From the
Rapid drug discovery is a pressing need in the pharmaceutical
industry. As new target proteins are proposed for therapeutic
intervention, the methods of random screening or rational drug design
may not be adequate to the challenge.
SELEX
We frequently seek chemicals with properties suitable for a
given task. In pharmaceutical, diagnostic, and biological research,
these chemicals often must bind tightly and specifically to some target
molecule, even though the target may be undefined during the search for
the appropriate chemical. Search strategies for novel binding compounds
historically depended on screening; the natural products found
in fermentation broths, plant extracts, and organisms without tumors or
infections have been a source of interesting compounds and new drugs.
In addition, medicinal chemists have created derivatives of many
natural compounds, seeking improved efficacy. Thus large libraries of
natural and synthetic chemicals are extant (perhaps comprising nearly
10
SELEX (Systematic Evolution of Ligands by EXponential
enrichment
(1) ) is a combinatorial chemistry methodology in
which vast numbers of oligonucleotides (DNA, RNA, or unnatural
compounds) are screened rapidly for specific sequences that have
appropriate binding affinities and specificities toward any target.
SELEX also has been used to identify new ribozymes and
deoxyribozymes
(4, 5) .
When a high affinity ligand has been
identified using a particular set of monomers (ribonucleotides,
deoxyribonucleotides, 2`-aminopyrimidine ribonucleotides, or
5-iodopyrimidine ribonucleotides), full replacement of the monomers in
the winners by other nucleotide monomers degrades the affinity and
specificity of the ligands (winning RNA ligands are not potent and
specific ligands when made as DNA and vice versa). We
usually front-load SELEX with the modifications intended for final use,
but we also perform post-SELEX modifications
(15) . Substitutions
that enhance the ligand performance can be made at some positions in
SELEX-derived ligands. Again, there are too few experiments and not
enough data to form rules for acceptable or enhancing post-SELEX
modifications.
The targets that have been used for SELEX include nucleic
acid-binding proteins, other proteins (including growth factors,
proteases, antibodies, and small peptides), and small
molecules
(4) . The protein targets that do not function in
vivo as nucleic acid-binding proteins include large and small
proteins, glycosylated and non-glycosylated proteins, enzymes and
structural proteins, and complex mixtures of target proteins found as
aggregates or on the outside of cells. The small molecule targets
include biologically interesting redox-sensitive molecules
(16) ,
dyes
(12) , drugs
(17) , amino
acids
(18, 19, 20) , and
nucleotides
(21, 22) .
Nucleic acid-binding proteins
have yielded ligands after SELEX with high specificity and dissociation
constants in the low nanomolar range. With respect to specificity,
oligonucleotides selected to bind to one retroviral reverse
transcriptase do not bind with high affinity to other reverse
transcriptases
(23, 24) . SELEX has been used for
proteins whose biological targets are known; the SELEX-derived ligands
usually bind more tightly than the natural sequences. Often the
selected ligands have significant structural or sequence relations to
the natural ligands
(25) . No extremely high affinity
SELEX-derived ligands (low picomolar, for example) have been reported
thus far for known nucleic acid-binding proteins.
Protein targets
that are not thought to bind nucleic acids naturally give
ligands after SELEX with high specificity and dissociation constants in
the low nanomolar range and, frequently, in the picomolar range. SELEX
against members of the fibroblast growth factor family gives ligands
with extraordinary specificity for the exact protein used as the target
during SELEX
(26) . Ligands identified for vascular endothelial
growth factor bind within the polyanionic recognition site that is
functionally aimed at heparin in vivo; again, those ligands do
not bind to the other heparin-binding proteins that were
tested
(27) . Non-nucleic acid-binding proteins appear to give
extremely high affinity ligands more frequently than known nucleic
acid-binding proteins. Antagonists are found without difficulty. It was
unexpected that ordinary proteins would be suitable targets for
SELEX
(28) , and it is quite shocking that those ordinary
proteins could be among the better protein targets.
The dissociation
constants for oligonucleotides aimed at low molecular weight targets
are usually in the high micromolar range, with an occasional target
yielding a ligand with a K
Oligonucleotides
identified through SELEX are as potent as antibodies with respect to
affinities and specificities
(29) . SELEX bypasses the obvious
issues of immune tolerance and thus seems suitable for many experiments
in which antibodies are inappropriate. For peptide libraries,
constrained or not, and the early versions of small molecule
combinatorial libraries, the affinities and specificities of the
winners do not approach the affinities and specificities of molecules
identified through SELEX
(2, 3) .
SELEX may work as
well as it does because of the stable structural motifs and restrained
``loops''
(9) that are formed by many random,
single-stranded nucleic acid sequences. The perfect binding partner for
any target object would complement its molecular shape, ionic
character, and hydrophobicity; such molecules could provide astounding
affinities and specificities, even for rather small contact surfaces.
