U.S. patent application number 10/705300 was filed with the patent office on 2004-04-15 for high affinity nucleic acid ligands to lectins.
Invention is credited to Bridonneau, Philippe, Gold, Lary, Hicke, Brian, Parma, David H..
Application Number | 20040072234 10/705300 |
Document ID | / |
Family ID | 27560031 |
Filed Date | 2004-04-15 |
United States Patent
Application |
20040072234 |
Kind Code |
A1 |
Parma, David H. ; et
al. |
April 15, 2004 |
High affinity nucleic acid ligands to lectins
Abstract
This invention discloses high-affinity oligonucleotide ligands
to lectins, specifically nucleic acid ligands having the ability to
bind to the lectins, wheat germ agglutinin, L-selectin, E-selectin
and P-selectin. Also disclosed are the methods for obtaining such
ligands. This invention discloses high-affinity oligonucleotide
ligands to lectins, specifically nucleic acid ligands having the
ability to bind to the lectins, wheat germ agglutinin, L-selectin,
E-selectin and P-selectin. Also disclosed are the methods for
obtaining such ligands.
Inventors: |
Parma, David H.; (Boulder,
CO) ; Hicke, Brian; (Boulder, CO) ;
Bridonneau, Philippe; (Boulder, CO) ; Gold, Lary;
(Boulder, CO) |
Correspondence
Address: |
SWANSON & BRATSCHUN L.L.C.
1745 SHEA CENTER DRIVE
SUITE 330
HIGHLANDS RANCH
CO
80129
US
|
Family ID: |
27560031 |
Appl. No.: |
10/705300 |
Filed: |
November 10, 2003 |
Related U.S. Patent Documents
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10705300 |
Nov 10, 2003 |
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10409627 |
Apr 7, 2003 |
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Apr 7, 2003 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12; 530/370 |
Current CPC
Class: |
C12Q 1/6811 20130101;
C40B 40/00 20130101; C07K 14/001 20130101; C12N 2310/53 20130101;
G01N 33/532 20130101; G01N 33/68 20130101; G01N 2333/966 20130101;
C12Q 1/6804 20130101; C07H 19/06 20130101; C12N 2310/322 20130101;
G01N 2333/4724 20130101; G01N 2333/974 20130101; C07H 21/00
20130101; C12Q 2525/101 20130101; C12Q 1/37 20130101; C12Q 1/70
20130101; C12N 15/115 20130101; C12Q 1/6811 20130101; G01N
2333/96433 20130101; G01N 33/76 20130101; G01N 33/535 20130101;
G01N 2333/8125 20130101; C12N 9/1276 20130101; C12N 2310/13
20130101; G01N 2333/575 20130101; G01N 33/53 20130101; G01N
2333/9726 20130101; G01N 2333/163 20130101; A61K 47/547 20170801;
G01N 33/56988 20130101; G01N 2333/62 20130101; G01N 2333/96455
20130101; A61K 47/549 20170801; C12N 15/1048 20130101; G01N
2333/976 20130101; B82Y 5/00 20130101; F02B 2075/027 20130101; G01N
2333/7056 20130101; G01N 33/531 20130101; C07H 19/10 20130101 |
Class at
Publication: |
435/006 ;
530/370 |
International
Class: |
C12Q 001/68; C07K
014/415 |
Claims
We claim:
1. A method for identifying nucleic acid ligands and nucleic acid
ligand sequences to a lectin comprising: a) contacting a candidate
mixture of nucleic acids with a lectin, wherein nucleic acids
having an increased affinity to said lectin 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 mixture
of nucleic acids enriched for nucleic acid sequences with
relatively higher affinity and specificity for binding to said
lectin, whereby nucleic acid ligands to said lectin may be
identified.
2. The method of claim 1 further comprising: d) repeating steps a),
b) and c).
3. The method of claim 1 wherein said candidate mixture is
comprised of single-stranded nucleic acids.
4. The method of claim 3 wherein said single-stranded nucleic acids
are ribonucleic acids.
5. The method of claim 4 wherein said nucleic acids comprise
modified ribonucleic acids.
6. The method of claim 5 wherein said nucleic acids comprise
modified ribonucleic acids selected from the group consisting of
2'-amino (2'- NH.sub.2) modified ribonucleic acids and 2'-fluoro
(2'-F) modified ribonucleic acids.
7. The method of claim 3 wherein said single-stranded nucleic acids
are deoxyribonucleic acids.
8. The method of claim 2 further comprising e) forming a
multivalent Complex comprising two nucleic acid ligands identified
in step c).
9. The method of claim 5 further comprising e) substituting
2'-O-methyl ribonucleic acids for 2'-OH ribonucleic acids in the
nucleic acid ligands identified in step c).
10. The method of claim 1 wherein said lectin is selected from the
group consisting of a mammalian lectin, a plant lectin, a microbial
lectin and a viral lectin.
11. The method of claim 1 wherein said lectin is wheat germ
agglutinin.
12. The method of claim 1 wherein said lectin is a selectin.
13. The method of claim 12 wherein said selectin is selected from
the group consisting of L-selectin, E-selectin, and P-selectin.
14. The method of claim 1 wherein said lectin is serum mannose
binding protein.
15. A purified and isolated non-naturally occurring nucleic acid
ligand to a lectin.
16. The nucleic acid ligand of claim 15 which is a non-naturally
occurring nucleic acid ligand having a specific binding affinity
for said lectin, such lectin being a three dimensional chemical
structure other than a polynucleotide that binds to said nucleic
acid ligand through a mechanism which predominantly depends on
Watson/Crick base pairing or triple helix binding, wherein said
nucleic acid ligand is not a nucleic acid having the known
physiological function of being bound by said lectin.
17. The nucleic acid ligand of claim 15 wherein said lectin is
selected from the group consisting of a mammalian lectin, a plant
lectin, a microbial lectin and a viral lectin.
18. The nucleic acid ligand of claim 15 wherein said lectin is
selected from the group consisting of wheat germ agglutinin,
L-selectin, E-selectin and P-selectin.
19. The nucleic acid ligand of claim 15 wherein said lectin is
wheat germ agglutinin.
20. The nucleic acid ligand to wheat germ agglutinin of claim 19
wherein said nucleic acid ligand is a ribonucleic acid (RNA)
ligand.
21. The nucleic acid ligand of claim 20 which comprises a modified
ribonucleic acid.
22. The nucleic acid ligand of claim 21 wherein said modified
ribonucleic acid is a 2'-amino (NH.sub.2) modified ribonucleic
acid.
23. The nucleic acid ligand to wheat germ agglutinin of claim 22
wherein said ligand is an RNA ligand selected from the group
consisting of the nucleotide sequences set forth in Table 2.
24. The nucleic acid ligand of claim 23 wherein said ligand is
selected from the group consisting of SEQ ID NOS: 455.
25. The nucleic acid ligand of claim 20 wherein said ligand
comprises sequences selected from the group consisting of SEQ ID
NOS: 5-63.
26. The nucleic acid ligand to wheat germ agglutinin of claim 19
wherein said ligand is substantially homologous to and has
substantially the same ability to bind said wheat germ agglutinin
as a ligand selected from the group consisting of the sequences set
forth in Table 2.
27. The nucleic acid ligand to wheat germ agglutinin of claim 19
wherein said ligand has substantially the same structure and the
same ability to bind said wheat germ agglutinin as a ligand
selected from the group consisting of the sequences set forth in
Table 2.
28. The nucleic acid ligand of claim 15 wherein said lectin is a
selectin.
29. The nucleic acid ligand of claim 28 wherein said selectin is
selected from the group consisting of L-selectin, E-selectin and
P-selectin.
30. The nucleic acid ligand of claim 29 wherein said selectin is
L-selectin.
31. The nucleic acid ligand to L-selectin of claim 30 wherein said
nucleic acid ligand is ribonucleic acid (RNA) ligand.
32. The nucleic acid ligand of claim 31 which comprises a modified
ribonucleic acid.
33. The nucleic acid ligand of claim 32 wherein said modified
ribonucleic acid is selected from the group consisting of a
2'-amino (2'-NH.sub.2) modified ribonucleic acid and a 2'-fluoro
(2'-F) modified ribonucleic acid.
34. The nucleic acid ligand to L-selectin of claim 33 wherein said
ligand is an RNA ligand selected from the group consisting of the
nucleotide sequences set forth in Tables 8 and 16.
35. The nucleic acid ligand of claim 34 wherein said ligand is
selected from the group consisting of SEQ ID NOS: 67-117 and
293-388.
36. The nucleic acid ligand of claim 31 wherein said ligand
comprises sequences selected from the group consisting of SEQ ID
NOS: 118-125.
37. The nucleic acid ligand to L-selectin of claim 30 wherein said
ligand is substantially homologous to and has substantially the
same ability to bind said L-selectin as a ligand selected from the
group consisting of the sequences set forth in Tables 8, 12 and
16.
38. The nucleic acid ligand to L-selectin of claim 30 wherein said
ligand has substantially the same structure and the same ability to
bind said L-selectin as a ligand selected from the group consisting
of the sequences set forth in Tables 8, 12 and 16.
39. The nucleic acid ligand to L-selectin of claim 30 wherein said
nucleic acid ligand is deoxyribonucleic acid (DNA).
40. The nucleic acid ligand to L-selectin of claim 39 wherein said
ligand is an DNA ligand selected from the group consisting of the
nucleotide sequences set forth in Table 12.
41. The nucleic acid ligand of claim 40 wherein said ligand is
selected from the group consisting of SEQ ID NOS: 129-180 and
185-196.
42. The nucleic acid ligand of claim 39 wherein said ligand
comprises sequences selected from the group consisting of SEQ ID
NOS: 181-184.
43. The nucleic acid ligand of claim 29 wherein said selectin is
P-selectin.
44. The nucleic acid ligand to P-selectin of claim 43 wherein said
nucleic acid ligand is ribonucleic acid (RNA) ligand.
45. The nucleic acid ligand of claim 44 which comprises a modified
ribonucleic acid.
46. The nucleic acid ligand of claim 45 wherein said modified
ribonucleic acid is selected from the group consisting of a
2'-amino (2'-NH.sub.2) modified ribonucleic acid, a 2'-fluoro
(2'-F) modified ribonucleic acid, and a 2'-O-Methyl (2'-O-Me)
modified ribonucleic acid.
47. The nucleic acid ligand to P-selectin of claim 46 wherein said
ligand is an RNA ligand selected from the group consisting of the
nucleotide sequences set forth in Tables 19, 21 and 25.
48. The nucleic acid ligand of claim 47 wherein said ligand is
selected from the group consisting of SEQ ID NOS: 199-219 and
236-290.
49. The nucleic acid ligand of claim 44 wherein said ligand
comprises sequences selected from the group consisting of SEQ ID
NO: 291.
50. The nucleic acid ligand to P-selectin of claim 43 wherein said
ligand is substantially homologous to and has substantially the
same ability to bind said P-selection as a ligand selected from the
group consisting of the sequences set forth in Tables 19, 21 and
25.
51. The nucleic acid ligand to P-selectin of claim 43 wherein said
ligand has substantially the same structure and the same ability to
bind said P-selectin as a ligand selected from the group consisting
of the sequences set forth in Tables 19, 21 and 25.
52. The nucleic acid ligand to P-selectin of claim 46 wherein said
nucleic acid ligand is deoxyribonucleic acid (DNA).
53. The nucleic acid ligand of claim 15 wherein said ligand is a
ribonucleic acid ligand.
54. The nucleic acid ligand of claim 53 which comprises a modified
ribonucleic acid.
55. The nucleic acid ligand of claim 54 wherein said modified
ribonucleic acid is selected from the group consisting of 2'-amino
(2'-NH.sub.2) modified ribonucleic acids, 2'-fluoro (2'-F) modified
ribonucleic acids and 2'-O-Methyl (2'-O-Me) modified ribonucleic
acids.
56. The nucleic acid ligand of claim 15 wherein said ligand is a
deoxyribonucleic acid.
57. The nucleic acid ligand of claim 15 wherein said ligand has
been further chemically modified at the sugar and/or phosphate
and/or base.
58. A multivalent Complex comprising a plurality of ligands of
claim 15.
59. A nucleic acid ligand to a lectin identified according to the
method comprising: a) contacting a candidate mixture of nucleic
acids with a lectin, wherein nucleic acids having an increased
affinity to said lectin 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 mixture of nucleic acids enriched
for nucleic acid sequences with relatively higher affinity and
specificity for binding to said lectin, whereby nucleic acid
ligands of said lectin may be identified.
60. A method for treating a lectin-mediated disease comprising
administering a pharmaceutically effective amount of a nucleic acid
ligand to a lectin.
61. The method of claim 60 wherein said nucleic acid ligand to a
lectin is identified according to the method of claim 1.
62. The method of claim 60 wherein said lectin is a selectin.
63. The method of claim 62 wherein said selectin is L-selectin.
64. The method of claim 62 wherein said selectin is P-selectin.
Description
FIELD OF THE INVENTION
[0001] Described herein are methods for identifying and preparing
high-affinity nucleic acid ligands to lectins. Lectins are
carbohydrate binding proteins. The method utilized herein for
identifying such nucleic acid ligands is called SELEX, an acronym
for Systematic Evolution of Ligands by EXponential enrichment.
Specifically disclosed herein are high-affinity nucleic acid
ligands to wheat germ agglutinin (WGA), L-selectin, E-selectin, and
P-selectin.
BACKGROUND OF THE INVENTION
[0002] The biological role of lectins (non-enzymatic
carbohydrate-binding proteins of non-immune origin; I. J. Goldstein
et al., 1980, Nature 285:66) is inextricably linked to that of
carbohydrates. One function of carbohydrates is the modification of
physical characteristics of glyco-conjugates (i.e., solubility,
stability, activity, susceptibility to enzyme or antibody
recognition), however, a more interesting and relevant aspect of
carbohydrate biology has emerged in recent years; the carbohydrate
portions of glyco-conjugates are information rich molecules (N.
Sharon and H. Lis, 1989, Science 246:227-234; K. Drickamer and M.
Taylor, 1993, Annu. Rev. Cell Biol. 9:237-264; A. Varki, 1993,
Glycobiol. 3:97-130). Within limits, the binding of carbohydrates
by lectins is specific (i.e., there are lectins that bind only
galactose or N-acetylgalactose; other lectins bind mannose; still
others bind sialic acid and so on; K. Drickamer and M. Taylor,
supra). Specificity of binding enables lectins to decode
information contained in the carbohydrate portion of
glyco-conjugates and thereby mediate many important biological
functions.
[0003] Numerous mammalian, plant, microbial and viral lectins have
been described (I. Ofek and N. Sharon, 1990, Current Topics in
Microbiol.and Immunol. 151:91-113; K. Drickamer and M. Taylor,
supra; I. J. Goldstein and R. D. Poretz, 1986, in The Lectins, p.p.
33-247; A. Varki, supra). These proteins mediate a diverse array of
biological processes which include: trafficking of lysosomal
enzymes, clearance of serum proteins, endocytosis, phagocytosis,
opsonization, microbial and viral infections, toxin binding,
fertilization, immune and inflammatory responses, cell adhesion and
migration in development and in pathological conditions such as
metastasis. Roles in symbiosis and host defense have been proposed
for plant lectins but remain controversial. While the functional
role of some lectins is well understood, that of many others is
understood poorly or not at all.
[0004] The diversity and importance of processes mediated by
lectins is illustrated by two well documented mammalian lectins,
the asialoglycoprotein receptor and the serum mannose binding
protein, and by the viral lectin, influenza virus hemagglutinin.
