Nucleic Acids in Chemistry and Biology / Edition 3

by Martin Egli, Andy Flavell, Anna Marie Pyle, W David Wilson, S Ihtshamul Haq

Overview

The structure, function and reactions of nucleic acids are central to molecular biology and are crucial for the understanding of complex biological processes involved. Revised and updated Nucleic Acids in Chemistry and Biology 3rd Edition discusses in detail, both the chemistry and biology of nucleic acids and brings RNA into parity with DNA. Written by leading experts, with extensive teaching experience, this new edition provides some updated and expanded coverage of nucleic acid chemistry, reactions and interactions with proteins and drugs. A brief history of the discovery of nucleic acids is followed by a molecularly based introduction to the structure and biological roles of DNA and RNA. Key chapters are devoted to the chemical synthesis of nucleosides and nucleotides, oligonucleotides and their analogues and to analytical techniques applied to nucleic acids. The text is supported by an extensive list of references, making it a definitive reference source. This authoritative book presents topics in an integrated manner and readable style. It is ideal for graduate and undergraduates students of chemistry and biochemistry, as well as new researchers to the field.

Product Details

ISBN-13:	9780854046546
Publisher:	RSC
Publication date:	11/03/2006
Edition description:	Third Edition,New edition
Pages:	503
Product dimensions:	7.44(w) x 9.69(h) x (d)

About the Author

Mike Blackburn has been active in chemical biology research for over 50 years, beginning with chemical synthesis of a substrate analogue for ribosomal peptide synthesis (J. Mol. Biol., 1965, 13, 617-628). He studied enzyme mechanisms with W. P. Jencks (Brandeis University, 1966-7) and launched into the use of stable analogues of phosphate esters and anhydrides for studies on nucleotides and glycolysis intermediates from the late ‘70s. This has culminated in the current use of metal fluorides to generate transition state analogues for phosphoryl transfer reactions by kinases, mutases, and isomerases. He has authored some 300 research papers, several patents, one book (“Nucleic Acids in Chemistry and Biology, 3rd Edition, RSC 2005), and is a co-author for more than 70 structures in the Protein Data Bank.

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Nucleic Acids in Chemistry and Biology

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By G. Michael Blackburn, Michael J. Gait, David Loakes, David M. Williams

The Royal Society of Chemistry

Copyright © 2006 The Royal Society of Chemistry
All rights reserved.
ISBN: 978-0-85404-654-6

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CHAPTER 1

Introduction and Overview

CONTENTS

1.1 The Biological Importance of DNA

1.2 The Origins of Nucleic Acids Research

1.3 Early Structural Studies on Nucleic Acids

1.4 The Discovery of the Structure of DNA

1.5 The Advent of Molecular Biology

1.6 The Partnership of Chemistry and Biology

1.7 Frontiers in Nucleic Acids Research

References

1.1 THE BIOLOGICAL IMPORTANCE OF DNA

From the beginning, the study of nucleic acids has drawn together, as though by a powerful unseen force, a galaxy of scientists of the highest ability. Striving to tease apart its secrets, these talented individuals have brought with them a broad range of skills from other disciplines while many of the problems they have encountered have proved to be soluble only by new inventions. Looking at their work, one is constantly made aware that scientists in this field appear to have enjoyed a greater sense of excitement in their work than is given to most. Why?

For over 60 years, such men and women have been fascinated and stimulated by their awareness that the study of nucleic acids is central to the knowledge of life. Let us start by looking at Fred Griffith, who was employed as a scientific civil servant in the British Ministry of Health investigating the nature of epidemics. In 1923, he was able to identify the difference between a virulent, S, and a non-virulent, R, form of the pneumonia bacterium. Griffith went on to show that this bacterium could be made to undergo a permanent, hereditable change from non-virulent to virulent type. This discovery was a bombshell in bacterial genetics.

Oswald Avery and his group at the Rockefeller Institute in New York set out to identify the molecular mechanism responsible for the change Griffith had discovered, now technically called bacterial transformation. They achieved a breakthrough in 1940 when they found that nonvirulent R pneumococci could be transformed irreversibly into a virulent species by treatment with a pure sample of high molecular weight DNA. Avery had purified this DNA from heat-killed bacteria of a virulent strain and showed that it was active at a dilution of 1 part in 10.

