U.S. patent application number 11/029949 was filed with the patent office on 2005-07-21 for nucleic acid ligands to the prostate specific membrane antigen.
This patent application is currently assigned to GILEAD SCIENCES, INC.. Invention is credited to Coffey, Donald S., Hicke, Brian J., Lin, Yun, Lupold, Shawn E..
Application Number | 20050158780 11/029949 |
Document ID | / |
Family ID | 26933702 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158780 |
Kind Code |
A1 |
Lupold, Shawn E. ; et
al. |
July 21, 2005 |
Nucleic acid ligands to the prostate specific membrane antigen
Abstract
Methods are provided for generating nucleic acid ligands of
Prostate Specific Membrane Antigen (PSMA). The methods of the
invention use the SELEX method for the isolation of nucleic acid
ligands. The invention also includes nucleic acid ligands to PSMA,
and methods and compositions for the treatment and diagnosis of
disease using the nucleic acid ligands.
Inventors: |
Lupold, Shawn E.;
(Alexandria, VA) ; Lin, Yun; (Louisville, CO)
; Hicke, Brian J.; (Boulder, CO) ; Coffey, Donald
S.; (Lutheriville, MD) |
Correspondence
Address: |
SWANSON & BRATSCHUN L.L.C.
1745 SHEA CENTER DRIVE
SUITE 330
HIGHLANDS RANCH
CO
80129
US
|
Assignee: |
GILEAD SCIENCES, INC.
Foster City
CA
|
Family ID: |
26933702 |
Appl. No.: |
11/029949 |
Filed: |
January 4, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11029949 |
Jan 4, 2005 |
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09978969 |
Oct 16, 2001 |
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60278830 |
Mar 26, 2001 |
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60240781 |
Oct 16, 2000 |
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Current U.S.
Class: |
435/6.14 ;
534/11; 536/23.1 |
Current CPC
Class: |
A61P 35/00 20180101;
C12Q 1/6886 20130101; C12N 2310/322 20130101; C07K 14/705 20130101;
C12N 15/115 20130101 |
Class at
Publication: |
435/006 ;
536/023.1; 534/011 |
International
Class: |
C12Q 001/68; C07F
005/00; C07H 021/02 |
Claims
What is claimed is:
1. A complex comprised of a purified and non-naturally occurring
RNA ligand to prostate specific membrane antigen (PSMA) and a
marker, wherein said ligand is selected from the group consisting
of SEQ ID NOS:5, 6, 15, 16, 17, and 18.
2. The complex of claim 1 further comprising a linker between said
RNA ligand and said marker.
3. The complex of claim 1 wherein said marker is a
radionuclide.
4. The complex of claim 3 wherein said radionuclide is selected
from the group consisting of Tc-99m, Re-188, Cu-64, Cu-67, F-18,
.sup.121I, .sup.131I, .sup.32P, .sup.186Re and .sup.111In.
5. A method for detecting the presence of a disease that is
expressing prostate specific membrane antigen (PSMA) in a
biological tissue which may contain said disease, the method
comprising exposing said biological tissue to the complex of claim
1 and detecting the presence of the complex in said biological
tissue.
6. The method of claim 5 wherein said complex further comprises a
linker between said RNA ligand and said marker.
7. The method of claim 5 wherein said marker is a radionuclide.
8. The method of claim 7 wherein said radionuclide is selected from
the group consisting of Tc-99m, Re-188, Cu-64, Cu-67, F-18,
.sup.125I, .sup.131I, .sup.32P, .sup.186Re and .sup.111In.
9. A complex comprised of a purified and non-naturally occurring
RNA ligand to prostate specific membrane antigen (PSMA) and a
therapeutic compound, wherein said ligand is selected from the
group consisting of SEQ ID NOS:5, 6, 15, 16, 17, and 18.
10. The complex of claim 9 wherein said therapeutic compound is
selected from the group consisting of cytotoxic compounds, immune
enhancing substances, and therapeutic radionuclides.
11. A method for delivering a therapeutic compound to a biological
tissue expressing prostate specific membrane antigen (PSMA) in an
organism, the method comprising administering the complex of claim
9 to said organism.
12. The method of claim 11 wherein said therapeutic compound is
selected from the group consisting of cytotoxic compounds, immune
enhancing substances, and therapeutic radionuclides.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/978,969, filed Oct. 16, 2001, which claims
the benefit of U.S. Provisional Patent Application Ser. No.
60/278,830, filed Mar. 26, 2001 "Nucleic Acid Ligands to the
Prostate Specific Membrane Antigen" and which also claims the
benefit of U.S. Provisional Patent Application Ser. No. 60/240,781,
filed Oct. 16, 2000, entitled "Nucleic Acid Ligands to the Prostate
Specific Membrane Antigen". Each of the aforementioned applications
is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] Described herein are high affinity nucleic acid ligands to
Prostate Specific Membrane Antigen (PSMA). Also described herein
are methods for identifying and preparing high affinity nucleic
acid ligands to PSMA. The method used herein for identifying such
nucleic acid ligands is called SELEX, an acronym for Systematic
Evolution of Ligands by Exponential enrichment. Further disclosed
are RNA ligands to PSMA. Also included are oligonucleotides
containing nucleotide derivatives chemically modified at the 2'
positions of pyrimidines. Additionally disclosed are RNA ligands to
PSMA containing 2'-F modifications. The invention also includes
high affinity nucleic acid ligand inhibitors of PSMA. The
oligonucleotides of the present invention are useful as diagnostic
agents and/or therapeutic agents.
BACKGROUND OF THE INVENTION
[0003] The Prostate Specific Membrane Antigen (PSMA) is a 750-amino
acid type II transmembrane protein. PSMA is expressed by prostatic
epithelial cells and extraprostatic expression has been detected in
the brain, kidney, salivary gland and duodenum. (See e.g.,
Renneberg et al. (1999) Urol. Res. 27(1):23-7; Troyer et al. (1995)
Int. J. Cancer 62(5):552-8; Israel et al. (1994) Cancer Res.
54(7):1807-11; Israel et al. (1993) Cancer Res. 53(2):227-30). PSMA
is a carboxypeptidase which cleaves N-acetyl-asp-glu. PSMA has
three domains: a 19-amino acid cytoplasmic domain, a 24-amino acid
transmembrane domain, and a 707-amino acid extracellular domain. A
monoclonal antibody specific to the cytoplasmic domain, 7E11.C5,
has been adapted for in vivo imaging of prostatic cancer through
radiolabeling with indium-111. (Elgamal et al. (1998) Prostate
37(4):261-9; Lamb and Faulds (1998) Drugs Aging 12(4):293-304).
[0004] Since its discovery in 1987 (Horoszewicz et al. (1987)
Anticancer Res. 7:927-35), PSMA has been considered an excellent
prostate tumor cell marker. PSMA expression is primarily prostate
specific, with barely detectable levels seen in the brain, salivary
glands, and small intestine (Israeli et al. (1994) Cancer Res.
54:1807-11). Additionally, PSMA expression is high in malignant
prostate cells, with the highest expression in androgen resistant
cells due to negative regulation by androgens (Wright et al. (1996)
Urology 48:326-34). Furthermore, PSMA is alternatively spliced,
where normal prostate cells predominantly express a cytosolic form
named PSM' and malignant cells express the characteristic
full-length membrane bound form (Su et al. (1995) Cancer Res.
55:1441-3). This full-length PSMA is a type II membrane
glycoprotein, in which the majority of the protein is extracellular
and available as a target for diagnostic and therapeutic agents.
These properties have made PSMA an ideal target for prostate cancer
immunotherapy (Murphy et al. (1999) Prostate 39:54-9); monoclonal
antibody imaging (Sodee et al. (1998) Prostate 37:140-8); and
therapy (McDevitt et al. (2000) Cancer Res. 60:6095-100). The first
anti-PSMA antibody was quickly modified into an imaging agent
(Lopes et al. (1990) Cancer Res. 50:6423-6429), which is currently
used clinically to diagnose metastatic prostate tumors.
Additionally, PSMA is expressed by neovascular endothelial cells in
a variety of cancers (Chang et al. (1999) Clin. Cancer Res.
5:2674-81; Liu et al. (1997) Cancer Res. 57:3629-34), making it a
candidate target for tumor vascular imaging and anti-angiogenesis
therapy.