The hepatic asialoglycoprotein receptor specifically binds
galactose and N-acetylgalactose and thereby mediates the clearance
of serum glycoproteins that present terminal N-acetylgalactose or
galactose residues, exposed by the prior removal of a terminal
sialic acid. The human mannose-binding protein (MBP) is a serum
protein that binds terminal mannose, fucose and N-acetylglucosamine
residues. These terminal residues are common on microbes but not
mammalian glyco-conjugates. The binding specificity of MBP
constitutes a non-immune mechanism for distinguishing self from
non-self and mediates host defense through opsonization and
complement fixation. Influenza virus hemagglutinin mediates the
initial step of infection, attachment to nasal epithelial cells, by
binding sialic acid residues of cell-surface receptors.
[0005] The diversity of lectin mediated functions provides a vast
array of potential therapeutic targets for lectin antagonists. Both
lectins that bind endogenous carbohydrates and those that bind
exogenous carbohydrates are target candidates. For example,
antagonists to the mammalian selecting, a family of endogenous
carbohydrate binding lectins, may have therapeutic applications in
a variety of leukocyte-mediated disease states. Inhibition of
selectin binding to its receptor blocks cellular adhesion and
consequently may be useful in treating inflammation, coagulation,
transplant rejection, tumor metastasis, rheumatoid arthritis,
reperfusion injury, stroke, myocardial infarction, burns,
psoriasis, multiple sclerosis, bacterial sepsis, hypovolaemic and
traumatic shock, acute lung injury, and ARDS.
[0006] The selecting, E-, P- and L-, are three homologous C-type
lectins that recognize the tetrasaccharide, sialyl-Lewis.sup.X (C.
Foxall et al, 1992, J. Cell Biol. 117,895-902). Selectins mediate
the initial adhesion of neutrophils and monocytes to activated
vascular endothelium at sites of inflammation (R. S. Cotran et al.,
1986, J. Exp. Med. 164, 661-; M. A. Jutila et al., 1989, J.
Immunol. 143,3318-; J. G. Geng et al., 1990, Nature, 757; U. H. Von
Adrian et al., 1994, Am. J. Physiol. Heart Circ. Physiol. 263,
H1034-H1044). In addition, L-selectin is responsible for the homing
of lymphocytes to peripheral and mesenteric lymph nodes (W. M.
Gallatin et al., 1983, Nature 304,30; T. K. Kishimoto et al., 1990,
Proc. Natl. Acad. Sci. 87,2244-) and P-selectin mediates the
adherence of platelets to neutrophils and monocytes (S- C. Hsu-Lin
et al., 1984, J. BioL Chem. 259,9121).
[0007] Selectin antagonists (antibodies and carbohydrates) have
been shown to block the extravasation of neutrophils at sites of
inflammation (P. Piscueta and F. W. Luscinskas, 1994, Am. J.
Pathol. 145, 461-469), to be efficacious in animal models of
ischemia/reperfusion (A. S. Weyrich et al., 1993, J. Clin. Invest.
91,2620-2629; R. K. Winn et al., 1993, J. Clin. Invest. 92,
2042-2047), acute lung injury (M. S. Mulligan et al., 1993, J.
Immunol. 151, 6410-6417; A. Seekamp et al., 1994, Am. J. Pathol.
144, 592-598), insulitis/diabetes (X. D. Yang et al., 1993, Proc.
Natl. Acad. Sci. 90,10494-10498), meningitis (C. Granet et al.,
1994, J. Clin. Invest. 93, 929-936), hemorrhagic shock (R. K. Winn
et al., 1994, Am J. Physiol. Heart Circ. Physiol. 267, H2391-H2397)
and transplantation. In addition, selectin expression has been
documented in models of arthritis (F. Jamar et al., 1995, Radiology
194, 843-850), experimental allergic encephalomyelitis (J. M. Dopp
et al., 1994, J. Neuroimmunol. 54, 129-144), cutaneous inflammation
(A. Siber et al., 1994, Lab. Invest. 70, 163-170)
glomerulonephritis (P. G. Tipping et al., 1994, Kidney Int. 46,
79-88), on leukaemic cells and colon carcinomas (R. M. Lafrenie et
al., 1994, Eur. J. Cancer [A] 30A, 2151-2158) and L-selectin
receptors have been observed in myelinated regions of the central
nervous system (K. Huang et al., 1991, J. Clin. Invest. 88,
1778-1783). These animal model data strongly support the
expectation of a therapeutic role for selectin antagonists in a
wide variety of disease states in which host tissue damage is
neutrophil-mediated.
[0008] Other examples of lectins that recognize endogenous
carbohydrates are CD22.beta., CD23, CD44 and sperm lectins (A.
Varki, 1993, Glycobiol.3, 97-130; P. M. Wassarman, 1988, Ann. Rev.
Biochem. 57, 415-442). CD22.beta. is involved in early stages of B
lymphocyte activation; antagonists may modulate the immune
response. CD23 is the low affinity IgE receptor; antagonists may
modulate the IgE response in allergies and asthma. CD44 binds
hyaluronic acid and thereby mediates cell/cell and cell/matrix
adhesion; antagonists may modulate the inflammatory response. Sperm
lectins are thought to be involved in sperm/egg adhesion and in the
acrosomal response; antagonists may be effective contraceptives,
either by blocking adhesion or by inducing a premature, spermicidal
acrosomal response.
[0009] Antagonists to lectins that recognize exogenous
carbohydrates may have wide application for the prevention of
infectious diseases. Many viruses (influenza A, B and C; Sendhi,
Newcastle disease, coronavirus, rotavirus, encephalomyelitis virus,
enchephalomyocarditis virus, reovirus, paramyxovirus) use lectins
on the surface of the viral particle for attachment to cells, a
prerequisite for infection; antagonists to these lectins are
expected to prevent infection (A. Varki, 1993, Glycobiol.3,
97-130). Similarly colonization/infection strategies of many
bacteria utilize cell surface lectins to adhere to mammalian cell
surface glyco-conjugates. Antagonists to bacterial cell surface
lectins are expected to have therapeutic potential for a wide
spectrum of bacterial infections, including: gastric (Helicobacter
pylori), urinary tract (E. coli), pulmonary (Klebsiella pneumoniae,
Stretococcus pneumoniae, Mycoplasma pneumoniae) and oral
(Actinomyces naeslundi and Actinomyces viscosus)
colonization/infection (S. N. Abraham, 1994, Bacterial Adhesins, in
The Handbook of Immunopharmacology: Adhesion Molecules, C. D.
Wegner, ed; B. J. Mann et al., 1991, Proc. Natl. Acad. Sci. 88,
3248-3252). A specific bacterial mediated disease state is
Pseudomonas aeruginosa infection, the leading cause of morbidity
and mortality in cystic fibrosis patients. The expectation that
high affinity antagonists will have efficacy in treating P.
aeruginosa infection is based on three observations. First, a
bacterial cell surface, GalNAc.beta.1-4Gal binding lectin mediates
infection by adherence to asialogangliosides (.alpha.GM1 and
.alpha.GM2) of pulmonary epithelium (L. Imundo et al., 1995, Proc.
Natl. Acad. Sci 92, 3019-3023). Second, in vitro, the binding of P.
aeruginosa is competed by the gangliosides' tetrasaccharide moiety,
Gal.beta.1-3GalNAc.beta.1-4Gal.beta.1-4Glc. Third, in vivo,
instillation of antibodies to Pseudomonas surface antigens can
prevent lung and pleural damage (J. F. Pittet et al., 1993, J.
Clin. Invest. 92, 1221-1228).
[0010] Non-bacterial microbes that utilize lectins to initiate
infection include Entamoeba histalytica (a Gal specific lectin that
mediates adhesion to intestinal mucosa; W. A. Petri, Jr., 1991, AMS
News 57:299-306) and Plasmodium faciparum (a lectin specific for
the terminal Neu5Ac(a2-3)Gal of glycophorin A of erthrocytes; P. A.
Orlandi et al., 1992, J. Cell Biol. 116:901-909). Antagonists to
these lectins are potential therapeutics for dysentery and
malaria.
[0011] Toxins are another class of proteins that recognize
exogenous carbohydrates (K -A Karlsson, 1989, Ann. Rev. Biochem.
58:309-350). Toxins are complex, two domain molecules, composed of
a functional and a cell recognition/adhesion domain. The adhesion
domain is often a lectin (i.e., bacterial toxins: pertussis toxin,
cholera toxin, heat labile toxin, verotoxin and tetanus toxin;
plant toxins: ricin and abrin). Lectin antagonists are expected to
prevent these toxins from binding their target cells and
consequently to be useful as antitoxins.
[0012] There are still other conditions for which the role of
lectins is currently speculative. For example, genetic mutations
result in reduced levels of the serum mannose-binding protein
(MBP). Infants who have insufficient levels of this lectin suffer
from severe infections, but adults do not. The high frequency of
mutations in both oriental and Caucasian populations suggests a
condition may exist in which low levels of serum mannose-binding
protein are advantageous. Rheumatoid arthritis (RA) may be such a
condition. The severity of RA is correlated with an increase in IgG
antibodies lacking terminal galactose residues on Fc region
carbohydrates (A. Young et al., 1991, Arth. Rheum. 34, 1425-1429;
I. M. Roitt et al., 1988, J. Autoimm. 1, 499-506). Unlike their
normal counterpart, these gal-deficient carbohydrates are
substrates for MBP. MBP/IgG immunocomplexes may contribute to host
tissue damage through complement activation. Similarly, the
eosinophil basic protein is cytotoxic. If the cytotoxicity is
mediated by the lectin activity of this protein, then a lectin
antagonist may have therapeutic applications in treating eosinophil
mediated lung damage.
[0013] Lectin antagonists may also be useful as imaging agents or
diagnostics. For example; E-selectin antagonists may be used to
image inflamed endothelium. Similarly antagonists to specific serum
lectins, i.e. mannose-binding protein, may also be useful in
quantitating protein levels.
[0014] Lectins are often complex, multi-domain, multimeric
proteins. However, the carbohydrate-binding activity of mammalian
lectins is normally the property of a carbohydrate recognition
domain or CRD. The CRDs of mammalian lectins fall into three
phylogenetically conserved classes: C-type, S-type and P-type (K.
Drickamer and M. E. Taylor, 1993, Annu. Rev. Cell Biol. 9,
237-264). C-type lectins require Ca.sup.++ for ligand binding, are
extracellular membrane and soluble proteins and, as a class, bind a
variety of carbohydrates. S-type lectins are most active under
reducing conditions, occur both intra- and extracellularly, bind
.beta.-galactosides and do not require Ca.sup.++. P-type lectins
bind mannose 6-phosphate as their primary ligand.
[0015] Although lectin specificity is usually expressed in terms of
monosaccharides and/or oligosacchrides (i.e., MBP binds mannose,
fucose and N-acetylglucosamine), the affinity for monosaccharides
is weak. The dissociation constants for monomeric saccharides are
typically in the millimolar range (Y. C. Lee, 1992, FASEB J.
6:3193-3200; G. D. Glick et al., 1991, J Biol.Chem.
266:23660-23669; Y. Nagata and M. M. Burger, 1974, J. Biol. Chem.
249:116-3122).
[0016] Co-crystals of MBP complexed with mannose oligomers offer
insight into the molecular limitations on affinity and specificity
of C-type lectins (W. I. Weis et al., 1992, Nature 360:127-134; K.
Drickamer, 1993, Biochem. Soc. Trans. 21:456-459). The 3- and
4-hydroxyl groups of mannose form coordination bonds with bound
Ca.sup.++ ion #2 and hydrogen bonds with glutamic acid (185 and
193) and asparagine (187 and 206). The limited contacts between the
CRD and bound sugar are consistent with its spectrum of
monosaccharide binding; N-acetylglucosamine has equatorial 3- and
4-hydroxyls while fucose has similarly configured hydroxyls at the
2 and 3 positions.
[0017] The affinity of the mannose-binding protein and other
lectins for their natural ligands is greater than that for
monosaccharides. Increased specificity and affinity can be
accomplished by establishing additional contacts between a protein
and its ligand (K. Drickamer, 1993, supra) either by 1) additional
contacts with the terminal sugar (i.e., chicken hepatic lectin
binds N-acetylglucose amine with greater affinity than mannose or
fucose suggesting interaction with the 2-substituent); 2)
clustering of CRDs for binding complex oligosaccharides (i.e., the
mammalian asialylglycoprotein receptor); 3) interactions with
additional saccharide residues (i.e., the lectin domain of
selectins appears to interact with two residues of the
tetrasaccharide sialyl-Lewis.sup.X: with the charged terminal
residue, sialic acid, and with the fucose residue; wheat germ
agglutinin appears to interact with all three residues of trimers
of N-acetylglucosamine); or by 4) contacts with a non-carbohydrate
portion of a glyco-protein.
[0018] The low affinity of lectins for mono- and oligo-saccharides
presents major difficulties in developing high affinity antagonists
that may be useful therapeutics. Approaches that have been used to
develop antagonists are similar to those that occur in nature: 1)
addition or modification of substituents to increase the number of
interactions; and 2) multimerization of simple ligands.
[0019] The first approach has had limited success. For example,
homologues of sialic acid have been analyzed for affinity to
influenza virus hemagglutinin (S. J. Watowich et al. 1994,
Structure 2:719-731). The dissociation constants of the best
analogues are 30 to 300 .mu.M which is only 10 to 100-fold better
than the standard monosaccharide.
[0020] Modifications of carbohydrate ligands to the selectins have
also had limited success. In static ELISA competition assays,
sialyl-Lewis.sup.a and sialyl-Lewis.sup.X have IC.sub.50s of 220
.mu.M and 750 .mu.M, respectively, for the inhibition of the
binding of an E-selectin/IgG chimera to immobilized
sialyl-Lewis.sup.X (R. M. Nelson et al., 1993, J. Clin. Invest. 91,
1157-1166). The IC.sub.50 of a sialyl-Lewis.sup.a derivative
(addition of an aliphatic aglycone to the GlcNAc and replacement of
the N-acetyl with an NH.sub.2 group) improved 10-fold to 21 .mu.M.
Similarly, removal of the N-acetyl from sialyl-lewis.sup.X improves
inhibition in an assay dependent manner (C. Foxall et al., 1992, J.
Cell Biol. 117, 895-902; S. A. DeFrees et al., 1993, J. Am. Chem.
Soc. 115, 7549-7550).
[0021] The second approach, multimerization of simple ligands, can
lead to dramatic improvements in affinity for lectins that bind
complex carbohydrates (Y. C. Lee, supra). On the other hand, the
approach does not show great enhancement for lectins that bind
simple oligosaccharides; dimerizing sialyl-Lewis.sup.X, a minimal
carbohydrate ligand for E-selectin, improves inhibition
approximately 5-fold (S. A. DeFrees et al., supra).
[0022] An alternative approach is to design compounds that are
chemically unrelated to the natural ligand. In the static ELISA
competition assays inositol polyanions inhibit L- and P-selectin
binding with IC.sub.50s that range from 1.4 .mu.M to 2.8 mM (O.
Cecconi et al., 1994, J. Biol. Chem. 269, 15060-15066). Synthetic
oligopeptides, based on selectin amino acid sequences, inhibit
neutrophil binding to immobilized P-selectin with IC.sub.50s
ranging from 50 .mu.M to 1 mM (J -G Geng et al., 1992, J of Biol.
Chem. 267, 19846-19853).
[0023] Lectins are nearly ideal targets for isolation of
antagonists by SELEX technology described below. The reason is that
oligonucleotide ligands that are bound to the carbohydrate binding
site can be specifically eluted with the relevant sugar(s).
