Note: Descriptions are shown in the official language in which they were submitted.
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METHOD AND APPARATUS FOR THE AUTOMATED
GENERATION OF NUCLEIC ACID LIGANDS
Field of the Invention
This invention is directed to a method for the generation of nucleic acid
ligands
having specific functions against target molecules using the SELEX process.
The methods
described herein enable nucleic acid ligands to be generated in dramatically
shorter times and
with much less operator intervention than was previously possible using prior
art techniques.
The invention includes a device capable of generating nucleic acid ligands
with little or no
operator intervention.
Background of the Invention
The dogma for many years was that nucleic acids had primarily an informational
role.
Through a method known as Systematic Evolution of Ligands by EXponential
enrichment,
termed the SELEX process, it has become clear that nucleic acids have three
dimensional
structural diversity not unlike proteins. The SELEX process is a method for
the in vitro
evolution of nucleic acid molecules with highly specific binding to target
molecules and is
described in United States Patent Application Serial No. 07/536,428, filed
June 11, 1990,
entitled "Systematic Evolution of Ligands by EXponential Enrichment," now
abandoned,
United States Patent Application Serial No. 07/714,131, filed June 10, 1991,
entitled
"Nucleic Acid Ligands," now United States Patent No. 5,475,096, United States
Patent
Application Serial No. 07/931,473, filed August 17, 1992, entitled "Methods
for Identifying
Nucleic Acid Ligands," now United States Patent No. 5,270,163 (see also WO
91/19813),
each of which is specifically incorporated by reference herein. Each of these
applications,
collectively referred to herein as the SELEX Patent Applications, describes a
fundamentally
novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX
process provides a class of products which are referred to as nucleic acid
ligands, each ligand
having a unique sequence, and which has the property of binding specifically
to a desired
target compound or molecule. Each SELEX-identified nucleic acid ligand is a
specific ligand
of a given target compound or molecule. The SELEX process is based on the
unique insight
that nucleic acids have sufficient capacity for forming a variety of two- and
three-
dimensional structures and sufficient chemical versatility available within
their monomers to
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act as ligands (form specific binding pairs) with virtually any chemical
compound, whether
monomeric or polymeric. Molecules of any size or composition can serve as
targets.
The SELEX method applied to the application of high affinity binding involves
selection from a mixture of candidate oligonucleotides and step-wise
iterations of binding,
partitioning and amplification, using the same general selection scheme, to
achieve virtually
any desired criterion of binding affinity and selectivity. Starting from a
mixture of nucleic
acids, preferably comprising a segment of randomized sequence, the SELEX
method
includes steps of contacting the mixture with the target under conditions
favorable for
binding, partitioning unbound nucleic acids from those nucleic acids which
have bound
specifically to target molecules, dissociating the nucleic acid-target
complexes, amplifying
the nucleic acids dissociated from the nucleic acid-target complexes to yield
a ligand-
enriched mixture of nucleic acids, then reiterating the steps of binding,
partitioning,
dissociating and amplifying through as many cycles as desired to yield highly
specific high
affinity nucleic acid ligands to the target molecule.
It has been recognized by the present inventors that the SELEX method
demonstrates
that nucleic acids as chemical compounds can form a wide array of shapes,
sizes and
configurations, and are capable of a far broader repertoire of binding and
other functions than
those displayed by nucleic acids in biological systems.
The present inventors have recognized that SELEX or SELEX-like processes could
be used to identify nucleic acids which can facilitate any chosen reaction in
a manner similar
to that in which nucleic acid ligands can be identified for any given target.
In theory, within
a candidate mixture of approximately 10'3 to 10'8 nucleic acids, the present
inventors
postulate that at least one nucleic acid exists with the appropriate shape to
facilitate each of a
broad variety of physical and chemical interactions.
The basic SELEX method has been modified to achieve a number of specific
objectives. For example, United States Patent Application Serial No.
07/960,093, filed
October 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of
Structure,"
(See United States Patent No. 5,707,796), describes the use of the SELEX
process in
conjunction with gel electrophoresis to select nucleic acid molecules with
specific structural
characteristics, such as bent DNA. United States Patent Application Serial No.
08/123,935,
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filed September 17, 1993, entitled "Photoselection of Nucleic Acid Ligands,"
describes a
SELEX based method for selecting nucleic acid ligands containing photoreactive
groups
capable of binding and/or photocrosslinking to and/or photoinactivating a
target molecule.
United States Patent Application Serial No. 08/134,028, filed October 7, 1993,
entitled
"High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and
Caffeine," now abandoned (See United States Patent No. 5,580,737), describes a
method for
identifying highly specific nucleic acid ligands able to discriminate between
closely related
molecules, which can be non-peptidic, termed Counter-SELEX. United States
Patent
Application Serial No. 08/143,564, filed October 25, 1993, entitled
"Systematic Evolution of
Ligands by EXponential Enrichment: Solution SELEX," now abandoned (See United
States
Patent No. 5,567,588), describes a SELEX-based method which achieves highly
efficient
partitioning between oligonucleotides having high and low affinity for a
target molecule.
The SELEX method encompasses the identification of high-affinity nucleic acid
ligands containing modified nucleotides conferring improved characteristics on
the ligand,
such as improved in vivo stability or improved delivery characteristics.
Examples of such
modifications include chemical substitutions at the ribose and/or phosphate
and/or base
positions. SELEX process-identified nucleic acid ligands containing modified
nucleotides
are described in United States Patent Application Serial No. 08/117,991, filed
September 8,
1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified
Nucleotides," now
abandoned (See United States Patent No. 5,660,985), that describes
oligonucleotides
containing nucleotide derivatives chemically modified at the 5- and 2'-
positions of
pyrimidines. United States Patent Application Serial No. 08/134,028, supra,
describes highly
specific nucleic acid ligands containing one or more nucleotides modified with
2'-amino (2'-
NHz), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). United States Patent
Application Serial
No. 08/264,029, filed June 22, 1994, entitled "Novel Method of Preparation of
Known and
Novel 2' Modified Nucleosides by Intramolecular Nucleophilic Displacement,"
describes
oligonucleotides containing various 2'-modified pyrimidines.
The SELEX method encompasses combining selected oligonucleotides with other
selected oligonucleotides and non-oligonucleotide functional units as
described in United
States Patent Application Serial No. 08/284,063, filed August 2, 1994,
entitled "Systematic
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Evolution of Ligands by EXponential Enrichment: Chimeric SELEX," now United
States
Patent No. 5,637,459 and United States Patent Application Serial No.
08/234,997, filed April
28, 1994, entitled "Systematic Evolution of Ligands by EXponential Enrichment:
Blended
SELEX," now United States Patent No. 5,683,867, respectively. These
applications allow
the combination of the broad array of shapes and other properties, and the
efficient
amplification and replication properties, of oligonucleotides with the
desirable properties of
other molecules.
The SELEX method further encompasses combining selected nucleic acid ligands
with lipophilic compounds or non-immunogenic, high molecular weight compounds
in a
diagnostic or therapeutic complex as described in United States Patent
Application Serial No.
08/434,465, filed May 4, 1995, entitled "Nucleic Acid Ligand Complexes." Each
of the
above described patent applications which describe modifications of the basic
SELEX
procedure are specifically incorporated by reference herein in their entirety.
Given the unique ability of SELEX to provide ligands for virtually any target
molecule, it would be highly desirable to have an automated, high-throughput
method for
generating nucleic acid ligands. The methods and instruments described herein,
collectively
termed automated SELEX, enable the generation of large pools of nucleic acid
ligands with
little or no operator intervention. In particular, the methods provided by
this invention will
allow high affinity nucleic acid ligands to be generated routinely in just a
few days, rather
than over a period of weeks or even months as was previously required. The
highly parallel
nature of automated SELEX process allows the simultaneous isolation of ligands
against
diverse targets in a single automated SELEX process experiment. Similarly, the
automated
SELEX process can be used to generate nucleic acid ligands against a single
target using
many different selection conditions in a single experiment. The present
invention greatly
enhances the power of the SELEX process, and will make SELEX the routine
method for the
isolation of ligands.