Avery concluded that 'DNA is responsible for the transforming activity' and published that analysis in 1944, just 3 years after Griffith had died in a London air-raid. The staggering implications of Avery's work turned a searchlight on the molecular nature of nucleic acids and it soon became evident that ideas on the chemistry of nucleic acid structure at that time were wholly inadequate to explain such a momentous discovery. As a result, a new wave of scientists directed their attention to DNA and discovered that large parts of the accepted tenets of nucleic acid chemistry had to be set aside before real progress was possible. We need to examine some of the earliest features of that chemistry to fully appreciate the significance of later progress.

1.2 THE ORIGINS OF NUCLEIC ACIDS RESEARCH

Friedrich Miescher started his research career in Tübingen by looking into the physiology of human lymph cells. In 1868, seeking a more readily available material, he began to study human pus cells, which he obtained in abundant supply from the bandages discarded from the local hospital. After defatting the cells with alcohol, he incubated them with a crude preparation of pepsin from pig stomach and so obtained a grey precipitate of pure cell nuclei. Treatment of this with alkali followed by acid gave Miescher a precipitate of a phosphorus-containing substance, which he named nuclein. He later found this material to be a common constituent of yeast, kidney, liver, testicular and nucleated red blood cells.

After Miescher moved to Basel in 1872, he found the sperm of Rhine salmon to be a more plentiful source of nuclein. The pure nuclein was a strongly acidic substance, which existed in a salt-like combination with a nitrogenous base that Miescher crystallized and called protamine. In fact, his nuclein was really a nucleoprotein and it fell subsequently to Richard Altman in 1889 to obtain the first protein-free material, to which he gave the name nucleic acid.

Following William Perkin's invention of mauveine in 1856, the development of aniline dyes had stimulated a systematic study of the colour-staining of biological specimens. Cell nuclei were characteristically stained by basic dyes, and around 1880, Walter Flemming applied that property in his study of the rod-like segments of chromatin (called so because of their colour-staining characteristic), which became visible within the cell nucleus only at certain stages of cell division. Flemming's speculation that the chemical composition of these chromosomes was identical to that of Miescher's nuclein was confirmed in 1900 by E.B. Wilson who wrote

Now chromatin is known to be closely similar to, if not identical with, a substance known as nuclein which analysis shows to be a tolerably definite chemical compound of nucleic acid and albumin. And thus we reach the remarkable conclusion that inheritance may, perhaps, be affected by the physical transmission of a particular compound from parent to offspring.

While this insight was later to be realized in Griffith's 1928 experiments, all of this work was really far ahead of its time. We have to recognize that, at the turn of the century, tests for the purity and identity of substances were relatively primitive. Emil Fischer's classic studies on the chemistry of high molecular weight, polymeric organic molecules were in question until well into the twentieth century. Even in 1920, it was possible to argue that there were only two species of nucleic acids in nature: animal cells were believed to provide thymus nucleic acid(DNA), while nuclei of plant cells were thought to give pentose nucleic acid (RNA).

1.3 EARLY STRUCTURAL STUDIES ON NUCLEIC ACIDS

Accurate molecular studies on nucleic acids essentially date back to 1909 when Levene and Jacobs began a reinvestigation of the structure of nucleotides at the Rockefeller Institute. Inosinic acid, which Liebig had isolated from beef muscle in 1847, proved to be hypoxanthineriboside 5'-phosphate. Guanylic acid, isolated from the nucleoprotein of pancreas glands, was identified as guanine-riboside 5'-phosphate (Figure 1.1). Each of these nucleotides was cleaved by alkaline hydrolysis to give phosphate and the corresponding nucleosides, inosine and guanosine, respectively. Since then, all nucleosides are characterized as the condensation products of a pentose and a nitrogenous base while nucleotides are the phosphate esters of one of the hydroxyl groups of the pentose.

Thymus nucleic acid, which was readily available from calf tissue, was found to be resistant to alkaline hydrolysis. It was only successfully degraded into deoxynucleosides in 1929 when Levene adopted enzymes to hydrolyse the deoxyribonucleic acid followed by mild acidic hydrolysis of the deoxynucleotides. He identified its pentose as the hitherto unknown 2-deoxy-Dribose. These deoxynucleosides involved the four heterocyclic bases, adenine, cytosine, guanine and thymine, with the latter corresponding to uracil in ribonucleic acid.

Up to 1940, most groups of workers were convinced that hydrolysis of nucleic acids gave the appropriate four bases in equal relative proportions. This erroneous conclusion probably resulted from the use of impure nucleic acid or from the use of analytical methods of inadequate accuracy and reliability. It led, naturally enough, to the general acceptance of a tetranucleotide hypothesis for the structure of both thymus and yeast nucleic acids, which materially retarded further progress on the molecular structure of nucleic acids.

