Download Nucleic Acid Structures, Energetics, and Dynamics

J. Phys. Chem. 1996, 100, 13311-13322
13311
Nucleic Acid Structures, Energetics, and Dynamics
Ignacio Tinoco, Jr.
Department of Chemistry, UniVersity of California at Berkeley, and Structural Biology DiVision,
Lawrence Berkeley National Laboratory, Berkeley, California 94720-1460
ReceiVed: October 16, 1995; In Final Form: January 2, 1996X
In 1953 DNA was shown to be a double helix of hydrogen-bonded complementary bases. Since then,
knowledge of nucleic acid structures, thermodynamic stabilities, and dynamics of conformational changes
has grown exponentially. This knowledge has led to the development of the biotechnology industry, the
identification of plants and animals from a few cells, and many advances in the diagnosis and treatment of
diseases. All the methods of physical chemistry have been used to characterize the primary structures
(sequences), the secondary structures (base pairing), and the tertiary structures (folded 3D conformations) of
the nucleic acids. The interactions of nucleic acids with themselves, with proteins, and with small-molecule
ligands control their many functions. Novel methods are being developed to probe the structures and functions
of nucleic acids. These include methods to study a single molecule and methods to select, amplify, and
characterize one sequence among 1017 different sequences.
Introduction
The April 25, 1953, issue of Nature presented three short
reports on the structure of nucleic acids. The first, by Watson
and Crick,1 proposed that DNA was a double helix of antiparallel
strands held together by hydrogen bonds between complementary bases: guanine and cytosine or adenine and thymine. Their
model followed from the fact that this was the only way that
pairs of hydrogen-bonded bases in their correct keto tautomers
could fit into a continuous, regular helix. The G‚C and A‚T
base pairs each have a twofold axis in the plane of the base
pair; rotation by 180° around this axis interchanges their attached
sugars and phosphates. Thus a G‚C pair is interchangeable with
a C‚G, and A‚T and T‚A are interchangeable. As the distances
between the C1′ carbons where the bases attach to the sugars
are the same for G‚C and A‚T, any sequence of bases can form
a uniform double helix of constant pitch and radius. The other
two reports2 gave detailed X-ray diffraction data on B-form
DNA fibers which established the 10 base pairs per turn of 34
Å length with the phosphate groups on the outside of a double
helix of 20 Å diameter. The report by Rosalind Franklin showed
the best diffraction data and also described the crystalline
A-form DNA fiber seen at low humidity. Unfortunately, she
died before the Nobel Prize for this work was awarded. The
Nobel committee would have had a very difficult task to choose
who shared the award with Watson and Crick. By rule only
three people can enjoy one award; they were Crick, Watson,
and Wilkins.
The discovery of the DNA double helix was accomplished
by a biologist (Watson), two physicists (Crick and Wilkins),
and a physical chemist (Franklin). This combination of
knowledge seems ideal for solving nearly any problem in
science. As present and future students of physical chemistry,
we should all learn some biology and some physics in order to
solve the important problems of the next 100 years.
The goal of scientists studying structures, energetics, and
dynamics of nucleic acids should be to understand, predict, and
control their functions. The goal has been amply fulfilled
following the 1953 publications on the structure of DNA. The
complementary Watson-Crick3 base pairs immediately exX
Abstract published in AdVance ACS Abstracts, July 1, 1996.
S0022-3654(95)03053-X CCC: $12.00
plained how genes duplicated. The biotechnology industry, the
identification and characterization of plants or animals from a
few cells, advances in diagnosis and treatment of disease, and
many other applications have all followed. Is the fun over now?
Have all the important discoveries been made? When I arrived
in Berkeley in 1956 and mentioned to a biologist that I was
planning to study DNA structure, he responded, “Hasn’t that
already been done?” I do not remember the answer I gave then,
but the answer I would give now is that every important
discovery reveals new questions. The fun is just beginning.
Some of the progress made since 1953 in determining the
structures of nucleic acidssand understanding their functionsswill
be described here. Useful knowledge of a structure means
knowing where the atoms are, how much energy it takes to move
them, and how fast they can move. Thus, structures, thermodynamics, and kinetics of nucleic acids will be considered.
Structures
Primary Structures. Deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) are polynucleotides. Each nucleotide
contains a sugar (D-2′-deoxyribose for DNA and D-ribose for
RNA), a phosphate, and a base (A, C, G, T for DNA and A, C,
G, U for RNA). The primary structures of a DNA oligonucleotide, an RNA oligonucleotide, and the five bases are shown
in Figure 1; detailed structural information is available in
textbooks.4 All the biological, chemical, and physical properties
of a nucleic acid depend on its primary structuresits sequence
of nucleotides. Clearly, it is important to be able to determine
the sequence of any nucleic acid and then to deduce all its
properties from the sequence. We will start with the easier
problem, the determination of sequence.
Determination of Sequence. The Human Genome Project has
as its goal the sequence determination of a complete set of
human chromosomes, about 3 billion base pairs. The first
complete DNA sequence for a free living organism has been
published:5 Haemophilus influenza (1 830 137 base pairs). A
complete yeast genome of 14 million base pairs is expected in
1996. All the sequence information has come from a method
invented by Fred Sanger; he shared the chemistry Nobel Prize
in 1980 with Walter Gilbert for nucleic acid sequencing. It
was Sanger’s second Nobel; his first was for protein sequencing.
© 1996 American Chemical Society
13312 J. Phys. Chem., Vol. 100, No. 31, 1996
Tinoco
Figure 1. Structures of the four naturally occurring bases in DNA (A, C, G, T) and RNA (A, C, G, U) are given along with a portion of a
poly(deoxyribonucleotide) chain and of a poly(ribonucleotide) chain. The sequence of a chain is read from the 5′-end to the 3′-end.
In Sanger’s method6 DNA polymerase is used to synthesize a
DNA strand complementary to the strand being sequencedsthe
template strand. A primer oligonucleotide is used to select the
site where the polymerase starts synthesizing the complementary
strand. Four deoxynucleoside triphosphates (deoxy-NTPs) are
the monomer units polymerized by the enzyme. Normally the
enzyme will continue synthesis until it runs out of deoxy-NTPs
or of template strands. However, if a few percent of a dideoxyNTP is added to the reaction mixture, the dideoxy-NTP acts as
a strand termination monomer. For example, if dideoxy-GTP
is added, all strands will end in G. Most of the time a natural
G will be incorporated opposite C on the template strand, and
the new strand will continue. However, eventually a dideoxy-G
is added, and the strand terminates. The position of G’s relative
to the starting position for synthesis is read from the chain
lengths of the synthesized strands. The experiment is repeated
with each of the four bases acting as the chain termination
monomer. Gilbert’s method7 also uses chain length to obtain
the sequence, but base-specific chemical cleavage produces the
ends. A DNA or RNA strand is labeled at the 5′-end (see Figure
1) and then specifically, but lightly, cleaved at G’s, for example.