Oligonucleotide ligands with affinities that are several orders of
magnitude greater than that of the competing sugar can be obtained
by the appropriate manipulation of the nucleic acid ligand to
competitor ratio. Since the carbohydrate binding site is the active
site of a lectin, essentially all ligands isolated by this
procedure will be antagonists. In addition, these SELEX ligands
will exhibit much greater specificity than monomeric and oligomeric
saccharides.
[0024] A method for the in vitro evolution of nucleic acid
molecules with highly specific binding to target molecules has been
developed. This method, Systematic Evolution of Ligands by
EXponential enrichment, termed SELEX, is described in U.S. patent
application Ser. No. 07/536,428, entitled 37 Systematic Evolution
of Ligands by Exponential Enrichment," now abandoned, U.S. patent
application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled
"Nucleic Acid Ligands," now U.S. Pat. No. 5,475,096, U.S. patent
application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled
"Nucleic Acid Ligands," now U.S. Pat. No. 5,270,163 (see also
PCT/US91/04078), each of which is herein specifically incorporated
by reference. 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.
[0025] The SELEX method 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.
[0026] 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, entitled "Method for Selecting
Nucleic Acids on the Basis of Structure," describes the use of
SELEX 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"
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.
U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993,
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. patent application Ser. No. 08/143,564, filed
Oct. 25, 1993, 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. patent application Ser. No. 07/964,624, filed Oct.
21, 1992, entitled "Methods of Producing Nucleic Acid Ligands"
describes methods for obtaining improved nucleic acid ligands after
SELEX has been performed. U.S. patent application Ser. No.
08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of
Ligands by EXponential Enrichment: Chemi-SELEX," describes methods
for covalently linking a ligand to its target.
[0027] 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.
patent application Ser. No. 08/117,991, filed Sep. 8, 1993,
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. patent application Ser. No. 08/134,028, 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 2' Modified Pyrimidine Intramolecular Nucleophilic
Displacement," describes novel methods for making 2'-modified
nucleosides.
[0028] The SELEX method encompasses combining selected
oligonucleotides with other selected oligonucleotides as described
in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994,
entitled "Systematic Evolution of Ligands by Exponential
Enrichment: Chimeric SELEX". The SELEX method also includes
combining the selected nucleic acid ligands with
non-oligonucleotide functional units and U.S. patent application
Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic
Evolution of Ligands by Exponential Enrichment: Blended SELEX" and
U.S. patent application Ser. No. 08/434,465, filed May 4, 1995,
entitled "Nucleic Acid Ligand Complexes". 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.
Each of the above described patent applications which describe
modifications of the basic SELEX procedure are specifically
incorporated by reference herein in their entirety.
[0029] The present invention applies the SELEX methodology to
obtain nucleic acid ligands to lectin targets. Lectin targets, or
lectins, include all the non-enzymatic carbohydrate-binding
proteins of non-immune origin, which include, but are not limited
to, those described above.
[0030] Specifically, high affinity nucleic acid ligands to wheat
germ agglutinin, and various selectin proteins have been isolated.
For the purposes of the invention the terms wheat germ agglutinin,
wheat germ lectin and WGA are used interchangeably. Wheat germ
agglutinin (WGA) is widely used for isolation, purification and
structural studies of glyco-conjugates. As outlined above, the
selectins are important anti-inflammatory targets. Antagonists to
the selectins modulate extravasion of leukocytes at sites of
inflammation and thereby reduce neutrophil caused host tissue
damage. Using the SELEX technology, high affinity antagonists of
L-selectin, E-selectin and P-selectin mediated adhesion are
isolated.
BRIEF SUMMARY OF THE INVENTION
[0031] The present invention includes methods of identifying and
producing nucleic acid ligands to lectins and the nucleic acid
ligands so identified and produced. More particularly, nucleic acid
ligands are provided that are capable of binding specifically to
Wheat Germ Agglutinin (WGA), L-Selectin, E-selectin and
P-selectin.
[0032] Further included in this invention is a method of
identifying nucleic acid ligands and nucleic acid ligand sequences
to lectins comprising the steps of (a) preparing a candidate
mixture of nucleic acids, (b) partitioning between members of said
candidate mixture on the basis of affinity to said lectin, 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 said lectin.
[0033] More specifically, the present invention includes the
nucleic acid ligands to lectins identified according to the
above-described method, including those ligands to Wheat Germ
Agglutinin listed in Table 2, those ligands to L-selectin listed in
Tables 8, 12 and 16, and those ligands to P-selectin listed in
Tables 19 and 25. Additionally, nucleic acid ligands to E-selectin
and serum mannose binding protein are provided. Also included are
nucleic acid ligands to lectins that are substantially homologous
to any of the given ligands and that have substantially the same
ability to bind lectins and antagonize the ability of the lectin to
bind carbohydrates. Further included in this invention are nucleic
acid ligands to lectins that have substantially the same structural
form as the ligands presented herein and that have substantially
the same ability to bind lectins and antagonize the ability of the
lectin to bind carbohydrates.
[0034] The present invention also includes modified nucleotide
sequences based on the nucleic acid ligands identified herein and
mixtures of the same.
[0035] The present invention also includes the use of the nucleic
acid ligands in therapeutic, prophylactic and diagnostic
applications.
BRIEF DESCRIPTION OF THE FIGURES
[0036] FIG. 1 shows consensus hairpin secondary structures for WGA
2'-NH.sub.2 RNA ligands: (a) family 1, (b) family 2 and (c) family
3. Nucleotide sequence is in standard one letter code. Invariant
nucleotides are in bold type. Nucleotides derived from fixed
sequence are in lower case.
[0037] FIG. 2 shows binding curves for the L-selectin SELEX second
and ninth round 2'-NH.sub.2 RNA pools to peripheral blood
lymphocytes (PBMCs).
[0038] FIG. 3 shows binding curves for random 40N7 2'-NH.sub.2 RNA
(SEQ ID NO: 64) and the cloned L-selectin ligand, F14.12 (SEQ ID
NO: 78), to peripheral blood lymphocytes (PBMC).
[0039] FIG. 4 shows the results of a competition experiment in
which the binding of 5 nM .sup.32P-labeled F14.12 (SEQ ID NO: 78)
to PBMCs (10.sup.7/ml) is competed with increasing concentrations
of unlabeled F14.12 (SEQ ID NO: 78). RNA Bound equals
100.times.(net counts bound in the presence of competitor/net
counts bound in the absence of competitor).
[0040] FIG. 5 shows the results of a competition experiment in
which the binding of 5 nM .sup.32P-labeled F14.12 (SEQ ID NO: 78)
to PBMCs (10.sup.7/ml) is competed with increasing concentrations
of the blocking monoclonal anti-L-selectin antibody. DREG-56, or an
isotype matched, negative control antibody. RNA Bound equals
100.times.(net counts bound in the presence of competitor/net
counts bound in the absence of competitor).
[0041] FIG. 6 shows the results of a competitive ELISA assay in
which the binding of soluble LS-Rg to immobilized
sialyl-Lewis.sup.X/BSA conjugates is competed with increasing
concentrations of unlabeled F14.12 (SEQ ID NO: 78). Binding of
LS-Rg was monitored with an HRP conjugated anti-human IgG antibody.
LS-Rg Bound equals 100.times.(OD.sub.450 in the presence of
competitor)/(OD.sub.450 in the absence of competitor). The observed
OD.sub.450 was corrected for nonspecific binding by subtracting the
OD.sub.450 in the absence of LS-Rg from the experimental values. In
the absence of competitor the OD.sub.450 was 0.324 and in the
absence of LS-Rg 0.052. Binding of LS-Rg requires divalent cations;
in the absence of competitor, replacement of Ca.sup.++/Mg.sup.++
with 4 mM EDTA reduced the OD.sub.450 to 0.045.
[0042] FIG. 7 shows hairpin secondary structures for representative
L-selectin 2'NH.sub.2 RNA ligands: (a) F13.32 (SEQ. ID NO: 67),
family I; (b) 6.16 (SEQ. ID NO: 84), family III; and (c) F14.12
(SEQ. ID NO: 78), family II. Nucleotide sequence is in standard one
letter code. Invariant nucleotides are in bold type. Nucleotides
derived from fixed sequence are in lower case.
[0043] FIG. 8 shows a schematic representation of each dimeric and
mutimeric oligonucleotide complex: (a) dimeric branched
oligonucleotide; (b) multivalent streptavidin/bio-oligonucleotide
complex (A: streptavidin; B: biotin); (c) dimeric dumbell
oligonucleotide; (d) dimeric fork oligonucleotide.
[0044] FIG. 9 shows binding curves for the L-selectin SELEX
fifteenth round ssDNA pool to PBMCs (10.sup.7/ml).
[0045] FIG. 10 shows the results of a competition experiment in
which the binding of 2 nM .sup.32P-labeled round 15 ssDNA to PBMCs
(10.sup.7/ml) is competed with increasing concentrations of the
blocking monoclonal anti-L-selectin antibody, DREG-56, or an
isotype matched, negative control antibody. RNA Bound equals
100.times.(net counts bound in the presence of competitor/net
counts bound in the absence of competitor).
[0046] FIG. 11 shows L-selectin specific binding of LD201T1 (SEQ ID
NO: 185) to human lymphocytes and granulocytes in whole blood a,
FITC-LD201T1 binding to lymphocytes is competed by DREG-56,
unlabeled LD201T1, and inhibited by EDTA. b, FITC-LD201T1 binding
to granulocytes is competed by DREG-56, unlabeled LD201T1, and
inhibited by EDTA. All samples were stained with 0.15 mM
FITC-LD201T1; thick line: FITC-LD201T1 only; thick dashed line:
FITC-LD201T1 with 0.3 mM DREG-56; medium thick line: FITC-LD201T1
with 7 mM unlabeled NX280; thin line: FITC-LD201T1 stained cells,
reassayed after addition of 4 mM EDTA; thin dashed line:
autofluorescence.
[0047] FIG. 12 shows the consensus hairpin secondary structures for
family 1 ssDNA ligands to L-selectin. Nucleotide sequence is in
standard one letter code. Invariant nucleotides are in bold type.
The base pairs at highly variable positions are designated N--N'.
To the right of the stem is a matrix showing the number of
occurances of particular base pairs for the position in the stem
that is on the same line.
[0048] FIG. 13 shows that in vitro pre-treatment of human PBMC with
NX288 (SEQ ID NO: 193) inhibits lymphocyte trafficking to SCID
mouse PLN. Human PBMC were purified from heparinised blood by a
Ficoll-Hypaque gradient, washed twice with HBSS (calcium/magnesium
free) and labeled with .sup.51Cr (Amersham). After labeling, the
cells were washed twice with HBSS (containing calcium and
magnesium) and 1% bovine serum albumin (Sigma). Female SCID mice
(6-12 weeks of age) were injected intravenously with
2.times.10.sup.6 cells. The cells were either untreated or mixed
with either 13 pmol of antibody (DREG-56 or MEL-14), or 4, 1, or
0.4 nmol of modified oligonucleotide. One hour later the animals
were anaesthetised, a blood sample taken and the mice were
euthanised. PLN, MLN, Peyer's patches, spleen, liver, lungs,
thymus, kidneys and bone marrow were removed and the counts
incorporated into the organs determined by a Packard gamma counter.
Values shown represent the mean.+-.s.e. of triplicate samples, and
are representative of 3 experiments.
[0049] FIG. 14 shows that pre-injection of NX288 (SEQ ID NO: 193)
inhibits human lymphocyte trafficking to SCID mouse PLN and MLN.
Human PBMC were purified, labeled, and washed as described above.
Cells were prepared as described in FIG. 13. Female SCID mice (6-12
weeks of age) were injected intravenously with 2.times.10.sup.6
cells. One to 5 min prior to injecting the cells, the animals were
injected with either 15 pmol DREG-56 or 4 nmol modified
oligonucleotide. Animals were scarificed 1 hour after injection of
cells. Counts incorporated into organs were quantified as described
in FIG. 13. Values shown represent the mean.+-.s.e. of triplicate
samples, and are representative of 2 experiments.
[0050] FIG. 15 shows the consensus hairpin secondary structures for
2'-F RNA ligands to L-selectin. Nucleotide sequence is in standard
one letter code. Invariant nucleotides are in bold type. The base
pairs at highly variable positions are designated N-N'. To the
right of the stem is a matrix showing the number of occurances of
particular base pairs for the position in the stem that is on the
same line.
[0051] FIG. 16 shows the consensus hairpin secondary structures for
2'-F RNA ligands to P-selectin. Nucleotide sequence is in standard
one letter code. Invariant nucleotides are in bold type. The base
pairs at highly variable positions are designated N-N'. To the
right of the stem is a matrix showing the number of occurances of
particular base pairs for the position in the stem that is on the
same line.
DETAILED DESCRIPTION OF THE INVENTION
[0052] This application describes high-affinity nucleic acid
ligands to lectins identified through the method known as SELEX.
SELEX is described in U.S. patent application Ser. No. 07/536,428,
entitled "Systematic Evolution of Ligands by EXponential
Enrichment", now abandoned; U.S. patent application Ser. No.
07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands",
now U.S. Pat. No. 5,475,096; U.S. patent application Ser. No.
07/931,473, filed Aug. 17, 1992, entitled "Nucleic Acid Ligands",
now U.S. Pat. No. 5,270,163, (see also PCT/US91104078). These
applications, each specifically incorporated herein by reference,
are collectively called the SELEX Patent Applications.
[0053] In its most basic form, the SELEX process may be defined by
the following series of steps:
[0054] 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).
[0055] 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
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 0.05-50%) are
retained during partitioning.
[0057] 4) Those nucleic acids selected during partitioning as
having the 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.
[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 SELEX Patent Applications describe and elaborate on this
process in great detail. Included are targets that can be used in
the process; methods for partitioning nucleic acids within a
candidate mixture; and methods for amplifying partitioned nucleic
acids to generate enriched candidate mixture. The SELEX Patent
Applications also describe ligands obtained to a number of target
species, including both protein targets where the protein is and is
not a nucleic acid binding protein.
[0060] This invention also includes the ligands as described above,
wherein certain chemical modifications are made in order to
increase the in vivo stability of the ligand or to enhance or
mediate the delivery of the ligand. Examples of such modifications
include chemical substitutions at the sugar and/or phosphate and/or
base positions of a given nucleic acid sequence. See, e.g., U.S.
patent application Ser. No. 08/117,991, filed Sep. 9, 1993,
entitled "High Affinity Nucleic Acid Ligands Containing Modified
Nucleotides" which is specifically incorporated herein by
reference. Additionally, in co-pending and commonly assigned U.S.
patent application Ser. No. 07/964,624, filed Oct. 21, 1992 ('624),
now U.S. Pat. No. 5,496,938, methods are described for obtaining
improved nucleic acid ligands after SELEX has been performed. The
'624 application, entitled "Methods of Producing Nucleic Acid
Ligands," is specifically incorporated herein by reference. Further
included in the '624 patent are methods for determining the
three-dimensional structures of nucleic acid ligands. Such methods
include mathematical modeling and structure modifications of the
SELEX-derived ligands, such as chemical modification and nucleotide
substitution. Other modifications are known to one of ordinary
skill in the art. Such modifications may be made post-SELEX
(modification of previously identified unmodified ligands) or by
incorporation into the SELEX process. Additionally, the nucleic
acid ligands of the invention can be complexed with various other
compounds, including but not limited to, lipophilic compounds or
non-immunogenic, high molecular weight compounds. Lipophilic
compounds include, but are not limited to, cholesterol, dialkyl
glycerol, and diacyl glycerol. Non-immunogenic, high molecular
weight compounds include, but are not limited to, polyethylene
glycol, dextran, albumin and magnetite. The nucleic acid ligands
described herein can be complexed with a lipophilic compound (e.g.,
cholesterol) or attached to or encapsulated in a complex comprised
of lipophilic components (e.g., a liposome). The complexed nucleic
acid ligands can enhance the cellular uptake of the nucleic acid
ligands by a cell for delivery of the nucleic acid ligands to an
intracellular target. The complexed nucleic acid ligands can also
have enhanced pharmacokinetics and stability. U.S. patent
application Ser. No. 08/434,465, filed May 4, 1995, entitled
"Nucleic Acid Ligand Complexes," which is herein incorporated by
reference describes a method for preparing a therapeutic or
diagnostic complex comprised of a nucleic acid ligand and a
lipophilic compound or a non-immunogenic, high molecular weight
compound.