Several of these tetranucleotide structures were proposed. They all had four nucleosides (one for each of the bases) with an arbitrary location of the two purines and two pyrimidines. They were joined together by four phosphate residues in a variety of ways, among which there was a strong preference for phosphodiester linkages. In 1932, Takahashi showed that yeast nucleic acid contained neither pyrophosphate nor phosphomonoester functions and so disposed of earlier proposals in preference for a neat, cyclic structure which joined the pentoses exclusively using phosphodiester units (Figure 1.2). It was generally accepted that these bonded 5'- to 3'-positions of adjacent deoxyribonucleosides, but the linkage positions in ribonucleic acid were not known.

One property stuck out like a sore thumb from this picture: the molecular mass of nucleic acids was greatly in excess of that calculated for a tetranucleotide. The best DNA samples were produced by Einar Hammarsten in Stockholm and one of his students, Torjbörn Caspersson, who showed that this material was greater in size than protein molecules. Hammarsten's DNA was examined by Rudolf Signer in Bern whose flow-birefringence studies revealed rod-like molecules with a molecular mass of 0.5–1.0 × 106 Da. The same material provided Astbury in Leeds with X-ray fibre diffraction measurements that supported Signer's conclusion. Finally, Levene estimated the molecular mass of native DNA to be between 200,000 and 1 × 106 Da, based on ultracentrifugation studies.

The scientists compromised. In his Tilden Lecture of 1943, Masson Gulland suggested that the concept of nucleic acid structures of polymerized, uniform tetranucleotides was limited, but he allowed that they could 'form a practical working hypothesis'.

This then was the position in 1944 when Avery published his great work on the transforming activity of bacterial DNA. One can sympathize with Avery's hesitance to press home his case. Levene, in the same Institute, and others were strongly persuaded that the tetranucleotide hypothesis imposed an invariance on the structure of nucleic acids, which denied them any role in biological diversity. In contrast, Avery's work showed that DNA was responsible for completely transforming the behaviour of bacteria. It demanded a fresh look at the structure of nucleic acids.

1.4 THE DISCOVERY OF THE STRUCTURE OF DNA

From the outset, it was evident that DNA exhibited greater resistance to selective chemical hydrolysis than did RNA. So, the discovery in 1935 that DNA could be cut into mononucleotides by an enzyme doped with arsenate was invaluable. Using this procedure, Klein and Thannhauser obtained the four crystalline deoxyribonucleotides, whose structures (Figure 1.3) were later put beyond doubt by total chemical synthesis by Alexander Todd and the Cambridge school he founded in 1944. Todd established the D-configuration and the glycosylic linkage for ribonucleosides in 1951, but found the chemical synthesis of the 2'-deoxyribonucleosides more taxing. The key to success for the Cambridge group was the development of methods of phosphorylation, for example for the preparation of the 3'- and 5'-phosphates of deoxyadenosine

All the facts were now available to establish the primary structure of DNA as a linear polynucleotide in which each deoxyribonucleoside is linked to the next by means of a 3'- to 5'-phosphate diester (see Figure 2.15). The presence of only diester linkages was essential to explain the stability of DNA to chemical hydrolysis, since phosphate triesters and monoesters, not to mention pyrophosphates, are more labile. The measured molecular masses for DNA of about 1 × 106 Da meant that a single strand of DNA would have some 3000 nucleotides. Such a size was much greater than that of enzyme molecules, but entirely compatible with Staudinger's established ideas on macromolecular structure for synthetic and natural polymers. But by the mid-twentieth century, chemists could advance no further with the primary structure of DNA. Neither of the key requirements for sequence determination was to hand: there were no methods for obtaining pure samples of DNA with homogeneous base sequence nor were methods available for the cleavage of DNA strands at a specific base residue. Consequently, all attention came to focus on the secondary structure of DNA.

Two independent experiments in biophysics showed that DNA possesses an ordered secondary structure. Using a sample of DNA obtained from Hammarsten in 1938, Astbury obtained an X-ray diffraction pattern from stretched, dry fibres of DNA. From the rather obscure data he deduced '... A spacing of 3.34 [angstrom] along the fibre axis corresponds to that of a close succession of flat or flattish nucleotides standing out perpendicularly to the long axis of the molecule to form a relatively rigid structure.' These conclusions roundly contradicted the tetranucleotide hypothesis.