The chain length of each labeled strand after cleavage provides
the sequence of G’s.
Determination of sequence has thus been changed to determination of chain length of single strands with a resolution of
one nucleotide. The standard method is electrophoresis in a
denaturing poly(acrylamide) gel to separate chains on the basis
of charge. At neutral pH there is one negatively charged
phosphate group per nucleotide, so strands can be separated
according to chain length. Gel electrophoresis is also used to
separate large fragments of double-stranded DNAsthousands
to millions of base pairssfor coarse-grained characterization:
so-called mapping. The speed and resolution of separation
depend on the pore size of the gel (controlled by gel concentration and cross-linking), the space and time distribution of the
electric field,8 the solvent, and so forth. To optimize conditions,
it is important to understand how chain macromolecules travel
during gel electrophoresis. Chains are thought to migrate
through gels like a snake through a warren of interconnected
rabbit holes. This reptation model9,10 approximately represents
the motion of the chain molecule, but it is necessary to watch
a DNA molecule move through a gel to appreciate the
complexity of the real behavior. Individual DNA molecules
stained with fluorophores can be followed in a fluorescence
microscope11 and displayed in a video.12 The DNA acts like a
successful kickoff returner in football. It moves fast in spurts,
is transiently slowed or stopped, but suddenly changes direction,
and continues. This is its motion in a constant electric field;
the erratic motion is caused by its choices at different points in
the gel warren. For optimum separation of different DNA size
ranges the electric field is pulsed so as to make the DNA move
forward, backward, or sideways with rest periods in between
(with the electric field off).13 It is obviously very difficult to
model the motion with great fidelity. One model uses charged
beads connected by entropic springs. The beads are acted on
by the electric field, viscous drag, Brownian diffusion, and
interbead elasticity.14 Reasonable agreement between simulation
and experiment is obtained, but there is clearly much room for
improvement.
In the future, faster separations with higher resolution on
smaller amounts of material are urgently required. One human
chromosome with about 100 million base pairs is a worthy target
for sequencing. Of course, methods of chain length measurement other than gel electrophoresis, such as mass spectroscopy,
are possible. Silicon mazes can replace poly(acrylamide) gels
as electrophoresis materials. Finally, completely different
sequencing techniques can be used. Among the many methods
elicited by the Human Genome Project, sequence determination
by hybridization stands out. The logic of the method is that an
N-mer sequence is a set of overlapping shorter sequences. From
knowledge of these shorter sequences the N-mer sequence can
be deduced.15 How long the shorter sequences need to be to
obtain a unique sequence for the N-mer depends on the
sequence. Long repeating sequences are the most difficult. For
example, in a 100-mer there are two overlapping 99-mers, three
overlapping 98-mers, and (101 - n) overlapping n-mers. Thus,
a sequence of 100 nucleotides will contain 93 overlapping
8-mers, not necessarily all different. The 8-mers that occur in
the target sequence, and how often each occurs, can be used to
deduce the target sequence or a number of possible target
sequences. There are 48 ) 65 536 possible 8-mers, so only a
small fraction can occur in any 100-mer. If long repeating
Nucleic Acid Structures, Energetics, and Dynamics
sequences occur in the target, a longer n-mer sequence can be
used to resolve ambiguities. To implement the method, a
complete set of 4n oligonucleotides of length n is immobilized
on a solid support, and a target strand is presented. The target
strand hybridizes by Watson-Crick base pairing to each n-mer
contained in its sequence. From the n-mers hybridized the
sequence of the target is reconstructed.16 The 4n oligonucleotides can be synthesized photochemically on a chip using
photolithographic methods; hybridization is detected by fluorescence of the fluorescently labeled target.17 Determining
sequence by hybridization is very useful for testing sequences
determined by the standard Sanger method. It may be most
used in detecting small changes in sequence, such as singlebase mutations in some genetic diseases.
The ultimate goal is to be able to quickly sequence any DNA
or RNA from a single molecule without first amplifying it by
the polymerase chain reaction (PCR).18 The method will be
left as an exercise for the reader.
Analysis of DNA Sequence. The Human Genome Project is
supported by NIH and DOE to identify all human genes and
thus to revolutionize the diagnosis, prevention, and treatment
of disease. Less than 10% of the human DNA codes for genes;
most of the rest has no known function, although some is
involved in the regulation of gene expression. The DNA that
codes for genes is first transcribed into RNA with a one-to-one
code based on Watson-Crick base pairs. The RNA can either
function directly, for example as the transfer RNAs and
ribosomal RNAs involved in protein synthesis, or the RNAs
messenger RNAscan be translated into protein. Here the code
is three nucleotides to one amino acid. The 64 trinucleotide
sequences code redundantly for 20 amino acids, except for three
trinucleotides (UAA, UAG, UGA) that are stop signals, which
cause protein synthesis to end at that point in the messenger
RNA. The start signals for protein synthesis are a nontranslated
RNA sequence followed by an AUG triplet; this triplet, which
also codes for methionine, is the first triplet translated. The
noncoding DNA may have long runs of repeating sequences.
Some repeating trinucleotide sequences are causes (and diagnostics) for human diseases such as fragile X syndrome.19
We will emphasize here the structural and physical aspects
of sequence. DNA is normally double stranded, but chromosomes have single-stranded ends necessary for complete replication of the chromosome. DNA polymerase needs a primer to
start making a complement from a template DNA strand. The
primer should bind to the DNA strand before the important
sequences in the DNA that are replicated. This is accomplished
by attaching a repeating sequence, such as a variable number
of copies of the sequence T2G4, to the 5′-end of each strand of
the double helix. The ends of chromosomes are called telomeres, and the enzyme that synthesizes the single strands is
telomerase. The telomeres have a unique structure as revealed
by NMR20 and X-ray diffraction.21 The single-strand ends fold
back to form cubelike structures made of stacked G-quartets
(Figure 2). They are very sensitive thermodynamically and
kinetically to the nature of the counterions; potassium ions
specifically stabilize the structure presumably because they fit
well in the central hole of the quartet.22 This unique, nonWatson-Crick interaction of guanines in G-quartets was first
seen by fiber diffraction in guanosine aggregates and in guanine
polynucleotides (poly-G) in the 1960s.23 At that time biologists
and biochemists thought that the finding was not biologically
relevant. Their thought was more or less that in an X-ray
capillary or NMR tube anything can happen, but in a living
organism proteins and other biological molecules limit the
structures to the correctsWatson-Cricksones. Now G-quar-
J. Phys. Chem., Vol. 100, No. 31, 1996 13313
Figure 2. Structures of the G-quartets formed by GGGGTTTTGGGG
in solution20 (top) and in a crystal21 (bottom). Reproduced with
permission from ref 22. Copyright 1994 Annual Reviews.
tets are not only relevant to biology, they are being studied as
a possible AIDS prophylactic.24
In the next sections we will discuss the measurement and
the prediction of DNA and RNA Watson-Crick and nonWatson-Crick structures.