[0061] The methods described herein and the nucleic acid ligands
identified by such methods are useful for both therapeutic and
diagnostic purposes. Therapeutic uses include the treatment or
prevention of diseases or medical conditions in human patients.
Many of the therapeutic uses are described in the background of the
invention, particularly, nucleic acid ligands to selectins are
useful as anti-inflammatory agents. Antagonists to the selectins
modulate extravasion of leukocytes at sites of inflammation and
thereby reduce neutrophil caused host tissue damage. Diagnostic
utilization may include both in vivo or in vitro diagnostic
applications. The SELEX method generally, and the specific
adaptations of the SELEX method taught and claimed herein
specifically, are particularly suited for diagnostic applications.
SELEX identifies nucleic acid ligands that are able to bind targets
with high affinity and with surprising specificity. These
characteristics are, of course, the desired properties one skilled
in the art would seek in a diagnostic ligand.
[0062] The nucleic acid ligands of the present invention may be
routinely adapted for diagnostic purposes according to any number
of techniques employed by those skilled in the art. 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 also be able to adapt any nucleic acid ligand by
procedures known in the art to incorporate a labeling tag in order
to track the presence of such ligand. Such a tag could be used in a
number of diagnostic procedures. The nucleic acid ligands to
lectin, particularly selectins, described herein may specifically
be used for identification of the lectin proteins.
[0063] SELEX provides high affinity ligands of a target molecule.
This represents a singular achievement that is unprecedented in the
field of nucleic acids research. The present invention applies the
SELEX procedure to lectin targets. Specifically, the present
invention describes the identification of nucleic acid ligands to
Wheat Germ Agglutinin, and the selecting, specifically, L-selectin,
P-selectin and E-selectin. In the Example section below, the
experimental parameters used to isolate and identify the nucleic
acid ligands to lectins are described.
[0064] In order to produce nucleic acids desirable for use as a
pharmaceutical, it is preferred that the nucleic acid ligand (1)
binds to the target in a manner capable of achieving the desired
effect on the target; (2) be as small as possible to obtain the
desired effect; (3) be as stable as possible; and (4) be a specific
ligand to the chosen target. In most situations, it is preferred
that the nucleic acid ligand have the highest possible affinity to
the target.
[0065] In the present invention, a SELEX experiment was performed
in search of nucleic acid ligands with specific high affinity for
Wheat Germ Agglutinin from a degenerate library containing 50
random positions (50N). This invention includes the specific
nucleic acid ligands to Wheat Germ Agglutinin shown in Table 2 (SEQ
ID NOS: 4-55), identified by the methods described in Examples 1
and 2. Specifically, RNA ligands containing 2'-NH.sub.2 modified
pyrimidines are provided. The scope of the ligands covered by this
invention extends to all nucleic acid ligands of Wheat Germ
Agglutinin, modified and unmodified, identified according to the
SELEX procedure. More specifically, this invention includes nucleic
acid sequences that are substantially homologous to the ligands
shown in Table 2. By substantially homologous it is meant a degree
of primary sequence homology in excess of 70%, most preferably in
excess of 80%. A review of the sequence homologies of the ligands
of Wheat Germ Agglutinin shown in Table 2 shows that sequences with
little or no primary homology may have substantially the same
ability to bind Wheat Germ Agglutinin. For these reasons, this
invention also includes nucleic acid ligands that have
substantially the same ability to bind Wheat Germ Agglutinin as the
nucleic acid ligands shown in Table 2. Substantially the same
ability to bind Wheat Germ Agglutinin means that the affinity is
within a few 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 substantially the same ability to bind Wheat Germ
Agglutinin.
[0066] In the present invention, SELEX experiments were performed
in search of nucleic acid ligands with specific high affinity for
L-selectin from degenerate libraries containing 30 or 40 random
positions (30N or 40N). This invention includes the, specific
nucleic acid ligands to L-selectin shown in Tables 8, 12 and 16
(SEQ ID NOS: 67-117, 129-180, 185-196 and 293-388), identified by
the methods described in Examples 7, 8, 13, 14, 22 and 23.
Specifically, RNA ligands containing 2'-NH.sub.2 or 2'-F
pyrimidines and ssDNA ligands are provided. The scope of the
ligands covered by this invention extends to all nucleic acid
ligands of L-selectin, modified and unmodified, identified
according to the SELEX procedure. More specifically, this invention
includes nucleic acid sequences that are substantially homologous
to the ligands shown in Tables 8, 12 and 16. By substantially
homologous it is meant a degree of primary sequence homology in
excess of 70%, most preferably in excess of 80%. A review of the
sequence homologies of the ligands of L-selectin shown in Tables 8,
12 and 16 shows that sequences with little or no primary homology
may have substantially the same ability to bind L-selectin. For
these reasons, this invention also includes nucleic acid ligands
that have substantially the same ability to bind L-selectin as the
nucleic acid ligands shown in Tables 8, 12 and 16. Substantially
the same ability to bind L-selectin means that the affinity is
within a few 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 substantially the same ability to bind L-selectin.
[0067] In the present invention, SELEX experiments were performed
in search of nucleic acid ligands with specific high affinity for
P-selectin from degenerate libraries containing 50 random positions
(SON). This invention includes the specific nucleic acid ligands to
P-selectin shown in Tables 19 and 25 (SEQ ID NOS: 199-247 and
251-290), identified by the methods described in Examples 27, 28,
35 and 36. Specifically, RNA ligands containing 2'-NH.sub.2 and
2'-F pyrimidines are provided. The scope of the ligands covered by
this invention extends to all nucleic acid ligands of P-selectin,
modified and unmodified, identified according to the SELEX
procedure. More specifically, this invention includes nucleic acid
sequences that are substantially homologous to the ligands shown in
Tables 19 and 25. By substantially homologous it is meant a degree
of primary sequence homology in excess of 70%, most preferably in
excess of 80%. A review of the sequence homologies of the ligands
of P-selectin shown in Tables 19 and 25 shows that sequences with
little or no primary homology may have substantially the same
ability to bind P-selectin. For these reasons, this invention also
includes nucleic acid ligands that have substantially the same
ability to bind P-selectin as the nucleic acid ligands shown in
Tables 19 and 25. Substantially the same ability to bind P-selectin
means that the affinity is within a few 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 substantially the same ability to bind
P-selectin.
[0068] In the present invention, a SELEX experiment was performed
in search of nucleic acid ligands with specific high affinity for
E-selectin from a degenerate library containing 40 random positions
(40N). This invention includes specific nucleic acid ligands to
E-selectin identified by the methods described in Example 40. The
scope of the ligands covered by this invention extends to all
nucleic acid ligands of E-selectin, modified and unmodified,
identified according to the SELEX procedure.
[0069] Additionally, the present invention includes multivalent
Complexes comprising the nucleic acid ligands of the invention. The
mulivalent Complexes increase the binding energy to facilitate
better binding affinities through slower off-rates of the nucleic
acid ligands. The multivalent Complexes may be useful at lower
doses than their monomeric counterparts. In addition, high
molecular weight polyethylene glycol was included in some of the
Complexes to decrease the in vivo clearance rate of the Complexes.
Specifically, nucleic acid ligands to L-selectin were placed in
multivalent Complexes.
[0070] As described above, because of their ability to selectively
bind lectins, the nucleic acid ligands to lectins described herein
are useful as pharmaceuticals. This invention, therefore, also
includes a method for treating lectin-mediated diseases by
administration of a nucleic acid ligand capable of binding to a
lectin.
[0071] 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 nonaqueous 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.
[0072] 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.
[0073] Well established animal models exist for many of the disease
states which are candidates for selectin antagonist therapy. Models
available for testing the efficacy of oligonucleotide selectin
antagonists include:
[0074] 1) mouse models for peritoneal inflammation (P. Pizcueta and
F. W. Luscinskas, 1994, Am. J. Pathol. 145, 461-469), diabetes (A.
C. Hanninen et al., 1992, J. Clin. Invest. 92, 2509-2515),
lymphocyte trafficking (L. M. Bradley et al., 1994, J. Exp. Med.,
2401-2406), glomerulonephritis (P. G. Tipping et al., 1994, Kidney
Int. 46, 79-88), experimental allergic encephalomyelitis (J. M.
Dopp et al., 1994, J. Neuroimmunol. 54: 129-144), acute
inflammation in human/SCID mouse chimera (H. -C. Yan et al., 1994,
J. Immunol. 152, 3053-3063), endotoxin-mediated inflammation (W. E.
Sanders et al., 1992, Blood 80, 795-800);
[0075] 2) rat models for acute lung injury (M. S. Milligan et al.,
1994, J. Immunol. 152, 832-840), hind limb ischemia/reperfusion
injury (A. Seekamp et al., 1994, Am. J. Pathol 144, 592-598),
remote lung injury (A. Seekamp et al., 1994, supra; D. L. Carden et
al., 1993, J. Appl. Physiol 75, 2529-2543), neutrophil rolling on
mesenteric venules (K. Ley et al., 1993, Blood 82, 1632-1638),
myocardial infarction ischemia reperfusion injury (D. Altavilla et
al., 1994, Eur. J. Pharmacol. Environ. Toxicol. Pharmacol. 270,
45-51);
[0076] 3) rabbit models for hemorrhagic shock (R. K. Winn et al.,
1994, Am. J. Physiol. Heart Circ. Physiol. 267, H2391-H2397), ear
ischemia reperfusion injury (D. Mihelcic et al., 1994, Bollod 84,
2333-2328) neutrophil rolling on mesenteric venules (A. M. Olofsson
et al., Blood 84, 2749-2758), experimental meningitis (C. Granert
et al., 1994, J. Clin. Invest. 93, 929-936); lung, peritoneal and
subcutaneous bacterial infection (S. R. Sharer et al., 1993, J.
Immunol. 151, 4982-4988), myocardial ischemia/repefusion (G.
Montrucchio et al., 1989, Am. J. Physiol. 256, H1236-H1246),
central nervous system ischemic injury (W. M. Clark et al., 1991,
Stroke 22, 877-883);
[0077] 4) cat models for myocardial infraction ischemia reperfusion
injury (M. Buerke et al., 1994, J. Pharmacol. Exp. Ther. 271,
134-142);
[0078] 5) dog models for myocardial infarction ischemia reperfusion
injury (D. J. Lefer et al., 1994, Circulation 90, 2390-2401);
[0079] 6) pig models for arthritis (F. Jamar et al., 1995,
Radiology 194, 843-850);
[0080] 7) rhesus monkey models for cutaneous inflammation (A.
Silber et al., Lab. Invest. 70, 163-175);
[0081] 8) cynomolgus monkey models for renal transplants (S. -L.
Wee, 1991, Transplant. Prod. 23, 279-280); and
[0082] 9) baboon models for dacron grafts (T. Palabrica et al,
1992, Nature 359, 848-851), septic, traumatic and hypovolemic shock
(H. Redl et al., 1991, Am. J. Pathol. 139, 461466).
[0083] The nucleic acid ligands to lectins described herein are
useful as pharmaceuticals and as diagnostic reagents.
EXAMPLES
[0084] The following examples are illustrative of certain
embodiments of the invention and are not to be construed as
limiting the present invention in any way. Examples 1-6 describe
identification and characterization of 2'-NH.sub.2 RNA ligands to
Wheat Germ Agglutinin. Examples 7-12 described identification and
characterization of 2'-NH.sub.2 RNA ligands to L-selectin. Examples
13-21 describe identification and characterization of ssDNA ligands
to L-selectin. Examples 22-25 describe identification and
characterization of 2'-F RNA ligands to L-selectin. Example 26
describes identification of ssDNA ligands to P-selectin. Examples
27-39 describes identification and characterization of 2'-NH.sub.2
and 2'-F RNA ligands to P-selectin. Example 40 describes
identification of nucleic acid ligands to E-selectin.
Example 1
Nucleic Acid Ligands to Wheat Germ Agglutinin
[0085] The experimental procedures outlined in this Example were
used to identify and characterize nucleic acid ligands to wheat
germ agglutinin (WGA) as described in Examples 2-6.
Experimental Procedures
A) Materials
[0086] Wheat Germ Lectin (Triticum vulgare) Sepharose 6MB beads
were purchased from Pharmacia Biotech. Wheat Germ Lectin, Wheat
Germ Agglutinin, and WGA are used interchangeably herein. Free
Wheat Germ Lectin (Triticum vulgare) and all other lectins were
obtained from E Y Laboratories; methyl-.alpha.-D-mannopyranoside
was from Calbiochem and N-acetyl-D-glucosamine, GlcNAc, and the
trisaccharide N N'N'-triacetylchitotriose, (GlcNAc).sub.3, were
purchased from Sigma Chemical Co. The 2'-NH.sub.2 modified CTP and
UTP were prepared according to Pieken et. al. (1991, Science
253:314-317). DNA oligonucleotides were synthesized by Operon. All
other reagents and chemicals were purchased from commercial
sources. Unless otherwise indicated, experiments utilized Hanks'
Balanced Salt Solutions (HBSS; 1.3 mM CaCl.sub.2, 5.0 mM KCl, 0.3
mM KH.sub.2PO.sub.4, 0.5 mM MgCl.sub.2.6H.sub.2O, 0.4 mM
MgSO.sub.4.7H.sub.2O, 138 mM NaCl, 4.0 mM NaHCO.sub.3, 0.3 mM
Na.sub.2HPO4, 5.6 mM D-Glucose; GibcoBRL).
B) SELEX
[0087] The SELEX procedure is described in detail in U.S. Pat. No.
5,270,163 and elsewhere. In the wheat germ agglutinin SELEX
experiment, the DNA template for the initial RNA pool contained 50
random nucleotides, flanked by N9 5' and 3' fixed regions (50N9) 5'
gggaaaagcgaaucauacacaaga-50N-gcuccgccagagaccaaccgagaa 3' (SEQ ID
NO: 1). All C and U have 2'-NH.sub.2 substituted for 2'-OH for
ribose. The primers for the PCR were the following: 5' Primer 5'
taatacgactcactatagggaaaagcgaatcatacacaaga 3' (SEQ ID NO: 2) and 3'
Primer 5' ttctcggttggtctctggcggagc 3' (SEQ ID NO: 3). The fixed
regions of the starting random pool include DNA primer annealing
sites for PCR and cDNA synthesis as well as the consensus T7
promoter region to allow in vitro transcription. These
single-stranded DNA molecules were converted into double-stranded
transcribable templates by PCR amplification. PCR conditions were
50 mM KCl, 10 mM Tris-Cl, pH 8.3, 0.1% TritonX-100, 7.5 mM
MgCl.sub.2, 1 mM of each dATP, dCTP, dGTP, and dTTP, and 25 U/ml of
Taq DNA polymerase. Transcription reactions contained 5 mM DNA
template, 5 units/.mu.l T7 RNA polymerase, 40 mM Tris-Cl (pH 8.0),
12 mM MgCl.sub.2, 5 mM DTT, 1 mM spermidine, 0.002% Triton X-100,
4% PEG 8000, 2 mM each of 2'-OH ATP, 2'-OH GTP, 2'-NH.sub.2 CTP,
2'-NH.sub.2 UTP, and 0.31 mM .alpha.-.sup.32P 2'-OH ATP.