Some years later, Gulland studied the viscosity and flow-birefringence of calf thymus DNA and thence postulated the presence of hydrogen bonds linking the purine–pyrimidine hydroxyl groups and some of the amino groups. He suggested that these hydrogen bonds could involve nucleotides either in adjacent chains or within a single chain, but he somewhat hedged his bets between these alternatives. Sadly, Astbury returned to the investigation of proteins and Gulland died prematurely in a train derailment in 1947. Both of them left work that was vital for their successors to follow, but each contribution contained a misconception that was to prove a stumbling block for the next half-a-dozen years. Thus, Linus Pauling's attempt to create a helical model for DNA located the pentose-phosphate backbone in its core and the bases pointing outwards – as Astbury had decided. Gulland had subscribed to the wrong tautomeric forms for the heterocyclic bases thymine and guanine, believing them to be enolic and having hydroxyl groups. The importance of the true keto forms was only appreciated in 1952.

Erwin Chargaff began to investigate a very different type of order in DNA structure. He studied the base composition of DNA from a variety of sources using the new technique of paper chromatography to separate the products of hydrolysis of DNA and employing one of the first commercial ultraviolet spectrophotometers to quantify their relative abundance. His data showed that there is a variation in base composition of DNA between species that is overridden by a universal 1:1 ratio of adenine with thymine and guanine with cytosine. This meant that the proportion of purines, (A + G), is always equal to the proportion of pyrimidines, (C + T). Although the ratio (G + C)/(A + T) varies from species to species, different tissues from a single species give DNA of the same composition. Chargaff's results finally discredited the tetranucleotide hypothesis, because it called for equal proportions of all four bases in DNA.

In 1951, Francis Crick and Jim Watson joined forces in the Cavendish Laboratory in Cambridge to tackle the problem of DNA structure. Both of them were persuaded that the model-building approach that had led Pauling and Corey to the α-helix structure for peptides should work just as well for DNA. Almost incredibly, they attempted no other line of direct experimentation but drew on the published and unpublished results of other research teams in order to construct a variety of models, each to be discarded in favour of the next until they created one which satisfied all the facts.

The best X-ray diffraction results were to be found in King's College, London. There, Maurice Wilkins had observed the importance of keeping DNA fibres in a moist state and Rosalind Franklin had found that the X-ray diffraction pattern obtained from such fibres showed the existence of an A-form of DNA at low humidity, which changed into a B-form at high humidity. Both forms of DNA were highly crystalline and clearly helical in structure. Consequently, Franklin decided that this behaviour required the phosphate groups to be exposed to water on the outside of the helix, with the corollary that the bases were on the inside of the helix.

Watson decided that the number of nucleotides in the unit crystallographic cell favoured a double-stranded helix. Crick's physics-trained mind recognized the symmetry implications of the space-group of the A-form diffraction pattern, monoclinic C2. There had to be local twofold symmetry axes normal to the helix, a feature, which called for a double-stranded helix, whose two chains must run in opposite directions.

Crick and Watson thus needed merely to solve the final problem: how to construct the core of the helix by packing the bases together in a regular structure. Watson knew about Gulland's conclusions regarding hydrogen bonds joining the DNA bases. This convinced him that the crux of the matter had to be a rule governing hydrogen bonding between bases. Accordingly, Watson experimented with models using the enolic tautomeric forms of the bases (Figure 1.3) and pairing like with like. This structure was quickly rejected by Crick because it had the wrong symmetry for B-DNA. Self-pairing had to be rejected because it could not explain Chargaff's 1:1 base ratios, which Crick had perceived were bound to result if you had complementary base pairing.

(Continues...)

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Excerpted from Nucleic Acids in Chemistry and Biology by G. Michael Blackburn, Michael J. Gait, David Loakes, David M. Williams. Copyright © 2006 The Royal Society of Chemistry. Excerpted by permission of The Royal Society of Chemistry.
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Table of Contents

