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1 Introduction

Nucleic acids are among the most important molecules for living creatures. Nucleic acids store the information about how to organize the body in early development, how to build the proteins needed for an organism to function, and the information that needs to be passed down to the next generation. However, nucleic acids are not simply storage devices. They can be dynamic as well, actively altering cell products and playing significant roles in gene expression. Some nucleic acids use these properties to rob life from other organisms, forming diseases that wreak havoc from the smallest to the largest living things.

Scientists have been studying nucleic acids for nearly 200 years. An enormous wealth of information about them has been gathered in that time. This chapter covers a portion of that history and how those early studies provide us with important information for modern lab work with nucleic acids.

2 A Brief History of the Measurement of Physical and Chemical Properties of Nucleic Acids

2.1 DNA

The earliest measurements of the physical and chemical properties of DNA were done by its discoverer, Friedrich Miescher. In his studies of pus cells in the 1860s, he discovered an unusual precipitate that represented a molecule unknown to science. This precipitate was DNA, which he named nuclein because it came from the cells’ nuclei. Miescher performed elementary analyses on his nuclein samples; he heated it along with various other chemicals which would react with the different elements that made up nuclein. This allowed him to determine the chemical makeup of the nuclein structure. It contained the usual elements of organic molecules such as carbon, oxygen, nitrogen, but also a large amount of phosphorus which suggested nuclein was not a protein. Miescher also determined that nuclein had a large molecular weight when attempts to diffuse it across a sheet of paper failed [1].

Following Miescher’s death, Albrecht Kossel continued to investigate the properties of DNA through hydrolysis analyses. In a hydrolysis reaction , chemical bonds are cleaved through the addition of water, breaking polymers down into their monomers [2]. By analyzing the pieces left over from hydrolysis of nuclein, Kossel was able to discover the identity of the four nitrogenous bases that make up DNA: adenine, guanine, thymine, and cytosine. Kossel’s student Ascoli discovered uracil working in his lab [3]. This information about the identity of the bases of DNA led Phoebus Levene to incorrectly form his hypothesis that the bases formed a tetramer that repeated itself, killing scientific interest in DNA. The tetramer hypothesis led scientists to believe that DNA could not have the variability necessary to code for genetic information and its study declined. It was not until Erwin Chargraff disproved the tetranucleotide hypothesis that interest in DNA was rekindled in the mid-twentieth century [1].

As interest in DNA spread throughout the scientific community after Chargraff, a variety of methods were employed to elucidate its properties. Electron microscopy was used to determine its diameter. Optical measurements performed by Torbjörn Caspersson, Einar Hammarsten, and Rudolf Signer determined that the bases were perpendicular to the axis of the helix. J.M. Gullan and D.O. Jordan performed amperometric titrations, a method which uses electrical current to determine the equivalence point, to determine that hydrogen bonding kept the DNA helix together. William Astbury made use of X-ray crystallography to study the structure of DNA, followed by Maurice Wilkins, Raymond Gosling, and Rosalind Franklin. Watson and Crick put all this information together and concluded that the structure of DNA was a double helix. X-ray diffraction data would later show that DNA could exist in many different conformations other than the first one that they had discovered [4].

By the time the structure of DNA had been discovered, it had also been shown that the function of DNA was that of a carrier of genetic information. This relationship between DNA and genetic material had been suggested as early as 1881 by botanist Eduard Zacharius [1]. It was Oswald Avery, Colin MacLeod, and Maclyn McCarty who provided the first proof that DNA contained the genetic information in 1944. They studied this property of DNA by treating extracts of DNA with DNases, which break down DNA. The experiment converted a non-virulent strain of Pneumococcus to a virulent one by exposing it to an extract of the virulent strain’s DNA. After the extracts were treated with DNases they could no longer convert the non-virulent strain because the genetic information carrier had been destroyed [5]. This was strong proof that DNA held the genetic information.