Biotin-avidin springs to mind as an example of a ligand-target pair
that may approach what is possible for the atoms of biology. Perfect
binding partners are not found often in biology, probably because
finite dissociation rates are demanded in organismic evolution.
Proteins, which are macromolecules of distinct and exquisite shapes,
have surprising dynamic properties
(30, 31, 32) .
We know little of the dynamics of oligonucleotides and nothing about
the dynamics of those ordered oligonucleotides identified through SELEX
as potent and specific binding partners. Structural studies now under
way in many laboratories on these molecules will be informative and
will suggest if the entropic gains achieved by stable oligonucleotide
ligands rationalize the surprising potency and specificity of these
compounds.
We have been thinking about genomic
SELEX as a research tool to identify biological regulatory loops
that must exist. Imagine that the oligonucleotide library has been made
for SELEX by fragmentation of the DNA from an organism of interest
(with further modifications such that the library can be amplified and
used to seek RNA or DNA ligands). In an obvious use of genomic SELEX a
regulatory protein (the lac repressor, for example) is used to
find those sequences from the organism that are the most likely in
vivo targets. Genomic SELEX allows the natural targets for known
nucleic acid-binding proteins to be uncovered exhaustively, independent
of the particular genetic and biochemical experiments that identified a
specific target for that regulatory protein. Regulation of gene
expression might be even more intricate than we suspect.
More
interestingly, if every protein can be a SELEX target in
vitro, isn't it reasonable to wonder if many (even
all) proteins regulate cell activity by binding to some specific
nucleic acid in vivo? Any protein, housekeeping or not, may
contact a nucleic acid for a meaningful biological purpose. Genomic
SELEX experiments are under way and are formally similar to the
beautiful work of Fields and his colleagues aimed at uncovering the
protein-protein linkage map for an organism
(33) . Genomic SELEX
offers the opportunity to identify the protein-oligonucleotide linkage
map for an organism. We have no idea how common this phenomenon might
be, but the accidental discoveries of such regulatory loops are
provocative
(4, 34, 35) .
SELEX has
also been used to identify antagonists of blood clotting, some
antibodies, some essential viral proteins, and growth
factors
(4) . In all cases therapeutic uses of these fascinating
molecules will depend on in vivo data for various formulations
of the drug candidates. Finally, for those situations in which
permanent antagonists (analogous to suicide enzyme substrates) are
required, the SELEX technology has been modified so as to allow direct
selection of oligonucleotides that bind covalently to their
targets.
With these goals in mind I define a fully successful
combinatorial chemistry methodology as providing: 1) enough compounds
to provide rapidly very high affinity individual molecules for any
target, enough compounds to provide molecules aimed at several epitopes
on that target, and enough compounds to saturate shape space so
thoroughly that the library history is not relevant; 2) high
specificity of the emergent ligands toward their cognate targets and
weak binding to either related targets or other potentially interactive
molecules in the environment of intended use; 3) rapid,
cost-appropriate, generic methods to produce and use the ligands in the
intended environment; 4) rapid, generic formulation steps that retain
ligand activity with high bioavailability and low toxicity; and 5)
immediate testing in appropriate animal disease models if the intended
uses are therapeutic.
A fully successful combinatorial chemistry
methodology for therapeutics yields drug candidates rather than
compounds that require extensive further experimentation and
development, including reiterative medicinal chemistry accompanied by
tedious functional and toxicity assays. That is, combinatorial
chemistry should not seek lead compounds, ``hits''
for structural analysis and further development, and compounds that
with expensive and slow research may lead to a definitive experiment in
an animal. Combinatorial chemistry technologies should provide, if
possible, compounds for direct animal testing because in animals, and
finally in humans, the clinical outcome is all that matters. SELEX may
meet most, if not all, of the criteria for a robust, generic drug
discovery technology.
Biologists often think they have seen a set of
biological regulatory loops that, when perturbed, will lead to a
desired therapeutic outcome. Because of an incomplete biology data
base, target selection for such perturbation is a guess, sometimes
informed yet always biased by accidental observations that appear to
have been made just in time for the experimental scientist. Jacob
understood evolution as ``tinkerer''
(36) , leaving the
unavoidable reality of the human organism as a Rube Goldberg machine;
those machines are not predictable in detail from even substantial sets
of data. The deep value of combinatorial chemistry paradigms is that
many therapeutic interventions can be tried inexpensively in animal
models for the diseases of interest. For those of us concerned with
drug development, only robust combinatorial chemistry methodologies can
keep up with the rapid extension of our knowledge afforded by the
understanding of genes in pathogens, normal cells, and their altered
disease counterparts.