LIST OF CONTRIBUTORS; NOMENCLATURE; 1: INTRODUCTION AND OVERVIEW; 1.1: The Biological Importance of DNA; 1.2: The Origins of Nucleic Acids Research; 1.3: Early Structural Studies on Nucleic Acids; 1.4: The Discovery of the Structure of DNA; 1.5: The Advent of Molecular Biology; 1.6: The Partnership of Chemistry and Biology; 1.7: Frontiers in Nucleic Acids Research; 2: DNA AND RNA STRUCTURE; 2.1: Structures of Components; 2.2: Standard DNA Structures; 2.3: Real DNA Structures; 2.4: Structures of RNA Species; 2.5: Dynamics of Nucleic Acid Structures; 2.6: Higher-order DNA Structures; 3: NUCLEOSIDES AND NUCLEOTIDES; 3.1: Chemical Synthesis of Nucleosides; 3.2: Chemistry of Esters and Anhydrides of Phosphorus Oxyacids; 3.3: Nucleoside Esters of Polyphosphates; 3.4: Biosynthesis of Nucleotides; 3.5: Catabolism of Nucleotides; 3.6: Polymerisation of Nucleotides; 3.7: Therapeutic Applications of Nucleoside Analogues; 4: SYNTHESIS OF OLIGONUCLEOTIDES; 4.1: Synthesis of Oligodeoxyribonucleotides; 4.2: Synthesis of Oligoribonucleotides; 4.3: Enzymatic Synthesis of Oligonucleotides; 4.4: Synthesis of Modified Oligonucleotides; 5: NUCLEIC ACIDS IN BIOTECHNOLOGY; 5.1: DNA Sequence Determination; 5.2: Gene Cloning; 5.3: Enzymes Useful in Gene Manipulation; 5.4: Gene Synthesis; 5.5: The Detection of Nucleic Acids Sequences by Hybridization; 5.6: Gene Mutagenesis; 5.7: Oligonucleotides as Reagents and Therapeutics; 5.8: Footprinting; 6: GENES AND GENOMES; 6.1: Gene Structure; 6.2: Gene Families; 6.3: Intergenic DNA; 6.4: Chromosomes; 6.5: DNA Sequence and Bioinformatics; 6.6: Copying DNA; 6.7: DNA Mutation and Genome Repair; 6.8: DNA Recombination; 7: RNA STRUCTURE AND FUNCTION; 7.1: Overview of RNA Structural Motifs; 7.2: RNA Processing and Modification; 7.3: RNAs in the Protein Factory: Translation; 7.4: RNAs Involved in Export and Transport; 7.5: RNAs and Epigenetic Phenomena; 7.6: RNA Structure and Function in Viral Systems; 8: COVALENT INTERACTIONS OF NUCLEIC ACIDS WITH SMALL MOLECULES AND THEIR REPAIR; 8.1: Hydrolysis of Nucleosides, Nucleotides, and Nucleic Acids; 8.2: Reduction of Nucleosides; 8.3: Oxidation of Nucleosides, Nucleotides, and Nucleic Acids; 8.4: Reactions with Nucleophiles; 8.5: Reactions with Electrophiles; 8.6: Reactions with Metabolically Activated Carcinogens; 8.7: Reactions with Anti-cancer Drugs; 8.8: Photochemical Modification of Nucleic Acids; 8.9: Effects of Ionising Radiation on Nucleic Acids; 8.10: Biological Consequences of DNA Alkylation; 8.11: DNA Repair; 9: REVERSIBLE SMALL MOLECULE-NUCLEIC ACID INTERACTIONS; 9.1: Introduction; 9.2: Binding Modes and Sites of Interaction; 9.3: Counter-ion Condensation and Polyelectrolyte Theory; 9.4: Non-specific Outside-edge Interactions; 9.5: Hydration Effects and Water-DNA Interactions; 9.6: DNA Intercalation; 9.7: Interactions in the Minor Groove; 9.8: Intercalation versus Minor Groove Binding; 9.9: Co-operativity in Ligand-DNA Interactions; 9.10: Small Molecule Interactions with Higher-order DNA; 10: PROTEIN-NUCLEIC ACID INTERACTIONS; 10.1: Structural Features of DNA Important in Protein Recognition; 10.2: The Physical Chemistry of Protein-Nucleic Acid Interactions; 10.3: Representative DNA Recognition Motifs; 10.4: Kinetic and Thermodynamic Aspects of Protein-Nucleic Acid Interactions; 10.5: The Specificity of DNA Enzymes; 10.6: DNA Packaging; 10.7: Polymerases; 10.8: Machines that Manipulate Duplex DNA; 10.9: RNA-Protein Interactions and RNA-mediated Assemblies; 11: PHYSICAL AND STRUCTURAL TECHNIQUES APPLIED TO NUCLEIC ACIDS; 11.1: Spectroscopic Techniques; 11.2: Nuclear Magnetic Resonance; 11.3: X-ray Crystallography; 11.4: Hydrodynamic and Separation Methods; 11.5: Microscopy; 11.6: Mass Spectrometry; 11.7: Molecular Modelling and Dynamics

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From the Publisher

An excellent overview of the chemistry and biology of nucleic acids, at a level that is suitable for use in university teaching, but with enough detail to be useful as a reference source for chemists, biochemists and biologists involved in current research of nucleic acids.