, RNA and proteins) and functions as the instruction manual for our body. These instructions are, however, not written in the 26 alphabets of English, but in a code which has only four alphabets — Adenine, Cytosine, Guanine and Thymine — collectively known as nitrogenous bases. As can be seen in the figure, DNA has a very interesting structure. It is a double helix formed by two anti-parallel single strands made up of these nitrogenous bases. The backbone to these nitrogenous bases is provided by alternating sugar and phosphate groups. These two single strands are held together by hydrogen bonding, or in other words, the attraction between the positively charged hydrogen atom on one strand and another negatively charged atom such as nitrogen, oxygen or fluorine on the other. The two strands twist around each other with an offset pairing, resulting in the formation of two kinds of grooves – major and minor: these are structurally opposite to one another and run alternately along the entire length of the DNA. This double helix of the DNA is provided structural support by a single layer of water molecules, referred to as the ‘spine of hydration’, which remain hydrogen-bonded to the DNA in its minor groove. Another way in which water lends structural support to the DNA is via the ‘cone of hydration’ — small clusters of water that surround the backbone of the DNA, the alternating sugar and phosphate groups.
Interestingly, the same water molecules fail to stabilise the DNA when it comes to its covalent bonding. They disrupt these bonds, breaking the DNA down to smaller fragments which are again amenable to further break down right up to the monomeric components, the nitrogenous bases. This is called hydrolysis. In order to circumvent hydrolysis, two strategies are attempted in DNA storage — it is either stored in a dry state, i.e., it is dehydrated, or in water but frozen, so that the rate of hydrolysis diminishes rapidly. However, both the methods are not foolproof. With every dehydration attempt or a freeze-thaw cycle, DNA, being made up of extremely long, thin strands, tends to break, leading to structural damage.
From the study by Dr. Ranganathan sprouted many unanswered questions that excited Prof. Sanjib and group. They figured out that MD simulations could serve as highly valuable tools to explore what ionic liquids are doing to the DNA to be able to have such a stabilising effect on it. They found that on introducing ionic liquids into a setup of DNA surrounded by water, the ionic liquid molecules gradually start replacing water, and after about 100 to 200 nanoseconds, there are very few water molecules left surrounding the DNA (Fig. 1).
Now, this was both a cause for relief as well as worry because water is both a friend and a foe to DNA. Interestingly, ionic liquids replace both the spine and the cone of hydration in the DNA. This should actually lead to the entire DNA structure collapsing; but with ionic liquids it does not, because of their ability to hydrogen-bond with the DNA the same way that water does. The result is the formation of a spine and a cone of ionic liquids that now support the DNA structure instead of a spine and a cone of hydration (Fig. 2). So, we have something that gets rid of most of the water and, therefore, reduces the rate of hydrolysis to almost negligible while at the same time mimicking the supporting role of water. It’s a win-win situation!
At this point, Debostuti acknowledges one of the drawbacks of using MD simulations for a study like this – that a technique with a timescale of nanoseconds cannot really predict whether the DNA will remain stable for years to come or not. So, to substantiate the findings of their MD simulations, the group stored the DNA in a large number of ionic liquids at different concentrations and at room temperature. The DNA remained stable even after a year of storage at room temperature.
Despite such positive findings, the use of ionic liquids as storage molecules is a field that has not yet been embraced by the scientific community. A major reason for this is that while ionic liquids have been found to be fantastic hosts for DNA, their track record with proteins has been patchy. Protein structure involves a great amount of diversity and complexity as compared to DNA structure. Given this, while researchers are now able to recommend different ionic liquids for storing different kinds of proteins, they have still not reached a stage where they have been able to pinpoint a single universal ionic liquid as being apt for protein storage. Nevertheless, the search is surely on.
More importantly, the field of study is still in its infancy, where people have figured out the pros of the technique, but are still not sure about the cons — such as any likely adverse effects due to the long-term storage of bio-molecules in ionic liquids. Debostuti’s current job is screening out as many ionic liquids from as many different classes as possible for their potency in stable storage of DNA. The goal is to be able to make a confident statement one day: “Yes, all ionic liquids are good for long-term DNA storage.”
Meanwhile, the group is also exploring what happens to DNA in an ionic liquid under conditions of environmental stress such as high temperature, which is known to melt DNA. The results of their preliminary study are exciting — the melting temperature of DNA in ionic liquids is way higher than its melting temperature in water, which means that DNA in ionic liquids is resistant to higher temperatures.
Besides all this, Debostuti also sees applications of ionic liquids in many other fields, including extraction and separation technologies and, of course, the hot topic of drug delivery. However, the high viscosity of ionic liquids and their lack of specificity are proving to be challenges in the way, which scientists are trying to overcome in order to help these miracle solvents make their mark.
Debostuti is now on the verge of her thesis submission. She admits that when she was offered the project initially, she was sceptical; DNA was never her ‘comfort-zone’, she was more of a ‘protein’ person. But after five years of exciting work on ionic liquids and DNA, she seems to have changed her mind. She says in a very cheerful tone, “Today, if somebody asks me what I would like to do my post-doctoral research on, I would say DNA nanotechnology. Because, now, I can only think of DNA and there is so much more to be done!” And we wish her all the best with that!
Prof. Sanjib Senapati received his M.Sc. degree in Physical Chemistry from University of Calcutta, Kolkata. He obtained his PhD from IIT Kanpur. He has worked as a Post Doctoral Fellow at University of North Carolina, Chapel Hill and University of California, San Diego, USA. In 2005, Prof. Senapati joined the Department of Biotechnology, IITM where he is currently a full Professor. Apart for ionic liquids, his other research interests include identifying drug targets and designing novel drugs for HIV and heart disease.
Kiranmayi is a PhD student in the Department of Biotechnology. As part of her thesis, she studies the involvement of DNA changes in the development of hypertension and diabetes in Indian populations. A great admirer of the English language ever since she can remember, she aspires to be a technical writer after she completes her PhD and tries to find as much time as possible between her late nights at lab and her research seminars to keep up with this passion.
Cover image : A watercolor painting of DNA, courtesy Caitie Magraw Art