Once the structure and function of DNA had been determined, research on DNA went in many directions, using many different techniques that had not yet been applied to it. One of these techniques is centrifugation, which applies a large gravitational field on the sample, causing the particles in the solution to precipitate out. This was used to study the density and base composition of DNA. Many different types of chromatography have been used as well to separate and identify the bases. Measuring UV absorbance is another technique used to study DNA. It can be used to determine structure such as conformations, as well as learning about DNA’s absorbance of light and base composition [6].

The advance of using UV absorbance measurements in DNA also provided information about the thermodynamics of the molecule by using calorimetry. Work with calorimetry and biological molecules began in the 1960s. This technique measures the heat capacity of a sample and compares it to the temperature. It has been used to generate a wealth of thermodynamic information about DNA as well as RNA. Transitions from one conformation to another, unbending, or the melting of a single strand of a nucleic acid have been studied through calorimetric data. Energy, enthalpy, as well as ligand binding have also been studied with calorimetry [7]. This technique has also been used to determine the absorbance melting curve of nucleic acids. The curve is constructed by measuring the transition between the ordered native state of the nucleic acid to the disordered, denatured state using ultraviolet (UV) absorbance, which can also be used to study DNA structure. The curves plotted with this information can be used to measure the melting temperature of the sample, as well as conformation of the nucleic acid [8].

DNA study is not limited to laboratory tests. DNA has also been studied by applying calculations to determine its properties. Pullman and Pullman studied the electronic structure of DNA in this way. They used these calculations to study interactions of bases, atom charges, and interaction energies in the 1960s. These helped them understand how the bases were held together and gave information about the stability of the molecule [6]. More modern calculations in the 1990s involved the use of quantum mechanics . These calculations allowed scientists to derive properties of dipole moments, proton affinities, and vibrational spectra. These calculations, too, explore interactions within the molecule and with its solvent [9].

The determination of these properties of DNA throughout the many different techniques explored has led the molecule to an extremely prominent place in our society [10]. Its use for higher purposes is also being investigated. In the 1980s, Nadrian Seeman proposed that DNA could be a useful material to incorporate into nanotechnology due to a number of its physical and chemical properties [11]. This field has grown in importance with great potential by incorporating some of DNA’s intrinsic properties : base pairings and sequence. The properties of DNA are exceedingly important in both past and future research.

2.2 RNA

Early in the study of nucleic acids, scientists believed that there were two types of nucleic acids; those that were extracted from animal cell nuclei and a second type which was extracted from plant cell nuclei. It was eventually proven that both types of nucleic acids were present in both animal and plant cells . The distinction between the nucleic acids was chemical: one is made of ribose sugar, the other with deoxyribose [12]. The two types of nucleic acids were DNA and RNA.

Early research on RNA was limited to the use of dyes and stains. These gave important information on where the molecules were located, but left scientists with little information about what RNA did. Research on RNA improved with the increased use of the electron microscope to image RNA and cell compartments, as well as cell fractionation. Cell fractionation is done by destroying the cells and using differential centrifugation to separate the cell components. RNA is also widely studied by examining sedimentation rates to gather information on the size of the molecules [13]. Many of the same techniques used to determine the early properties of DNA have also been applied to RNA. Hydrolysis in conjunction with chromatography liberated the bases for study. The 1940s saw the use of chromatography to separate the samples as well as remove substances for further study. The data yielded from these experiments helped to quantify RNA makeup, such as the quantity of each of the four bases [14]. Like DNA, RNA has also been studied with X-ray diffraction. The X-ray data showed that RNA, like DNA, was a helix [4].