I thank my many colleagues at the University of
Colorado and NeXstar Pharmaceuticals Inc. for useful and stimulating
conversations.
INTRODUCTION
Combinatorial Libraries
SELEX: Oligonucleotide Combinatorial Libraries
Summary Statements about Oligonucleotides
Identified by SELEX
Uses of Molecules Derived from SELEX
Prospects
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
-derived oligonucleotides, possessing
shapes that interact with target molecules, may provide a surprising
alternative to other ligand discovery technologies. In addition, the
technology leads to a novel research idea that may enrich our
understanding of the mechanisms by which cells and organisms integrate
their metabolic activities.
compounds in sum), awaiting ever faster, ever more
automated ``high throughput'' screening methodologies. Since
screening begins with libraries made for past screenings, usually
unrelated to the present search, library histories constrain the
breadth of available compounds.
Rational Drug Design
This is an integrated set of
methodologies that include structural analysis of target molecules,
powerful synthetic chemistries, advanced computational tools, and
reiterative structural analyses of compound-target complexes. Rational
drug design, when aimed at protein targets, depends critically on the
depth of x-ray (or NMR) information and on calculations of
electrostatics, hydrophobicities, and solvent accessibility for those
targets. Since new potential target proteins are being identified
rapidly by human and pathogen genomic sequencing, rational drug design
faces a requirement for high throughput compound identification similar
to the demands on traditional, random screening.
Combinatorial Chemistry
The creation and
simultaneous screening of large libraries of synthetic molecules appear
to meet the requirements for rapid and successful compound
identification for many research, diagnostic, and therapeutic purposes.
In this review I focus on SELEX, the combinatorial chemistry technology
that uses oligonucleotides as the source of compounds
(1) . Two
very recent reviews describe and evaluate a variety of other
combinatorial chemistry methodologies. One review aims to elaborate the
various ways in which peptide and protein libraries are constructed and
screened and does not deal with oligonucleotide libraries
(2) .
The other review attempts to be complete and surprisingly does not deal
with oligonucleotide libraries
(3) , probably because the author
and most scientists are unaware of the useful qualities of
oligonucleotides. This review of SELEX might be read with the other two
reviews in hand for comparative purposes.
Strategy
The basic strategy is to prepare a library of
nucleic acid sequences that can be amplified, to challenge the library
for either binding or catalytic properties, to selectively amplify
those members of the library that were best at the challenge, and then
(if necessary) to rechallenge the amplified subset and continue the
process. One cycle of challenge and selective amplification is a SELEX
``round''; selective amplification usually is preceded by
physical partitioning of the better oligonucleotides prior to
amplification but can be accomplished with ``partitionless''
amplification that is dependent upon a positive response to the
challenge (for example, the ligation of an oligonucleotide sequence
that is used directly for amplification
(6) ). Virtually all
SELEX experiments have been reviewed in more depth than is possible
here
(4) .
Philosophy
Janda
(3) concludes his review
by noting the following. ``It appears that the next wave of
combinatorial research will be directed at the design of libraries that
are devoid of the repetitive backbone linkage found within peptides or
nucleotides. It is here that structural/stereoelectronic variation and
unconstrained motifs will be allowed to expand to unparalleled
combinatorial chemical diversity.'' And yet no binding challenge
thus far has been beyond oligonucleotides
(4) , in spite of the
repetitive backbone linkage. Single-stranded nucleic acids fold into a
bewildering number of stable structures, available in the vast starting
libraries of SELEX. SELEX provides ``unparalleled combinatorial
chemical diversity'' now, and modified nucleotides that are
compatible with SELEX
(7) add features beyond what may be
already ``unparalleled.'' Nucleic acids can be shapes, not
tapes
(8) ; biology has utilized nucleic acids as tapes for the
creation of genomes and for some (no longer
obvious
(4, 9) ) reason utilized proteins for most
recognition (and catalytic) phenomena.
Library Size
Nucleic acids are synthetically
randomized by condensing a mixture of activated monomers over some
specified number of positions (n), where the sequence
diversity for a nucleic acid with four different bases is
n; the availability of novel, additional base
pairs could allow even more random sequences over the same
length
(10) . Commercially available DNA synthesis machines are
approaching millimole scale, that is on a single column in a single run
(with 15% yield) about 10
molecules can be made. For SELEX
experiments in the literature the number of molecules in the starting
library has been 10
-10
, but this number
could be higher if necessary. Large lengths of randomness quickly
provide sequence diversity potential that dwarfs even the number
10
, so two libraries made at different times with, say,
100 randomized nucleotide positions, will share essentially no exact
sequences longer than 25-35 nucleotides. Thus SELEX will always
provide winners that reflect the best oligonucleotides in a specific
library, and the same sequences will be found in repeat selections only
when the best sequences have modest information content
(11) . In
the SELEX experiments reviewed
(4) the winning ligands were most
often found to represent between 1 in 10
and 1 in 10
of the starting library.