[0005] An aptamer that recognizes PSMA's extracellular domain has
potential utility as a therapeutic entity, via inhibition of PSMA
enzymatic activity, as an in vivo imaging agent, and additionally
as a targeting agent for therapeutic delivery of cytotoxic
chemicals and radionuclides. The use of proteins as drugs and
reagents is often limited by the activity of proteases, the size of
the protein, transport and the ability of an organism to make
antibodies against that protein. Many of these limitations can be
circumvented by the use of aptamers, made of synthesized RNA, that
are stabilized against nuclease activity. Relative to antibodies,
aptamers are small (7-20 kDa), clear very rapidly from blood, and
are chemically synthesized. Rapid blood clearance is important for
in vivo diagnostic imaging, where blood levels are a primary
determinant of background that obscures an image. Rapid blood
clearance may also be important in therapy, where blood levels may
contribute to toxicity. SELEX technology allows rapid aptamer
isolation, and chemical synthesis enables facile and site-specific
conjugation of aptamers to a variety of inert and bioactive
molecules. An aptamer to PSMA would therefore be useful for tumor
therapy or in vivo or ex vivo diagnostic imaging and/or for
delivering a variety of therapeutic agents complexed with the PSMA
nucleic acid ligand for treatment of disease conditions in which
PSMA is expressed.
[0006] The development of the Systematic Evolution of Ligands by
Exponential Enrichment (SELEX) process has provided a new
alternative, nuclease-resistant oligonucleotides that can be
selected to bind tightly and specifically to almost any ligand.
(Tuerk and Gold (1990) Science 249:505-10; Ellington and Szostak
(1990) Nature 346:818-22; Lin et al. (1994) Nucleic Acids Res.
22:5229-34; Gold (1995) J. Biol. Chem. 270:13581-4); for example:
organic dyes, antibiotics, amino acids, and cells (Ellington and
Szostak (1990) Nature 346:818-22; Wang and Rando (1995) Chem. Biol.
2:281-90; Connell et al. (1993) Biochemistry 32:5497-502; Morris et
al. (1998) Proc. Natl Acad. Sci. USA 95:2902-7). These synthetic
oligonucleotide sequences, termed "RNA aptamers," have been made to
bind over 100 target ligands and are emerging as a new class of
molecules that contest antibodies in therapeutics, imaging, and
diagnostics (Hicke and Stephens (2000) J. Clin. Invest. 106:923-8;
Jayasena (1999) Clin. Chem. 45:1628-50).
[0007] 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 U.S. patent application Ser. No.
07/536,428, filed Jun. 11, 1990, entitled "Systematic Evolution of
Ligands by EXponential Enrichment," now abandoned, U.S. Pat. No.
5,475,096, entitled "Nucleic Acid Ligands," and U.S. Pat. No.
5,270,163 (see also WO 91/19813), entitled "Methods for Identifying
Nucleic Acid Ligands," each of which is specifically incorporated
herein by reference in its entirety. 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.
[0008] The SELEX process provides a class of products which are
referred to as nucleic acid ligands or aptamers, each 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 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.
[0009] 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.
[0010] The basic SELEX method has been modified to achieve a number
of specific objectives. For example, U.S. patent application Ser.
No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat.
No. 5,707,796, both entitled "Method for Selecting Nucleic Acids on
the Basis of Structure," describe the use of the SELEX process in
conjunction with gel electrophoresis to select nucleic acid
molecules with specific structural characteristics, such as bent
DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17,
1993, entitled "Photoselection of Nucleic Acid Ligands," now
abandoned, U.S. Pat. No. 5,763,177 and U.S. Pat. No. 6,011,577,
both entitled "Systematic Evolution of Ligands by Exponential
Enrichment: Photoselection of Nucleic Acid Ligands and Solution
SELEX," describe 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.
U.S. Pat. No. 5,580,737, entitled "High-Affinity Nucleic Acid
Ligands That Discriminate Between Theophylline and Caffeine,"
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. U.S. Pat. No.
5,567,588, entitled "Systematic Evolution of Ligands by EXponential
Enrichment: Solution SELEX," describes a SELEX-based method which
achieves highly efficient partitioning between oligonucleotides
having high and low affinity for a target molecule.
[0011] 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 U.S. Pat. No. 5,660,985, entitled "High Affinity Nucleic Acid
Ligands Containing Modified Nucleotides," that describes
oligonucleotides containing nucleotide derivatives chemically
modified at the 5- and 2'-positions of pyrimidines. U.S. Pat. No.
5,580,737, supra, describes highly specific nucleic acid ligands
containing one or more nucleotides modified with 2'-amino
(2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S.
patent application Ser. No. 08/264,029, filed Jun. 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.
[0012] The SELEX method encompasses combining selected
oligonucleotides with other selected oligonucleotides and
non-oligonucleotide functional units as described in U.S. Pat. No.
5,637,459, entitled "Systematic Evolution of Ligands by EXponential
Enrichment: Chimeric SELEX," and U.S. Pat. No. 5,683,867, entitled
"Systematic Evolution of Ligands by EXponential Enrichment: Blended
SELEX," 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.
[0013] 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 U.S. Pat. No. 6,011,020, 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.
[0014] Since the first discovery of RNA aptamers as ligand binding
agents (Tuerk and Gold (1990) Science 249:505-10; Ellington and
Szostak (1990) Nature 346:818-22), an enormous diversity of target
molecules have been identified (Famulok et al. (2000) Acc. Chem.
Res. 33:591-9). The diversity of structures employed by an aptamer
library allows tight binding RNA ligands from targets as simple as
a single amino acid (Connell et al. (1993) Biochemistry
32:5497-502), to complex targets such as red blood cells (Morris et
al. (1998) Proc. Natl Acad. Sci. USA 95:2902-7). Despite the
success of this technique, however, there are no reported RNA
aptamers to membrane bound tumor antigens. Therefore, the
possibility of identifying and producing nuclease stable RNA
aptamers that bind to and inhibit the enzymatic activity of the
well-known prostate tumor cell surface antigen, PSMA was
explored.
[0015] It is an object of the present invention to provide methods
that can be used to identify nucleic acid ligands that bind with
high specificity and affinity to PSMA.
[0016] It is a further object of the present invention to obtain
nucleic acid ligands to PSMA that inhibit the activity of PSMA when
bound.
[0017] It is a further object of the present invention to provide a
complex for use in in vivo or ex vivo diagnostics comprising one or
more PSMA nucleic acid ligands and one or more markers.
[0018] It is a further object of this invention to provide a method
for delivering therapeutic agents for the treatment or prophylaxis
of disease conditions in which PSMA is expressed.
SUMMARY OF THE INVENTION
[0019] The present invention includes methods for identifying and
producing nucleic acid ligands to the Prostate Specific Membrane
Antigen (PSMA) and the nucleic acid ligands so identified and
produced. The method uses the SELEX process for the Systematic
Evolution of Ligands by EXponential enrichment. In particular,
novel nuclease resistant RNA sequences are provided which are
capable of binding specifically to the extracellular portion of
PSMA using a Baculovirus-purified PSMA fusion protein as the target
protein. The method described herein is the first application of
SELEX to a membrane tumor antigen. Also included are
oligonucleotides containing nucleotide derivatives modified at the
2' position of the pyrimidines. Specifically included in the
invention are the RNA ligand sequences shown in Table 3 (SEQ ID
NOS:3-27). The high affinity to PSMA of two of these unique aptamer
sequences, xPSM-A9 and xPSM-A10 (SEQ ID NOS:5 & 15), was
demonstrated by their ability to inhibit native PSMA
N-acetyl-alpha-linked-acid dipeptidase (NAALADase) activity. These
aptamers bind to the extracellular portion of PSMA and inhibit
native PSMA enzymatic activity with low nanomolar K.sub.i's. The
nucleic acid ligands of the invention can be used clinically to
inhibit PSMA enzymatic activity or can be modified to carry agents
for imaging or delivery of therapeutic agents to prostate cancer
cells.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 illustrates the design of in vitro selection target,
the extracellular portion of PSMA. Recombinant baculovirus
expressing the fusion protein secrete Tag-xPSM via the gp64
secretion signal. This fusion protein is purified from the media
using a cellulose column or S-protein agarose beads. A protein
coding for only the extracellular portion of PSMA (xPSM) is
released by enterokinase cleavage.
[0021] FIG. 2 depicts a silver stain of the purified xPSM protein.
The purity of xPSM is evident by silver staining. The negative
control shows that no protein is released in the absence of
enterokinase. The size of purified xPSM has been calculated as
approximately 90 kD, suggesting glycosylation of the expected 79.5
kD product.
[0022] FIG. 3 depicts the NAALADase activity of the xPSM fusion
protein. Purified xPSM displays native NAALADase activity with a
K.sub.m of 16.1 nM and V.sub.max of 13 mmoles/mg*min.