Discovering these basic properties allowed research to begin on the function of the molecule that had been found. Research on RNA after 1960 progressed rapidly. In a few decades, a wealth of important information was gained on the variety of RNA and how they work in the cell, which are described later in this chapter [13]. Radioactive labeling was used to help determine the function of certain RNAs. Messenger RNA was studied with pulse-chase experiments in the 1950s and 1960s [15]. A pulse-chase experiment tracks the movement of a compound through a system by labeling the molecule of interest and assaying its location over time. This can be done with radioactive labeling [2]. Labeling also was key in the discovery of transfer RNA by Zamecnik [16]. In addition to labeling techniques, calorimetry can again be used to study the properties of RNA. It was used to determine the folding pathway of RNA molecules by examining the transitions from one state to another. Researchers were also able to observe the differences in energy that occur when mutations in the sequence were introduced. Calorimetric data can also be used to study RNA-ligand binding and the stability of certain conformations of RNA [17]. Thermodynamic studies on the melting temperature of RNA also confirmed that RNA binds to single-stranded DNA where their base sequences align [13]. Many techniques have been put to use to discover the nuances of the various RNAs.

In addition to research on the functions of RNA, research efforts have recently turned to nanotechnology. Because RNA has many similar properties to DNA, it has the potential to be an incredibly useful nano-material. It is flexible, it is easy to program its structure, and unlike DNA, RNA can perform catalytic functions [11]. There is an incredible amount of potential for future uses of RNA as we continue to learn more about it and its properties (Table 1.1).

Table 1.1 Physical and chemical properties of DNA
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Watson and Crick’s [24] model of DNA only scratched the surface of DNA structure. Since then more conformations of DNA have been, and likely will continue to be, discovered. The Watson and Crick model has become known as the B model of DNA. Here, three of the conformations of DNA are discussed, although there are many others [25].

RNAs are incredibly varied in size, structure, and function across many different types of organisms and cell types. The following Tables 1.2, 1.3, 1.4, 1.5, and 1.6 display the vast amount of specialization RNAs have achieved. The types of RNAs discussed here have been broken down by function into several tables that cover the different categories of RNA thus far discovered.

Table 1.2 Protein synthesis RNAs
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Table 1.3 Post-transcriptional modification or DNA replication RNAs
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Table 1.4 Regulatory RNAs
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Table 1.5 Parasitic RNAs
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Table 1.6 Other RNAs
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RNS is one of the key components of protein synthesis in cells. RNA is used to convert the genetic information stored in the DNA into a form that can be made into proteins. This process is critical to normal functioning of all life forms. This table describes types of RNA that are involved in protein synthesis.

In addition to helming the process of protein synthesis , RNAs can also function as modifying elements to the various types of RNA in the cell. Several types of RNA cannot function without the additional processing provided by the RNAs in this table. In addition, RNA can be used in the cell to modify DNA and manipulate its replication.

RNA can also regulate many aspects of cellular function. RNA can stop translation, prevent viral infections, and modify gene expression.

Not all RNA present in a cell is a product of that cell. The following table describes types of RNAs that can invade other cells and cause disease.

There is still much to discover about RNA that may not fall within the typical groupings that are currently understood. Other types of RNA are being discovered, although not much may be known about them at the present.

3 Chemical Properties as Applied for Sample Preparation

Utilizing nucleic acids in experiments requires careful planning and consideration of the chemical properties of the nucleic acid being used. An improper understanding of the properties at work can severely undermine the success of a particular project. Many properties require special attention for work with nucleic acids. The following examples are used to showcase the importance of a few of these properties in sample preparation, although there may be other properties that are more pertinent to each specific experiment being done.

One example is the use of DNA in biosensors. A biosensor combines a biological sensing unit with a transducer to generate an electronic signal, the intensity of which is proportional to the concentration of what the sensing unit is capturing. Many different biological units have been incorporated into biosensors: tissues, cells, membranes, enzymes, antibodies, and many others [60]. DNA is highly appealing for this purpose in multiple fields. Using nucleic acids in biosensors has obvious applications in medical diagnostics as well as potential uses in forensics, agriculture, the environment, and the food industry [61, 62]. There are several properties of DNA that make it a good biosensor. DNA has a unique sequence of nucleotides that bind to their complementary strands. Because of this, these sensors have high specificity as they will only bind to the probe for the specific analyte that is being searched for. Furthermore, DNA can be manipulated easily to create layers on a transducer and these layers are thermally stable [61]. The properties of DNA can be manipulated in many ways to create these new technologies.