Summary of the Invention
The present invention includes methods and apparatus for the automated
generation
of nucleic acid ligands against virtually any target molecule. This process is
termed the
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automated SELEX process. In its most basic embodiment, the method uses a
robotic
manipulator to move reagents to one or more work stations on a work surface
where the
individual steps of the SELEX process are performed. The individual steps
include: 1 )
contacting the candidate nucleic acid ligands with the target molecules) of
interest
immobilized on a solid support; 2) partitioning the nucleic acid ligands that
have interacted in
the desired way with the target molecule on the solid support away from those
nucleic acids
that have failed to do so; and 3) amplifying the nucleic acid ligands that
have interacted with
the target molecule. Steps 1-3 are performed for the desired number of cycles
by the
automated SELEX process and apparatus; the resulting nucleic acid ligands are
then isolated
and purified.
Detailed Description of the Figures
Figure 1 demonstrates the effect of blocking reagents on background binding of
RNA
to microtiter plates. The total number of RNA molecules remaining in wells of
an Immulon
1 polystyrene plate, quantified with QPCR as described below are displayed for
wells treated
with various blocking reagents, (1) SHMCK alone, (2) SuperBlock, (3) SCHMK +
Iblock,
(4) SCHMK + SuperBlock, (5) SCHMK + Casein, (6) SCHMK + BSA.
Figure 2 demonstrates the effect of buffer reagents on background binding of
RNA to
microtiter plates. The total number of RNA molecules remaining in unblocked
wells of an
Immulon 1 polystyrene plate, quantified with QPCR as described below are
displayed for
wells incubated and washed with solutions containing various buffer reagents,
(1) SHMCK +
0.1 % Iblock + 0.05% Tween 20 (SIT), (2) SHMCK + 0.01 % HAS (SA), (3) SCHMK +
0.05% Tween 20 (ST), (4) SCHMK + 0.01 % HSA+ 0.05% Tween 20 (SAT), (5) SCHMK.
Figure 3 depicts the binding and EDTA elution of aptamer 1901 from marine PS-
Rg
passively hydrophobically attached to an Immulon 1 polystyrene plate. Total
binding of 32P
labeled aptamer 1901 to wells coated with marine PS-Rg, loaded at 4.0 mg/ml,
is plotted as a
function of total aptamer concentration (filled circles). The amount of eluted
aptamer for
each of these concentrations is shown by filled triangles, and the amount of
aptamer
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remaining in the protein coated wells after elution is shown by open squares.
All samples
were quantified by scintillation counting of 3zP.
Figure 4 depicts the quantification of passive adsorption of PS-Rg to Immulon
1
polystyrene plates. The amount of PS-Rg capable of binding aptamer 1901 after
protein
immobilization through hydrophobic interactions (filled circles) is displayed
as a function of
input protein concentration. The amount of active protein was obtained from
the plateau
values of aptamer binding curves.
Figure 5 depicts the progress of the automated in vitro selection process. The
number of RNA molecules eluted from plate wells for both manual (squares) and
automated
(circles) experiments are displayed for each of five rounds of SELEX
performed. The
amount of RNA eluted from protein coated wells is denoted by the filled
markers and
background binding RNA is denoted by open markers, and the amount of coated
protein used
in each round is denoted by x markers.
Figure 6 depicts the solution phase binding curves of round 5 RNA pools to
murine
PS-Rg protein. The binding curve measured for the enriched round five RNA pool
generated
with the automated SELEX process (+) is compared to the manual process (filled
circles) as
well as the starting random RNA pool (filled diamonds).
Figure 7 shows a perspective view of an embodiment of an apparatus for
performing
automated SELEX according to the present invention.
Figure 8 shows a plan elevation view of an embodiment of an apparatus for
performing automated SELEX according to the present invention.
Figure 9 shows a front elevation view of an embodiment of an apparatus for
performing automated SELEX according to the present invention.
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Figure 10 shows a right side elevation view of an embodiment of an apparatus
for
performing automated SELEX according to the present invention.
Detailed Description of the Invention
Definitions
Various terms are used herein to refer to aspects of the present invention. To
aid in
the clarification of the description of the components of this invention, the
following
definitions are provided:
As used herein, "nucleic acid ligand" is a non-naturally occurring nucleic
acid having
a desirable action on a target. Nucleic acid ligands are also sometimes
referred to in this
applications as "aptamers" or "clones." A desirable action includes, but is
not limited to,
binding of the target, catalytically changing the target, reacting with the
target in a way which
modifies/alters the target or the functional activity of the target,
covalently attaching to the
target as in a suicide inhibitor, facilitating the reaction between the target
and another
molecule. In the preferred embodiment, the action is specific binding affinity
for a target
molecule, such target molecule being a three dimensional chemical structure
other than a
polynucleotide that binds to the nucleic acid ligand through a mechanism which
predominantly depends on Watson/Crick base pairing or triple helix binding,
wherein the
nucleic acid ligand is not a nucleic acid having the known physiological
function of being
bound by the target molecule. Nucleic acid ligands include nucleic acids that
are identified
from a candidate mixture of nucleic acids, said nucleic acid ligand being a
ligand of a given
target, by the method comprising: a) contacting the candidate mixture with the
target,
wherein nucleic acids having an increased affinity to the target relative to
the candidate
mixture may be partitioned from the remainder of the candidate mixture; b)
partitioning the
increased affinity nucleic acids from the remainder of the candidate mixture;
and c)
amplifying the increased affinity nucleic acids to yield a ligand-enriched
mixture of nucleic
acids, whereby nucleic acid ligands of the target molecule are identified.
As used herein, "candidate mixture" is a mixture of nucleic acids of differing
sequence from which to select a desired ligand. The source of a candidate
mixture can be
from naturally-occurring nucleic acids or fragments thereof, chemically
synthesized nucleic
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acids, enzymatically synthesized nucleic acids or nucleic acids made by a
combination of the
foregoing techniques. In this invention, candidate mixture is also referred to
as "40N8
RNA," or as "RNA pool." In a preferred embodiment, each nucleic acid has fixed
sequences
surrounding a randomized region to facilitate the amplification process.
As used herein, "nucleic acid" means either DNA, RNA, single-stranded or
double-
stranded, and any chemical modifications thereof. Modifications include, but
are not limited
to, those which provide other chemical groups that incorporate additional
charge,
polarizability, hydrogen bonding, electrostatic interaction, and fluxionality
to the nucleic acid
ligand bases or to the nucleic acid ligand as a whole. Such modifications
include, but are not
limited to, 2'-position sugar modifications, 5-position pyrimidine
modifications, 8-position
purine modifications, modifications at exocyclic amines, substitution of 4-
thiouridine,
substitution of 5-bromo or 5-iodo-uracil; backbone modifications,
methylations, unusual
base-pairing combinations such as the isobases isocytidine and isoguanidine
and the like.
Modifications can also include 3' and 5' modifications such as capping.
"SELEX" methodology involves the combination of selection of nucleic acid
ligands
which interact with a target in a desirable manner, for example binding to a
protein, with
amplification of those selected nucleic acids. Optional iterative cycling of
the
selection/amplification steps allows selection of one or a small number of
nucleic acids
which interact most strongly with the target from a pool which contains a very
large number
of nucleic acids. Cycling of the selection/amplification procedure is
continued until a
selected goal is achieved. The SELEX methodology is described in the SELEX
Patent
Applications.
"SELEX target" or "target" means any compound or molecule of interest for
which a
ligand is desired. A target can be a protein, peptide, carbohydrate,
polysaccharide,
glycoprotein, hormone, receptor, antigen, antibody, virus, substrate,
metabolite, transition
state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, etc.
without limitation.
As used herein, "solid support" is defined as any surface to which molecules
may be
attached through either covalent or non-covalent bonds. This includes, but is
not limited to,
membranes, plastics, paramagnetic beads, charged paper, nylon, Langmuir-
Bodgett films,
functionalized glass, germanium, silicon, PTFE, polystyrene, gallium arsenide,
gold and
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silver. Any other material known in the art that is capable of having
functional groups such
as amino, carboxyl, thiol or hydroxyl incorporated on its surface, is also
contemplated. This
includes surfaces with any topology, including, but not limited to, spherical
surfaces grooved
surfaces, and cylindrical surfaces.