Secondary Structures, Energetics, and Dynamics. Soon after
Watson’s and Crick’s paper appeared, physical chemists tried
to test whether adenine and thymine, or guanine and cytosine,
actually formed hydrogen-bonded coplanar pairs in solution
(Figure 3). They found instead that the bases, or bases
connected to sugars (nucleosides), stacked in aqueous solution.
The donor and acceptor groups on the bases hydrogen bond to
water, and the aromatic rings stack on top of each other. In
chloroform25 or in vacuum,26 hydrogen-bonded pairs do form,
but not just Watson-Crick pairs. This is not surprising because
the crucial discovery of Watson and Crick was that only their
pairs would fit into a uniform helix in any sequence. Without
the geometrical constraint each base can hydrogen bond to every
other base. This was emphasized by the fact that the first crystal
structure of A‚T base pairs did not have Watson-Crick
hydrogen bonding.27 In fact, there are 28 possible pairings
between the neutral bases that involve at least two hydrogen
bonds;4 protonation increases the number of possible pairings.
Many have been found in natural nucleic acids, synthetic
oligonucleotides, and crystals. Eight pairs involving A‚T, G‚C,
and G‚C+ are shown in Figure 3.
In order to study Watson-Crick base pairing, it is necessary
to use polynucleotides or oligonucleotides. The important
questions are the following: What are the thermodynamics (and
statistical thermodynamics) of forming a DNA, RNA, or hybrid
double helix from any sequence in any solvent? What are the
kinetics of formation? These questions are far from answered.
We leave for the following sections the questions about the
structures of the helices and the formation of more complex
structures than simple helices.
13314 J. Phys. Chem., Vol. 100, No. 31, 1996
Figure 3. Hydrogen-bonded base pairs. Only Watson-Crick pairs can
form in any order to produce a continuous, uniform double helix. The
other structures illustrate a few of the 28 other hydrogen-bonded pairs
that can occur.
Thermodynamics of Double-Helix Formation. Double-helix
formation is generally measured by absorption vs temperature
curves, so-called melting curves. The absorbance in the
ultraviolet region (260 nm) increases as the double helix “melts”
to two single strands. This dependence of molar absorptivity
on the conformation of a nucleic acid was very puzzling
originally. The molar absorptivity of a DNA (absorbance per
mole of nucleotide) depended on the previous history of the
DNA. For example, heating and quick cooling of a DNA could
cause a 40% increase in absorbance without any obvious
chemical changes. Hydrolyzing the polynucleotide to mononucleotides increased the absorbance by 60% over the absorbance of the native double helix. All these changes in
magnitude of absorbance occurred without significant changes
in shape of the broad absorption band. New bands were not
being produced, nor were significant changes in energy levels
occurring.
The hypochromismsthe decrease in absorbance of chromophores arranged in an ordered arrayscould be explained by
electronic interaction between chromophore units in the array.28
No electron exchange in the ground state and no charge transfer
in the excited state were considered, so a simple exciton model
sufficed. A semiquantitative account of the hypochromism
resulted. The electronic transition dipoles of the stacked
chromophores interact so as to shift absorption intensity to the
higher energy bands. The bands at 200 nm and lower
wavelengths borrow intensity from the observed 260 nm band.
The absorption intensity integrated over all bandssthe sum over
oscillator strengthssdoes not change. This understanding of
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the cause of hypochromism meant that the changes in absorption
of a polynucleotide (or polypeptide) could be interpreted
structurally. A more sensitive optical signal for changes in
conformation is given by circular dichroismsthe differential
absorbance of left and right circularly polarized light. The same
sort of theory applies, but now rotational strengths instead of
oscillator strengths are calculated; both electric and magnetic
transition dipoles are used.29 Recent reviews30,31 on absorption
and circular dichroism of nucleic acids and proteins can be found
in a book entitled Biochemical Spectroscopy.32
Now that we knew how to interpret the absorption, we could
confidently analyze the absorbance melting curves in terms of
the standard Gibbs free energy, ∆G°, standard enthalpy, ∆H°,
and standard entropy, ∆S°, of the double-helix-to-single-strands
(helix-coil) reactions. A systematic study of double-helix
formation in RNA was begun by measurements on 19 oligonucleotides ranging in length from 6 to 14 base pairs.33 The
reactions were assumed to be all-or-none equilibria; that is, only
completely base-paired duplex and single strands were assumed
present. The absorbance at any temperature of the strands and
the helix could be obtained by extrapolation from the high- and
low-temperature base lines, respectively. Thus, from the
measured absorbance (and the known total concentration) the
equilibrium constant for double-helix formation was obtained
at each temperature. The equilibrium constant is an effective
one based on concentrations, instead of activities, in the solvent
chosen, 1 M NaCl (pH 7). From the equilibrium constant and
its temperature dependence (van’t Hoff equation), the thermodynamic parameters were obtained.
Even the earliest experiments showed that the thermodynamics depended not only on the chain length and the number of
G‚C and A‚U pairs, as expected, but also on the sequence. The
simplest interpretation was to assume nearest-neighbor-only
contributions of the sequence. In this model the thermodynamics of double-helix formation from single strands for any
sequence and chain length can be calculated from an initiation
parameter for forming the first base pair plus 10 nearest-neighbor
parameters for the 10 possible Watson-Crick nearest neighbors.
The Watson-Crick pairing requirement reduces the 16 possible
single-strand nearest neighbors to 10 possible double-strand
neighbors. The nearest-neighbor thermodynamic parameters
have been obtained by a least-squares fit to an overdetermined
set of measured values for double helices of 4-9 base pairs in
length.34 The parameters provide useful predictions of doublestrand formation for RNA in 1 M NaCl.35
In order to have a qualitative understanding of the thermodynamic stability of an RNA (or DNA) double helix relative to
its single strands, it is sufficient to remember the following
data.35 The standard free energy at 37 °C for forming the first
base pair is about +3.4 kcal mol-1. This positive free energy
is mainly loss of translational entropy in bringing the two strands
together. There are 10 different nearest-neighbor values for
adding a base pair. They range from -0.9 to -3.4 kcal mol-1
for ∆G°37. The average values are ∆G°37 ) -1.9 kcal mol-1,
∆H° ) -9.8 kcal mol-1, and ∆S° ) -24.9 cal mol-1 K-1.
RNA is synthesized in nature as a single strand from a DNA
template. The RNA then folds into a compact form that
involves intramolecular double helices of Watson-Crick base
pairs, plus many other hydrogen-bonded and stacked secondary
structural elements. These elements are36 single strands, double
helices (also called stems), hairpin loops (the strand makes a
U-turn), internal loops (non-Watson-Crick base appositions
within a stem), bulge loops (one or more bases are not paired
on one strand, but pairing is continuous on the other), and
junctions (the joining region of three or more stems). The
Nucleic Acid Structures, Energetics, and Dynamics
contributions of these structural elements to the thermodynamics
of the folded RNA are much more complex than the simple
formation of a double helix. We can still assume that the
thermodynamics of the different elements are additive, but each
type of loop or junction has its own sequence and length
dependence. The loops’ sequence dependences are not nearestneighbor properties, so many experiments are required to
establish a sufficient database for accurate predictions. Nevertheless, progress is being made by estimating the effect of
loop length from the configurational entropy loss of forming
loops and taking sequence into account if data are available.