[0023] FIG. 4 illustrates schematically in vitro selection as
described in Example 1. The applied RNA aptamer library template
consists of a 5'-terminal fixed region containing a T7 promoter, an
internal random region of 40 consecutive nucleotides, followed by a
final fixed primer region. A typical round of selection involves
transcription of the RNA library with 2'-fluoro (2'-F) modified
pyrimidines, followed by a partitioning step where ligand-bound RNA
is separated from non-ligand-bound RNA. The bound RNA is then
amplified by RT-PCR and in vitro transcription. Several rounds of
in vitro selection are completed until the affinity of the RNA
aptamer pool for the target ligand has peaked. The resultant dsDNA
is then cloned into a plasmid vector and sequenced. Individual
aptamers are then tested for their affinity for the target
ligand.
[0024] FIG. 5 depicts the inhibition of xPSM activity by SELEX-RNA
pools. As illustrated in FIG. 5 in vitro selection rounds inhibit
NAALADase activity, whereas the initial pool shows no inhibition.
Round six of xPSM binding selection shows the best IC50 when
compared to both early and late round selections. The original
random RNA has no effect on NAALADase activity in these ranges.
(.smallcircle.) Random RNA; (.box-solid.) Round 3;
(.tangle-solidup.) Round 6; (.diamond-solid.) Round 8; (*) Round
9.
[0025] FIG. 6 depicts the 40N7 library which is the complement of
SEQ ID NO:1 and individual aptamer sequences from round 6. The
original diversity of .about.10.sup.14 RNA sequences was selected
to essentially two aptamer sequences, xPSM-A9 (SEQ ID NO:5 and
xPSM-A10 (SEQ ID NO:15).
[0026] FIGS. 7A and B illustrate graphically that the two aptamers,
xPSM-A9 and xPSM-A10 have separate types of inhibition, indicating
two separate epitopes. In FIG. 7A, 30 nM of aptamer xPSM-A10 shows
competitive inhibition, with a calculated K.sub.I of 11.9 nM.
Alternatively, in FIG. 7B, 1 nM of aptamer xPSM-A9 shows
noncompetitive inhibition, with a calculated K.sub.I of 1.1 nM. In
both graphs: (.box-solid.) is xPSM and (.smallcircle.) is xPSM plus
aptamer inhibitor. R.sup.2 values A:xPSM 0.7932, A:xPSM-A10 0.887,
B:xPSM 0.8155, B:xPSM-A9 0.7248.
[0027] FIG. 8 depicts graphically NAALADase inhibition by the
56-nucleotide xPSM-A10 truncate. Aptamer xPSM-A10-3 (SEQ ID NO:18)
is the 15 nucleotide truncate of xPSM-A10. This shorter nucleotide
shows competitive inhibition, raising the K.sub.m with no effect on
V.sub.max. Average K.sub.I=20.46+/-7.8 nM. R.sup.2 values: xPSM
0.8748, xPSM-A10-3 0.7861.
[0028] FIG. 9 depicts graphically the NAALADase inhibition of
native PSMA by aptamers xPSM-A9 (SEQ ID NO:5) and xPSM-A10 (SEQ ID
NO:15).
[0029] FIG. 10 depicts the ability of aptamer A10-3 to specifically
bind native PSMA expressed on the cell surface. Fluorescently
labeled A10-3 (50 nM) or A10-3-rndm (A10-3 sequence scrambled) was
incubated with formalin fixed LNCaP cells (PSMA positive) and PC-3
cells (PSMA negative) for 12 minutes, washed, and visualized by
fluorescent microscopy.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The central method utilized herein for identifying nucleic
acid ligands to PSMA is called the SELEX process, an acronym for
Systematic Evolution of Ligands by Exponential enrichment. The
SELEX method involves: (a) contacting the candidate mixture of
nucleic acids with PSMA, or expressed domains or peptides
corresponding to PSMA; (b) partitioning between members of said
candidate mixture on the basis of affinity to PSMA; and (c)
amplifying the selected molecules to yield a mixture of nucleic
acids enriched for nucleic acid sequences with a relatively higher
affinity for binding to PSMA.
[0031] The invention includes RNA ligands to PSMA. This invention
further includes the specific RNA ligands to PSMA shown in Table 3
(SEQ ID NOS:3-27). More specifically, this invention includes
nucleic acid sequences that are substantially homologous to and
that have substantially the same ability to bind PSMA as the
specific nucleic acid ligands shown in Table 3. By substantially
homologous it is meant a degree of primary sequence homology in
excess of 70%, most preferably in excess of 80%, and even more
preferably in excess of 90%, 95%, or 99%. The percentage of
homology as described herein is calculated as the percentage of
nucleotides found in the smaller of the two sequences which align
with identical nucleotide residues in the sequence being compared
when 1 gap in a length of 10 nucleotides may be introduced to
assist in that alignment. Substantially the same ability to bind
PSMA means that the affinity is within one or two orders of
magnitude of the affinity of the ligands described herein. It is
well within the skill of those of ordinary skill in the art to
determine whether a given sequence--substantially homologous to
those specifically described herein--has the same ability to bind
PSMA.
[0032] A review of the sequence homologies of the nucleic acid
ligands of PSMA shown in Table 3 shows that sequences with little
or no primary homology may have substantially the same ability to
bind PSMA. For this reason, this invention also includes nucleic
acid ligands that have substantially the same postulated structure
or structural motifs and ability to bind PSMA as the nucleic acid
ligands shown in Table 3. Substantially the same structure or
structural motifs can be postulated by sequence alignment using the
Zukerfold program (see Zuker (1989) Science 244:48-52). As would be
known in the art, other computer programs can be used for
predicting secondary structure and structural motifs. Substantially
the same structure or structural motif of nucleic acid ligands in
solution or as a bound structure can also be postulated using NMR
or other techniques as would be known in the art.
[0033] Also included in this invention is a method for detecting
the presence of a disease that is expressing PSMA in a biological
tissue which may contain the disease by the method of: (a)
identifying a nucleic acid ligand from a candidate mixture of
nucleic acids, the nucleic acid ligand being a ligand of PSMA, by
the method comprising (i) contacting a candidate mixture of nucleic
acids with PSMA, wherein nucleic acids having an increased affinity
to PSMA relative to the candidate mixture may be partitioned from
the remainder of the candidate mixture; (ii) partitioning the
increased affinity nucleic acids from the remainder of the
candidate mixture; (iii) amplifying the increased affinity nucleic
acids to yield a mixture of nucleic acids with relatively higher
affinity and specificity for binding to PSMA, whereby a nucleic
acid ligand of PSMA is identified; (b) attaching a marker that can
be used in in vivo or ex vivo diagnostics to the nucleic acid
ligand identified in step (iii) to form a marker-nucleic acid
ligand complex; (c) exposing a tissue which may contain the disease
to the marker-nucleic acid ligand complex; and (d) detecting the
presence of the marker-nucleic acid ligand in the tissue, whereby a
disease expressing PSMA is identified.
[0034] Further included in this invention is a complex for use in
in vivo or ex vivo diagnostics comprising one or more PSMA nucleic
acid ligands and one or more markers. Still further included in
this invention is a method for delivering therapeutic agents for
the treatment or prophylaxis of disease conditions in which PSMA is
expressed.
[0035] Definitions
[0036] 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.
[0037] As used herein a "nucleic acid ligand" is a non-naturally
occurring nucleic acid having a desirable action on a target.
Nucleic acid ligands are often referred to as "aptamers." 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 a 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 does not have the known
physiological function of being bound by the target molecule. In
the present invention, the target is PSMA, or regions thereof.
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.
[0038] As used herein a "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
acids, enzymatically synthesized nucleic acids or nucleic acids
made by a combination of the foregoing techniques. In a preferred
embodiment, each nucleic acid has fixed sequences surrounding a
randomized region to facilitate the amplification process.
[0039] 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.
[0040] "SELEX" methodology involves the combination of selection of
nucleic acid ligands that 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. In the present
invention, the SELEX methodology is employed to obtain nucleic acid
ligands to PSMA.
[0041] The SELEX methodology is described in the SELEX Patent
Applications.
[0042] "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. In this application, the
SELEX target is PSMA. In particular, the SELEX targets in this
application include purified PSMA, and fragments thereof, and short
peptides or expressed protein domains comprising PSMA.
[0043] 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, microtiter plates, magnetic beads, charged paper, nylon,
Langmuir-Bodgett films, functionalized glass, germanium, silicon,
PTFE, polystyrene, gallium arsenide, gold, and 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
[0044] Note, that throughout this application various citations are
provided. Each citation is specifically incorporated herein in its
entirety by reference.
[0045] In the preferred embodiment, the nucleic acid ligands of the
present invention are derived from the SELEX methodology. The SELEX
process is described in U.S. patent application Ser. No.