"Partitioning" means any process whereby ligands bound to target molecules can
be
separated from nucleic acids not bound to target molecules. More broadly
stated, partitioning
allows for the separation of all the nucleic acids in a candidate mixture into
at least two pools
based on their relative affinity to the target molecule. Partitioning can be
accomplished by
various methods known in the art. Nucleic acid-protein pairs can be bound to
nitrocellulose
filters while unbound nucleic acids are not. Columns which specifically retain
nucleic acid-
target complexes can be used for partitioning. For example, oligonucleotides
able to
associate with a target molecule bound on a column allow use of column
chromatography for
separating and isolating the highest affinity nucleic acid ligands. Beads upon
which target
molecules are conjugated can also be used to partition nucleic acid ligands in
a mixture.
Surface plasmon resonance technology can be used to partition nucleic acids in
a mixture by
immobilizing a target on a sensor chip and flowing the mixture over the chip,
wherein those
nucleic acids having affinity for the target can be bound to the target, and
the remaining
nucleic acids can be washed away. Liquid-liquid partitioning can be used as
well as filtration
gel retardation, and density gradient centrifugation.
In its most basic form, the SELEX process may be defined by the following
series of
steps:
1) A candidate mixture of nucleic acids of differing sequence is prepared. The
candidate mixture generally includes regions of fixed sequences (i.e., each of
the members of
the candidate mixture contains the same sequences in the same location) and
regions of
randomized sequences. The fixed sequence regions are selected either: a) to
assist in the
amplification steps described below; b) to mimic a sequence known to bind to
the target; or
c) to enhance the concentration of a given structural arrangement of the
nucleic acids in the
candidate mixture. The randomized sequences can be totally randomized (i.e.,
the probability
of finding a base at any position being one in four) or only partially
randomized (e.g., the
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probability of finding a base at any location can be selected at any level
between 0 and 100
percent).
2) The candidate mixture is contacted with the selected target under
conditions
favorable for binding between the target and members of the candidate mixture.
Under these
5 circumstances, the interaction between the target and the nucleic acids of
the candidate
mixture can be considered as forming nucleic acid-target pairs between the
target and those
nucleic acids having the strongest affinity for the target.
3) The nucleic acids with the highest affinity for the target are partitioned
from
those nucleic acids with lesser affinity to the target. Because only an
extremely small
10 number of sequences (and possibly only one molecule of nucleic acid)
corresponding to the
highest affinity nucleic acids exist in the candidate mixture, it is generally
desirable to set the
partitioning criteria so that a certain amount of the nucleic acids in the
candidate mixture are
retained during partitioning.
4) Those nucleic acids selected during partitioning as having relatively
higher
affinity to the target are then amplified to create a new candidate mixture
that is enriched in
nucleic acids having a relatively higher affinity for the target.
5) By repeating the partitioning and amplifying steps above, the newly formed
candidate mixture contains fewer and fewer unique sequences, and the average
degree of
affinity of the nucleic acids to the target will generally increase. Taken to
its extreme, the
SELEX process will yield a candidate mixture containing one or a small number
of unique
nucleic acids representing those nucleic acids from the original candidate
mixture having the
highest affinity to the target molecule.
The SELEX Patent Applications describe and elaborate on this process in great
detail.
Included are targets that can be used in the process; methods for the
preparation of the initial
candidate mixture; methods for partitioning nucleic acids within a candidate
mixture; and
methods for amplifying partitioned nucleic acids to generate enriched
candidate mixtures.
The SELEX Patent Applications also describe ligand solutions obtained to a
number of target
species, including protein targets wherein the protein is or is not a nucleic
acid binding
protein.
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In one embodiment, the automated SELEX method uses one or more computer
controlled Cartesian robotic manipulators to move solutions to and from a work
station
located on a work surface. The individual steps of the SELEX process are
carried out at the
work station. In some embodiments, each robotic manipulator is a movable arm
that is
capable of carrying tools in both horizontal and vertical planes. One tool
contemplated is a
pipetting tool. A robotic manipulator uses the pipetting tool to pick up
liquid from a defined
location on the work surface and then dispense the liquid at a different
location. The
pipetting tool can also be used to mix liquids by repeatedly picking up and
ejecting the liquid
i.e. "sip and spit" mixing. The robotic manipulator is also able to eject a
disposable tip from
the pipetting tool into a waste container, and then pick up a fresh tip from
the appropriate
station on the work surface.
In preferred embodiments, the pipetting tool is connected to one or more fluid
reservoirs that contain some of the various buffers and reagents needed in
bulk for the
SELEX process. A computer controlled valve determines which solution is
dispensed by the
pipetting tool. The pipetting tool is further able to eject liquid at desired
locations on the
work surface without the outside of the tip coming in contact with liquid
already present at
that location. This greatly reduces the possibility of the pipette tip
becoming contaminated at
each liquid dispensing step, and reduces the number of pipette tip changes
that must be made
during the automated SELEX process.
In some embodiments, tips that are used at certain steps of the automated
SELEX
process can be reused. For example, a tip can be reused if it is used in each
cycle of the
SELEX process to dispense the same reagent. The tip can be rinsed after each
use at a rinse
station, and then stored in a rack on the work surface until it is needed
again. Reusing tips in
this way can drastically reduce the number of tips used during the automated
SELEX
process.
In preferred embodiments, a vacuum aspiration system is also attached to a
separate
robotic manipulator. This system uses a fine needle connected to a vacuum
source to
withdraw liquid from desired locations on the work surface without immersing
the needle in
that liquid. In embodiments where the pipetting tool and the vacuum aspirator
are associated
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with separate robotic manipulators, the pipetting tool and the aspiration
system can work
simultaneously at different locations on the work surface.
In preferred embodiments, a robotic manipulator is also capable of moving
objects to
and from defined locations on the work surface. Such objects include lids for
multi-well
plates, and also the various pieces of apparatus used in the embodiments
outlined below. In
one embodiment of the invention, the robotic manipulator uses a "gripper" to
mechanically
grasp objects. In other embodiments, the vacuum aspiration system described
above is also
used to power a suction cup that can attach to the object to be moved. For
example, the fine
needle described above can pick up a suction cup, apply a vacuum to the cup,
pick up an
object using the suction cup, move the object to a new location, release the
object at the new
location by releasing the vacuum, then deposit the suction cup at a storage
location on the
work surface.
Suitable robotic systems contemplated in the invention include the
MultiPROBETM
system (Packard), the Biomek 200TM (Beckman Instruments) and the TecanTM
(Cavro). In
the embodiment depicted in FIGURES 7-10, the system uses three robotic
manipulators: one
carries the pipetting tool, one carries a vacuum aspirator, and one carnes the
fluorometry
cover (see below).
In its most basic embodiment, the automated SELEX process method involves:
(a) contacting a candidate mixture of nucleic acid ligands in a containment
vessel
with a target molecule that is associated with a solid support;
(b) incubating the candidate mixture and the solid support in the containment
vessel at a predetermined temperature to allow candidate nucleic acid ligands
to interact with
the target;
(c) partitioning the solid support with bound target and associated nucleic
acid
ligands away from the candidate mixture;
(d) optionally washing the solid support under predetermined conditions to
remove nucleic acid that are associated non-specifically with the solid
support or the
containment vessel;
(e) releasing from the solid support the nucleic acid ligands that interact
specifically with the target;
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(f) amplifying, purifying and quantifying the released nucleic acid ligands;
(g) repeating steps (a)-(f) a predetermined number of times; and
(h) isolating the- resulting nucleic acid ligands.
Steps (a)-(g) are performed automatically by the computer-controlled robotic
manipulator.
The computer also measures and stores information about the progress of the
automated
SELEX process procedure, including the amount of nucleic acid ligand eluted
from the target
molecule prior to each amplification step. The computer also controls the
various heating
and cooling steps required for the automated SELEX process.
In preferred embodiments, the work surface comprises a single work station
where the
individual SELEX reactions take place. This station comprises heating and
cooling means
controlled by the computer in order to incubate the reaction mixtures at the
required
temperatures. One suitable heating and cooling means is a Peltier element. The
work station
preferably also comprises a shaking mechanism to insure that SELEX reaction
components
are adequately mixed. The work surface also comprises stations in which the
enzymes
necessary for SELEX are stored under refrigeration, stations where wash
solutions and
buffers are stored, stations where tools and apparatus are stored, stations
where tools and
apparatus may be rinsed, and stations where pipette tips and reagents are
discarded. The
work surface may also comprise stations for archival storage of small aliquots
of the SELEX
reaction mixtures. These mixtures may be automatically removed from the work
station by
the pipetting tool at selected times for later analysis. The work surface may
also comprise
reagent preparation stations where the robotic manipulator prepares batches of
enzyme
reagent solutions in preparation vials immediately prior to use.