Extensive discussion of RNA structures, properties, and functions is given in The RNA World and in the many references
given therein.37
Nearest-neighbor thermodynamic parameters for DNA doublehelix formation from two single strands were obtained from a
combination of 19 oligonucleotides and nine polynucleotides.38
Direct microcalorimetric measurements of enthalpies of doublehelix formation were measured. Clearly, for the polynucleotides
a van’t Hoff method based on a single equilibrium constant is
not possible. An important application of the DNA nearestneighbor data is to calculate thermodynamic stabilities for
oligonucleotide probes or primers to hybridize to natural DNAs.
Radioactive probes are used to identify specific sequences that
are diagnostic of disease or that identify a unique person.
Primers determine the beginning of complementary strand
synthesis for sequencing or for PCR amplification. For these
applications it is very important to know how much one basebase mismatch will destabilize the hybridization. The specificity
of the hybridization is crucial for diagnostics, where one base
change can mean the difference between a normal gene and a
cancer gene. Some results on mismatches have been obtained,39
but a complete data set is not available.
The nearest-neighbor assumption does not seem to be as
useful in DNA as it is in RNA. One reason may be that the
conformation of DNA is more sequence dependent than RNA
is. There is evidence for this from measurements of optical
properties such as absorption and circular dichroism. The
circular dichroism of the polynucleotide with all A’s on one
strand and all T’s on the other (polyA‚polyT) can not be used
for a nearest-neighbor calculation of sequences containing two
or three consecutive A’s.40,41 More direct evidence of sequencedependent conformation is that consecutive A‚T base pairs cause
the DNA to bend.42
Thermodynamic analyses of transitions from single strands
to double helices, or to folded single strands, require the
identification of the initial and final states of the system. For
nucleic acids the single-stranded states in particular are very
temperature dependent and not easily defined. Any partially
single-stranded region of a folded RNA, or a partially melted
DNA double helix, has this complication. What is needed is a
partition function to characterize the nucleic acid at each
temperature and a statistical thermodynamic interpretation of
the system. Theoretical equations were derived to explain the
melting of long double helices, and the main features of the
melting were correctly reproduced. The theory is described in
Theory of Helix-Coil Transitions in Biopolymers,43 and many
of the original papers are included there.
Kinetics of Double-Helix Formation. The rates of formation
and dissociation of a double helix are crucial for the correct
functioning of nucleic acids. There is competition between rates
of conformational changes of macromolecules and rates of
chemical reactions in a biological cell. The kinetics of doublehelix formation were studied systematically by temperaturejump kinetics at the Max Planck Institute in Göttingen,
J. Phys. Chem., Vol. 100, No. 31, 1996 13315
Germany. Many of the same oligonucleotides used for the early
thermodynamic studies were used for the kinetics.44-46 For
RNA oligonucleotides in the range from 6 to 14 base pairs the
second-order association rate constant for forming a double helix
at room temperature is of order 1 × 106 M-1 s-1. This is slower
than a diffusion-limited reaction, but it is not very sensitive to
sequence or chain length (as expected for diffusion-limited
reactions). The activation energy is sequence dependent.
Molecules with G‚C pairs have an activation energy in the range
+5 to +10 kcal mol-1. For molecules with no G‚C pairs the
activation energy is negative! The explanation is that a stable
nucleus of two or three A‚U base pairs must be formedswith
a negative free energysbefore a fast zippering of the remaining
base pairs occurs.46 The dissociation rate constants and activation energies for forming single strands from the double helix
are very sequence and length dependent as expected. The
dissociation activation energies are closely related to the
equilibrium enthalpies of dissociation.
The double-helix to single-strand reactions for oligonucleotides have relaxation times in the range of milliseconds as
measured by conventional temperature-jump methods using an
electric discharge from a capacitor to raise the temperature. A
much faster signal is also seen that is ascribed to single-strand
conformational changessstacking-unstacking of the bases.
These dynamics in the nanosecond to microsecond range have
been measured by laser temperature-jump methods.47 NMR
measurements can also be used to measure the kinetics of
double-helix association in the millisecond range and the
nanosecond dynamics of individual nucleotides.48
The rate constants for formation and dissociation of helices
are directly applicable to understanding the kinetics of
ribozymessRNA catalystssthat cleave RNA.49 The RNA
ribozyme binds a substrate RNA, cleaves the RNA strand, and
releases the products to bind a new substrate. The efficiency
of the enzyme depends on the chemical steps of breaking
phosphodiester bonds and on the physical steps of binding
substrate and releasing products. Any of these steps can be
rate limiting. Ribozymes are being investigated as antiviral
agents that will cleave the RNA of a target virus. Specificity
can be increased by using a larger binding sequence on the target
RNA, but this clearly decreases the rate of product release after
cleavage. Knowledge of the oligonucleotide rate constants
allows a rational optimization of specificity and of kinetic
efficiency.
Prediction of Base Pairing. The folded, three-dimensional
conformations of RNA molecules have evolved to perform their
many functions effectively. The first step in assessing the RNA
conformations is to learn which bases form Watson-Crick base
pairs. This is far from knowing a 3D structure, but it greatly
limits the conformational space possible. The imino NMR
spectrum can provide useful information about the base pairing
in small nucleic acids (less than 50 nucleotides). Guanine and
uracil (or thymine) each have one imino proton (see Figure 1)
on the base. These protons resonate in a region (15 to 10 ppm)
well separated from the nonexchangeable protons (below 10
ppm). Furthermore, only the slowly exchanging protons are
seen in the spectrum; protons exchanging rapidly (lifetimes less
than about 1 ms) will not be seen.50 Thus, in H2O a proton
spectrum in the imino region will show one peak for each base
pair (or slowly exchanging imino proton). As the rate of
exchange increases due to increasing temperature, or due to
change of pH or added catalyst, the proton spectrum will
broaden and eventually disappear.51 Base pairs at the ends of
helices, or next to perturbations of the helix, broaden first.
13316 J. Phys. Chem., Vol. 100, No. 31, 1996
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TABLE 1: A-, B-, and Z-Form Double-Stranded Nucleic Acids4
sense of helix
sugar puckera
glycosidic angleb
repeating unit
base pairs per turn
major groovec
A-form
B-form
Z-form
right-handed
C3′-endo
anti
1 base pair
11
deep and narrow
right-handed
C2′-endo
anti
1 base pair
10
wide
left-handed
alternating C3′-endo and C2′-endo
alternating anti and syn
2 base pairs
12
very shallow
a
In nucleic acids the deoxyribose and ribose five-membered rings have either the 2′-carbon (C2′-endo), r the 3′-carbon (C3′-endo) above the
approximate plane of the other four carbons. The base is attached above the 1′-carbon. b The glycosidic angle is the torsion angle around the C-N
bond between the sugar ring and the base. Syn and anti torsion angles differ by 180°. c The major groove is the side of the double helix the bases
face; the minor groove is the side the sugars are on. The main interactions between proteins and DNA occur through the major groove.