07/536,428, entitled "Systematic Evolution of Ligands by
Exponential Enrichment," now abandoned, U.S. Pat. No. 5,475,096,
entitled "Nucleic Acid Ligands," and U.S. Pat. No. 5,270,163 (see
also WO 91/19813), entitled "Methods for Identifying Nucleic Acid
Ligands." These applications, each specifically incorporated herein
by reference, are collectively called the SELEX Patent
Applications.
[0046] The SELEX process provides a class of products that are
nucleic acid molecules, each having a unique sequence, and each of
which has the property of binding specifically to a desired target
compound or molecule. Target molecules are preferably proteins, but
can also include among others carbohydrates, peptidoglycans and a
variety of small molecules. SELEX methodology can also be used to
target biological structures, such as cell surfaces or viruses,
through specific interaction with a molecule that is an integral
part of that biological structure.
[0047] In its most basic form, the SELEX process may be defined by
the following series of steps.
[0048] 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 probability of finding a base at any location
can be selected at any level between 0 and 100 percent).
[0049] 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 circumstances,
the interaction between the target and the nucleic acids of the
also contemplated. This includes surfaces with any topology,
including, but not limited to, spherical surfaces and grooved
surfaces.
[0050] "Complex" as used herein means the molecular entity formed
by the covalent linking of one or more PSMA nucleic acid ligands
with one or more markers. In certain embodiments of the present
invention, the complex is depicted as A-B-Y, wherein A is a marker;
B is optional, and comprises a linker; and Y is a PSMA nucleic acid
ligand. "Marker" as used herein is a molecular entity or entities
that when complexed with the PSMA nucleic acid ligand, either
directly or through a linker(s) or spacer(s), allows the detection
of the complex in an in vivo or ex vivo setting through visual or
chemical means. Examples of markers include, but are not limited to
radionuclides, including Tc-99m, Re-188, Cu-64, Cu-67, F-18,
.sup.125I, .sup.131I, .sup.32P, .sup.186Re; .sup.111In; all
fluorophores, including fluorescein, rhodamine, Texas Red;
derivatives of the above fluorophores, including Rhodamine-Red-X;
magnetic compounds; and biotin.
[0051] As used herein, "linker" is a molecular entity that connects
two or more molecular entities through covalent bond or
non-covalent interactions, and can allow spatial separation of the
molecular entities in a manner that preserves the functional
properties of one or more of the molecular entities. A linker can
also be known as a spacer. Examples of a linker include, but are
not limited to, the (CH.sub.2CH.sub.2O).sub.- 6 and hexylamine
structures shown in FIG. 2 of U.S. patent application Ser. No.
09/364,902, filed Jul. 29, 1999, entitled "Tenascin-C Nucleic Acid
Ligands," which is incorporated herein by reference in its
entirety.
[0052] "Therapeutic" as used herein, includes treatment and/or
prophylaxis. When used, therapeutic refers to humans, as well as,
other animals.
[0053] "Covalent Bond" is the chemical bond formed by the sharing
of electrons.
[0054] "Non-covalent interactions" are means by which molecular
entities are held together by interactions other than Covalent
Bonds including ionic interactions and hydrogen bonds.
[0055] As used herein "PSMA" refers to purified protein, the
extracellular, including xPSM, cytoplasmic, or intracellular
domains of the protein or any allelic variants thereof. "PSMA" as
used herein also includes the protein isolated from a species other
than humans. 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.
[0056] 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 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 significant amount of the nucleic
acids in the candidate mixture (approximately 5-50%) are retained
during partitioning.
[0057] 4. Those nucleic acids selected during partitioning as
having the relatively higher affinity for 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.
[0058] 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.
[0059] The basic SELEX method has been modified to achieve a number
of specific objectives. For example, U.S. patent application Ser.
No. 07/960,093, filed Oct. 14, 1992, now abandoned, and U.S. Pat.
No. 5,707,796, both entitled "Method for Selecting Nucleic Acids on
the Basis of Structure," describe the use of the SELEX process in
conjunction with gel electrophoresis to select nucleic acid
molecules with specific structural characteristics, such as bent
DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17,
1993, entitled "Photoselection of Nucleic Acid Ligands," now
abandoned, U.S. Pat. No. 5,763,177 and U.S. Pat. No. 6,001,577,
both entitled "Systematic Evolution of Ligands by Exponential
Enrichment: Photoselection of Nucleic Acid Ligands and Solution
SELEX," all describe 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. U.S. Pat. No. 5,580,737, entitled "High-Affinity Nucleic
Acid Ligands That Discriminate Between Theophylline and Caffeine,"
describes a method for identifying highly specific nucleic acid
ligands able to discriminate between closely related molecules,
termed Counter-SELEX. U.S. Pat. No. 5,567,588, entitled "Systematic
Evolution of Ligands by Exponential Enrichment: Solution SELEX,"
describes a SELEX-based method which achieves highly efficient
partitioning between oligonucleotides having high and low affinity
for a target molecule. U.S. Pat. No. 5,496,938, entitled "Nucleic
Acid Ligands to HIV-RT and HIV-1 Rev," describes methods for
obtaining improved nucleic acid ligands after SELEX has been
performed. U.S. Pat. No. 5,705,337, entitled "Systematic Evolution
of Ligands by Exponential Enrichment: Chemi-SELEX," describes
methods for covalently linking a ligand to its target.
[0060] 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-identified nucleic
acid ligands containing modified nucleotides are described in U.S.
Pat. No. 5,660,985, entitled "High Affinity Nucleic Acid Ligands
Containing Modified Nucleotides," that describes oligonucleotides
containing nucleotide derivatives chemically modified at the 5- and
2'- positions of pyrimidines. U.S. Pat. No. 5,637,459, supra,
describes highly specific nucleic acid ligands containing one or
more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2
.degree. F.), and/or 2'-O-methyl (2'-OMe). U.S. patent application
Ser. No. 08/264,029, filed Jun. 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.
[0061] The SELEX method encompasses combining selected
oligonucleotides with other selected oligonucleotides and
non-oligonucleotide functional units as described in U.S. Pat. No.
5,637,459, entitled "Systematic Evolution of Ligands by Exponential
Enrichment: Chimeric SELEX," and U.S. Pat. No. 5,683,867, entitled
"Systematic Evolution of Ligands by Exponential Enrichment: Blended
SELEX," 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.
[0062] In U.S. Pat. No. 5,496,938, methods are described for
obtaining improved nucleic acid ligands after the SELEX process has
been performed. This patent, entitled "Nucleic Acid Ligands to
HIV-RT and HIV-1 Rev," is specifically incorporated herein by
reference.
[0063] One potential problem encountered in the diagnostic use of
nucleic acids is that oligonucleotides in their phosphodiester form
may be quickly degraded in body fluids by intracellular and
extracellular enzymes, such as endonucleases and exonucleases,
before the desired effect is manifest. Certain chemical
modifications of the nucleic acid ligand can be made to increase
the in vivo stability of the nucleic acid ligand or to enhance or
to mediate the delivery of the nucleic acid ligand. See, e.g., U.S.
patent application Ser. No. 08/117,991, filed Sep. 8, 1993, now
abandoned and U.S. Pat. No. 5,660,985, both entitled "High Affinity
Nucleic Acid Ligands Containing Modified Nucleotides," and U.S.
patent application Ser. No. 09/362,578, filed Jul. 28, 1999,
entitled "Transcription-free SELEX," each of which is specifically
incorporated herein by reference in its entirety. Modifications of
the nucleic acid ligands contemplated in this invention include,
but are not limited to, those which provide other chemical groups
that incorporate additional charge, polarizability, hydrophobicity,
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, phosphorothioate
or alkyl phosphate 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. In preferred embodiments of the
instant invention, the nucleic acid ligands are RNA molecules that
are 2'-fluoro (2'-F) modified on the sugar moiety of pyrimidine
residues.
[0064] The modifications can be pre- or post-SELEX process
modifications. Pre-SELEX process modifications yield nucleic acid
ligands with both specificity for their SELEX target and improved
in vivo stability. Post-SELEX process modifications made to 2'-OH
nucleic acid ligands can result in improved in vivo stability
without adversely affecting the binding capacity of the nucleic
acid ligand.
[0065] Other modifications are known to one of ordinary skill in
the art. Such modifications may be made post-SELEX process
(modification of previously identified unmodified ligands) or by
incorporation into the SELEX process.
[0066] The nucleic acid ligands of the invention are prepared
through the SELEX methodology that is outlined above and thoroughly
enabled in the SELEX applications incorporated herein by reference
in their entirety.