Structural properties are essential to DNA manipulation for technology, but other properties are key as well. The adsorption of DNA to surfaces is extremely important for its use in biosensors because the ability to immobilize the molecule onto surfaces allows it to function as a surface for a signal to be produced. One way this can be done is by adsorbing DNA to gold surfaces. Many properties come into play to make this happen. The electronic charges present in the backbone and bases of the molecule can interact with the metals to keep the DNA attached. This property can be tweaked to increase absorption in different ways. Researchers have managed to adsorb DNA to gold surfaces by attaching thiol groups to the nucleic acids, which then formed gold-thiolate bonds in addition to the bonds formed between the gold and charged particles in the DNA itself [63]. Another property of DNA that affects its adsorption is the base sequence. The nucleotide bases do not all adsorb equally well to surfaces. Sequences with adenines adsorbed to gold surfaces better than other bases, or even to hybrid bases that included adenine. More importantly, this increased affinity of adenine over the other bases can denature the oligonucleotides used [64]. These properties of DNA’s charge and sequence can be manipulated to adsorb it to desired surfaces, but they require careful sequence planning and potential annotations to the sequence to include molecules that increase adsorption even further.

DNA’s electronic properties can create other problems as well. Nucleic acids evolved in the cell; they are adapted to existing in a polar, aqueous environment [65, 66]. This is why DNA is soluble in water and little else. This causes problems because a limited number of usable solvents reduce the number of applications nucleic acids can be put to. DNA research in medicine and nanotechnology may require DNA to dissolve in an apolar solvent or cross an apolar barrier such as a cell membrane. The polar nature of DNA presents difficulties moving the molecule to a solvent that is not polar. When the environment changes from polar aqueous to something else, DNA is subject to changes. It can change in conformation (B to A form for example), or even denature entirely. The behavior of DNA in apolar solvents due to its charge is something that has begun to be studied. Computer simulations of an ordered DNA molecule being transferred to an apolar solvent from an aqueous one suggests that the molecule can in fact hold its shape when placed in a vastly different solvent. The simulation also showed that DNA that attempted to cross a barrier between polar and apolar solvents would travel in a hydrated state if it were to keep its structural integrity [66]. These examples show how a property of DNA can be limiting and needs to be factored into research projects.

Just as DNA has difficulty dissolving in a solvent that is nonpolar, it is also difficult to dissolve it in an organic one since it is not evolved to persist in that environment. However, it is possible to manipulate DNA into becoming soluble in organic liquids. It can be complexed with ammonium salts or mixed with molecules that help it dissolve in organic solvents. DNA can also be made to be soluble in organic solvents by attaching a polyethylene glycol to one end of the DNA. Importantly, dissolving DNA with polyethylene glycol leaves it functional to carry out certain catalytic activities [65]. One of the reagents required to synthesize polyethylene glycol is ethylene glycol, which has similar applications with DNA and solubility. Ethylene glycol can be mixed with other compounds to make ionic solvents known as DESs that can dissolve DNA. Over 50 times more DNA can be dissolved in DES solvents compared to other ionic liquids used for nucleic acids. DNA stored in this liquid retained its structure and the solvent could be recycled multiple times. The researchers theorized that the cations in the solution were interacting with the phosphates of the DNA backbone to help keep it dissolved. The spectra they examined of the DNA in solution supported this [67]. In multiple ways, solubility, a very important part of using DNA for research, was dependent on the molecule’s properties.

These examples show how physical properties have concrete consequences on the ability of researchers to use nucleic acids in experiments. However, the more the information that is gathered on DNA’s properties, the more the options that become open to scientists who wish to work with nucleic acids.