Sequence Correlation (Phylogenetic) Methods. The most
common method for establishing base pairing is to compare
the sequences of a large group of RNAs that all have the same
function. For example, all cells contain ribosomes, part of the
machinery that synthesizes proteins. Hundreds of 16S ribosomal
RNAs have been sequenced; their sequences of about 1500 bases
are similar but not identical. By finding covariation of complementary bases, one can identify base-paired regions.52
Possible helical regions of four or more consecutive base pairs
are identified first. Then one looks, for example, for a change
from an A‚U to a G‚C pair in a possible helix. A helix is
assumed proven if there are at least two covariations of sequence
that leave the number of base pairs unchanged in a possible
double helix. Clearly, if the helix sequence is completely
conserved, this method cannot identify it. However, the most
highly conserved sequences seem to be in single-strand regions.
This phylogenetic method has been very accurate when tested
by chemical and physical methods.53
Thermodynamic Methods. A free energy can be estimated
for any folded RNA molecule relative to the unfolded single
strand by adding the free energies of its stems (double helices),
loops, and junctions. The free energies of these secondary
structural elements can be obtained experimentally. We realize,
of course, that additivity is only a first approximation because
interactions among the secondary structural elements may also
be significant. That is, the folded RNA may contain tertiary
structural elements such as pseudoknots,54 kissing hairpins,55,56
and so forth. We will ignore tertiary structure temporarily and
attempt to predict secondary structure by finding the lowest free
energy structure. We clearly need to search among a great many
possible structures.
A dynamic programming algorithm was devised to find the
lowest free energy structure among all possible Watson-Crick
paired secondary structures.57 The strategy is to calculate the
minimum free energy for a secondary structure element that
has base i paired to base j. The formation of this base pair
separates the linear sequence into two parts: the sequence
between i and j and all the rest. Adding base pairs to one part
has no effect on the free energy of the other part. We first
examine all base pairs separated by three unpaired bases
(assuming that this is the minimum hairpin loop size). Other
base pairs are added, and the minimum free energy is calculated.
At the end of the process the calculated global free energy is
obtained. The key to the algorithm is that by neglecting
interactions between loops the global minimum is a sum of local
minimasthe free energies of the component optimal secondary
structure elements.
Unfortunately, it quickly became evident that the calculated
folded RNA structure was often inconsistent with experiment.
Some of the calculated structural elements were correct, and
some were not. It was important to calculate not just the lowest
free energy, but also other low free energy structures. Experiments, such as chemical reactivity of certain bases or enzymatic
cleavage of single-strand regions, could be used to select among
the predicted structures. Even if the correct structure was
obtained from the calculated global free energy, alternate
structures could be important during the biological functioning
of the RNA. To obtain structures of low free energy, but not
the lowest free energy, it was necessary to extend the original
algorithm57 to save suboptimal structural elements.58,59 It was
necessary to identify significantly different low free energy
structures. Otherwise, too many structures would be obtained
that only differ from the global minimum by breaking one base
pair, for example.
In the Zuker program59 the experimental thermodynamic
parameters have been expanded over the years to include G‚U
base pairs, specific base-base mismatches, extrastable tetraloops, dangling bases at the ends of helices, coaxial stacking
of helices, and so forth.60 The predictive accuracy has improved
significantly, making the program very useful in identifying
possible secondary structures. For example, when designing
an oligonucleotide sequence to form a particular structure, it is
important to avoid alternate structures. The Zuker program is
very helpful in choosing a likely sequence. Similarly, in studies
of natural RNAs the program can identify secondary structures
to test by direct experiment.
Three-Dimensional Conformations. A-, B-, and Z-Form
Antiparallel Double-Stranded Helices. DNA fibers were first
found in two forms: a well-ordered A-form at low relative
humidity and a more disordered B-form at humidities above
90%. In aqueous solution (and presumably in biological cells)
DNA is B-form. Decreasing the water activitysby adding
ethanol, for examplescan induce a B to A transition. The
conformation of the DNA double helix is sensitive to sequence,
solvent, and temperature; therefore, it is best to think about
families of structures: A-type and B-type. However, the two
families are distinct and can undergo a first-order transition from
one family to the other. The structural differences among the
right-handed A- and B-families and the left-handed Z-form are
summarized in Table 1. All three forms have antiparallel
strands. Left-handed Z-DNA was first seen in 4 M NaCl by
circular dichroism measurements on synthetic DNA polynucleotides containing alternating deoxyribo-C’s and deoxyriboG’s on each strand (polydCdG‚polydCdG).61 The lefthandedness and the detailed structure were determined by X-ray
crystallography of a six-base-pair helix.62 In solution the
conformations of DNA are not identical to those seen in crystals.
The number of base pairs per turn for B-DNA in solution is
near 10.3 (depending on sequence, solvent, etc.) rather than 10.0.
This number can be measured very accurately in solution by
exploiting the topological constraints in a covalently closed
circular DNA to count the number of strand crossings.63
RNA is A-form in fibers independent of relative humidity
and is A-form in most aqueous solutions. In 6 M NaClO4 RNA
polynucleotides of alternating ribo-C’s and ribo-G’s (polyrCrG‚
polyrCrG) undergo a transition to left-handed Z-form.64 NMR
Nucleic Acid Structures, Energetics, and Dynamics
studies showed that the conformation was very close to that of
Z-form DNA.65 Attempts to produce B-form RNA have failed
so far; the A-form helix melts to single strands without passing
through a B-form geometry.
In biological cells, the predominant form of DNA is B-form
and that of RNA is A-form. However, Z-form DNA and Z-form
RNA exist naturally as shown by in ViVo binding of antibodies
specific for these forms.66,67 There are strong indications that
Z-DNA is involved in gene regulation; the function of Z-RNA,
if any, is unknown.
The structures and thermodynamics of the A-, B- and
Z-families of nucleic acids and the statistical mechanics and
kinetics of their interconversions provide a challenging arena
for developing and testing theories of interactions in charged
polymers. Of course, the different conformations are not static
entities. The double helices undergo bending and torsional
vibrations; each nucleotide has its own dynamics. The global
and local motions can be followed by such methods as dynamic
light scattering, anisotropy of fluorescence, nuclear magnetic
resonance, and electron paramagnetic resonance.68
Parallel Double-Stranded Helices.69 Watson-Crick double
helices have the two strands with the 5′-end of one next to the
3′-end of the other (see Figure 1); they are antiparallel.