In other embodiments, the work surface comprises more than one work station.
In
this way, it is possible to perform the individual steps of the automated
SELEX process
asynchronously. For example, while a first set of candidate nucleic acid
ligands is being
amplified on a first work station of step (f), another set from a different
experiment may be
contacted with the support-bound target molecule of step (b) on a different
work station.
Using multiple work stations minimizes the idle time of the robotic
manipulator.
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In still other embodiments, the individual steps of the automated SELEX
process are
carried out at discrete work stations rather than at a single mufti-functional
work station. In
these embodiments, the solutions of candidate nucleic acid mixtures are
transferred from one
work station to another by the robotic manipulator. Separate work stations may
be provided
for heating and cooling the reaction mixtures.
In preferred embodiments, the individual steps of the automated SELEX process
are
carried out in a containment vessel that is arranged in an array format. This
allows many
different SELEX reactions--using different targets or different reaction
conditions--to take
place simultaneously on a single work station. For example, in some
embodiments the
individual steps may be performed in the wells of microtitre plates, such as
Immulon 1 plates.
In other embodiments, an array of small plastic tubes is used. Typical tube
arrays comprise
96 0.5 ml round-bottomed, thin-walled polypropylene tubes laid out in a 8 x 12
format.
Arrays can be covered during the heating and cooling steps to prevent liquid
loss through
evaporation, and also to prevent contamination. Any variety of lids, including
heated lids,
can be placed over the arrays by the robotic manipulator during these times.
Furthermore,
arrays allow the use of multipipettor devices, which can greatly reduce the
number of
pipetting steps required. For the purposes of this specification, the term
"well" will be used
to refer to an individual containment vessel in any array format.
Solid supports suitable for attaching target molecules are well known in the
art. Any
solid support to which a target molecule can be attached, either covalently or
non-covalently,
is contemplated by the present invention. Covalent attachment of molecules to
solid supports
is well known in the art, and can be achieved using a wide variety of
derivatization
chemistries. Non-covalent attachment of targets can depend on hydrophobic
interactions;
alternatively, the solid support can be coated with streptavidin which will
bind strongly to a
target molecule that is conjugated to biotin.
In particularly preferred embodiments, the solid support is a paramagnetic
bead.
When target molecules are attached to paramagnetic beads, complexes of target
molecules
and nucleic acid ligands can be rapidly partitioned from the candidate mixture
by the
application of a magnetic field to the wells. In preferred embodiments, the
magnetic field is
applied by an array of electromagnets adjacent to the walls of each well; when
the
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electromagnets are activated by the computer, paramagnetic target beads are
held to the sides
of the wells. The magnets can either be an integral part of the work station,
or they can be
attached to a cover that is lowered over the work station by the robotic
manipulator. In this
latter embodiment, the magnetic separator cover allows the magnets to be
placed adjacent to
5 the wells without blocking access to the wells themselves. In this way, the
wells are
accessible by the pipetting and aspirating units when the cover is in place.
Following magnet
activation, liquid can be aspirated from the wells, followed by the addition
of wash solutions.
When the electromagnets are deactivated, or when the cover is removed, the
beads become
resuspended in the solution. The magnetic separator cover can be stored on the
work surface.
10 In other embodiments, the magnets in the separator cover are permanent
magnets. In this
case, withdrawing the cover removes the influence of the magnets, and allows
the beads to go
into suspension.
In still further embodiments, the magnets used for bead separation are
attached to a
series of bars that can slide between adjacent rows of wells. Each bar has
magnets regularly
15 spaced along its length, such that when the bar is fully inserted between
the wells, each well
is adjacent to at least one magnet. For example, an 8x12 array of wells would
have 8 magnet
bars, each bar with 12 magnets. In this embodiment, bead separation is
achieved by inserting
the bars between the wells; bead release is accomplished by withdrawing the
bars from
between the wells. The array of bars can be moved by a computer-controlled
stepper motor.
The paramagnetic target beads used in the above embodiments are preferably
stored
on the work surface in an array format that mirrors the layout of the array
format on the work
station. The bead storage array is preferably cooled, and agitated to insure
that the beads
remain in suspension before use.
Beads can be completely removed from the wells of the work station using a
second
array of magnets. In preferred embodiments, this second array comprises an
array of
electromagnets mounted on a cover that can be placed by the robotic
manipulator over the
surface of the individual wells on the work station. The electromagnets on
this bead removal
cover are shaped so that they project into the liquid in the wells. When the
electromagnets
are activated, the beads are attracted to them. By then withdrawing the bead
removal cover
away from the wells, the beads can be efficiently removed from the work
station. The beads
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16
can either be discarded, or can be deposited back in the bead storage array
for use in the next
cycle of automated SELEX. The bead removal cover can then be washed at a wash
station
on the work surface prior to the next bead removal step.
In a typical embodiment involving paramagnetic beads, the automated SELEX
process begins when the pipetting tool dispenses aliquots of the beads--with
their bound
target--to the individual wells of a microtitre plate located on the work
station. Each well
already contains an aliquot of a candidate mixture of nucleic acid ligands
previously
dispensed by the robotic manipulator. After dispensing the beads, the robot
optionally "sips
and spits" the contents of each well up and down several times to facilitate
thorough mixing.
The microtitre plate is then incubated at a preselected temperature on the
work station in
order to allow nucleic acid ligands in the candidate mixture to bind to the
bead-bound target
molecule. Agitation of the plate insures that the beads remain in suspension.
After incubation for a suitable time, the magnetic separator cover is placed
over the
microtitre plate by the robotic manipulator. The beads are then held to the
sides of the wells,
and the aspirator tool removes the solution containing unbound candidate
nucleic acids from
the wells. A washing solution, such as a low salt solution, can then be
dispensed into each
well by the pipetting tool. The beads are released from the side of the wells
by withdrawing
the magnetic separator cover or deactivating the electromagnets, then
resuspended in the
wash solution by agitation and "sip and spit" mixing. The magnetic separator
cover is placed
over the plate again, and the wash solution is aspirated. This wash loop can
be repeated for a
pre-selected number of cycles. At the end of the wash loop, the beads are held
by the
magnets to the sides of the empty wells.
The beads can then be resuspended in a solution designed to elute the nucleic
acid
ligands from the target molecule, such as dH20. The dissociation of nucleic
acid ligand from
target can also be achieved by heating the beads to a high temperature on the
work station.
After dissociation of the nucleic acid ligands from the bead-bound target, the
pipetting tool can dispense into the wells the enzyme and buffer components
necessary to
perform amplification of the candidate nucleic acid ligands. After
amplification, purification
and quantification (see below), a predetermined amount of the amplified
candidate mixture
can then used in the next cycle of the automated SELEX process. At any point
during the
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17
cycles, the pipetting tool can remove an aliquot of the candidate mixture and
store it in an
archive plate for later characterization. Furthermore, during incubation
periods, the pipetting
tool can prepare reaction mixtures for other steps in the SELEX process.
As described above, the preferred embodiments of the automated SELEX process
method and apparatus use microtitre plates and magnetic beads to achieve
selection.
However, any other method for partitioning bound nucleic acid ligands from
unbound is
contemplated in the invention. For example, in some embodiments, the target
molecule is
coupled directly to the surface of the microtitre plate. Suitable methods for
coupling in this
manner are well known in the art.
In other embodiments, the target molecule is coupled to affinity separation
columns
known in the art. The robotic device would dispense the candidate mixture into
such a
column, and the bound nucleic acid ligands could be eluted into the wells of a
microtitre plate
after suitable washing steps.