Length of Random Region
Random regions of roughly
30 nucleotides were used in many of the first experiments. The known
simple single-stranded oligonucleotide motifs can be built from 30
nucleotides
(9) : hairpins, bulges within helices, pseudoknots,
and G-quartets. Thus the major structural frameworks for
single-stranded oligonucleotides are reached easily with randomization
over the lengths that can be searched thoroughly; the experimentalist
need not specify a motif in building the initial library. The same
targets have been used for a few SELEX experiments in which the
libraries had different lengths of randomized sequences; while the same
motifs and sequences emerged, it seems likely that large and very
precise structural motifs will emerge idiosynchratically if long
randomized domains are used. In some instances the experimental goal is
to identify a rather short oligonucleotide drug candidate, and so very
long random regions have been avoided.
RNA, DNA, or Non-natural Single-stranded
Oligonucleotides
The first SELEX experiments were done with
RNA
(1, 12) . In the 50 SELEX experiments reviewed in
Gold et al.(4) , 36 used RNA, 5 used DNA, and 9 used
libraries containing modified nucleotides
(7) . The number of
experiments is small, and other variables (including partitioning
methods and the length of the randomized region) are numerous. At this
moment I can see no obvious pattern with respect to binding affinity or
specificity that correlates with the choice of oligonucleotide
chemistry. Binding affinity and specificity do seem correlated with the
number of SELEX rounds performed, but even for this parameter
(13) the correlation is driven by the small number of
experiments done in which weakly bound ligands were identified after a
small number of SELEX rounds. For many purposes the winning ligands
should be nuclease-resistant, and modifications toward that end can be
incorporated into the entire library
(14) . High affinity, high
specificity ligands have been found no matter what monomers have been
used to make the library.
Activities of SELEX-derived Oligonucleotides
At
first glance I have described a technology that yields binding reagents
and not bioactive compounds. While this would be interesting in its own
right and while SELEX thus contributes to discussions about an early
RNA world
(9) , in fact antagonists of a variety of protein
activities have been found without difficulty. Intracellular protein
targets that function naturally by interacting with nucleic acids have
been used to derive ligands; when studied in vitro or in
vivo those ligands are antagonists. The delivery issues for
ligands aimed at intracellular targets in a therapeutic setting include
all of the problems faced by antisense compounds. Extracellular
targets, including growth factors and secreted enzymes, have yielded
antagonists, although not every selected oligonucleotide is an
inhibitor of the target protein (nor would one expect that the dominant
SELEX epitope for every protein would overlap the active site). Rapid
screening for inhibitory ligands is routinely part of the technology,
and that screening (of 100 or so oligonucleotides) has been
successful
(4) . Oligonucleotide agonists have not been reported
thus far
(4) , but such compounds are likely to emerge.
of about 100
nM. The specificity of these oligonucleotides can be
startling. Oligonucleotides have been found that bind to AMP and not to
simple variants
(21) . Oligonucleotides have been found that bind
to theophylline and not to caffeine
(17) . Oligonucleotides have
been found that bind to an amino acid and not its
enantiomer
(18) . Recently, oligonucleotides have been found that
distinguish between NAD and NADH
(16) .
Research Uses
SELEX-derived oligonucleotides are
an alternative to antibodies for research purposes. The common step of
preparing antibodies to a new protein for intracellular localization
experiments could be complemented or replaced with SELEX-derived
reagents; one could modify oligonucleotides with
visualization-enhancing adducts and reporters. (Care should be taken to
utilize target proteins in the same structural state as those proteins
will be during the localization experiments, since SELEX ligands often
bind to structural epitopes that are lost upon protein denaturation
(26, 27).) Similarly, SELEX-derived reagents could be used for affinity
purification of proteins.
Diagnostic Uses
Oligonucleotides could be viewed
as alternatives to antibodies for in vitro and in vivo diagnostic reagents. The strength of these ideas depends on low
nonspecific oligonucleotide binding to other proteins (which is already
reasonably well established) and to components found in biological
specimens or animals. Data are being accumulated that high
concentrations of non-target proteins do not degrade affinities or
specificities.(
)In vivo imaging will
depend on some of the same issues raised by the potential therapeutic
uses of oligonucleotides.
Therapeutic Uses
One major effort since the
invention of SELEX has been aimed at direct use of oligonucleotides as
drugs. For example, a target for oncology has been angiogenesis, the
process by which most tumors recruit new vasculature for the required
supply of nutrients
(26, 27) . Tumors secrete a number of
angiogenic factors, and the responsive endothelial cells express some
key receptors at elevated levels
(37, 38) . SELEX has
been used to make antagonists that block angiogenesis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.