However, by linking two strands with a loop containing a 5′5′ link or a 3′-3′ link, a hairpin (stem-loop) can be synthesized
in which the strands in the stem are parallel.70 Parallel doublestranded helices form containing A‚T pairs and G‚C pairs; the
helices are less stable than the identical sequence with antiparallel strands. The hydrogen bonding between bases is reverse
Watson-Crick pairing71 as shown in Figure 3. Parallel doublestranded helices can also be held together by A‚A pairs and
G‚G pairs.72,73 A parallel double-stranded helix of poly-A‚polyA+ has been known for many years,74 and G-quartets can have
either parallel or antiparallel strands.75 Single nucleotides that
reverse the direction of the chain occur in natural RNA
molecules.76,77
Triple-Stranded Helices.78,79 Synthetic double-stranded helices containing all A’s on one strand and all U’s or T’s on the
other can undergo a disproportionation to form a triple-stranded
helix and a single strand of U’s or T’s.80
polyA‚polyU + polyA‚polyU f
(polyA‚polyU)‚polyU + polyA
The reaction is favored by molar concentrations of univalent
cations or millimolar concentrations of divalent cations. Triple
strands also form with (polyG‚polyC)‚polyC+. Although the
pK of cytosine is about 4.2, stabilization of the protonated form
by triple-strand formation allows the helix to form near neutral
pH. The helix is made up of hydrogen-bonded base triples with
the purine (A or G) forming one Watson-Crick pair and one
Hoogsteen pair (see Figure 3). The Watson-Crick pair has
the usual antiparallel strand directions; the third strand (containing the pyrimidines U or C) is in the major groove and is
antiparallel to the pyrimidine strand. Triple-stranded helices
with a Watson-Crick pair plus a homopurine strand, such as
(polyA‚polyT)‚polyA and (polyG‚polyC)‚poly G, also form.
Again, the Watson-Crick strands are antiparallel, and the third
strand is in the major groove and antiparallel to the strand
containing the same type of base. Mixed-sequence triple strands
have been studied in many combinations to determine their
structures and thermodynamic stabilities; a small sampling of
references is given.81-85 Very little work has been done on the
kinetics of the formation of triple helices.86
DNA triple-helix regions exist in biological cells. Sequences
of homopurines-homopyrimidines in natural DNAs can dis-
J. Phys. Chem., Vol. 100, No. 31, 1996 13317
Figure 4. Two topoisomers of a double-stranded DNA. On the left is
shown a molecule with linking number, Lk ) 20; twist, Tw ) 20; and
writhe, Wr ) 0. On the right is a topological isomer with Lk ) 18,
Tw ) 20, and Wr ) -2. Twist and writhe need not be integers, but
their sum must be an integer. The Watson-Crick windings are righthanded (Tw is positive), so a negative writhe means that left-handed
supercoiling is present. The topoisomers can be separated by gel
electrophoresis. Reproduced by permission from ref 75. Copyright 1994
Academic Press.
proportionate as above to form triple helices and unpaired single
strands.78,87 There is great interest in using triple-helix formation
in an antigene strategy to control gene function for medical use.
The idea is to treat a patient with an oligonucleotide that forms
a triple helix and prevents a specific DNA, such as a bacterial
DNA or a cancer-causing gene, from being expressed.88,89
Understanding the thermodynamics of which base triples form
and how mismatches decrease stabilities is obviously of crucial
importance for the clinical applications of antigene therapy.
Supercoiled DNA and Topological Isomers of DNA.90 The
ends of a double-stranded DNA can be be covalently linked to
form a circle. Once this is done the number of times one strand
crosses the other is permanently fixed as long as neither strand
is broken. The number of strand crossings (counted by
projecting the strands on a plane) is a topological constant called
the linking numbersan integer. Natural DNAs often occur as
covalently closed circles. For example, plasmids that provide
bacteria with antibiotic resistance are naturally occurring covalently closed circles. DNA in higher organisms are sometimes
topologically constrained by proteins that hold two regions in
a fixed relation. In either case this means topological
isomersstopoisomerssexist.
Topoisomers were first identified and characterized in
polyoma viral DNA by ultracentrifugation and electron microscopy.91 Double-stranded DNA can have “sticky ends” with a
5′ single strand overhang on one end complementary to a 3′
single strand overhang on the other end. The enzyme DNA
ligase can covalently close the sticky ends to form intramolecular
circles or intermolecular dimers, trimers, etc. The circles formed
are a mixture of topoisomers having linking numbers that differ
by one (see Figure 4). When the ends are ligated, the mean
number of strand crossings depends on the number of base pairs
(one strand crossing for every ≈10.3 base pairs), but there will
be a distribution of linking numbers. For example, for 1000
base pairs there could be a linking number of 1000/10.3 ) 97
and also 95, 96, 98, and 99. The distribution is at equilibrium
(the ligase action is slow compared to the fluctuations in
conformations); therefore, the relative concentrations of topoisomers provides the free energy diffferences among topoisomers.92 The topological constraints characterizing the topoisomers are represented by90
13318 J. Phys. Chem., Vol. 100, No. 31, 1996
Lk ) Tw + Wr
The integer linking number, Lk, is a constant for each topoisomer as long as no bonds are broken. It is equal to the sum
of twist, Tw, and writhe, Wr. Twist is the number of double
helical turns in the DNA, and writhe is a measure of the
superhelical turns as indicated in Figure 4. Neither twist nor
writhe is necessarily an integer, but their sum is. The
hydrodynamic properties of the DNA depend on its writhe. This
means that molecules with different writhes can be separated
by ultracentrifugation or, most conveniently, by gel electrophoresis. We now have an extremely sensitive method of
studying twistsa direct measure of the number of base pairs
per turnsin DNA. The number of 10.3 base pairs per turn used
above came from studies of topoisomers.63 Of course, the exact
number will depend on the sequence, the pH, the ionic strength,
etc. Instead of base pairs per turn, we can consider the winding
angle (360°/base pairs per turn = 35°); it decreases about 0.01°
per °C rise in temperature.92 The effect of any molecule that
unwinds the DNA double strand, such as proteins that bind or
antibiotics that intercalate, can be characterized precisely.
Conformational transitions from B to A, or particularly to lefthanded Z, can be easily monitored.93,94 Creative applications
of the simple topological equation above have provided a wealth
of information about structures and thermodynamics of DNA.