4 Nano-size Properties of Nucleic Acids

When Watson and Crick created their model of the structure of DNA, they realized that the two strands of the DNA helix are held together by base pairing: adenine to thymine, and guanine to cytosine. Their model also came to the conclusion that the two strands must then be complementary [24]. This makes DNA a powerful candidate for use in nanotechnologies, as two single strands of DNA with complementary sequences will bind to each other. These sequences can be manipulated; scientists can create custom lengths of DNA that will spontaneously bind in predictable ways in a process known as self-assembly [68].

Nanotechnology using self-assembling DNA is based largely on the principles of base pairing and “sticky ends.” Sticky ends is a phrase which describes the end of a DNA double helix where one strand ends before the other, leaving orphan nucleotides that are not bound to their base pair. One sticky end can bind to a different sticky end, provided that the two have a complementary sequence of bases. This creates hybrid DNA. Sticky ends are not limited to simply making one hybrid DNA helices. Sticky ends can be combined to form three-armed and four-armed branching structures. These can even be used to build lattices and cubes [20, 68]. These forms can be made so long as the DNA is programmed to have a sequence that will exhibit the proper complementarity needed to fold into the desired shape. The potential for using DNA as a nano-material is clear.

Although base pairing and auto-assembly are important properties of DNA in regard to constructing nano-materials, they are not the only ones significant enough to be considered. The electronic state of DNA and its conductivity are important things to keep in mind when working with DNA. For example, there has previously been demand to create nano-sized wires out of DNA to supplant the use of silicon in electronics. Obviously the success of such a project depended on whether or not DNA could conduct electricity. Multiple studies have yielded different answers: it has been seen functioning as an insulator, a semiconductor, and a conductor. Other researchers have rebuffed the studies vouching for the conductivity of DNA, finding that it was ions, contamination, or water dipoles attached to the DNA conducting the current, not electrons in the DNA [20].

Electrical conduction in DNA was prevented by a number of nano-sized properties of the molecule. First, the shape of the orbitals. DNA orbitals of one base engage in σ-bonding with the orbitals of the next base, whereas the orbitals of non-DNA organic conductors engage in π-bonding. The σ bonds fall several Å short of the amount of overlap the π bonds can achieve, which provides a much smaller surface for electricity conduction. Second, the sequence of DNA. Conducting organic polymers have periodic sequences. DNA does not. The bases are arranged in a random order and each one has different electronic properties that may not mesh with the next base in the helix. This lack of structure prevents electrical conduction. Third, as has already been discussed in the tables, DNA has multiple conformations. These conformations change depending on the environment the DNA finds itself in. Switching from one conformation to another alters the electronic structure and its potential to conduct electricity [20]. These roadblocks to making functional DNA nano-wires show how critical it is to consider the physical and chemical properties of nucleic acids when attempting to work with them.

It is also important to note that DNA is not the only nucleic acid being applied toward nanotechnology. RNA is also emerging as a useful nano-material. It has many physical and chemical properties that make it an ideal piece for construction of nanotechnologies. Many of the same properties of DNA apply here, such as base pairing. However, there are many other advantages to using RNA. It is more flexible than DNA, allowing a wider array of possible loops and motifs to finely tune the shapes achieved. RNA can also perform catalytic functions. The rules of base pairing are more lax in RNA, allowing for more options in sequence and form. A double helix made of RNA is also more thermodynamically stable. Another important factor is that unlike proteins, RNA does not cause the immune system to generate antibodies, allowing for great possibility in applications for medicine [11].

The physical and chemical properties on a nanoscale of DNA and RNA give both molecules tremendous potential, and should be considered in research. A better understanding of these properties will advance our knowledge greatly and allow us to better apply nanotechnology in a number of important fields.

5 Conclusions

The physical and chemical properties of nucleic acids have proven themselves to be of incredible importance in research, from the earliest experiments over a century ago to the cutting-edge research done in the present. A diverse array of technologies has been developed in that time to better study nucleic acids and to characterize its properties and functions. Many of those discoveries have been summarized in this chapter in the tables, or discussed in detail with regard to sample preparation. Consideration of these properties must be done with any research that is to be done with nucleic acids for successful science to be done.