[0088] The strategy for partitioning WGA/RNA complexes from unbound
RNA was 1) to incubate the RNA pool with WGA immobilized on
sepharose beads; 2) to remove unbound RNA by extensive washing; and
3) to specifically elute RNA molecules bound at the carbohydrate
binding site by incubating the washed beads in buffer containing
high concentrations of (GlcNAc).sub.3. The SELEX protocol is
outlined in Table 1.
[0089] The WGA density on Wheat Germ Lectin Sepharose 6MB beads is
approximately 5 mg/ml of gel or 116 .mu.M (manufacturer's
specifications). After extensive washing in HBSS, the immobilized
WGA was incubated with RNA at room temperature for 1 to 2 hours in
a 2 ml siliconized column with constant rolling (Table 1). Unbound
RNA was removed by extensive washing with HBSS. Bound RNA was
eluted as two fractions; first, nonspecifically eluted RNA was
removed by incubating and washing with 10 mM
methyl-.alpha.-D-mannopyranoside in HBSS (Table 1; rounds 1-4) or
with HBSS (Table 1; rounds 5-11); second, specifically eluted RNA
was removed by incubating and washing with 0.5 to 10 mM
(GlcNAc).sub.3 in HBSS (Table 1). The percentage of input RNA that
was specifically eluted is recorded in Table 1.
[0090] The specifically eluted fraction was processed for use in
the following round. Fractions eluted from immobilized WGA were
heated at 90.degree. C. for 5 minutes in 1% SDS, 2%
.beta.-mercaptoethanol and extracted with phenol/chloroform. RNA
was reverse transcribed into cDNA by AMV reverse transcriptase at
48.degree. C. for 60 min in 50 mM Tris-Cl pH (8.3), 60 mM NaCl, 6
mM Mg(OAc).sub.2, 10 mM DTT, 100 pmol DNA primer, 0.4 mM each of
dNTPs, and 0.4 unit/.mu.l AMV RT. PCR amplification of this cDNA
resulted in approximately 500 pmol double-stranded DNA, transcripts
of which were used to initiate the next round of SELEX.
D) Nitrocellulose Filter Binding Assay
[0091] As described in SELEX Patent Applications, a nitrocellulose
filter partitioning method was used to determine the affinity of
RNA ligands for WGA and for other proteins. Filter discs
(nitrocellulose/cellulose acetate mixed matrix, 0.45 .mu.m pore
size, Millipore; or pure nitrocellulose, 0.45 .mu.m pore size,
Bio-Rad) were placed on a vacuum manifold and washed with 4 ml of
HBSS buffer under vacuum. Reaction mixtures, containing .sup.32P
labeled RNA pools and unlabeled WGA, were incubated in HBSS for 10
min at room temperature, filtered, and then immediately washed with
4 ml HBSS. The filters were air-dried and counted in a Beckman
LS6500 liquid scintillation counter without fluor.
[0092] WGA is a homodimer, molecular weight 43.2 kD, with 4 GlcNAc
binding sites per dimer. For affinity calculations, we assume one
RNA ligand binding site per monomer (two per dimer). The monomer
concentration is defined as 2 times the dimer concentration. The
equilibrium dissociation constant, K.sub.d, for an RNA pool or
specific ligand that binds monophasically is given by the
equation
K.sub.d=[P.sub.f][R.sub.f]/[RP]
[0093] where, [Rf]=free RNA concentration
[0094] [Pf]=free WGA monomer concentration
[0095] [RP]=concentration of RNA/WGA monomer complexes
[0096] K.sub.d=dissociation constant
[0097] A rearrangement of this equation, in which the fraction of
RNA bound at equilibrium is expressed as a function of the total
concentration of the reactants, was used to calculate Kds of
monophasic binding curves:
q=(P.sub.T+R.sub.T+K.sub.d-((P.sub.T+R.sub.T+K.sub.d).sup.2-4
P.sub.T R.sub.T).sup.1/2)
[0098] q=fraction of RNA bound
[0099] [P.sub.T=total WGA monomer concentration
[0100] [R.sub.T=total RNA concentration
[0101] K.sub.ds were determined by least square fitting of the data
points using the graphics program Kaleidagraph (Synergy Software,
Reading, Pa.).
E) Cloning and Sequencing
[0102] The sixth and eleventh round PCR products were re-amplified
with primers which contain a BamH1 or a EcoR1 restriction
endonuclease recognition site. Using these restriction sites the
DNA sequences were inserted directionally into the pUC18 vector.
These recombinant plasmids were transformed into E. coli strain
JM109 (Stratagene, La Jolla, Calif.). Plasmid DNA was prepared
according to the alkaline hydrolysis method (Zhou et al., 1990
Biotechniques 8:172-173) and about 72 clones were sequenced using
the Sequenase protocol (United States Biochemical Corporation,
Cleveland, Ohio). The sequences are provided in Table 2.
F) Competitive Binding Studies
[0103] Competitive binding experiments were performed to determine
if RNA ligands and (GlcNAc).sub.3 bind the same site on WGA. A set
of reaction mixtures containing .alpha..sup.32P labeled RNA ligand
and unlabeled WGA, each at a fixed concentration (Table 5), was
incubated in HBSS for 15 min at room temperature with
(GlcNAc).sub.3. Individual reaction mixtures were then incubated
with a (GlcNAc).sub.3 dilution from a 2-fold dilution series for 15
minutes. The final (GlcNAc).sub.3 concentrations ranged from 7.8
.mu.M to 8.0 mM (Table 5). The reaction mixtures were filtered,
processed and counted as described in "Nitrocellulose Filter
Binding Assay," paragraph D above.
[0104] Competition titration experiments were analyzed by the
following equation to determine the concentration of free protein
[P] as a function of the total concentration of competitor added,
[C.sub.T]:
0=[P](1+K.sub.L[L.sub.T]/(1+K.sub.L[P])+K.sub.C[C.sub.T]/(1+K.sub.C[P]))-P-
.sub.T
[0105] where L.sub.T is the concentration of initial ligand,
K.sub.L is the binding constant of species L to the protein
(assuming 1:1 stiochiometry) and Kc is the binding constant of
species C to the protein (assuming 1:1 stiochiometry). Since it is
difficult to obtain a direct solution for equation 1, iteration to
determine values of [P] to a precision of 1.times.10.sup.-15 was
used. Using these values of [P], the concentration of
protein-ligand complex [PL] as a function of [C.sub.T] was
determined by the following equation:
[P.sub.L]=K.sub.L[L.sub.T][P](1+K.sub.L[P])
[0106] Since the experimental data is expressed in terms of % [PL],
the calculated concentration of [PL] was normalized by the initial
concentration of [PLo] before addition of the competitor. ([PLo]
was calculated using the quadratic solution for the standard
binding equation for the conditions used. The maximum (M) and
minimum (B) % [PL] was allowed to float during the analysis as
shown in the following equation.
% [P.sub.L]=[PL]/[PLo]*(M-B)+B
[0107] A non-linear least-squares fitting procedure was used as
described by P. R. Bevington (1969) Data Reduction and Error
Analysis for the Physical Sciences, McGraw-Hill publishers. The
program used was originally written by Stanley J. Gill in MatLab
and modified for competition analysis by Stanley C. Gill. The data
were fit to equations 1-3 to obtain best fit parameters for
K.sub.C, M and B as a function of [C.sub.T] while leaving K.sub.L
and P.sub.T fixed.
G) Inhibition of WGA Agglutinating Activity
[0108] Agglutination is a readily observed consequence of the
interaction of a lectin with cells and requires that individual
lectin molecules crosslink two or more cells. Lectin mediated
agglutination can be inhibited by sugars with appropriate
specificity. Visual assay of the hemagglutinating activity of WGA
and the inhibitory activity of RNA ligands, GlcNAc and
(GlcNAc).sub.3 was made in Falcon round bottom 96 well microtiter
plates, using sheep erythrocytes. Each well contained 54 .mu.l of
erythrocytes (2.5.times.10.sup.8 cells/ml) and 54 .mu.l of test
solution.
[0109] To titrate WGA agglutinating activity, each test solution
contained a WGA dilution from a 4-fold dilution series. The final
WGA concentrations ranged from 0.1 .mu.M to 0.5 .mu.M. For
inhibition assays, the test solutions contained 80 nM WGA (monomer)
and a dilution from a 4-fold dilution series of the designated
inhibitor. Reaction mixtures were incubated at room temperature for
2 hours, after which time no changes were observed in the
precipitation patterns of erythrocytes. These experiments were
carried out in Gelatin Veronal Buffer (0.15 nM CaCl.sub.2, 141 mM
NaCl, 0.5 mM MgCl.sub.2, 0.1% gelatin, 1.8 mM sodium barbital, and
3.1 mM barbituric acid, pH 7.3-7.4; Sigma #G-6514).
[0110] In the absence of agglutination, erythrocytes settle as a
compact pellet. Agglutinated cells form a more diffuse pellet.
Consequently, in visual tests, the diameter of the pellet is
diagnostic for agglutination. The inhibition experiments included
positive and negative controls for agglutination and appropriate
controls to show that the inhibitors alone did not alter the normal
precipitation pattern.
Example 2
RNA Ligands to WGA
A. SELEX
[0111] The starting RNA library for SELEX, randomized 50N9 (SEQ ID
NO: 1), contained approximately 2.times.10.sup.15 molecules (2 nmol
RNA). The SELEX protocol is outlined in Table 1. Binding of
randomized RNA to WGA is undetectable at 36 .mu.M WGA monomer. The
dissociation constant of this interaction is estimated to be >4
mM.
[0112] The percentage of input RNA eluted by (GlcNAc).sub.3
increased from 0.05% in the first round, to 28.5% in round 5 (Table
1). The bulk K.sub.d of round 5 RNA was 600 nM (Table 1). Since an
additional increase in specifically eluted RNA was not observed in
round 6a (Table 1), round 6 was repeated (Table 1, round 6b) with
two modifications to increase the stringency of selection: the
volume of gel, and hence the mass of WGA, was reduced ten fold; and
RNA was specifically eluted with increasing concentrations of
(GlcNAc)3, in stepwise fashion, with only the last eluted RNA
processed for the following round. The percentage of specifically
eluted RNA increased from 5.7% in round 6b to 21.4% in round 8,
with continued improvement in the bulk Kd (260 nM, round 8 RNA,
Table 1).
[0113] For rounds 9 through 11 the WGA mass was again reduced ten
fold to further increase stringency. The K.sub.d of round 11 RNA
was 68 nM. Sequencing of the bulk starting RNA pool and sixth and
eleventh round RNA revealed some nonrandomness in the variable
region at the sixth round and increased nonrandomess at round
eleven.
[0114] To monitor the progess of SELEX, ligands were cloned and
sequenced from round 6b and round 11. From each of the two rounds,
36 randomly picked clones were sequenced. Sequences were aligned
manually and are shown in Table 2.
B. RNA Sequences
[0115] From the sixth and eleventh rounds, respectively, 27 of 29
and 21 of 35 sequenced ligands were unique. The number before the
"." in the ligand name indicates whether it was cloned from the
round 6 or round 11 pool. Only a portion of the entire clone is
shown in Table 2 (SEQ ID NOS: 4-55). The entire evolved random
region is shown in upper case letters. Any portion of the fixed
region is shown in lower case letters. By definition, each clone
includes both the evolved sequence and the associated fixed region,
unless specifically stated otherwise. A unique sequence is
operationally defined as one that differs from all others by three
or more nucleotides. In Table 2, ligands sequences are shown in
standard single letter code (Cornish-Bowden, 1985 NAR 13:
3021-3030). Sequences that were isolated more than once are
indicated by the parenthetical number, (n), following the ligand
isolate number. These clones fall into nine sequence families (1-9)
and a group of unrelated sequences (Orphans).
[0116] The distribution of families from round six to eleven
provides a clear illustration of the appearance and disappearance
of ligand families in response to increased selective pressure
(Table 2). Family 3, predominant (11/29 ligands) in round 6, has
nearly disappeared (2/35) by round 11. Similarly, minor families 6
through 9 virtually disappear. In contrast, only one (family 1) of
round eleven's predominant families (1, 2, 4 and 5) was detected in
round six. The appearance and disappearance of families roughly
correlates with their binding affinities.
[0117] Alignment (Table 2) defines consensus sequences for families
14 and 6-9 (SEQ ID NOS: 56-63). The consensus sequences of families
1-3 are long (20, 16 and 16, respectively) and very highly
conserved. The consensus sequences of families 1 and 2 contain two
sequences in common: the trinucleotide TCG and the pentanucleotide
ACGAA. A related tetranucleotide, AACG, occurs in family 3. The
variation in position of the consensus sequences within the
variable regions indicates that the ligands do not require a
specific sequence from either the 5' or 3' fixed region.
[0118] The consensus sequences of family 1 and 2 are flanked by
complementary sequences 5 or more nucleotides in length. These
complementary sequences are not conserved and the majority include
minor discontinuities. Family 3 also exhibits flanking
complementary sequences, but these are more variable in length and
structure and utilize two nucleotide pairs of conserved
sequence.
[0119] Confidence in the family 4 consensus sequence (Table 2) is
limited by the small number of ligands, the variability of spacing
and the high G content. The pentanucleotide, RCTGG, also occurs in
families 5 and 8. Ligands of family 5 show other sequence
similarities to those of family 4, especially to ligand 11.28.
C. Affinities
[0120] The dissociation constants for representative members of
families 1-9 and orphan ligands were determined by nitrocellulose
filter binding experiments and are listed in Table 3. These
calculations assume one RNA ligand binding site per WGA monomer. At
the highest WGA concentration tested (36 .mu.M WGA monomer),
binding of random RNA is not observed, indicating a K.sub.d at
least 100-fold higher than the protein concentration or >4
mM.
[0121] The data in Table 3 define several characteristics of ligand
binding. First, RNA ligands to WGA bind monophasically. Second, the
range of measured dissociation constants is 1.4 nM to 840 nM.
Third, the binding for a number of ligands, most of which were
sixth round isolates, was less than 5% at the highest WGA
concentration tested. The dissociation constants of these ligands
are estimated to be greater than 20 .mu.M. Fourth, on average
eleventh round isolates have higher affinity than those from the
sixth round. Fifth, the SELEX probably was not taken to completion;
the best ligand (11.20)(SEQ ID NO: 40) is not the dominant species.
Since the SELEX was arbitrarily stopped at the 11th round, it is
not clear that 11.20 would be the ultimate winner. Sixth, even
though the SELEX was not taken to completion, as expected, RNA
ligands were isolated that bind WGA with much greater affinity than
do mono- or oligosaccharides (ie., the affinity of 11.20 is
5.times.10.sup.5 greater than that of GlcNAc, Kd=760 .mu.M, and 850
better than that of (GlcNAc).sub.3, Kd=12 .mu.M; Y. Nagata and M.
Burger, 1974, supra). This observation validates the proposition
that competitive elution allows the isolation of oligonucleotide
ligands with affinities that are several orders of magnitude
greater than that of the competing sugar.