[0067] In preferred embodiments, the SELEX process is carried out
using fragments of PSMA that are bound to magnetic beads through
hydrophobic interactions. A candidate mixture of single stranded
RNA molecules is then contacted with the magnetic beads in a
microfuge tube. After incubation for a predetermined time at a
selected temperature, the beads are held to the sides of the tube
by a magnetic field, and the microfuge tube is washed to remove
unbound candidate nucleic acid ligands. The nucleic acid ligands
that bind to the PSMA are then released into solution in the
microfuge tube, then reverse transcribed by reverse transcriptase
and amplified using the Polymerase Chain Reaction (PCR). The
amplified candidate mixture is then used to begin the next round of
the SELEX process.
[0068] In certain embodiments of the present invention, the nucleic
acid ligands to PSMA described herein are useful for diagnostic
purposes and can be used to image pathological conditions (such as
human tumor imaging). In addition to diagnosis, the PSMA nucleic
acid ligands are useful in the prognosis and monitoring of disease
conditions in which PSMA is expressed.
[0069] Diagnostic agents need only be able to allow the user to
identify the presence of a given target at a particular locale or
concentration. Simply the ability to form binding pairs with the
target may be sufficient to trigger a positive signal for
diagnostic purposes. Those skilled in the art would be able to
adapt any PSMA nucleic acid ligand by procedures known in the art
to incorporate a marker in order to track the presence of the
nucleic acid ligand. Such a marker could be used in a number of
diagnostic procedures, such as detection of primary and metastatic
tumors. In one embodiment the labeling marker is technetium-99 m;
however, other markers such as additional radionuclides, magnetic
compounds, fluorophores, biotin, and the like can be conjugated to
the PSMA nucleic acid ligand for imaging in an in vivo or ex vivo
setting disease conditions in which PSMA is expressed. The marker
may be covalently bound to a variety of positions on the PSMA
nucleic acid ligand, such as to an exocyclic amino group on the
base, the 5-position of a pyrimidine nucleotide, the 8-position of
a purine nucleotide, the hydroxyl group of the phosphate, or a
hydroxyl group or other group at the 5' or 3' terminus of the PSMA
nucleic acid ligand. In embodiments where the marker is
technetium-99m, preferably it is bonded to the 5' or 3' hydroxyl of
the phosphate group thereof or to the 5 position of a modified
pyrimidine. In the most preferred embodiment, the marker is bonded
to the 5' hydroxyl of the phosphate group of the nucleic acid
ligand with or without a linker. In another embodiment, the marker
is conjugated to the nucleic acid ligand by incorporating a
pyrimidine containing a primary amine at the 5 position, and use of
the amine for conjugation to the marker. Attachment of the marker
can be done directly or with the utilization of a linker. In the
embodiment where technetium-99 m is used as the marker, the
preferred linker is a hexylamine linker.
[0070] In other embodiments, the PSMA nucleic acid ligands are
useful for the delivery of therapeutic compounds (including, but
not limited to, cytotoxic compounds, immune enhancing substances
and therapeutic radionuclides) to tissues or organs expressing
PSMA. Disease conditions in which PSMA may be expressed include
cancer. Those skilled in the art would be able to adapt any PSMA
nucleic acid ligand by procedures known in the art to incorporate a
therapeutic compound in a complex. The therapeutic compound may be
covalently bound to a variety of positions on the PSMA nucleic acid
ligand, such as to an exocyclic amino group on the base, the
5-position of a pyrimidine nucleotide, the 8-position of a purine
nucleotide, the hydroxyl group of the phosphate, or a hydroxyl
group or other group at the 5' or 3' terminus of the PSMA nucleic
acid ligand. In the preferred embodiment, the therapeutic agent is
bonded to the 5' amine of the nucleic acid ligand. Attachment of
the therapeutic agent can be done directly or with the utilization
of a linker. In embodiments in which cancer is the targeted
disease, 5-fluorodeoxyuracil or other nucleotide analogs known to
be active against tumors can be incorporated internally into
existing U's within the PSMA nucleic acid ligand or can be added
internally or conjugated to either terminus either directly or
through a linker. In addition, both pyrimidine analogues
2',2'-difluorocytidine and purine analogues (deoxycoformycin) can
be incorporated. In addition, U.S. application Ser. No. 08/993,765,
filed Dec. 18, 1997, entitled "Nucleotide Based Prodrugs,"
incorporated herein by reference in its entirety, describes, inter
alia, nucleotide-based prodrugs comprising nucleic acid ligands
directed to tumor cells for precisely localizing
chemoradiosensitizers, and radiosensitizers and radionuclides and
other radiotherapeutic agents to the tumor.
[0071] It is also contemplated that both the marker and therapeutic
agent may be associated with the PSMA nucleic acid ligand such that
detection of the disease condition and delivery of the therapeutic
agent is accomplished together in one aptamer or as a mixture of
two or more different modified versions of the same aptamer. It is
also contemplated that either or both the marker and/or the
therapeutic agent may be associated with a non-immunogenic, high
molecular weight compound or lipophilic compound, such as a
liposome. Methods for conjugating nucleic acid ligands with
lipophilic compounds or non-immunogenic compounds in a diagnostic
or therapeutic complex are described in U.S. Pat. No. 6,011,020,
filed May 4, 1995, entitled "Nucleic Acid Ligand Complexes," which
is incorporated herein in its entirety.
[0072] Therapeutic compositions of the nucleic acid ligands may be
administered parenterally by injection, although other effective
administration forms, such as intraarticular injection, inhalant
mists, orally active formulations, transdermal iontophoresis or
suppositories, are also envisioned. One preferred carrier is
physiological saline solution, but it is contemplated that other
pharmaceutically acceptable carriers may also be used. In one
preferred embodiment, it is envisioned that the carrier and the
ligand constitute a physiologically-compatible, slow release
formulation. The primary solvent in such a carrier may be either
aqueous or non-aqueous in nature. In addition, the carrier may
contain other pharmacologically-acceptable excipients for modifying
or maintaining the pH, osmolarity, viscosity, clarity, color,
sterility, stability, rate of dissolution, or odor of the
formulation. Similarly, the carrier may contain still other
pharmacologically-acceptable excipients for modifying or
maintaining the stability, rate of dissolution, release, or
absorption of the ligand. Such excipients are those substances
usually and customarily employed to formulate dosages for parental
administration in either unit dose or multi-dose form.
[0073] Once the therapeutic composition has been formulated, it may
be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or dehydrated or lyophilized powder. Such
formulations may be stored either in a ready to use form or
requiring reconstitution immediately prior to administration. The
manner of administering formulations containing nucleic acid
ligands for systemic delivery may be via subcutaneous,
intramuscular, intravenous, intranasal or vaginal or rectal
suppository.
[0074] The following examples are provided to explain and
illustrate the present invention and are not to be taken as
limiting of the invention.
[0075] Example 1 describes the materials and experimental
procedures used for the generation of RNA ligands to PSMA. Purified
PSMA protein was required for the in vitro selection of aptamers.
Because the ultimate application of these aptamers is to bind
prostate cancer cells in vivo, only the extracellular portion of
PSMA was considered a sufficient target. A vector was therefore
designed to express only the extracellular portion of PSMA, with
removable affinity tags.
[0076] A baculovirus expression vector encoding only the
extracellular portion of PSMA, termed xPSM was designed as
described in Example 1 and illustrated schematically in FIG. 1.
With reference to FIG. 1, a fragment of PSMA cDNA, coding only for
the 706 extracellular amino acids of full length PSMA, was cloned
into the multiple cloning site of the baculoviral transfer vector,
pBACgus-10. This vector was designed to provide high levels fusion
protein in the growth medium, which can be purified by affinity
tags and released by enterokinase cleavage. The resulting transfer
plasmid, pBACgus-PSM, was sequenced to confirm correct coding frame
and sequence integrity. Both pBACgus-PSM and BACvector3000 linear
DNA were co-transfected into Sf-9 cells and resulting recombinant
viral plaques were purified and screened for expression of
Tag-xPSM. A single recombinant baculovirus was then used for
large-scale infections under serum free conditions.
[0077] Infected cell media was harvested 72-80 hours post infection
and incubated with S-protein agarose to capture Tag-xPSM. A
recombinant enterokinase was then used to free xPSM. Following
digestion, the enterokinase was captured with affinity resin,
leaving only pure xPSM in the supernatant. The purity of the
protein was determined by silver staining, with no Tag-xPSM evident
by minus enterokinase control (FIG. 2). The size of purified xPSM
has been calculated as .about.90 kD, suggesting glycosylation of
the expected 79.5 kD product.