In still other embodiments, the solid support used in the automated SELEX
process
1 S method is a surface plasmon resonance (SPR) sensor chip. The use of SPR
sensor chips in
the isolation of nucleic acid ligands is described in WO 98/33941, entitled
"Flow Cell
SELEX," incorporated herein by reference in its entirety. In the Flow Cell
SELEX method, a
target molecule is coupled to the surface of a surface plasmon resonance
sensor chip. The
refractive index at the junction of the surface of the chip and the
surrounding medium is
extremely sensitive to material bound to the surface of the chip. In one
embodiment of the
present invention, a candidate mixture of nucleic acid ligands is passed over
the chip by the
robotic device, and the kinetics of the binding interaction between the chip-
bound target and
nucleic acid ligands is monitored by taking readings of the resonance signal
from the chip.
Such readings can be made using a device such as the BIACore 2000TM (BIACore,
Inc.).
Bound nucleic acid ligands can then be eluted from the chip; the kinetics of
dissociation can
be followed by measuring the resonance signal. In this way it is possible to
program the
computer that controls the automated SELEX process to automatically collect
nucleic acid
ligands which have a very fast association rate with the target of interest
and a slow off rate.
The collected nucleic acid ligands can then be amplified and the automated
SELEX process
cycle can begin again.
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In still other embodiments, the solid support is a non-paramagnetic bead.
Solutions
can be removed from the wells containing such beads by aspirating the liquid
through a hole
in the well that is small enough to exclude the passage of the beads. For
example, a vacuum
manifold with a 0.2 p.M filter could be used to partition 100 ~M beads.
At the end of the automated SELEX process, the resulting nucleic acid ligands
can be
isolated from the automated SELEX process apparatus for sequence analysis and
cloning.
Amplification of the Candidate Nucleic Acid Ligands
At the end of each binding and partitioning step in the automated SELEX
process
method, the candidate nucleic acid ligands must be amplified. In preferred
embodiments, the
amplification is achieved using the Polymerase Chain Reaction (PCR). As the
candidate
nucleic acid ligands in the automated SELEX process method preferably all have
fixed 5' and
3' regions, primers that bind to these regions are used to facilitate PCR.
In embodiments that use target beads, the beads are removed from the wells
before
beginning the amplification procedure. When paramagnetic beads are used, this
can be done
using the magnetic removal system described above.
Candidate nucleic acid ligands can be single-stranded DNA molecules, double-
stranded DNA molecules, single-stranded RNA molecules, or double-stranded RNA
molecules. In order to amplify RNA nucleic acid ligands in a candidate
mixture, it is
necessary to first reverse transcribe the RNA to cDNA, then perform the PCR on
the cDNA.
This process, known as RT-PCR, can be carned out using the automated SELEX
process
method by dispensing the necessary enzymes, primers and buffers to the wells
on the work
station containing the eluted ligand. The resulting reaction mixtures are then
first incubated
on the work station at a temperature that promotes reverse transcription.
After reverse
transcription, the work station thermally-cycles the reaction mixtures to
amplify the cDNA
products. The amount of amplified product is then measured to give a value for
the amount
of candidate nucleic acid ligand eluted from the target (see below).
For RNA ligands, the amplified DNA molecules must be transcribed to regenerate
the
pool of candidate RNA ligands for the next cycle of automated SELEX. This can
be
achieved by using primers in the amplification step that contain sites that
promote
transcription, such as the T7 polymerase site. These primers become
incorporated into the
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19
amplification product during the PCR step. Transcription from these sites can
be achieved
simply by dispensing the appropriate enzymes and buffer components into the
amplified
mixtures and then incubating at the appropriate temperature. A predetermined
amount of the
amplified mixture is then used in the next cycle of the automated SELEX
process.
S Purification of RNA Li~ands from Amplification Mixtures
In some embodiments, amplified RNA ligands are purified from their DNA
templates
before beginning the next cycle of automated SELEX. This can be done using a
second set
of paramagnetic beads to which primers complementary to the 3' constant region
of the RNA
ligands are attached. When these primer beads are added to the transcribed
amplification
mixture, the newly transcribed full length RNA ligands hybridize to the bead-
bound primer,
whereas the amplified double-stranded DNA molecules remain in solution. The
beads can be
separated from the reaction mixture by applying a magnetic field to the wells
and aspirating
the liquid in the wells, as described above. The beads can then be washed in
the appropriate
buffer at a preselected temperature, and then the RNA ligands may be eluted
from the beads
by heating in an elution buffer (typically dH20). Finally, the beads may be
removed from the
wells on the work station, as described above to leave only a solution of
candidate RNA
ligands remaining in the wells. This point marks the completion of one cycle
of the
automated SELEX procedure.
The amount of primer bead added determines the amount of RNA ligand that is
retained in the wells. Therefore, the amount of RNA ligand that is used in the
next cycle of
the automated SELEX procedure can be controlled by varying the amount of
primer bead that
is added to the amplification mixture. The amount of RNA ligand that is to be
used can be
determined through quantitation of the amount of PCR product (see below).
Calculation of the Amount of Eluted Nucleic Acid Li~andin Each Amplification
Mixture
In certain embodiments, it may be important to measure the amount of candidate
nucleic acid ligand eluted from the target before beginning the next cycle of
the automated
SELEX process. Such measurements yield information about the efficiency and
progress of
the selection process. The measurement of eluted nucleic acid ligand--which
serves as
template for the amplification reaction--can be calculated based on
measurements of the
amount of amplification product arising out of each PCR reaction.
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In preferred embodiments, the automated SELEX process method uses a novel
system
for the automated real-time quantitation of PCR products during amplification.
This, in turn,
permits the progress of the selection experiment to be monitored in real time
during the
automated SELEX process. In preferred embodiments, the automated SELEX process
5 method uses a fluorophore/quencher pair primer system. This system is used
to calculate
automatically the amount of eluted nucleic acid ligand introduced into the
reaction mixture
by measuring the fluorescence emission of the amplified mixture. In one such
embodiment
of the invention, the PCR reaction is carried out using primers that have a
short hairpin
region attached to their 5' ends. The stem of the hairpin has a fluorophore
attached to one
10 side and a quencher attached on the other side opposite the fluorophore.
The quencher and
the fluorophore are located close enough to one another in the stem that
efficient energy
transfer occurs, and so very little fluorescent signal is generated upon
excitation of the
fluorophore. Examples of such primers are described in Example 2. During the
PCR
reaction, polymerase extension of the 3' end of DNA molecules that anneal to
the primer
15 disrupts the stem of the hairpin. As a result, the distance between the
quencher and the
fluorophore increases, and the efficiency of quenching energy transfer drops
dramatically.
An incorporated primer therefore has a much higher fluorescence emission
signal than an
unincorporated primer. By monitoring the fluorescence signal as a function of
the PCR cycle
number, PCR reaction kinetics can be monitored in real time. In this way, the
amount of
20 candidate nucleic acid ligand eluted from target in each reaction can be
quantitated. This
information in turn is used to follow the progress of the selection process.
In other embodiments, the candidate nucleic acid ligand templates are
quantitated
using the TaqManTM probe PCR system available from Roche Molecular Systems.
Briefly, a
TaqManTM probe is an oligonucleotide with a sequence complementary to the
template being
detected, a fluorophore on the 5' end, and a quencher on the 3' end. The probe
is added to a
standard PCR reaction and anneals to the template between the primer binding
sites during
the annealing phase of each PCR cycle. During the extension phase, the probe
is degraded by
the 5'~ 3' exonuclease activity of Taq Polymerase, separating the fluorophore
from the
quencher and generating a signal. Before PCR begins, the probe is intact and
the excitation
energy of the fluorophore is non-radioactively transferred to the quencher.
During PCR, as
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21
template is amplified, the probe is degraded and the amount of fluorescent
signal generated is
directly proportional to the amount of PCR product formed.
The current invention contemplates the use of fluorometry instruments that can
monitor the fluorescence emission profile of the reaction mixtures) on the
work station
during thermal-cycling. Suitable instruments contemplated comprise a source
for excitation
of the fluorophore, such as a laser, and means for measuring the fluorescence
emission from
the reaction mixture, such as a Charge Coupled Device (CCD) camera.
Appropriate filters
are used to select the correct excitation and emission wavelengths. Especially
preferred
embodiments use a fluorometry instrument mounted on an optically-transparent
cover that
can be placed over the wells on the work station by the robotic manipulator.