[0122] In addition these data show that even under conditions of
high target density, 116 pmol WGA dimer/.mu.l of beads, it is
possible to overcome avidity problems and recover ligands with
nanomolar affinities. From the sixth to the eleventh round (Table
2), in response to increased selective pressure as indicated by the
improvement in bulk K.sub.d (Table 1), sequence families with lower
than average affinity (Table 3) are eliminated from the pool.
Example 3
Specificity of RNA Ligands to WGA
[0123] The affinity of WGA ligands 6.8, 11.20 and 11.24 (SEQ ID
NOS: 13, 40, and 19) for GlcNAc binding lectins from Ulex
europaeus, Datura stramonium and Canavalia ensiformis were
determined by nitrocellulose partitioning. The results of this
determination are shown in Table 4. The ligands are highly specific
for WGA. For example, the affinity of ligand 11.20 for WGA is
1,500, 8,000 and >15,000 fold greater than it is for the U.
europaeus, D. stramonium and C. ensiformis lectins, respectively.
The 8,000 fold difference in affinity for ligand 11.20 exhibited by
T. vulgare and D. stramonium compares to a 3 to 10 fold difference
in their affinity for oligomers of GlcNAc and validates the
proposition that competitive elution allows selection of
oligonucleotide ligands with much greater specificity than
monomeric and oligomeric saccharides (J. F. Crowley et al., 1984,
Arch. Biochem. and Biophys. 231:524-533; Y. Nagata and M. Burger,
1974, supra; J -P. Privat et al., FEBS Letters 46:229-232).
Example 4
Competitive Binding Studies
[0124] If an RNA ligand and a carbohydrate bind a common site, then
binding of the RNA ligand is expected to be competitively inhibited
by the carbohydrate. Furthermore, if the oligonucleotide ligands
bind exclusively to carbohydrate binding sites, inhibition is
expected to be complete at high carbohydrate concentrations. In the
experiments reported in Table 5, dilutions of unlabeled
(GlcNAc).sub.3, from a 2-fold dilution series, were added to three
sets of binding reactions that contained WGA and an .alpha..sup.32P
labeled RNA ligand (6.8, 11.20 or 11.24 (SEQ ID NOS: 13, 40 and
19); [RNA] final=[WGA]final=15 nM). After a 15 minute incubation at
room temperature, the reactions were filtered and processed as in
standard binding experiments.
[0125] Qualitatively, it is clear that RNA ligands bind only to
sites at which (GlcNAc).sub.3 binds, since inhibition is complete
at high (GlcNAc).sub.3 concentrations (Table 5). These data do not
rule out the possibility that (GlcNAc).sub.3 binds one or more
sites that are not bound by these RNA ligands.
[0126] Quantitatively, these data fit a simple model of competitive
inhibition (Table 5) and give estimates of 8.4, 10.9 and 19.4 .mu.M
for the Kd of (GlcNAc).sub.3. These estimates are in good agreement
with literature values (12 .mu.M@4 C, Nagata and Burger, 1974,
supra; 11 .mu.M@10.8 C, Van Landschoot et al., 1977, Eur. J.
Biochem. 79:275-283; 50 .mu.M, M. Monsigny et al., 1979, Eur J.
Biochem. 98:39-45). These data confirm the proposition that
competitive elution with a specific carbohydrate targets the
lectin's carbohydrate binding site.
Example 5
Inhibition of WGA Agglutinating Activity
[0127] At 0.5 .mu.M, RNA ligands 6.8 and 11.20 (SEQ ID NO: 13 and
40) completely inhibit WGA mediated agglutination of sheep
erythrocytes (Table 6). Ligand 11.24 (SEQ ID NO: 19) is not as
effective, showing only partial inhibition at 2 .mu.M, the highest
concentration tested (Table 6). (GlcNAc)3 and GlcNAc completely
inhibit agglutination at higher concentrations, 8 .mu.M and 800
.mu.M, respectively, (Table 6; Monsigny et al., supra). The
inhibition of agglutination varifies the proposition that ligands
isolated by this procedure will be antagonists of lectin function.
Inhibition also suggests that more than one RNA ligand is bound per
WGA dimer, since agglutination is a function of multiple
carbohydrate binding sites.
[0128] An alternative interpretation for the inhibition of
agglutination is that charge repulsion prevents negatively charged
WGA/RNA complexes from binding carbohydrates (a necessary condition
for agglutination) on negatively charged cell surfaces. This
explanation seems unlikely for two reasons. First, negatively
charged oligonucleotide ligands selected against an immobilized
purified protein are known to bind to the protein when it is
presented in the context of a cell surface (see Example 10,
L-selectin cell binding). Second, negatively charged (pI=4)
succinylated WGA is as effective as native WGA (pI=8.5) in
agglutinating erythrocytes (M. Monsigny et al., supra).
Example 6
Secondary Structure of High Affinity WGA Ligands
[0129] In favorable instances, comparative analysis of aligned
sequences allows deduction of secondary structure and
structure-function relationships. If the nucleotides at two
positions in a sequence covary according to Watson-Crick base
pairing rules, then the nucleotides at these positions are apt to
be paired. Nonconserved sequences, especially those that vary in
length are not apt to be directly involved in function, while
highly conserved sequence are likely to be directly involved.
[0130] Comparative analyses of both family 1 and 2 sequences each
yield a hairpin structure with a large highly conserved loop (FIGS.
1a and 1b). Interactions between loop nucleotides are likely but
they are not defined by these data. The stems of individual ligands
vary in sequence, length and structure (i.e., a variety of bulges
and internal loops are allowed; Table 2). Qualitatively it is clear
that the stems are validated by Watson/Crick covariation and that
by the rules of comparative analysis the stems are not directly
involved in binding WGA. Family 3 can form a similar hairpin in
which 2 pairs of conserved nucleotides are utilized in the stem
(FIG. 1c).
[0131] If it is not possible to fold the ligands of a sequence
family into homologous structures, their assignment to a single
family is questionable. Both ligand 11.7, the dominant member of
family 4, and ligand 11.28 can be folded into two plane G-quartets.
However, this assignment is speculative: 1) 11.28 contains five GG
dinucleotides and one GGGG tetranucleotide allowing other
G-quartets; and 2) ligands 11.2 and 11.33 cannot form G-quartets.
On the other hand, all ligands can form a hairpin with the
conserved sequence GAGRFTNCRT in the loop. However, the conserved
sequence RCTGGC (Table 2) does not have a consistent role in these
hairpins.
[0132] Multiple G-quartet structures are possible for Family 5. One
of these resembles the ligand 11.7 G-quartet. No convincing hairpin
structures are possible for ligand 11.20.
Example 7
2'-NH.sub.9 RNA Ligands to Human L-Selectin
[0133] The experimental procedures outlined in this Example were
used to identify and characterize the 2'-NH.sub.2 RNA ligands to
human L-selectin in Examples 8-12.
Experimental Procedures
A) Materials
[0134] LS-Rg is a chimeric protein in which the extracellular
domain of human L-selectin is joined to the Fc domain of a human G2
immunoglobulin (Norgard et al., 1993, PNAS 90:1068-1072). ES-Rg,
PS-Rg and CD22.beta.-Rg are analogous constructs of E-selectin,
P-selectin and CD22.beta. joined to a human G1 immunoglobulin Fc
domain (R. M. Nelson et al., 1993, supra; I. Stamenkovic et al.,
1991, Cell 66, 1133-1144). Purified chimera were provided by A.
Varki. Soluble P-selectin was purchased from R&D Systems.
Protein A Sepharose 4 Fast Flow beads were purchased from Pharmacia
Biotech. Anti-L-selectin monoclonal antibodies: SK11 was obtained
from Becton-Dickinson, San Jose, Calif.; DREG-56, an L-selectin
specific monoclonal antibody, was purchased from Endogen,
Cambridge, Mass. The 2'-NH.sub.2 modified CTP and UTP were prepared
according to Pieken et. al. (1991, Science 253:314-317). DNA
oligonucleotides were synthesized by Operon. All other reagents and
chemicals were purchased from commercial sources. Unless otherwise
indicated, experiments utilized HSMC buffer (1 mM CaCl.sub.2, 1 mM
MgCl.sub.2, 150 mM NaCl, 20.0 mM HEPES, pH 7.4).
B) SELEX
[0135] The SELEX procedure is described in detail in U.S. Pat. No.
5,270,163 and elsewhere. The nucleotide sequence of the synthetic
DNA template for the LS-Rg SELEX was randomized at 40 positions.
This variable region was flanked by N7 5' and 3' fixed regions
(40N7). 40N7 transcript has the sequence 5'
gggaggacgaugcgg-40N-cagacgacucgcccga 3' (SEQ ID NO: 64). All C and
U have 2'-NH.sub.2 substituted for 2'-OH on the ribose. The primers
for the PCR were the following:
[0136] N7 5' Primer 5' taatacgactcactatagggaggacgatgcgg 3' (SEQ ID
NO: 65)
[0137] N7 3' Primer 5' tcgggcgagtcgtcctg 3' (SEQ ID NO: 66)
[0138] The fixed regions include primer annealing sites for PCR and
cDNA synthesis as well as a consensus T7 promoter to allow in vitro
transcription. The initial RNA pool was made by first Klenow
extending 1 nmol of synthetic single stranded DNA and then
transcribing the resulting double stranded molecules with T7 RNA
polymerase. Klenow extension conditions: 3.5 nmols primer 5N7, 1.4
nmols 40N7, 1.times. Klenow Buffer, 0.4 mM each of dATP, dCTP, dGTP
and dTTP in a reaction volume of 1 ml.
[0139] For subsequent rounds, eluted RNA was the template for AMV
reverse transcriptase mediated synthesis of single-stranded cDNA.
These single-stranded DNA molecules were converted into
double-stranded transcription templates by PCR amplification. PCR
conditions were 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 7.5 mM
MgCl.sub.2, 1 mM of each dATP, dCTP, dGTP, and dTTP, and 25 U/ml of
Taq DNA polymerase. Transcription reactions contained 0.5 mM DNA
template, 200 nM T7 RNA polymerase, 80 mM HEPES (pH 8.0), 12 mM
MgCl.sub.2, 5 mM DTT, 2 mM spermidine, 2 mM each of 2'-OH ATP,
2'-OH GTP, 2'-NH.sub.2 CTP, 2'-NH.sub.2 UTP, and 250 nM
.alpha.-.sup.32P 2'-OH ATP.
[0140] The strategy for partitioning LS-Rg/RNA complexes from
unbound RNA is outlined in Tables 7a and 7b. First, the RNA pool
was incubated with LS-Rg immobilized on protein A sepharose beads
in HSMC buffer. Second, the unbound RNA was removed by extensive
washing. Third, the RNA molecules bound at the carbohydrate binding
site were specifically eluted by incubating the washed beads in
HMSC buffer containing 5 mM EDTA in place of divalent cations. The
5 mM elution was followed by a non-specific 50 mM EDTA elution.
LS-Rg was coupled to protein A sepharose beads according to the
manufacturer's instructions (Pharmacia Biotech).
[0141] The 5 mM EDTA elution is a variation of a specific site
elution strategy. Although it is not a priori as specific as
elution by carbohydrate competition, it is a general strategy for
C-type (calcium dependent binding) lectins and is a practical
alternative when the cost and/or concentration of the required
carbohydrate competitor is unreasonable (as is the case with
sialyl-Lewis.sup.X). This scheme is expected to be fairly specific
for ligands that form bonds with the lectin's bound Ca.sup.++
because the low EDTA concentration does not appreciably increase
the buffer's ionic strength and the conformation of C-type lectins
is only subtly altered in the absence of bound calcium (unpublished
observations cited by K. Drickamer, 1993, Biochem. Soc. Trans.
21:456-459).
[0142] In the initial SELEX rounds, which were performed at
4.degree. C., the density of immobilized LS-Rg was 16.7 pmols/.mu.l
of Protein A Sepharose 4 Fast Flow beads. In later rounds, the
density of LS-Rg was reduced (Tables 7a and 7b), as needed, to
increase the stringency of selection. At the seventh round, the
SELEX was branched and continued in parallel at 4.degree. C. (Table
7a) and at room temperature (Table 7b). Wash and elution buffers
were equilibrated to the relevant incubation temperature. Beginning
with the fifth round, SELEX was often done at more than one LS-Rg
density. In each branch, the eluted material from only one LS-Rg
density was carried forward.
[0143] Before each round, RNA was batch adsorbed to 100 .mu.l of
protein A sepharose beads for 1 hour in a 2 ml siliconized column.
Unbound RNA and RNA eluted with minimal washing (two volumes) were
combined and used for SELEX input material. For SELEX, extensively
washed, immobilized LS-Rg was batch incubated with pre-adsorbed RNA
for 1 to 2 hours in a 2 ml siliconized column with constant
rocking. Unbound RNA was removed by extensive batch washing (200 to
500 .mu.l HSMC/wash). Bound RNA was eluted as two fractions; first,
bound RNA was eluted by incubating and washing columns with 5 mM
EDTA in HSMC without divalent cations; second the remaining
elutable RNA was removed by incubating and/or washing with 50 mM
EDTA in HSMC without divalents. The percentage of input RNA that
was eluted is recorded in Tables 7a and 7b. In every round, an
equal volume of protein A sepharose beads without LS-Rg was treated
identically to the SELEX beads to determine background binding. All
unadsorbed, wash and eluted fractions were counted in a Beckman
LS6500 scintillation counter in order to monitor each round of
SELEX.
[0144] The eluted fractions were processed for use in the following
round (Tables 7a and 7b). After extracting with phenol/chloroform
and precipitating with isopropanol/ethanol (1:1, v/v), the RNA was
reverse transcribed into cDNA by AMV reverse transcriptase either
1) at 48.degree. C. for 15 minutes and then 65.degree. C. for 15
minutes or 2) at 37.degree. C. and 48.degree. C. for 15 minutes
each, in 50 mM Tris-Cl pH (8.3), 60 mM NaCl, 6 mM Mg(OAc).sub.2, 10
mM DTT, 100 pmol DNA primer, 0.4 mM each of dNTPs, and 0.4
unit/.mu.l AMV RT. Transcripts of the PCR product were used to
initiate the next round of SELEX.
C) Nitrocellulose Filter Binding Assay
[0145] As described in SELEX Patent Applications, a nitrocellulose
filter partitioning method was used to determine the affinity of
RNA ligands for LS-Rg and for other proteins. Filter discs
(nitrocellulose/cellulose acetate mixed matrix, 0.45 .mu.m pore
size, Millipore) were placed on a vacuum manifold and washed with 2
ml of HSMC buffer under vacuum. Reaction mixtures, containing
.sup.32P labeled RNA pools and unlabeled LS-Rg, were incubated in
HSMC for 10-20 min at 4.degree. C., room temperature or 37.degree.
C., filtered, and then immediately washed with 4 ml HSMC at the
same temperature. The filters were air-dried and counted in a
Beckman LS6500 liquid scintillation counter without fluor.