Theories and calculations of the shapes and dynamics of
supercoiled DNAsthe spatial manifestation of the writhesare
numerous; we cite two.95,96
The correct topological state of DNA molecules in living cells
is crucial for the replication of the DNA and the division of the
cells. Enzymes called topoisomerases and gyrases are continually unwinding and winding the DNA double strands during
replication. Topoisomerase inhibitors are being tested as
anticancer agents and as antibiotics.97,98
Folded RNA Structures. The first RNA structure determined
at atomic resolution was the crystal structure of phenylalanine
transfer RNA99sthe 76-nucleotide RNA that reads the threeletter codon on the messenger RNA and places the correct amino
acid on the growing polypeptide chain. In 1973, this RNA
already provided coordinates for many of the structural elements
that are now being investigated in other RNAs: G‚A nonWatson-Crick base pairs, base triples, hairpin loops, four-stem
junctions, and so forth. The number of RNA X-ray structures
published since then has not been large because of the difficulty
of obtaining useful crystals. However, NMR studies of RNA
oligonucleotides in solution are now providing coordinates and
dynamics for all kinds of non-Watson-Crick base pairs, hairpin
loops, internal loops, bulges, pseudoknots, junctions, and so
forth. Several reviews are available.36,100-102
Tetraloops are hairpin loops containing four nucleotides in
the loop. They occur often in natural RNA sequences, but
among the 256 possible permutations a few tetraloop sequences
are very common. These tetraloops are also more stable
thermodynamically relative to the unfolded single strand than
the less common loops. For example, the extrastable tetraloop
5′-GGAC(UUCG)GUCC-3′ has a standard free energy change
at 37 °C of -6.3 kcal mol-1 for forming four base pairs and a
UUCG loop from the single strand in aqueous solution in 1 M
NaCl.103 Changing the loop sequence to (UUUG) or (UUUU)
increases the standard free energy to -4.2 kcal mol-1. To
understand the reason for the difference in 2.1 kcal mol-1 in
loop stability, we began an NMR study of the (UUCG) and
(UUUG) tetraloops and furnished the oligonucleotides for
crystallization for an X-ray study.
The NMR structures of the tetraloop 12-mer oligonucleotides
were determined by a now standard method.104 The 1D imino
Tinoco
spectrum measured in H2O reveals the hydrogen-bonded, or
slowly exchanging, imino protons of guanine and uracil. There
are seven imino protons in the (UUCG) 12-mer; they are all
visible in the spectrum. The amino protons of adenine, cytosine,
and guanine are more difficult to observe because of rapid
rotation of the amino group around the C-N bond. Labeling
with 15N allows their identification.105 The 2′-hydroxyl hydrogens are the most difficult to see; most of them exchange too
fast with water. However, the 2′-hydroxyl of the first U of the
(UUCG) tetraloop is visible. It is present in a H2O spectrum
and vanishes in D2O, but 1H-15N correlated spectra prove it is
not bonded to nitrogen.106 Its identification and assignment were
confirmed by a three-bond coupling to the C3′ of the ribose
group of that U.107
The 92 nonexchangeable protons were assigned by a combination of two-dimensional correlated spectroscopy (COSY),
which gives through-bond scalar couplings, and 2D nuclear
Overhauser effect spectroscopy (NOESY), which gives throughspace dipolar couplings.108 The scalar coupling (J-coupling)
constants are related to torsion angles by Karplus-type equations;
the nuclear Overhauser enhancements are proportional to the
inverse sixth power of the interproton distances. These
constraints are added to standard bond lengths and bond angles
to obtain a conformationsor range of conformationssconsistent
with all the NMR data. The results for the (UUCG) tetraloop
are shown in Figure 5a.109 Reasons for the extra thermodynamic
stability of the loop apparent in the structure are the U‚G nonWatson-Crick base pair, which involves an unusual syn
guanosine and a hydrogen bond between the C amino group
and a nearby phosphate oxygen. Changing this C to a U in the
less stable (UUUG) tetraloop replaces the C amino by a U
carbonyl oxygen that repels the phosphate. The conformation
changes very slightly, but the stability markedly decreases. There
is an increase in ∆G° of +2.1 kcal mol-1. Of course, the
stabilities of the two tetraloops relative to the single strands
depend on many interactions other than the hydrogen bonds
mentioned. Stacking of the bases, hydrogen bonds to water,
locations of counterions, and so forth are all important. It would
be wonderful to be able to calculate all these effects and to be
able to predict structures and thermodynamics of all 256
tetraloops; it would leave so much more NMR time for all 1024
pentaloops.
To assess the quality of the NMR structures, we wanted to
compare NMR coordinates with coordinates from X-ray crystal
structures. Both the UUCG110 and UUUG111 oligonucleotides
were determined, but unfortunately neither form hairpin loops
in the crystal. They both form double-stranded helices with
four non-Watson-Crick base pairs in the center of each helix.
The crystals give high-resolution structures for G‚U, U‚C, and
U‚U base pairs and show that the mismatches do not disrupt
standard A-form helices very much. Presumably, helices rather
than hairpins form in the crystals because of the higher
symmetry of the helices. We tried to force double-stranded
helices to form in solution, but we only achieved aggregates
and precipitates.
A more complex structure is a pseudoknot;112,113 it is a
hairpinsa stem-loopswith base pairing of bases in the loop
with a single-strand sequence that can extend from either side
of the stem. Thus, two hairpins form in which bases in the
loop of each hairpin form part of the stem of the other. If each
stem had a complete turn (11 base pairs), a knot would be
formed if the ends were linked. At Berkeley we investigated
the effect of loop size on pseudoknot stability114 and used NMR
to show that the stems are coaxially stacked and that the two
loops are on the same side of the quasicontinuous helix.113 We
Nucleic Acid Structures, Energetics, and Dynamics
Figure 5. (top) NMR structure109 of a 12-nucleotide extrastable hairpin
tetraloop. There are four Watson-Crick base pairs in the stem; the
loop sequence is UUCG. (bottom) NMR structure of a pseudoknot that
causes frame shifting in MMTV retrovirus.115 There is an A nucleotide
between the two stems that favors the bent structure shown. Deletion
of the A leads to a linear pseudoknot and eliminates the frameshifting.
have recently determined the structure of a type of pseudoknot
necessary for the infectivity and replication of many retroviruses.115 The pseudoknot facilitates a programmed frameshift
during the translation of the retroviral RNA that leads to the
synthesis of such vital viral enzymes as reverse transcriptase
and integrase.116 The structure115 of the 34-nucleotide molecule
(Figure 5b) reveals a bent molecule with an intervening A
between the two stems. The loops are too short to bridge the
stems, so a bend occurs at the junction of the stems. The
intervening A provides the hinge. Removing this A eliminates
the frameshifting117 and produces a linear pseudoknot with
coaxially stacked stems.118 We are continuing study of the
structure-function relation of pseudoknots and retroviral frameshifting. We hope that it may suggest new methods to inhibit
viruses.
Prediction of Folded RNA Structures. Calculations of
three-dimensional structuressconformationssof RNA can be
done by the usual methods of molecular dynamics and free
energy calculations.119-121 However, the potential functions for
J. Phys. Chem., Vol. 100, No. 31, 1996 13319
intra- and intermolecular interactions are not good enough to
produce believable results in ionic solutions. If empirical data,
such as distance and torsion angle constraints from NMR
experiments, are used to find an approximate conformation, then
these methods can be used to improve and refine the structure.
The theoretical calculation provides the minimum free energy
structure in the local minimum chosen by experiment. The
NMR data determine the structure; the calculation just polishes
the result.