When placed
over the wells and then covered with a light shield, this fluorometry cover
can capture an
image of the entire array at pre-selected intervals. The computer interprets
this image to
calculate values for the amount of amplified product in each well at that
time. At the end of
the amplification step, the robotic manipulator removes the light shield and
fluorometry
cover and returns them to a storage station on the work surface.
In preferred embodiments, measurements of PCR product quantity are used to
determine a value for the amount of eluted nucleic acid ligand introduced as
template into the
amplification reaction mixture. This can be done by comparing the amount of
amplified
product with values stored in the computer that were previously obtained from
known
concentrations of template amplified under the same conditions. In other
embodiments, the
automated SELEX process apparatus automatically performs control PCR
experiments with
known quantities of template in parallel with the candidate nucleic acid
amplification
reactions. This allows the computer to re-calibrate the fluorescence detection
means
internally after each amplification step of the automated SELEX process.
The value for the amount of candidate nucleic acid ligand eluted from the
target is
used by the computer to make optimizing adjustments to any of the steps of the
automated
SELEX process method that follow. For example, the computer can change the
selection
conditions in order to increase or decrease the stringency of the interaction
between the
candidate nucleic acid ligands and the target. The computer can also calculate
how much of
the nucleic acid ligand mixture and/or target bead should be used in the next
SELEX cycle.
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In embodiments using primer beads (above), the computer uses this information
to determine
the amount of primer bead suspension to be added to each well on the work
station.
Similarly, the computer cawchange the conditions under which the candidate
nucleic acid
ligands are amplified. All of this can be optimized automatically without the
need for
operator intervention.
Figures 7-10 show various views of an embodiment of an apparatus for
performing
automated SELEX according to the present invention. This embodiment is based
on the
TecanTM (Cavro) robot system. Each view shows the apparatus during the PCR
amplification
stage of the automated SELEX process.
In FIGURE 7, a perspective view of this apparatus is shown. The system
illustrated
comprises a work surface 71 upon which the work station 72 is located (work
station is
partially obscured in this perspective view but can be seen in FIGURES 8, 9
and 10 as feature
72). The pipetting tool 74 and the aspirator 75 are attached to a central
guide rail 73 by
separate guide rails 77 and 78 respectively. The pipetting tool 74 can thus
move along the
long axis of guide rail 77; guide rail 77 can then move orthogonally to this
axis along the
long axis of central guide rail 73. In this way, the pipetting tool 74 can
move throughout the
horizontal plane; the pipetting tool can also be raised away from and lowered
towards the
work surface 71. Similarly, aspirator 75 is attached to guide rail 78, and
guide rail 78 is
attached to central guide rail 73 in such a way that aspirator 75 can move in
the horizontal
plane; aspirator 75 can also move in the vertical plane.
The fluorometry cover 76 is attached to guide rail 79 via bracket 710. Bracket
710
can move along the vertical axis of guide rail 79, thereby raising fluorometry
cover 76 above
the work station 72. When fluorometry cover 76 is positioned at the top of
guide rail 79, then
guide rails 77 and 78 can move underneath it to allow the pipetting tool 74
and the aspirator
75 to have access to work station 72. In this illustration, the fluorometry
cover 76 is shown
lowered into its working position on top of the work station 72.
Fluorometry cover 76 is attached to a CCD camera 71 la and associated optics
711b.
A source of fluorescent excitation light is associated with the cover 76 also
(not shown).
When positioned on top of the work station 72, the cover 76 allows the CCD
camera 711 a to
measure fluorescence emission from the samples contained on the work station
72 during
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23
PCR amplification. For clarity, the light shield--which prevents ambient light
from entering
the fluorometry cover--is omitted from the drawing. When PCR amplification is
finished,
fluorometry cover 76, with attached CCD camera 71 la and optics 71 lb, is
simply raised up
guide rail 79.
Also not visible in this view, but visible in FIGURES 9 and 10, is the heated
lid 91,
which is resting on top of the work station 72 underneath the fluorometry
cover 76.
The work surface 71 also comprises a number of other stations, including:
4°C
reagent storage stations 712, a -20°C enzyme storage station 713,
ambient temperature
reagent storage station 714, solution discard stations 715, pipette tip
storage stations 716 and
archive storage stations 717. Pipetting tool 74 is also associated with a
gripper tool 718 that
can move objects around the work surface 71 to these various storage
locations. Lid park
719 (shown unoccupied here) is for storage of the heated lid (see FIGURES 9
and 10).
FIGURE 8 shows the instrument of FIGURE 7 in a plan elevation view. Each
element of the instrument is labelled with the same nomenclature as in FIGURE
7.
FIGURE 9 is a front elevation view of the instrument in FIGURE 7. Note that
each
element of the instrument is labelled with the same nomenclature as in FIGURE
7 and
FIGURE 8. Note also that in this view, it can be seen that work station 72,
and chilled
enzyme and reagent storage stations 712 are each associated with shaking
motors 92.
Operation of these motors keeps the various reagents mixed during the
automated SELEX
process. The motors 92 are each under computer control, and can be momentarily
stopped to
allow reagent addition or removal, as appropriate, to the receptacle that is
being agitated.
Also visible in this view is heated lid 91 which is resting on top of work
station 72 to insure
uniform heating of the samples.
FIGURE 10 is a right side elevation view of the instrument shown in FIGURES 7,
8
and 9. Every element of the instrument is labelled with the same nomenclature
as in
FIGURES 7, 8, and 9.
Examples
The examples below are illustrative embodiments of the invention. They are not
to be taken
as limiting the scope of the invention.
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24
Example 1
The basis of the robotic workstation is a Packard MULTIProbe 204DTTM, a four
probe pipetting station that utilizes disposable pipette tips to minimize
nucleic acid
contamination. The workspace contains a 37°C constant temperature heat
block used for
equilibration of the binding reaction and in vitro transcription, a computer
controlled thermal
cycler for both RT and PCR reactions, a freezer unit for cold enzyme storage,
various vessels
for reagent storage, e.g., buffers, primers and mineral oil, and disposable
pipette tip racks.
The tip racks utilize the greatest area on the work surface and vary depending
on the number
of samples processed in parallel. All steps for in vitro selection take place
either on the heat
block or in the thermal cycler, liquids are transferred primarily between
these two stations,
although some enzyme buffers are premixed in an adjacent reagent block prior
to transfer to
the plate or thermal cycler.
The entire process is controlled by a PC with software developed in-house in
HAM
(High level Access Method), a DOS based C programming language interpreter
augmented
with liquid handling functions for the Packard MULTIProbe. In addition to
standard C
functionality and liquid handling such as aspirating, dispensing, and mixing
fluids, HAM
supports window based screen io, file handling, and RS-232 serial
communications. The
software automatically adjusts the process to run any number of samples
between one and 96,
preparing only those enzyme solutions necessary during the current run. Two
way
communication with the thermal cycler, established with an RS-232 connection,
allows the
main computer to perform lid opening/closing operations, initiate programs
stored on thermal
cycler and monitor thermal cycler programs for completion. The overall
software design
enables complete computer control of the process, from binding reaction
incubation through
transcription, to occur with no user intervention.
The process begins by placing a microtiter plate coated with protein on the
37°C
block. All subsequent liquid handling up to gel purification of the enriched
RNA pool is
controlled by the software. During the initial two hour incubation of RNA with
immobilized
protein target, dH,O is periodically added to the samples (to control
evaporative loss) and
each solution is mixed by repeated aspiration and dispensing, so-called sip-
and-spit. After
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the binding reaction has equilibrated, partitioning bound from free RNA is
easily
accomplished in this format by simply removing the RNA solution from each
well; bound
nucleic acid remains on the immobilized target and unbound molecules are
disposed.
Partitioning is followed by a series of wash steps, each wash comprised of
pipetting a wash
5 buffer solution into each well with subsequent repeated sip-and-spit mixing
and finally
disposal of the wash solution. The elution process begins by addition of EDTA
followed by
a 30 minute incubation with periodic sip-and-spit mixing. After incubation,
the solution is
transferred to the thermal cycler and the wells washed as described above,
with the exception
that each wash solution here is added to the eluted material in the cycler.
The sample is then
10 ready for enzymatic amplification.