[0146] LS-Rg is a dimeric protein that is the expression product of
a recombinant gene constructed by fusing the DNA sequence that
encodes the extracellular domains of human L-selectin to the DNA
that encodes a human IgG.sub.2 Fc region. For affinity
calculations, we assume one RNA ligand binding site per LS-Rg
monomer (two per dimer). The monomer concentration is defined as 2
times the LS-Rg dimer concentration. The equilibrium dissociation
constant, K.sub.d, for an RNA pool or specific ligand that binds
monophasically is given by the equation
Kd=[Pf][Rf]/[RP]
[0147] where, [Rf]=free RNA concentration
[0148] [Pf]=free LS-Rg monomer concentration
[0149] [RP]=concentration of RNA/LS-Rg complexes
[0150] Kd=dissociation constant
[0151] A rearrangement of this equation, in which the fraction of
RNA bound at equilibrium is expressed as a function of the total
concentration of the reactants, was used to calculate Kds of
monophasic binding curves:
q=(P.sub.T+R.sub.T+K.sub.d-((P.sub.T+R.sub.T+K.sub.d).sup.2-4
P.sub.T R.sub.T).sup.1/2)
[0152] q=fraction of RNA bound
[0153] [P.sub.T]=2.times.(total LS-Rg concentration)
[0154] [R.sub.T]=total RNA concentration
[0155] Many ligands and evolved RNA pools yield biphasic binding
curves. Biphasic binding can be described as the binding of two
affinity species that are not in equilibrium. Biphasic binding data
were evaluated with the equation
q=2P.sub.t+R.sub.t+Kd.sub.1+Kd.sub.2-[(P.sub.t+X.sub.1R.sub.1+K.sub.d1).su-
p.2-4P.sub.tX.sub.1R.sub.t].sup.1/2
-[(P.sub.t+X.sub.2R.sub.t+K.sub.d2).su-
p.2-4P.sub.tX.sub.2R.sub.t].sup.1/2,
[0156] where X.sub.1 and X.sub.2 are the mole fractions of affinity
species R.sub.1 and R.sub.2 and K.sub.d1 and K.sub.d2 are the
corresponding dissociation constants. K.sub.ds were determined by
least square fitting K.sub.ds were determined by least square
fitting of the data points using the graphics program Kaleidagraph
(Synergy Software, Reading, Pa.).
D) Cloning and Sequencing
[0157] Sixth, thirteenth (RT) and fourteenth (4.degree. C.) round
PCR products were re-amplified with primers which contain either a
BamHI or a HinDIII restriction endonuclease recognition site. Using
these restriction sites, the DNA sequences were inserted
directionally into the pUC9 vector. These recombinant plasmids were
transformed into E. coli strain DH5a (Life Technologies,
Gaithersburg, Md.). Plasmid DNA was prepared according to the
alkaline hydrolysis method (PERFECTprep, 5'-3', Boulder, Colo.).
Approximately 150 clones were sequenced using the Sequenase
protocol (Amersham, Arlington Heights, Ill.). The resulting ligand
sequences are shown in Table 8.
E) Cell Binding Studies
[0158] The ability of evolved ligand pools and cloned ligands to
bind to L-selectin presented in the context of a cell surface was
tested in experiments with isolated human peripheral blood
mononuclear cells (PBMCs). Whole blood, collected from normal
volunteers, was anticoagulated with 5 mM EDTA. Six milliliters of
blood were layered on a 6 ml Histopaque gradient in 15 ml
polypropylene tube and centrifuged (700 g) at room temperature for
30 minutes. The mononuclear cell layer was collected, diluted in 10
ml of Ca.sup.++/Mg.sup.++-free DPBS (DPBS(-); Gibco 14190-029) and
centrifuged (225 g) for 10 minutes at room temperature. Cell
pellets from two gradients were combined, resuspended in 10 ml of
DPBS(-) and recentrifuged as described above. These pellets were
resuspended in 100 .mu.l of SMHCK buffer supplemented with 1% BSA.
Cells were counted in a hemocytometer, diluted to 2.times.10.sup.7
cells/ml in SMHCK/1% BSA and immediately added to binding assays.
Cell viability was monitored by trypan blue exclusion.
[0159] For cell binding assays, a constant number of cells were
titrated with increasing concentrations of radiolabeled ligand. The
test ligands were serially diluted in DPBS(-)/1% BSA to 2-times the
desired final concentration approximately 10 minutes before use.
Equal volumes (25 .mu.l) of each ligand dilution and the cell
suspension (2.times.10.sup.7 cells/ml) were added to 0.65 ml
eppendorf tubes, gently vortexed and incubated on ice for 30
minutes. At 15 minutes the tubes were revortexed. The ligand/PBMC
suspension was layered over 50 .mu.l of ice cold phthalate oil
(1:1=dinonyl:dibutyl phthalate) and microfuged (14,000 g) for 5
minutes at 4.degree. C. Tubes were frozen in dry ice/ethanol,
visible pellets amputated into scintillation vials and counted in
Beckman LS6500 scintilation counter as described in Example 7,
paragraph C.
[0160] The specificity of binding to PBMCs was tested by
competition with the L-selectin specific blocking monoclonal
antibody, DREG-56, while saturability of binding was tested by
competition with unlabeled RNA. Experimental procedure and
conditions were like those for PBMC binding experiments, except
that the radiolabeled RNA ligand (final concentration 5 nM) was
added to serial dilutions of the competitor before mixing with
PBMCs.
F) Inhibition of Selectin Binding to sialyl-Lewis.sup.X
[0161] The ability of evolved RNA pools or cloned ligands to
inhibit the binding of LS-Rg to sialyl-Lewis.sup.X was tested in
competive ELISA assays (C. Foxall et al., 1992, supra). For these
assays, the wells of Coming (25801) 96 well microtiter plates were
coated with 100 ng of a sialyl-Lewis.sup.X/BSA conjugate, air dried
overnight, washed with 300 .mu.l of PBS(-) and then blocked with 1%
BSA in SHMCK for 60 min at room temperature. RNA ligands were
incubated with LS-Rg in SHMCK/1% BSA at room temperature for 15
min. After removal of the blocking solution, 50 .mu.l of LS-Rg (10
nM) or a LS-Rg (10 nM)/RNA ligand mix was added to the coated,
blocked wells and incubated at room temperature for 60 minutes. The
binding solution was removed, wells were washed with 300 .mu.l of
PBS(-) and then probed with HRP conjugated anti-human IgG, at room
temperature to quantitate LS-Rg binding. After a 30 minute
incubation at room temperature in the dark with OPD peroxidase
substrate (Sigma P9187), the extent of LS-Rg binding and percent
inhibition was determined from the OD.sub.450.
Example 8
2'-NH.sub.9 RNA Ligands to Human L-selectin
A. SELEX
[0162] The starting RNA pool for SELEX, randomized 40N7 (SEQ ID NO:
63), contained approximately 10.sup.15 molecules (1 nmol RNA). The
SELEX protocol is outlined in Tables 7a and 7b and Example 7. The
dissociation constant of randomized RNA to LS-Rg is estimated to be
approximately 10 .mu.M. No difference was observed in the RNA
elution profiles with 5 mM EDTA from SELEX and background beads for
rounds 1 and 2, while the 50 mM elution produced a 2-3 fold excess
over background (Table 7a). The 50 mM eluted RNA from rounds 1 and
2 were amplified for the input material for rounds 2 and 3,
respectively. Beginning in round 3, the 5 mM elution from SELEX
beads was significantly higher than background and was processed
for the next round's input RNA. The percentage of input RNA eluted
by 5 mM EDTA increased from 0.5% in the first round to 8.4% in
round 5 (Table 7a). An additional increase in specifically eluted
RNA from the 10 .mu.M LS-Rg beads was not observed in round 6
(Table 7a). To increase the stringency of selection, the density of
immobilized LS-Rg was reduced ten fold in round 5 with further
reductions in protein density at later rounds. The affinity of the
selected pools rapidly increased and the pools gradually evolved
biphasic binding characteristics.
[0163] Binding experiments with 6th round RNA revealed that the
affinity of the evolving pool for L-selectin was temperature
sensitive. Beginning with round 7, the SELEX was branched; one
branch was continued at 4.degree. C. (Table 7a) while the other was
conducted at room temperature (Table 7b). Bulk sequencing of 6th,
13th (rm temp) and 14th (4.degree. C.) RNA pools revealed
noticeable non-randomness at round six and dramatic non-randomess
at the later rounds. The 6th round RNA bound monophasically at
4.degree. C. with a dissociation constant of approximately 40 nM,
while the 13th and 14th round RNAs bound biphasically with high
affinity Kds of approximately 700 pM. The molar fraction of the two
pools that bound with high affinity were 24% and 65%, respectively.
The binding of all tested pools required divalent cations. In the
absence of divalent cations, the Kds of the 13th and 14th round
pools increased to 45 nM and 480 nM, respectively (HSMC, minus
Ca.sup.++/Mg.sup.++, plus 2 mM EDTA).
[0164] To monitor the progress of SELEX, ligands were cloned and
sequenced from rounds 6, 13 (rm temp) and 14 (4.degree. C.).
Sequences were aligned manually and with the aid of a computer
program that determines consensus sequences from frequently
occurring local alignments.
B. Sequences
[0165] In Table 8, ligand sequences are shown in standard single
letter code (Cornish-Bowden, 1985 NAR 13: 3021-3030). The
letter/number combination before the "." in the ligand name
indicates whether it was cloned from the round 6, 13 or 14 pools.
Only the evolved random region is shown in Table 8. Any portion of
the fixed region is shown in lower case letters. By definition,
each clone includes both the evolved sequence and the associated
fixed region, unless specifically stated otherwise. From the sixth,
thirteenth and fourteenth rounds, respectively, 26 of 48, 8 of 24
and 9 of 70 sequenced ligands were unique. A unique sequence is
operationally defined as one that differs from all others by three
or more nucleotides. Sequences that were isolated more than once,
are indicated by the parenthetical number, (n), following the
ligand isolate number. These clones fall into thirteen sequence
families (I-XIII) and a group of unrelated sequences (Orphans)(SEQ
ID NOs: 67-117).
[0166] Two families, I and III, are defined by ligands from
multiple lineages. Both families occur frequently in round 6, but
only one family m ligand was identified in the final rounds. Six
families (IV, V, VI, VII, VIII, and possibly II) are each defined
by just two lineages which limits confidence in their consensus
sequences. Five families (IX through XIII) are defined by a single
lineage which precludes determination of consensus sequences.
[0167] Ligands from family II dominate the final rounds: 60/70
ligands in round 14 and 9/24 in round 13. Family II is represented
by three mutational variations of a single sequence. One
explanation for the recovery of a single lineage is that the
ligand's information content is extremely high and was therefore
represented by a unique species in the starting pool. Family II
ligands were not detected in the sixth round which is consistent
with a low frequency in the initial population. An alternative
explanation is sampling error. Note that a sequence of questionable
relationship was detected in the sixth round.
[0168] The best defined consensus sequences are those of family I,
AUGUGUA (SEQ ID NO: 118), and of family III, AACAUGAAGUA (SEQ ID
NO: 120), as shown in Table 8. Family III has two additional,
variably spaced sequences, AGUC and ARUUAG, that may be conserved.
The tetranucleotide AUGW is found in the consensus sequence of
families I, III, and VII and in families II, VIII and IX. If this
sequence is significant, it suggests that the conserved sequences
of ligands of family VIII are circularly permuted. The sequence
AGAA is found in the consensus sequence of families IV and VI and
in families X and XIII.
C. Affinities
[0169] The dissociation constants for representative ligands from
rounds 13 and 14, including all orphans, were determined by
nitrocellulose filter binding experiments are described in Example
7 and the results are listed in Table 9. These calculations assume
two RNA ligand binding sites per chimera. The affinity of random
RNA cannot be reliably determined but is estimated to be
approximately 10 .mu.M.
[0170] In general, ligands bind monophasically with dissociation
constants ranging from 50 pM to 15 nM at 4.degree. C. Some of the
highest affinity ligands bind biphasically. Although ligands of
families I, VII, X and orphan F14.70 bind about equally well at
4.degree. C. and room temperature, in general the affinities
decrease with increasing temperature. The observed affinities
substantiate the proposition that it is possible to isolate
oligonucleotide ligands with affinities that are several orders of
magnitude greater than that of carbohydrate ligands.
Example 9
Specificity of 2'-NH.sub.9 RNA Ligands to L-Selectin
[0171] The affinity of L-selectin ligands to ES-Rg, PS-Rg and
CD22.beta.-Rg were determined by nitrocellulose partitioning as
described in Example 7. As indicated in Table 10, the ligands are
highly specific for L-selectin. In general, a ligand's affinity for
ES-Rg is 10.sup.3-fold lower and that for PS-Rg is about
10.sup.4-fold less than for LS-Rg. Binding above background is not
observed for CD22.beta.-Rg at the highest protein concentration
tested (660 nM), indicating that ligands do not bind the Fc domain
of the chimeric constructs nor do they have affinity for the sialic
acid binding site of an unrelated lectin. The specificity of
oligonucleotide ligand binding contrasts sharply with the binding
of cognate carbohydrates by the selectins and confirms the
proposition that SELEX ligands will have greater specificity than
carbohydrate ligands.
Example 10
Binding of L-Selectin 2'-NH.sub.7 RNA Ligands to Human PBMCs
[0172] Since the L-selectin ligands were isolated against purified,
immobilized protein, it is essential to demonstrate that they bind
L-selectin presented in the context of a cell surface. Comparison
of 2nd and 9th round RNAs (FIG. 2) shows that the evolved (9th
round) ligand pool binds isolated PBMCs with high affinity and, as
expected for specific binding, in a saturable fashion. The binding
of round 2 RNA appears to be non-saturable as is characteristic of
non-specific binding. The cloned ligand, F14.12 (SEQ ID NO: 78),
also binds in a saturable fashion with a dissociation constant of
1.3 mM, while random 40N7 (SEQ ID NO: 64) resembles round 2 RNA
(FIG. 3). The saturability of binding is confirmed by the data in
FIG. 4; >90% of 5 nNM .sup.32P-labeled F14.12 RNA binding is
competed by excess cold RNA. Specificity is demonstrated by the
results in FIG. 5; binding of 5 nM .sup.32P-labeled F14.12 RNA is
completely competed by the anti-L-selectin blocking monoclonal
antibody, DREG-56, but is unaffected by an isotype-matched
irrelevant antibody. These data validate the feasibility of using
immobilized, purified protein to isolate ligands against a cell
surface protein and the binding specificity of F14.12 to L-selectin
in the context of a cell surface.
Example 11
Inhibition of Binding to Sialyl-Lewis.sup.X
[0173] Oligonucleotide ligands, eluted by 2-5 mM EDTA, are expected
to derive part of their binding energy from contacts with the
lectin domain's bound Ca.sup.++ and consequently, are expected to
compete with sialyl-Lewis.sup.X for binding. The ability of ligand
F14.12 (SEQ ID NO: 78) to inhibit LS-Rg binding to immobilized
sialyl-Lewis.sup.X was determined by competition ELISA assays. As
expected, 4 mM EDTA reduced LS-Rg binding 7.4-fold, while 20 mM
round 2 RNA did not inhibit LS-Rg binding. Carbohydrate binding is
known to be Ca.sup.++ dependent; the affinity of round 2 RNA is too
low to bind 10 nM LS-Rg (Table 7).
[0174] In this assay F14.12 RNA inhibits LS-Rg binding in a
concentration dependent manner with an IC.sub.50 of about 10 nM
(FIG. 6). Complete inhibition is observed at 50 nM F14.12. The
observed inhibition is reasonable under the experimental
conditions; the Kd of F14.12 at room temperature is about 1 nM
(Table 9) and 10 nM LS-Rg is 20 nM binding sites. These data verify
that RNA ligands compete with sialyl-Lewis.sup.X for LS-Rg binding
and support the contention that low concentrations of EDTA
specifically elute ligands that bind the lectin domain's
carbohydrate binding site.