Other semiempirical methods use more qualitative experimental data. For example, thermodynamic and phylogenetic
methods can provide the secondary structuressingle-strand and
double-strand regions. Chemical and enzymatic reactivity of
each nucleotide and of different groups within each nucleotide
show which groups are available to solvent and which groups
are involved in other interactions. These data provide a starting
conformation for calculation of three-dimensional structures.
One method uses databases from crystal structures of mononucleotides and oligonuclceotides to limit the posssible calculated structures.122,123 The conformation of each nucleotide is
specified by seven torsion angles; only torsion angle values
found in known stuctures are allowed in the starting conformation. For simple, small hairpin root-mean-square deviations of
1.5-2.0 Å between calculated and observed can be expected.124
Modeling larger RNA containing hundreds of nucleotides can
give useful structures, although coordinates cannot be compared
with experiment.125-127 The predicted structures are vital for
designing experiments to test the predictions and to improve
the models.
Studies of Single DNA Molecules.128 The observation of the
mechanical properties and the viscoelastic behavior of a single
DNA molecule provides a new level of information about its
energetics and dynamics. Micron-sized fluorescent beads have
been attached to the ends of a single DNA molecule. The beads
serve both to manipulate the molecule and to visualize its ends.
Direct measurement of the elasticity of a DNA was obtained
by tethering one end of the DNA to a glass slide and using
flow and magnetic fields to apply forces in the range 10-14 to
10-10 N to a bead on the other end.129 The force vs extension
curves can be explained reasonably well in terms of entropic
elasticity.130 A receding meniscus has also been used to stretch
a DNA attached to a surface at both ends.131 Optical tweezers
can be used to manipulate beads attached to DNA to study
stretching, relaxation, and motion of single molecules.132-134
The ability to visualize and study a single nucleic acid
molecule in solution allows many new possibilities. Instead of
averaging physical properties over many molecules, we can
average one molecule over time. We trust the two methods
will give the same answer. Spectroscopic studies, such as linear
dichroism, circular dichroism, and hypochromism, of a single
molecule at different extensions and with different ligands bound
can be very informative. We can learn about structure and about
our understanding of nucleic acid optical properties.
Functions
Catalytic RNA Molecules: Ribozymes. Thomas Cech was
trying to isolate and purify the enzyme in the protozoan
Tetrahymena that catalyzes the excision of a 414-nucleotide
piece of its pre-ribosomal RNA. This RNA processing commonly occurs in pre-ribosomal RNAs, pre-transfer RNAs, and
pre-messenger RNAs. Cech found that during purification as
the amount of protein decreased the catalytic activity of the
enzyme increased. In fact, catalysis occurred in the complete
absence of protein. The RNA cut and spliced itself in the
presence of Mg2+ and a guanosine cofactor.135 The biochemists
13320 J. Phys. Chem., Vol. 100, No. 31, 1996
were convinced at first that the result was an artifact. They
thought that Cech did not know how to properly purify a sample
to remove all traces of contaminating proteinsthe real catalyst.
He was able to convince them that RNA catalysts did exist after
he synthesized the Tetrahymena RNA in another organism and
showed that it still was catalytic, although it had never been
exposed to Tetrahymena proteins.136 Cech and Sidney Altman,
who discovered a ribozyme that processes a pre-transfer RNA,137
shared the Nobel Prize for chemistry in 1989. A wide range of
catalytic RNA moleculessribozymesshave been discovered
and characterized since the original discovery in 1981.138 An
extensively characterized class of ribozymes, called hammerhead
or hairpin ribozymes, occur in plant viroids that self-cleave in
ViVo.139 They can be tailored to cleave or ligate external
substrates. Detailed kinetic studies of each step of their reactions
have been done,49 and two crystal structures have been
determined.140 NMR studies are underway, but no solution
structure has appeared yet. It is clear that the RNA is flexible
and that the structures obtained in the crystal or in solution will
not necessarily reveal the catalytic mechanism. Specific metal
ions are involved in the catalysis; their locations and interactions
are critical. The field of ribozyme catalysis is new (approximately 1980) and very important in understanding chemical
catalysis in solution, in providing new ideas about chemical
evolution of life,37 and in devising new methods of treating viral
diseases by sequence-specific cleavage. It is an active field as
shown by the 300 references found with the title word
“ribozyme” in the past six years of the Current Contents
database.
Random Synthesis and Selection of RNAs To Perform
Any Function.141 It is very easy to synthesize DNA or RNA
with random sequences; at each cycle of a commercial synthesis
machine all four nucleotides are introduced. The number of
different sequences produced depends on the number of
positions randomized and the total number of molecules present
in the amount of material synthesized. A 1 µmol synthesis could
produce about 1017 different sequences; this is the number of
possible sequences for a randomized 28-mer. Of course, you
can randomize more than 28 nucleotides; you will then get only
a small sampling of the total sequence space available. The
idea is to select the sequence among this large number that does
the function that you choose. For example, you can isolate a
sequence that binds a certain protein.142,143 The random mixture
of DNAs or RNAs is passed through a column containing the
target protein, and the tightest binding fraction is collected. This
fraction is amplified by PCR (for RNAs the fraction is first
reverse transcribed into DNA, amplified, and then transcribed
back to RNA), and the selection process is repeated. After about
10 cycles of selection and amplification, only a few strongbinding sequences are present. Each different sequence can be
cloned and characterized as to binding constant and structure.
From 1017 potential binders a small number of “best” molecule
have been selected. The functions and properties that can be
evolved are only limited by the originality of the experimenter.
Examples include RNA cleaving ribozymes,144 DNA cleaving
ribozymes,145 amide cleaving ribozymes,146 ligases,147 ATP
binders,148 a ribozyme that isomerizes a hindered biphenyl,149
and so forth. The ability to evolve RNA or DNA molecules to
have specific physical and chemical interactions provides a
unique method to test theories of molecular interactions. Of
course, it also can produce molecules of great medical and
practical use.
Conclusions
The Role of the Physical Chemist. Physical chemists
understand thermodynamics and kinetics. They can apply
Tinoco
quantum mechanics and statistical mechanics to increasingly
complex systems. They are comfortable with all the structural
methods including X-ray crystallography, nuclear magnetic
resonance, and other spectroscopies. So what do we lack that
prevents us from discovering and solving the important problems
in biology? It is lack of knowledge, and therefore lack of
interest, in the biological problems. We must take the time and
trouble to learn what questions the neurobiologists, the developmental biologists, the physiologists, the geneticists, and even
the physicians are asking. It may be that we can answer some
of their questions immediately. We will certainly learn about
the difficult, challenging, and vital problems that someone will
solve in the next 100 years.
Acknowledgment. Professor Carlos Bustamante, University
of Oregon, Dr. Ling X. Shen, University of California at Santa
Cruz, and Professor Douglas Turner, University of Rochester,
were kind enough to read the manuscript and to make very
helpful suggestions. Professor James Williamson, MIT, and
Professor Richard Sinden, Texas A&M, kindly supplied figures.
My research has been supported by grants from NIH and from
DOE; their support is gratefully acknowledged.
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