The first step for each of the three enzyme reactions requires the preparation
of a fresh
enzyme solution. This is done by pipetting an aliquot of enzyme from the
freezer to the
appropriate buffer located in the reagent block. The viscous enzyme solution
is mixed
carefully and thoroughly using slow sip-and-spit mixing to avoid foaming of
detergents in the
15 enzyme solution. An aliquot of the freshly prepared RT reaction mixture is
added to the dry
wells of the eluted plate for a wash to remove possible eluted RNA remaining
in the well.
The RT reaction mixture wash is then added to the appropriate well in the
thermal cycler and
capped with silicone oil to prevent evaporative loss during reaction
incubation at 48°C. The
thermal cycler lid is closed and a program initiated for the RT reaction. The
main computer
20 monitors the reaction progress and upon detecting program completion, the
lid is opened, a
Taq polymerase reaction mixture is prepared and added to each completed RT
reaction. This
is followed by lid closure, PCR program initiation, monitoring and lid opening
upon
completion of PCR. An aliquot of the amplified DNA is moved from the thermal
cycler to
appropriate wells in the 37°C plate for in vitro transcription of the
DNA template. A freshly
25 prepared T7 RNA polymerase solution is added to each well thoroughly mixed.
A layer of
silicone oil caps the reaction mixture that then incubates for 4 hours. This
completes the
automated process; the resulting transcribed RNA is gel purified off line and
added to a
microtiter plate with freshly coated protein wells for the next round of
SELEX.
Typical Automated SELEX Process Run
A typical automated SELEX process run using a multiwell plate begins with
loading
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26
the various reagents and materials needed to the appropriate locations on the
work surface.
The following steps then take place (each step performed by robot):
1 ) Pipette candidate nucleic acid mixture to each well of a 96 well plate on
work
station with one tip; tip disposed.
2) Pipette target paramagnetic beads to each well of the 96 well plate on work
station; tip disposed.
3) Binding
Plate incubated at 37°C with shaking for 30-120 minutes to allow
nucleic acid ligands
to interact with target on bead.
4) Bead Separation and Washing
Separate beads by placing magnetic separator cover on plate; aspirate liquid
from
wells; remove magnetic separator cover; dispense washing buffer to each well;
incubate at
37°C for 5 minutes with shaking.
5) Repeat step 4) for the desired number of wash cycles.
6) Elution 1
Separate beads by placing magnetic separator cover on plate and aspirate
liquid from
wells; remove magnetic separator cover and resuspend beads in each well in 90
p,L of dHzO;
heat plate to 90°C with shaking to elute nucleic acid ligands.
7) Cool plate to 48°C.
8) Prepare PCR reaction mixture in preparation vial on work surface using
buffers and reverse transcriptase.
9) Pipette aliquot of PCR reaction mixture to each well on work station.
10) Reverse Transcription
Incubate plate on work station at 48°C for 30 minutes with shaking to
allow reverse
transcription to take place.
11) Bead Removal 1
Place bead removal cover on plate to capture beads on magnets; move removal
cover
and attached beads to drop station; drop beads at drop station and wash cover
at wash station.
12) Place fluorometry cover over plate on work station; place light shield
over
work station.
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13) Amplification
27
Thermally cycle plate until fluorometry cover indicates that DNA saturation
has
occurred; calculate the amount of amplification product in each well using
fluorometer
readings.
14) Remove light shield and fluorometry cover; remove aliquot from each well
and dispense in an archive array for storage.
15) Prepare transcription mixture in preparation vial on work surface using
buffers
and RNA polymerase.
16) Pipette aliquot of transcription mixture to each well on work surface.
17) Transcription
Incubate plate on work surface at 37°C for 4 hours with shaking to
allow transcription
to take place.
18) Purification
Determine the volume of primer paramagnetic beads needed to retain the desired
1 S amount of RNA from each well; dispense the calculated quantity of beads to
each well on
work surface.
19) Incubate plate on work surface at 48°C for 5 minutes with shaking.
20) Bead Separation and Washing
Separate primer beads by placing magnetic separation cover on plate; aspirate
each
well; remove separation cover; pipette wash buffer to each well; incubate
plate at 48°C for 5
minutes with shaking.
21 ) Repeat step 20) for the desired number of wash cycles.
22) Elution 2
Separate beads by placing magnetic separation cover on plate; aspirate each
well;
remove separation cover; pipette 100 ~L dH20 to each well; incubate plate on
work station at
95°C for 3 minutes with shaking to elute RNA from primer beads.
23) Bead Removal 2
Place bead removal cover on plate to capture beads on magnets; move removal
cover
and attached beads to drop station; drop beads at drop station and wash cover
at wash station.
24) Begin at step 2) again for the desired number of cycles.
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Example 2
The following example describes the performance of automated SELEX on the
recombinant marine selectin/IgG fusion protein. For a description of manual
SELEX against
selection targets see Parma et al., United States Patent No. 5,780,228,
entitled, "High Affinity
Nucleic Acid Ligands to Lectins."
Automated Selection
Marine PS-Rg, a recombinant marine selectin/IgG fusion (purchased from D.
Vestweber) was manually coated in concentrations stated in results in 75 ~l
SHMCK buffer
(10 mM HEPES pH 7.3, 120 mM NaCI, 5 mM KCI, 1 mM MgCl2, 1 mM CaClz) for two
hours at room temperature (23°C) onto a round bottom Immulon 1
polystyrene 96 well
microtiter plate. Control wells were prepared by coating SHMCK alone. The
plate was then
washed six times with 150 pl SAT (SHMCK, 0.01% HSA (Sigma), 0.05% Tween 20
(Aldrich) and 200 pmoles of gel purified RNA pool was added in 75 ~,1 SAT
buffer. The
plate was placed on a 37°C heat block (USA Scientific) mounted on a
MultiPROBE 204DT
pipetting workstation (Packard) and samples were incubated uncovered at
37°C for two
hours. All subsequent steps were performed by the robotic workstation except
where noted.
Every twenty minutes during the incubation of the RNA with the plate, 5 pl of
dH20 was
added to compensate for evaporative loss (rate of loss measured at 14.5 + 0.4
p,l/hour) and to
mix the reactions. Plates were then washed six times with 150 ~1 SAT buffer.
To the dried plate 75 ~,l of SHKE (10 mM HEPES pH 7.3, 120 mM NaCI, 5 mM
KCI, 5 mM EDTA) was added to the plate and incubated at 37°C for 30
minutes with mixing
every ten minutes. The supernatant was then removed from the plate and added
to an MJ
Research thermocycler mounted on the work station with remote command
capabilities.
Automated Amplification
AMV reverse transcriptase(Boeringer Mannheim) stored in a pre-chilled
Styrofoam
cooler mounted on the work surface at below 0°C, was added to a
prepared RT buffer and
thoroughly mixed. 25 ~l of the resulting RT Mix (50 mM Tris-HCL pH 8.3, 60 mM
NaCI,
11 mM Mg(OAc)~, 10 mM DTT, 1 mM dATP, 1 mM dTTP, 1 mM dGTP, 1 mM dCTP, 400
pmoles 3P8, 20 units AMV-RT/reaction) was then added to the empty incubation
wells and
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29
mixed to provide a wash for the well. The RT mix was then moved into the
thermocycler,
added to the eluted RNA, and thoroughly mixed. To this 25 ~l of silicone oil
(Aldrich) was
added to prevent evaporation. The thermocycler was then remotely turned on by
the
computer. The lid was closed and the reaction incubated at 48°C for 30
minutes followed by
60°C for 5 minutes. Upon completion of the RT reaction the lid was
triggered to open and 10
~1 of the reaction was manually removed to be measured manually by
quantitative PCR
(qPCR).
Taq polymerase (Perkin Elmer) stored in the Styrofoam cooler, was added to a
prepared PCR buffer (Perkin Elmer Buffer 2 (50 mM KCL, 10 mM Tris-HCl pH 8.3),
7.5
mM MgClz, 400 pmoles SP8) and thoroughly mixed. 100 ~l of the Taq mix was then
added
to each well, the lid closed, and PCR was initiated. PCR was run under the
following
conditions: 93°C for 3 minutes followed by a loop consisting of
93°C for 1 minute, 53°C for 1
minute, and 72°C for 1 minute for n cycles where n was determined by
the input amount of
RNA to the RT reaction (see qPCR description). Upon completion of PCR the lid
was
opened and 50 ~l was removed and added to an empty plate well on the fixed
37°C heat
block.
