Table Of ContentSynthesis and Applications
of DNA and RNA
Edited by
Saran A. Narang
Division of Biological Sciences
National Research Council of Canada
Ottawa, Ontario, Canada
1987
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Library of Congress Cataloging in Publication Data
Synthesis and applications of DNA and RNA.
Includes index.
1. Nucleic acids—Synthesis. 2. Nucleic acids-
Analysis. 3. Molecular genetics. I. Narang, Saran A.
QP620.S96 1987 547.7'9 86-25936
ISBN 0-12-514030-4 (alk. paper)
PRINTED IN THE UNITED STATES OF AMERICA
87 88 89 90 9 8 7 6 5 4 3 2 1
To my inspiration Sandhya, my wife
Preface
About a billion years ago, Nature drafted the blueprints of life describ
ing our roots, heritage, and the process of evolution. The language was
DNA, its alphabet A, C, G, and T. For centuries mankind has been
curious about learning and decoding the language into meaningful infor
mation. In spite of tedious and painstaking efforts to understand the
chemistry and biology of DNA in the past century, unexpected complexi
ties continue to develop.
The ability to recreate the man-made version of the DNA molecule
offers a unique opportunity to learn about this molecule of life. During the
last decade the art of chemical synthesis has reached a maturity which
will play an invaluable role in advancing our knowledge. Many who have
played significant roles in the development of this field have contributed
to this book, whose main goals are to stimulate the interest of a younger
generation of scientists and to share our excitement in struggling through
the voyage of developing the synthetic route to DNA.
It is a pleasure to thank my colleague Dr. Roland Brousseau for his help
and advice during the preparation of this book and Dr. Wayne A. Jones
for his careful reading and editorial correction of the manuscript. The
main theme of this book, developed in my laboratory, would not have
been possible without the driving force and encouragement of my wife
Sandhya and my daughter Monica Ajoo, who have filled my life with joy.
Saran A. Narang
xi
Introduction
There can be excitement, adventure, challenge,
and there can be great art in organic synthesis.
Robert Woodward
To organic chemists, this statement is reason enough to justify their
efforts to advance scientific knowledge in their particular fields. In 1828,
Wohler's spirit of adventure led him to the crucial experiment in which
ammonium cyanate was converted to urea. This simple experiment,
which unified biology and chemistry, forever changed the fate of the
world. Released from dogmatic fetters about force vitale, chemistry
quickly became one of the driving forces of the industrial revolution,
adding immeasurably to its impetus.
During the past hundred years, chemists have synthesized several mil
lion different compounds, some related to natural products and some of
which had never before existed. The introduction of spectroscopic tech
niques such as infrared in the 1950s and nuclear magnetic resonance in the
1960s led to a golden age of organic synthesis, crowned by achievements
such as Robert Woodward's synthesis of vitamin Bi. A branch of science
2
which can accomplish the synthesis of chemical compounds possessing
extremely complex structures is clearly in a very healthy state. These
outstanding syntheses belong to the great cultural heritage of our age, no
less so than anything produced in literature or the arts.
Inevitably scientists continued to probe the nature of living cells at the
molecular level. The major achievement of this approach is undoubtedly
Watson and Crick's discovery of the double-helix structure of DNA in
1953, a discovery which began a golden era in molecular biology. Knowl
edge of how genetic information flows from DNA to mRNA to protein and
the elucidation of the genetic code and the confirmation of its quasi-
xisi
xiv Introduction
universality have also come about, at least in part, through a more rigor
ous application of the molecular approach to living systems.
These discoveries have wrought a radical change in our ideas about
microbes. We now view them as tiny biochemical factories and their long
familiar actions as highly sophisticated chemical transformations. A bac
terium such as Escherichia coli reproduces in 20 minutes under optimal
growth conditions—a new living cell is synthesized within one-third of an
hour. Not only must the bacterium replicate accurately (one base pair
mismatch can be lethal) the 2 million bases of its genome, it must also
synthesize the correct amounts of over 3500 different proteins, and all
within 20 minutes. The elucidation of the extremely precise chemical
control mechanisms involved will remain a fruitful field of study for
scientists for decades to come.
After 1953, it was clear that a rich field of DNA synthesis awaited
ambitious chemists, and H. G. Khorana was one to undertake the chal
lenge. Knowing DNA to be a macromolecule, he reasoned that with
chemical reagents alone meaningful lengths of it could be synthesized. By
using equally well-known enzymes and chemical reagents, he further
closed the gap between biology and chemistry, made a determinant con
tribution toward the elucidation of the genetic code, and showed the way
in which gene synthesis could eventually be undertaken.
In the 1970s, tremendous progress in the development of rapid chemical
synthesis of polynucleotides, DNA sequence analysis, and recombinant
DNA technology led to a genetic engineering revolution, one that has the
potential to touch nearly every phase of human life. Already recombinant
DNA production of human insulin, growth hormone, and interferon has
become a reality, and these are but small achievements in the face of
things to come.
As the twenty-first century approaches, we are faced with another chal
lenge of code elucidation, this one pertaining to protein folding. Globular
proteins consist of linear polypeptide chains which are synthesized from
20 different amino acids. During or after synthesis these chains fold spon
taneously to an exact three-dimensional structure. The spontaneity of the
folding is regarded as a generally valid principle because it can be demon
strated in renaturing experiments with several proteins. Yet the mathe
matics describing this process is staggering. For example, folding of a
chain of 100 amino acid residues requires the adjustment of about 300
parameters. Even if each parameter is restricted to only two values, some
2300 configurations become possible. Random adoption of each configura
tion until the chain stumbles onto the right one would require a time
exceeding the age of the earth even if only 10"13 seconds were assumed
for each trial. Hence, the parameter values must be run through in a
Introduction XV
directed manner, i.e., there must be a defined folding pathway so that
little time is lost with trial-and-error folding.