[0078] The xPSM fusion protein was then tested for enzymatic
activity to ensure native protein conformation. This is important
to avoid evolving aptamers that recognize an improperly
folded-fusion protein, but not the native enzyme. The purified xPSM
protein showed expected NAALADase activity, with a K.sub.m of 16.1
nM and a V.sub.max of 13 mmoles/mg*min as illustrated in FIG. 3.
The purified protein was immobilized on magnetic beads as a means
to partition bound RNA aptamers during selection. A fraction of the
xPSM remained NAALADase active while bound to the beads.
[0079] The in vitro selection strategy was designed to identify
aptamers that would be applicable under physiologic conditions. To
ensure nuclease stability, 2'-F modified pyrimidines were used in
all transcriptions. Fluoropyrimidine RNA aptamers have been
reported to be stable in serum for several hours. (Lin et al.
(1994) Nucleic Acids Res. 22:5229-34). Additionally, aptamers were
allowed to bind target only at 37.degree. C., pH 7.4.
[0080] A library of approximately 6.times.10.sup.14 different
nuclease stable RNA molecules was generated by transcription of a
random sequence synthetic template. The aptamer library template
consisted of a T7 promoter, two terminal fixed regions for PCR
amplification, and an internal random region of 40 nucleotides
(FIG. 4). Prior to selection, the target protein was bound to
magnetic beads, where it retained its enzymatic activity. The
random sequence library was incubated with xPSM-magnetic beads and
allowed 30 minutes to bind. The protein-bound population was
partitioned by magnetic separation, and amplified by reverse
transcription and quantitative PCR. The resulting templates were
transcribed to generate 2'-F modified RNA for the next cycle of
selection.
[0081] Six rounds of iterative selection were performed and
quantitated as illustrated in Table 1. The stringency of selection
was regulated by decreasing the amount of xPSM-magnetic beads
available for binding or by decreasing the amount of RNA. The
signal to noise ratio peaked at selection round six at 5700 fold,
and showed no further improvement up to nine total rounds of
selection. The signal/noise ratios depicted in Table 1 were
determined by comparison of RNA bound to xPSM beads versus beads
alone.
[0082] Enzyme assays provide a sensitive method to identify and
quantitate enzyme ligand interactions. Selected rounds of 2'-F
modified RNA were therefore tested for their ability to inhibit
xPSM NAALADase activity as described in Example 1. As a control
sequence for specificity, the original random sequence library was
tested and had no effect on xPSM NAALADase activity, where
micromolar aptamer inhibition could be seen as early as round three
in selected RNA populations (FIG. 5). The round six RNA aptamer
population showed the highest affinity for xPSM, and was therefore
used to isolate and sequence individual aptamer clones.
[0083] Round six RNA was amplified by RT-PCR and cloned. Sixty
randomly picked plasmid clones were sequenced. Ninety-five percent
of the sixty clones sequenced were represented by only two
sequences. The identified sequences, named xPSM-A9 (SEQ ID NO:5)
and xPSM-A10 (SEQ ID NO:15) (FIG. 6), are unique, sharing no
consensus sequences.
[0084] Each aptamer was tested for its affinity based on ability to
inhibit NAALADase activity. Aptamer xPSM-A9 displays
non-competitive inhibition with a K.sub.i of 1.1 nM (FIG. 7B),
whereas aptamer xPSM-A10 shows competitive inhibition with a
K.sub.i of 11.9 (FIG. 7A). These two separate modes of inhibition
suggest that each aptamer identifies a unique extracellular epitope
of PSMA. Both aptamers inhibit native NAALADase activity from LNCaP
cells with similar affinity.
[0085] FIGS. 9 and 10 demonstrate aptamer binding native PSMA on
the surface of LNCaP cells. This is significant as the data in the
previous figures demonstrates aptamer binding the synthetic PSMA,
xPSM, purified by baculovirus. The NAALADase assay depicted in FIG.
9 was performed as described in Example 1, except membrane extracts
from LNCaP were used instead of purified xPSM. This methodology is
described in Carter et al. (1996) Proc Natl Acad Sci USA
93(2):749-53). A known micromolar NAALADase inhibitor, quisqualic
acid, is included as a reference control. This demonstrates the
potency of the aptamers in comparison to known NAALADase
inhibitors.
[0086] The smallest aptamer, A10-3, was fluorescently labeled in
order to determine if these aptamers could specifically bind cells
expressing PSMA. A negative reference control, A10-3-rndm, was
developed by randomly scrambling the A10-3 sequence. Binding
specificity was demonstrated by fluorescent microscopy where
aptamer A10-3 specifically bound PSMA expressing LNCaP cells, but
not the negative control PC-3 cells (FIG. 10). As can be seen in
FIG. 10 the scrambled A10-3 sequence, A10-3-rndm, shows no
specificity for either cell line.
[0087] Example 2 describes the determination of minimal size
necessary for high affinity binding of two selected nucleic acid
ligands to xPSM. Aptamer, xPSM-A10 (SEQ ID NO:15), was successfully
truncated to fifty-six nucleotides, or 18.5 kD, while still
retaining its ability to inhibit PSMA activity (FIG. 8). These
aptamers can be used as inhibitors or be modified to carry agents
for imaging or therapeutic treatment.
EXAMPLES
Example 1.
Use of SELEX to Obtain Nucleic Acid Ligands to PSMA
[0088] Materials and Methods
[0089] Cloning PSMA cDNA from LNCaP.
[0090] First strand cDNA was synthesized from 2 .mu.g total LNCaP
RNA using Superscript II RNase H Reverse Transcriptase (Life
Technologies, Inc). Primers homologous to PSMA cDNA bases 134-152
and 2551-2567, flanking the entire full-length PSMA coding region,
were used for PCR amplification. Amplification was performed using
high fidelity Pfu DNA Polymerase (Stratagene, La Jolla, Calif.).
The isolated product was ligated into the pCR-2.1 vector
(Invitrogen Corporation, Carlsbad, Calif.). One successful clone,
pFULPSM-1, was sequenced and found identical to Genbank accession
number M99487. This clone represents the coding region for the full
length PSMA protein
[0091] Preparation of Recombinant PSMA Expressing Baculovirus.
[0092] Primers containing restriction enzyme cut sites were
designed to overlap the sequence of the entire extracellular
portion of PSMA plus a linking glycine, specifically bases 395-422
and 2491-2503. The PCR product was ligated into pBACgus-10 transfer
plasmid (Novagen Inc., Madison, Wis.), under the control of the
polyhedron promoter. The resulting plasmid, pBACgus-PSM, was
sequenced to confirm sequence integrity. Sf-9 cells (ATCC) were
co-transfected with pBACgus-PSM and linear high efficiency
BacVector-3000 Triple Cut Virus DNA (Novagen Inc) in Grace's Insect
Culture Media (Life Technologies, Inc) supplemented with 10% fetal
bovine serum. Individual recombinant viral plaques were picked and
assayed for recombinant protein expression by S-tag assays and
S-tag westerns (Novagen Inc) according to manufacturer's
instructions. A single positive clone expressing the entire
extracellular portion of PSMA, termed xPSM, was amplified to high
titer (.gtoreq.10.sup.8 PFU/mL) in 200 mL suspension cultures.
[0093] Large Scale xPSM Expression and Purification.
[0094] Sf-9 cells (Novagen Inc) were plated as monolayers in Sf-900
II Serum Free Media (Life Technologies Inc) and infected with
recombinant virus at an M.O.I of 5. Infected cell media was
harvested 72-80 hours post infection and tag-xPSM levels
quantitated by S-tag assay (Novagen Inc). Fusion protein was bound
by S-protein agarose, washed, and xPSM was released by recombinant
enterokinase (rEK) according to manufacturer's instructions
(Novagen Inc). Finally, rEK was bound by EKapture Agarose Beads
(Novagen Inc) and purified xPSM protein was concentrated by
Ultra-Free 15, MWCO 50 kD concentration spin columns (Millipore
Co., Bedford, Mass.). xPSM concentrations were determined by
Coomassie Plus Protein Assay Reagent (Pierce, Rockford, Ill.).
Protein purity was confirmed by silver stain analysis.
[0095] Silver Stains.
[0096] Approximately 100-500 ng of purified PSMA protein was
separated by 7.5% SDS-PAGE and stained using the Silver Stain Plus
Kit (Bio-Rad Laboratories, Hercules, Calif.). All purified xPSM
size and purity was checked by silver stained gels.
[0097] PSMA NAALADase Assays.