T7 RNA polymerase (Enzyco) stored in the Styrofoam cooler, was added to a
prepared Transcription buffer (40 mM Tris-HCl pH 8, 4% (w/v) PEG-8000,12 mM
MgCl2, 5
mM DTT, 1 mM Spermidine, 0.002% Triton X-100, 100 units/ml pyrophosphatase
(Sigma))
and thoroughly mixed. 200 ~l of the Transcription buffer was then added to the
PCR product
well and mixed. To this reaction a 25 ~,1 layer of silicone oil was added and
the reaction was
incubated for 4 hours at 37°C. The completed reaction was then removed
and purified
manually by PAGE.
Plate Characterization
1. Test of various blocking agents
To empty Immulon 1 wells, 150 ~1 of various buffers were incubated for 30
minutes
at room temperature including the following:
(1) SHMCK
(2) SuperBlock (Pierce)
(3) SHMCK + 0.1 % I-Block (Tropix)
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(4) SHMCK + 0.1 % Casein (Sigma)
(5) SHMCK + SuperBlock (1:1)
(6) SHMCK+1%BSA
5 The wells were then washed six times with 150 ~1 of SIT buffer. Then 200
pmoles of 40N8
RNA in SIT buffer were added to each well and incubated for 2 hours at
37°C. The wells
were washed six times with 150 ~1 SIT buffer. To each well 75 pl of dH20 was
added and
heated to 95°C for 5 minutes to elute the RNA from the plate. To this
25 ~,1 of an RT mix
was added and incubated as described. The eluant was then measured offline for
amount of
10 RNA present by qPCR. The results of this experiment are shown in Figure 1.
2. Role of buffer components in background on unblocked Immulon 1 plates
To empty Immulon 1 wells, 200 pmoles of 40N8 was added in 100 ~1 of the
15 following buffers and incubated at 37°C for 2 hours:
(1) SIT (SHMCK, 0.1% I-Block, 0.05% Tween 20)
(2) SHMCK
(3) SA (SHMCK, 0.01% HSA)
(4) ST (SHMCK, 0.05% Tween 20)
20 (5) SAT (SHMCK, 0.01% HSA, 0.05% Tween 20)
The wells were subsequently washed six times with 150 ~l of the appropriate
buffer and
eluted with SHKE as described. The eluant was then measured for the amount of
RNA
present by RT as described followed by qPCR. The results of this experiment
are shown in
25 Figure 2.
EDTA elution study with marine PS-R~
4 ~.g/ml marine PS-Rg was coated onto empty wells in 75 ~1 SHMCK for 2 hours
at
room temperature and washed as described. Then a titration of 3 fmoles to 20
pmoles of
RNA clone #395, isolated from a previous manual SELEX experiment, was coated
on two
30 sets of control and PS-Rg wells for 2 hours at 37°C and washed as
described. One set of
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31
control and PS-Rg wells were then removed and monitored for'ZP-RNA bound by
scintillation counting. 50 ~l of SHKE was then added to the other set of dry
wells and
incubated with mixing at 37°C for 30 minutes. The buffer was then
removed and 32P-labeled
RNA was measured by scintillation counting. The results of this experiment are
shown in
Figure 3.
SELEX Pro-press
Table 1 below outlines the progress of the PS-Rg SELEX experiment. PS-Rg
loading
is indicated in ~,g/ml concentrations. (Binding of PS-Rg to the plate surface
has been
measured by loading fixed amounts of PS-Rg, washing as described, and then
performing a
binding curve by titrating high affinity aptamer # 1901. This is done with
several protein
concentrations.
The plateau values of these binding curves then are taken as a representation
of the
amount of active protein bound to the surface, assuming a 1:1 stoichiometry.
See Figure 4.
Using these data, it was determined that the plate was near saturated
(calculated saturation is
220 fmol/well PS-Rg) when loading 4 pg/ml PS-Rg, representing 150 finoles of
bound PS-
Rg. The signal measured represents the number of RNA molecules bound to the
wells
containing PS-Rg as determined by qPCR for each sample. Similarly, noise is
representative
of the number of RNA molecules bound to control wells containing no protein.
Table 1. Progress of the PS-RG SELEX Experiment.
Round PS-Rg, Signal Noise SignaU Signal Noise SignaU
~.glml Manual Manual Noise Robot Robot Noise
Loaded Manual Robot
1 4 4.8e+8 1.8e+7 2.7 l.Se+9 1.8e+6 833
2 4 1.6e+10 6.6e+6 2424 4.2e+9 l.Se+6 2800
3 0.2 4e+7 1 a+7 4 2.8e+7 3.4e+6 8.2
4 0.2 l.le+8 4.Se+7 2.5 2e+8 l.Se+7 13.3
5 0.2 3.1 a+8 3.1 a+7 10 1.4e+8 1 a+6 140
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32
Example 3
Quantitative PCR
The following primers (SP7-FD2 and SP8-FD2) were designed wherein the
underlined portions are complementary to the N7 and N8 templates.
SP7-FD2
DABCYI.
I
(Cx2) s
A I
GCTCTAATACGACTCACTATAGGGAGGACGATGCGG-3'
A IIII 5P7 SEQ ID NO:1
CGAG-5'
G I
6-FAM
SP8-FD2
DABCYI~
(CH2) s
A I
GCTCTAATACGACTCACTATAGGGAGACAAGAATAAACGCTCAA-3'
A IIII 5P8 SEQ ID N0:2
CGAG-5'
G I
6-FAM
The hairpin in each primer has a Tm of ~85°C, and contains a
fluorophore (6-FAM) on its 5'
terminus and a quencher (DABCYL) opposite the fluorophore on its stem. Upon
excitation
at 495 nm, photons emitted by 6-FAM are absorbed by DABCYL by fluorescence
resonance
energy transfer. The efficiency of energy transfer is dependent on the sixth
power of the
distance between the fluorophore and quencher. Because the fluorophore and
quencher are in
very close proximity in the closed hairpin conformation, little signal is
generated by
unincorporated primer. However, as primer is incorporated into product during
PCR, the
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33
fluorophore and quencher are further separated by a distance of 10 base pairs,
and signal is
increased. The increase in signal is directly proportional to the amount of
product formed.
An example of a standard curve using 40N8 cDNA template, primers SP8 and 3P8
(80 pmoles) and the fluorescent primer SP8-FD2 (16 pmoles) in a PCR reaction
is illustrated
in Figure 5 on a linear plot and in Figure 6 in semi-log plot. Template
concentrations ranged
from 106 - 10'° copies/25 ~,L reaction. Fluorescein signal (normalized
to an internal reference
dye and background-subtracted) is plotted as a function of PCR cycle number.
In early PCR
cycles, product is being generated exponentially in all reactions; however,
background signal
exceeds product signal. The cycle at which product signal exceeds background
is dependent
on the starting template copy number. A signal threshold level (dashed line at
y = 0.03) is
chosen above the background level, and the cycle at which each reaction
crosses the
threshold (Ct) is plotted as a function of template copy number to generate a
standard curve.
The equation for the standard curve can then be used to calculate template
copy numbers in
unknowns based on the Ct values.
This quantitative PCR technique was used to measure signal to noise ratios and
absolute template copy number in a SELEX targeting PDGF adsorbed to
polystyrene plates.
Because very low protein loadings were used (<100 amol/reaction), quantitation
by radiation
was not possible. An amplification plot illustrates quantitation of 10 amol
RNA bound to the
background well and 600 amol RNA bound to the target well, for a signal-to-
noise ratio of
60.
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SEQUENCE LISTING
<110> Somalogic, Inc.
<120> Method and Apparatus for the Automated Generation of
Nucleic Acid Ligands
<130> NEX 77/CIP/PCT
<140>
<141>
<150> 09/356,233
<151> 1999-07-16
<150> 09/232,946
<151> 1999-O1-19
<160> 2
<170> PatentIn Ver. 2.0
<210> 1
<211> 44
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Nucleic Acid
Ligand
<400> 1
gagcggaagc tctaatacga ctcactatag ggaggacgat gcgg 44
<210> 2
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Nucleic Acid
Ligand
<400> 2
gagcgaagct ctaatacgac tcactatagg gagacaagaa taaacgctca a 51
1