There is hope that this mechanism can be elucidated and reproduced by
using an integrated approach involving modern computational tools, ex
tensive gene synthesis, and vast amounts of data drawn from X-ray crys
tallography, protein chemistry, protein spectroscopy, and genetic engi
neering technology. The knowledge gained will eventually allow scientists
to design custom-made enzymes, hormones, and structural proteins, and
will open the new era of protein engineering.
Saran A. Narang
1
Development of Chemical
Synthesis of Polynucleotides
SARAN A. NARANG
Division of Biological Sciences
National Research Council of Canada
Ottawa, Ontario, Canada K1A OR6
I. INTRODUCTION
Our roots and the history of evolution are written in terms of codes in
the base sequence of DNA. These facets of DNA have imposed the cod
ing concept on the field of modern biology. Thus, the goal of the study of
DNA is to decode and understand the biological information contained in
DNA sequences. Progress in the last 20 years has made us aware of the
role of various DNA sequences. We can now read and translate DNA
sequence into protein sequence and regulatory signals that control the
gene activity. The use of synthetic, tailor-made DNA has played a key
role in understanding the genetic code (Khorana et al., 1966) and various
regulatory signals such as the operator, promoter, ribosomal binding
sites, enhancers, transposable elements, and homeo-box. The synthetic
approach not only provides a final proof of the roles of various DNA
sequences but also offers an opportunity for further improvement in func
tion for practical application. The application of synthetic genes, linkers,
primers, and probes has become a powerful tool in the cloning, sequenc
ing, and isolation of genomic DNA. In the following sections, I have
outlined how various stages of development in the chemical synthesis of
polynucleotides helped to achieve the present status of this field.
1
Synthesis and Applications Copyright © 1987 by Academic Press, Inc.
of DNA and RNA All rights of reproduction in any form reserved.
2 Saran A. Narang
II. HISTORICAL BACKGROUND
The present foundation of synthetic work was laid by the prolonged and
intensive efforts of Todd and Khorana during the 1950s. Although
Michelson and Todd (1955) were the first to correctly link dinucleotides
by condensing 5'-acetylthymidine 3'-benzylphosphorochloridate with 3'-
acetylthymidine, nonetheless their method has limitations. The overall
yield of the final product was low because of various side reactions. Thus,
this approach was not useful in the synthesis of higher oligo- or polynucle
otides. The search continued in Todd's laboratory for a superior (that is,
milder and more effective) chemical phosphorylating method. In 1953,
Khorana and Todd described the use of a different phosphorylating re
agent, dicyclohexylcarbodiimide (DCC). Todd and associates published a
further study later in that same year using DCC for the synthesis of
coenzymes; (Christie et al., 1953) but it was Khorana who used this re
agent for polynucleotide synthesis. Between 1956 and 1958, Khorana and
co-workers in Vancouver employed this reagent extensively to establish a
phosphodiester approach of oligonucleotide synthesis which became rou
tine for the next two decades (Gilham and Khorana, 1958). In spite of the
fact that this approach was laborious and time-consuming, they did suc
ceed in accomplishing the total synthesis of a biologically active tyrosine
tRNA gene (Khorana, 1979).
By the mid-1960s, other organic chemists, such as Letsinger, Reese,
Eckstein, and Cramer, reinvestigated the phosphotriester approach first
introduced by Michelson and Todd (1955), in which the three phosphate
bonds were masked during synthesis. Although the synthesis of oligomers
up to octamers was obtained in a reasonable yield, it did not attract much
attention as compared to the established approach yielding a phospho
diester. Again it was the development of new phosphorylation and cou
pling reagents such as triazole and tetrazole derivatives by Narang (Kata-
giri et al.> 1974) which established the phosphotriester method on a firm
footing as an alternative to the phosphodiester approach. Using this
method, the first biologically active genetic element, the lac operator
DNA, was synthesized and cloned (Bahl et ai, 1976). Subsequently,
genes encoding other chemically important proteins such as insulin
(Goeddel et ai, 1979) and interferons (Edge et al., 1981) were synthesized
via the same technique.
In 1976, Letsinger and Lunsford introduced a phosphite triester ap
proach using phosphodichloridites to link nucleosides in a much shorter
time than before; this was further modified to the phosphoramidite ap
proach by Matteucci and Caruthers in 1981. This approach has been the
basis for several commercial solid-phase DNA synthesizers.
1. Development of Chemical Synthesis of Polynucleotides 3
III. CHEMICAL SYNTHESIS
The fundamental objective of oligonucleotide synthesis is the formation
of 3' —* 5' phosphate linkages between nucleoside units. This has been
achieved by the three reactions outlined in Fig. 1.
PHQSPHODIESTER APPROACH
f Κ f Κ
l\| |/l
ο ο ο
" I ^ Γ"Γ_·|·"| Phosphite group °*'d0tl0n». j-""I Phosphotriester group
"O— Ρ iRO-P l R 0 —Ρ = Ο!
I + ' I ' .
? On ?
OR" OR"
Fig. 1. Chemical approaches for the synthesis of a polynucleotide.