[0098] NAAG hydrolysis was performed essentially as described in
Robinson et al. (1987) J Biol Chem. 262:14498-506. LNCaP cell
extracts were prepared by sonication in the presence of 50 mM Tris,
pH 7.4, 0.5% Triton-X-100. Cell lysate or purified xPSM was
incubated in the presence of the radiolabeled substrate
N-acetyl-L-aspartyl-L-[3,4-.sup.3H]glutamat- e (NEN Life Science
Products, Boston, Mass.) at 37.degree. C. for 10-15 minutes. The
reaction was stopped with an equal volume of ice-cold 100 mM sodium
phosphate, 2 mM EDTA. Products were separated from intact substrate
using anion exchange chromatography and quantitated by
scintillation counting. In general, aptamer IC50's were determined
in the presence of 8 nM substrate. Aptamer K.sub.I's were
determined using 5-30 nM aptamer in serial dilutions of substrate.
In all cases less than 20% of substrate was cleaved.
[0099] In vitro Selection of PSMA Aptamers.
[0100] The SELEX-process has been described in detail in the SELEX
Patent Applications. In brief, double-stranded transcription
templates were prepared by Klenow fragment extension of 40N7a
ssDNA: 5'- TCGCGCGAGTCGTCTG[40N]CCGCATCGTCCTCCC-3' (SEQ ID NO:1)
using the 5N7 primer: 5'-TAATACGACTCACTATAGGGAGGACGATGCGG-3' (SEQ
ID NO:2) which contains the T7 polymerase promoter (underlined).
RNA was prepared with T7 RNA polymerase as described previously in
Fitzwater and Polisky (1996) Methods Enzymol. 267:275-301,
incorporated herein by reference in its entirety. All transcription
reactions were performed in the presence of pyrimidine nucleotides
that were 2'-fluoro (2'-F) modified on the sugar moiety. This
substitution confers enhanced resistance to ribonucleases that
utilize the 2'-hydroxyl moiety for cleavage of the phosphodiester
bond. Specifically, each transcription mixture contained 3.3 mM
2'-F UTP and 3.3 mM 2'-F CTP along with 1 mM GTP and ATP. The
initial randomized RNA library thus produced comprised
6.times.10.sup.14 molecules (with 1 nmole of RNA).
[0101] Nine rounds of the SELEX process were performed as described
below, and round six was chosen for cloning based on its ability to
inhibit PSMA enzymatic activity.
[0102] Target Bead Preparation.
[0103] Paramagnetic polystyrene beads were purchased from Dynal,
Inc. Dynabeads M-450, uncoated, 4.5 .mu.m, 2.4% w/v. Selections and
magnetic separations were performed in a 0.5 mL microfuge tube
using a Dynal MPC-E Separator. Prior to use the beads (100 .mu.L)
were washed with potassium phosphate (100 mM, 3.times.500 .mu.L, pH
8.0), 3.times.500 .mu.L Hepes buffered saline, pH 7.4, MgCl.sub.2
(1 mM), CaCl.sub.2 (1 mM) (HBSMC). The beads were then resuspend in
HBSMC (100 .mu.L) containing 10 .mu.g of the target protein and
rotated at 4.degree. C. overnight. The beads were then washed with
HBSMC (3.times.500 .mu.L), resuspended in HBSMC (400 .mu.L) and
washed with 3.times.500 .mu.L HBSMC, HSA (0.01%) and Tween 20
(0.05%) (HBSMCHT). The beads were then resuspended in HBSMCHIT
(where "I" refers to I-block) (300 .mu.L, 0.6% solids w/v) and
stored at 4.degree. C.
[0104] Selection and Partition.
[0105] The target beads (50 .mu.L) were pre-blocked for 30 minutes
in HBSMCIT at 37.degree. C. The target beads (50 .mu.L) were then
combined with the aptamer pool (1 nmole of RNA) in HBSMCHIT (100
.mu.L) buffer and the mixture was rotated for 30 minutes at
37.degree. C. The actual amounts of RNA and beads varied for each
round and were decreased in later rounds of selection. The beads
were then washed with HBSMCHT (5.times.500 .mu.L) at 37.degree. C.,
transferred to a new tube and the final wash (500 .mu.L) was
removed.
[0106] Elution and Reverse Transcription.
[0107] The washed beads were resuspended in 3' primer (20 .mu.L 5
.mu.M), incubated at 95.degree. C. for 5 minutes, and slowly cooled
to room temperature. 5.times.RT master mix (5 .mu.L) was added and
the mixture was incubated at 48.degree. C. for 30 minutes. The
reaction mixture was removed from the beads and Quantitative PCR
(QPCR) reaction mix was added. Reaction mixtures are summarized in
Table 2.
[0108] Quantative PCR (QPCR) and Transcription.
[0109] cDNA (25 .mu.L) was added to the QPCR master mix (75 .mu.L).
Quantitative reference cDNA's and non-template controls were also
included for each round. PCR was performed as follows: 35 cycles on
QPC machine (ABI 7700) at 95.degree. C. 15 sec, 55.degree. C. 10
sec, 72.degree. C. 30 sec, following an initial 3 minute incubation
at 95.degree. C. PCR product (50 .mu.L) was then added to the
transcription master mix (150 .mu.L) and the mixture was incubated
at 37.degree. C. from 4 to 16 hours, followed by a 10 minute DNAse
treatment, and finally gel purification of full length RNA
transcript.
[0110] Cloning and Sequencing.
[0111] Amplified sixth-round oligonucleotide pools were purified on
an 8% polyacrylamide gel, reverse transcribed into ssDNA and the
DNA amplified by the polymerase chain reaction (PCR) using primers
containing BamHI and HindEif restriction endonuclease sites. PCR
fragments were cloned, plasmids prepared and sequence analyses
performed according to standard techniques (Sambrook et al. (1989)
Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed. 3 vols., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor). Plasmids were
sequenced using DYEnamic ET-terminator cycle sequencing premix kit
(Amersham Pharmacia Biotech, Inc, Piscataway, N.J.) and ABI Prism
377 sequencer.
[0112] Fluorescent Staining.
[0113] 5'-Hexal-amine aptamers A10-3 and A10-3-scrambled
(caggcaugccuagcuaagcagcccauggcuuaugcgcggaauauuggcuuccguuc)
2'FY-aptamers were synthesized with a deoxy-T 3' cap. Aptamers were
end labeled with Rhodamine-Red-X succinimidyl ester *5 isomer*
(Molecular Probes) according to manufacturer's instructions.
Full-length rhodamine-labeled aptamers were gel purified and
quantitated. 5.times.10.sup.4 LNCaP Parent and PC-3 cells per well
were plated on 4 chamber glass slides (Becton Dickinson, Franklin
Lakes, N.J.). 24 hours after plating, slides were fixed in 10%
buffered formalin for 8-16 hours at room temperature and stored at
4.degree. C. in PBS without magnesium or calcium. Each well was
incubated in 50 nM labeled-aptamer in PBS without magnesium or
calcium for 10-15 minutes at room temperature. Slides were then
rinsed several times in PBS, coverslipped, and sealed. Slides were
imaged with a Zeiss Axioskop epifluoresence microscope equipped
with a short arc mercury lamp illumination (Carl Zeiss Inc,
Thornwood, N.Y.) and cooled CCD camera (Micro MAX Digital Camera,
Princeton Instruments, Trenton, N.J.). Images were equally
processed in Adobe Photoshop (Adobe Systems Inc., Seattle, Wash.).
The results are depicted in FIG. 10.
Example 2
Determination of Minimal Size of Aptamers A10 and A9
[0114] To determine minimal aptamer sequences, a series of 3' and
5' truncations were tested for IC.sub.50. Five nucleotides could be
removed from the 3' end of aptamer xPSM-A9 (SEQ ID NO:5) with
retention of activity, yielding aptamer A9-1 (SEQ ID NO:6). It was
found that at least 15 nucleotides could be deleted from the 3' end
of aptamer xPSM-A10 (SEQ ID NO:15) with retention of activity,
yielding aptamer A10-3 (SEQ ID NO:18). This 18.5 kD aptamer retains
the ability to inhibit xPSM NAALADase activity with a K.sub.I of
20.5 nM. (FIG. 8). The shorter A10-3 could not survive
5'-truncation.
1 TABLE 1 Round Signal/Noise 1 3.4 2 26.8 3 15.8 4 25.7 5 125.0 6
5688.0
[0115]
2TABLE 2 Reaction mixtures for reverse transcription (RT), QPCR and
Transcription 5X RT mix QPCR mix Transcription mix 2.5 .mu.L 10X RT
buffer 10 .mu.L 10X SQ buffer 40 .mu.L 5X nucleotides 1 .mu.L 25 mM
dNTP'S 1 .mu.L 3' primer 100 .mu.M 40 .mu.L 5X Ribomax 1 .mu.L
H.sub.2O 0.5 .mu.L 5' primer 100 .mu.M 10 .mu.L guanosine 100 mM
0.5 .mu.L AMV-RT 0.5 .mu.L 5' primer-FD2 100 .mu.M 6 .mu.L T7 RNA
Polymerase 1 .mu.L Taq Polymerase 2 .mu.L pyrophosphatase (ppase)
62 .mu.L H.sub.2O 52 .mu.L H.sub.2O
[0116]
3TABLE 3 xPSM-1 Aptamer Sequences. SEQ ID IC.sub.50 NO: Family #1
B8 (31)
GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUACCCAGACGACUCGCCC-
GA 3 A11 GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUA-
CGUUCUCAGACGACUCGCCCGA 4 A9 GGGAGGACGAUGCGGACCGAAAAAGACCU-
GACUUCUAUACUAAGUCUACGUUCCCAGACGACUCGCCCGA 5 nM 5 xPSM1.A9-1
GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUCCCAGACGAC- UCG
8 nM 6 xPSM1.A9-2 GGGAGGACGAUGCGGACCGAAAAAGACCUGACU-
UCUAUACUAAGUCUACGUUCCCAGACG 300 nM 7 xPSM1.A9-3
GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUCCC >300 nM
8 xPSM1.A9-3.1 GACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUAC- GUUCCC
9 Xpsm1.A9-3.2 UGCGGACCGAAAAAGACCUGACUUC- UAUACUAAGUCUACGUUCCC 10
xPSM1.A9-3.3 ACCGAAAAAGACCUGACUUCUAUACUAAGUCUACGUUCCC 11 xPSM1.A9-4
GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAAGUCUACG >300 nM 12
xPSM1.A9-5 GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUACUAAGU >300 nM
13 xPSM1.A9-6 GGGAGGACGAUGCGGACCGAAAAAGACCUGACUUCUAUAC >300 nM
14 Family #2 62-A10 (20)
GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUCAUCGGCAGACGACUCGCCCGA
80 nM 15 xPSM1.A10-1 GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACU-
CCUUGUCAAUCCUCAUCGGCAGACGACUCG 40 nM 16 xPSM1.A10-2
GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUCAUCGGCAGACG 17
xPSM1.A10-3 GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUCAUC-
GGC 50 nM 18 xPSM1.A10-4 GGGAGGACGAUGCGGAUCAGCCAUGUUUACG-
UCACUCCUUGUCAAUCCUCA >700 nM 19 xPSM1.A10-5
GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAU >700 nM 20
xPSM1.A10-6 GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUG >700 nM 21
64-A12 GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGUCAAUCCUCAUC-
GCCAGACGACUCGCCCGA 22 11-F9 GGGAGGACGAUGCGGAUCAGCCAUGUUUA-
CGUCACUCCUUGGUCAAUCCUCAUCGGCAGACGACUCGCCCGA 23 50-D10
GGGAGGACGAUGCGGAUAGCCAUQUUUACGUCACUCCUUGGUCAAUCCUCAUCGCCAGACGACUCGCCCGA
24 22-C8 GGGAGGACGAUGCGGAUCAGCCAUGUUUACGUCACUCCUUGACAAUCC-
UCAUCQGCAGACGACUCGCCCGA 25 Others 27-B3
GGGAGGACGATGCGGACGACACGCTCCTCTGATTAGACTAAAGACCACCGTGCCCAGACGACTCGCCCGA
26 36-H6 GGGAGGACGATCGGACCATCGAACAGTGGCTAAAAACCAAGGGCATCA-
TTCGCCCCAGACGACTCGCCCGA 27 *All pyrimidines are 2'-F, all purines
are 2'-OH. Bases corresponding to 5' and 3' fixed regions are
underlined.
[0117]
Sequence CWU 1
1
27 1 71 DNA Artificial Sequence Description of Artificial Sequence
Template 1 tcgcgcgagt cgtctgnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnccgc 60 atcgtcctcc c 71 2 32 DNA Artificial Sequence
Description of Artificial Sequence Primer 2 taatacgact cactataggg
aggacgatgc gg 32 3 70 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 3 gggaggacga
ugcggaccga aaaagaccug acuucuauac uaagucuacg uacccagacg 60
acucgcccga 70 4 70 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 4 gggaggacga
ugcggaccga aaaagaccug acuucuauac uaagucuacg uucucagacg 60
acucgcccga 70 5 70 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 5 gggaggacga
ugcggaccga aaaagaccug acuucuauac uaagucuacg uucccagacg 60
acucgcccga 70 6 65 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 6 gggaggacga
ugcggaccga aaaagaccug acuucuauac uaagucuacg uucccagacg 60 acucg 65
7 60 RNA Artificial Sequence Description of Artificial Sequence
Synthetic Nucleic Acid Ligand 7 gggaggacga ugcggaccga aaaagaccug
acuucuauac uaagucuacg uucccagacg 60 8 55 RNA Artificial Sequence
Description of Artificial Sequence Synthetic Nucleic Acid Ligand 8
gggaggacga ugcggaccga aaaagaccug acuucuauac uaagucuacg uuccc 55 9
50 RNA Artificial Sequence Description of Artificial Sequence
Synthetic Nucleic Acid Ligand 9 gacgaugcgg accgaaaaag accugacuuc
uauacuaagu cuacguuccc 50 10 45 RNA Artificial Sequence Description
of Artificial Sequence Synthetic Nucleic Acid Ligand 10 ugcggaccga
aaaagaccug acuucuauac uaagucuacg uuccc 45 11 40 RNA Artificial
Sequence Description of Artificial Sequence Synthetic Nucleic Acid
Ligand 11 accgaaaaag accugacuuc uauacuaagu cuacguuccc 40 12 50 RNA
Artificial Sequence Description of Artificial Sequence Synthetic
Nucleic Acid Ligand 12 gggaggacga ugcggaccga aaaagaccug acuucuauac
uaagucuacg 50 13 45 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 13 gggaggacga
ugcggaccga aaaagaccug acuucuauac uaagu 45 14 40 RNA Artificial
Sequence Description of Artificial Sequence Synthetic Nucleic Acid
Ligand 14 gggaggacga ugcggaccga aaaagaccug acuucuauac 40 15 71 RNA
Artificial Sequence Description of Artificial Sequence Synthetic
Nucleic Acid Ligand 15 gggaggacga ugcggaucag ccauguuuac gucacuccuu
gucaauccuc aucggcagac 60 gacucgcccg a 71 16 66 RNA Artificial
Sequence Description of Artificial Sequence Synthetic Nucleic Acid
Ligand 16 gggaggacga ugcggaucag ccauguuuac gucacuccuu gucaauccuc
aucggcagac 60 gacucg 66 17 61 RNA Artificial Sequence Description
of Artificial Sequence Synthetic Nucleic Acid Ligand 17 gggaggacga
ugcggaucag ccauguuuac gucacuccuu gucaauccuc aucggcagac 60 g 61 18
56 RNA Artificial Sequence Description of Artificial Sequence
Synthetic Nucleic Acid Ligand 18 gggaggacga ugcggaucag ccauguuuac
gucacuccuu gucaauccuc aucggc 56 19 51 RNA Artificial Sequence
Description of Artificial Sequence Synthetic Nucleic Acid Ligand 19
gggaggacga ugcggaucag ccauguuuac gucacuccuu gucaauccuc a 51 20 46
RNA Artificial Sequence Description of Artificial Sequence
Synthetic Nucleic Acid Ligand 20 gggaggacga ugcggaucag ccauguuuac
gucacuccuu gucaau 46 21 41 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 21 gggaggacga
ugcggaucag ccauguuuac gucacuccuu g 41 22 71 RNA Artificial Sequence
Description of Artificial Sequence Synthetic Nucleic Acid Ligand 22
gggaggacga ugcggaucag ccauguuuac gucacuccuu gucaauccuc aucgccagac
60 gacucgcccg a 71 23 72 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 23 gggaggacga
ugcggaucag ccauguuuac gucacuccuu ggucaauccu caucggcaga 60
cgacucgccc ga 72 24 71 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 24 gggaggacga
ugcggauagc cauguuuacg ucacuccuug gucaauccuc aucgccagac 60
gacucgcccg a 71 25 71 RNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 25 gggaggacga
ugcggaucag ccauguuuac gucacuccuu gacaauccuc aucggcagac 60
gacucgcccg a 71 26 71 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 26 gggaggacga
tgcggacgac acgctcctct gattagnact aaagaccacc gtgcccagac 60
gactcgcccg a 71 27 71 DNA Artificial Sequence Description of
Artificial Sequence Synthetic Nucleic Acid Ligand 27 gggaggacga
tcggaccatc gaacagtggc taaaaaccaa gggcatcatt cgccccagac 60
gactcgcccg a 71
* * * * *