Example 12
Secondary Structure of High Affinity 2'-NH.sub.2 Ligands to
L-Selectin
[0175] In favorable instances, comparative analysis of aligned
sequences allows deduction of secondary structure and
structure-function relationships. If the nucleotides at two
positions in a sequence covary according to Watson-Crick base
pairing rules, then the nucleotides at these positions are apt to
be paired. Nonconserved sequences, especially those that vary in
length are not apt to be directly involved in function, while
highly conserved sequence are likely to be directly involved.
[0176] Comparative analysis of the family I alignment suggests a
hairpin structure in which the consensus sequence, AUGUGUGA, is
contained within a variable size loop (FIG. 7a). The stem sequences
are not conserved and may be either 5' or 3'-fixed or variable
sequence. The one ligand that does not form a stem, F14.25 (SEQ ID
NO: 73), has a significantly lower affinity than the other
characterized ligands (Table 9).
[0177] The proposed structure for family III is also a hairpin with
the conserved sequence, AACAUGAAGUA, contained within a variable
length loop (FIG. 7b). The 5'-half of the stem is 5'-fixed sequence
which may account in part for the less highly conserved sequence,
AGUC.
[0178] Although there is no alignment data for family II, the
sequence folds into a pseudoknot (FIG. 7c). Three attractive
features of this model are 1) the helices stack on one another, 2)
the structure utilizes only variable sequence and 3) the structure
is compatible with the major variant sequences.
Example 13
ssDNA Ligands to Human L-Selectin
[0179] The experimental procedures outlined in this Example were
used to identify and characterize ssDNA ligands to human L-selectin
as described in Examples 14-21.
Experimental Procedures
A) Materials
[0180] Unless otherwise indicated, all materials used in the ssDNA
SELEX against the L-selectin/IgG2 chimera, LS-Rg, were identical to
those of Example 7, paragraph A. The buffer for SELEX experiments
was 1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 100 mM NaCl, 10.0 mM HEPES,
pH 7.4. The buffer for all binding affinity experiments differed
from the above in containing 125 mM NaCl, 5 mM KCl, and 20 nM
HEPES, pH 7.4.
B) SELEX
[0181] The SELEX procedure is described in detail in U.S. Pat. No.
5,270,163 and elsewhere. The strategy used for this ssDNA SELEX is
essentially identical to that described in Example 7, paragraph B
except as noted below. The nucleotide sequence of the synthetic DNA
template for the LS-Rg SELEX was randomized at 40 positions. This
variable region was flanked by BH 5' and 3' fixed regions. The
random DNA template was termed 40BH (SEQ ID NO: 126) and had the
following sequence:
5'-ctacctacgatctgactagc<40N>sgcttactctcatgtagttcc-3'. The
primers for the PCR were the following: 5' Primer:
5'-ctacctacgatctgactagc-3' (SEQ ID NO: 127) and 3' Primer:
5'-ajajaggaactacatgagagtaagc-3'; j=biotin (SEQ ID NO: 128). The
fixed regions include primer annealing sites for PCR amplification.
The initial DNA pool contained 500 pmols of each of two types of
single-stranded DNA: 1) synthetic ssDNA and 2) PCR amplified, ssDNA
from 1 nmol of synthetic ssDNA template.
[0182] For subsequent rounds, eluted DNA was the template for PCR
amplification. PCR conditions were 50 mM KCl, 10 mM Tris-Cl, pH
8.3, 7.5 mM MgCl.sub.2, 1 mM of each DATP, dCTP, dGTP, and dTTP and
25 U/ml of the Stoffel fragment of Taq DNA polymerase. After PCR
amplification, double stranded DNAs were end-labeled using
.gamma..sup.32P-ATP. Complementary strands were separated by
electrophoresis through an 8% polyacrylarnidenM urea gel. Strand
separation results from the molecular weight difference of the
strands due to biotintylation of the 3' PCR primer. In the final
rounds, DNA strands were separated prior to end labelling in order
to achieve high specific activity. Eluted fractions were processed
by ethanol precipitation.
[0183] The strategy for partitioning LS-Rg/ssDNA complexes from
unbound ssDNA was as described in Example 7, paragraph B, except
that 2 mM EDTA was utilized for specific elution. The SELEX
strategy is outlined in Table 11.
C) Nitrocellulose Filter Binding Assay
[0184] As described in SELEX Patent Applications and in Example 7,
paragraph C, a nitrocellulose filter partitioning method was used
to determine the affinity of ssDNA ligands for LS-Rg and for other
proteins. For these experiments a Gibco BRL 96 well manifold was
substituted for the 12 well Millipore manifold used in Example 7
and radioactivity was determined with a Fujix BAS100
phosphorimager. Binding data were analyzed as described in Example
7, paragraph C.
D) Cloning and Sequencing
[0185] Thirteenth, fifteenth and seventeenth round PCR products
were re-amplified with primers which contain either a BamHI or a
HinDIII restriction endonuclease recognition site. Approximately
140 ligands were cloned and sequenced using the procedures
described in Example 7, paragraph D. The resulting sequences are
shown in Table 12.
E) Cell Binding Studies
[0186] The ability of evolved ligand pools to bind to L-selectin
presented in the context of a cell surface was tested in
experiments with isolated human peripheral blood mononuclear cells
(PBMCs) as described in Example 7, paragraph E
Flow Cytometry
[0187] Binding of oligonucleotides to leukocytes was tested in flow
cytometry applications. Briefly, peripheral blood mononuclear cells
(PBMC) were purified on histoplaque by standard techniques. Cells
(500 cells/mL) were incubated with fluorescein labeled
oligonucleotide in 0.25 ML SMHCK buffer (140 mM NaCl, 1 mM
MgCl.sub.2, 1 mM CaCl.sub.2, 5 mM, KCl, 20 mM HEPES pH 7.4, 8.9 mM
NaOH, 0.1% (w/v) BSA, 0.1% (w/v) sodium azide) at room temperature
for 15 minutes. Fluorescent staining of cells was quantified on a
FACSCaliber fluorescent activated cell sorter (Becton Dickinson,
San Jose, Calif.).
[0188] To examine the ability of oligonucleotides to bind
leukocytes in whole blood, 25 .mu.l aliquots of heparinised whole
blood were stained for 30 min at 22.degree. C. with 2 .mu.g Cy5PE
labeled anti-CD45 (generous gift of Ken Davis, Becton-Dickinson)
and 0.15 .mu.M FITC-LD201T1 (synthesized with a 5'-Fluorescein
phosphoramidite by Operon Technologies, Alameda, Calif.; SEQ ID NO:
185). To determine specificity, other samples were stained with
FITC-LD201T1 in the presence of 0.3 .mu.M DREG-56 or 7 .mu.M
unlabeled LD201T1; or cells were reassayed after addition of 4 mM
EDTA. The final concentration of whole blood was at least 70%
(v/v). Stained, concentrated whole blood was diluted {fraction
(1/15)} in 140 mM NaCl, 5 mM KCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2,
20 mM HEPES pH 7.4, 0.1% bovine serum albumin and 0.1% NaN.sub.3
immediately prior to flow cytometry on a Becton-Dickinson FACS
Calibur. Lymphocytes and granulocytes were gated using side scatter
and CD45CyPE staining.
F) Synthesis and Characterization of Multimeric Oligonucleotide
Ligands
Synthesis of Branched Dimeric Oligonucleotide Complexes
[0189] Dimeric oligonucleotides were synthesized by standard solid
state processes, with initiation from a 3'-3' Symmetric Linking CPG
(Operon, Alameda, Calif.). Branched complexes contain two copies of
a truncated L-selectin DNA ligand, each of which is linked by the
3' end to the above CPG via a five unit ethylene glycol spacer
(FIG. 8A). Each ligand is labeled with a fluorescein
phosphoramidite at the 5' end (Glen Research, Sterling, Va.).
Branched dimers were made for 3 truncates of LD201T1 (SEQ ID NO:
142). The truncated ligands used were LD201T4 (SEQ ID NO: 187),
LD201T10 (SEQ ID NO: 187) and LD201T1 (SEQ ID NO: 185). Branched
dimers were purified by gel electrophoresis.
Synthesis of Multivalent Biotintylated-DNA Ligand/Streptavidin
Complexes
[0190] Multivalent oligonucleotide complexes were produced by
reacting biotintylated DNA ligands with either fluorescein or
phycoerythrin labeled streptavidin (SA-FITC, SA-PE, respectively)
(FIG. 8B). Streptavidin (SA) is a tetrameric protein, each subunit
of which has a biotin binding site. 5' and 3' biotintylated DNAs
were synthesized by Operon Technologies, Inc (Alameda, Calif.)
using BioTEG and BioTEG CPG (Glen Research, Sterling, Va.),
respectively. The expected stoichiometry is 2 to 4 DNA molecules
per complex. SA/bio-DNA complexes were made for 3 truncates of
LD201(SEQ ID NO: 142). The truncated ligands were LD201T4 (SEQ ID
NO: 187), LD201T10 (SEQ ID NO: 188) and LD201T1 (SEQ ID NO: 185).
The bio-DNA/SA multivalent complexes were generated by incubating
biotin modified oligonucleotide (1 mM) and fluoroscein labeled
streptavidin (0.17 mM) in 150 mM NaCl, 20 mM HEPES pH 7.4 at room
temperature for at least 2 hours. Oligonucleotide-streptavidin
complexes were used directly from the reaction mixture without
additional purification of the Complex from free streptavidin or
oligonucleotide.
Synthesis of a Dumbell Dimer Multivalent Complex
[0191] A "dumbell" DNA dimer complex was formulated from a
homobifunctional N-hydroxysuccinimidyl (or NHS) active ester of
polyethelene glycol, PEG 3400 MW, and a 29mer DNA oligonucleotide,
NX303 (SEQ ID NO: 196), having a 5' terminal Amino Modifier C6 dT
(Glen Research) and a 3'-3' terminal phosphodiester linkage (FIG.
8C). NX303 is a truncate of LD201 (SEQ ID NO: 142). The conjugation
reaction was in DMSO with 1% TEA with excess equivalents of the DNA
ligand to PEG. The PEG conjugates were purified from the free
oligonucleotide by reverse phase chromatography. The dimer was then
purified from the monomer by anion exchange HPLC. The
oligonucleotide was labeled at the 5' terminus with fluorescein as
previously described.
Synthesis of a Fork Dimer Multivalent Complex
[0192] To synthesize the fork dimer multivalent complex (FIG. 8D),
a glycerol was attached by its 2-position to one terminus of a
linear PEG molecule (MW 20 kD) to give the bis alcohol. This was
further modified to the bis succinate ester, which was activated to
the bis N-hydroxysuccinimidyl active ester. The active ester was
conjugated to the primary amine at the 5' terminus of the truncated
DNA nucleic acid ligand NX303 (SEQ ID NO: 196). The conjugation
reaction was in DMSO with 1% TEA with excess equivalents of the DNA
ligand to PEG. The PEG conjugates were purified away from the free
oligonucleotide by reverse phase chromatography. The dimer was then
purified away from the monomer by anion exchange HPLC. The
oligonucleotide was labeled at the 5' terminus with fluorescein as
previously described.
Characterization of Multimeric Oligonucleotide Ligands
[0193] The binding of dimeric and multimeric oligonucleotide
complexes to human peripheral blood mononuclear cells was analyzed
by flow cytometry as described in Example 13, paragraph D.
G) Photo-Crosslinking
[0194] A photo-crosslinking version of DNA ligand LD201T4 (SEQ ID
NO: 187) was synthesized by replacing nucleotide T15 (FIG. 12) with
5-bromo-deoxyuracil. 4 nmol of .sup.32P-labeled DNA was incubated
with 4 nmol L-selectin-Rg in 4 ml 1.times.SHMCK+0.01% human serni
albumin (w/v), then irradiated at ambient temperature with 12,500
pulses from an excimer laser at a distance of 50 cm and at 175
mJ/pulse. Protein and DNA were precipitated with 400 .mu.l 3 M
sodium acetate and 8.4 ml ethanol followed by incubation at -70
degrees C. Precipitated material was centrifuged, vacuum dried and
resuspended in 100 .mu.l 0.1 M Tris pH 8.0, 10 mM CaCl.sub.2.
Fourty-five fig chymotrypsin were added and after 20 min at 37
degrees C, the material was loaded onto an 8% polyacrylamide/7 M
ureal 1.times.TBE gel and electrophoresed until the xylene cyanole
had migrated 15 cm. The gel was soaked for 5 min in 1.times.TBE and
then blotted for 30 min at 200 mAmp in 1.times.TBE onto Immobilon-P
(Millipore). The membrane was washed for 2 min in water, air dried,
and an autoradiograph taken. A labeled band running slower than the
free DNA band, representing a chymotryptic peptide crosslinked to
LD201T4, was observed and the autoradiograph was used as a template
to excise this band from the membrane. The peptide was sequenced by
Edman degradation, and the resulting sequence was LEKTLP_SRSYY. The
blank residue corresponds to the crosslinked amino acid, F82 of the
lectin domain.
H) Lymphocyte Trafficking Experiments
[0195] Human PBMC were purified from heparinised blood by a
Ficoll-Hypaque gradient, washed twice with HBSS (calcium/magnesium
free) and labeled with .sup.51Cr (Amersham). After labeling, the
cells were washed twice with HBSS (containing calcium and
magnesium) and 1% bovine serum albumin (Sigma). Female SCID mice
(6-12 weeks of age) were injected intravenously with
2.times.10.sup.6 cells. The cells were either untreated or mixed
with either 13 pmol of antibody (DREG-56 or MEL-14), or 4, 1, or
0.4 nmol of modified oligonucleotide (synthesis described below).
One hour later the animals were anesthetized, a blood sample taken
and the mice were euthanised. PLN, MLN, Peyer's patches, spleen,
liver, lungs, thymus, kidneys and bone marrow were removed and the
counts incorporated into the organs determined by a Packard gamma
counter. In a second protocol, 2.times.10.sup.6 human PBMC,
purified, labeled, and washed as described above, were injected
intravenously into female SCID mice without antibody or
oligonucleotide pretreatment. One to 5 min prior to injecting the
cells, the animals were injected with either 15 pmol DREG-56 or 4
nmol modified oligonucleotide. Counts incorporated into organs were
quantified as described above.
[0196] Synthesis of modified nucleotides NX288 (SEQ ID NO: 193) and
NX303 (SEQ ID NO: 196) was initiated by coupling to a dT-5'-CE
polystyrene support (Glen Research), resulting in a 3'-3' terminal
phosphodiester linkage, and having a 5' terminal an Amino Modifier
C6 dT (Glen Research). Once NX288 and NX303 were synthesized, a
20,000 MW PEG2-NHS ester (Shearwater Polymers, Huntsville, Ala.)
was then coupled to the oligonucleotide through the 5' amine
moiety. The molar ratio, PEG:oligo, in the reactions was from 3:1
to 10:1. The reactions were performed in 80:20 (v:v) 100 mM borate
buffer pH 8: DMF at 37.degree. C. for one hour.
I) Inhibition of L-selectin Binding to Sialyl Lewis.sup.X
[0197] SLe.sup.X-BSA (Oxford GlycoSystems, Oxford, UK) in
1.times.PBS, without CaCl.sub.2 and MgCl.sub.2,' (GIBCO/BRL) was
immobilized at 100 ng/well onto a microtiter plate by overnight
incubation at 22.degree. C. The wells were blocked for 1 h with the
assay buffer consisting of 20 mM HEPES, 111 mM NaCl, 1 mM
CaCl.sub.2, 1 mM MgCl.sub.2, 5 mM KCl, 8.9 mM NaOH, final pH 8, and
1% globulin-free BSA (Sigma). The reaction mixtures, incubated for
90 min with orbital shaking, contained 5 nM L-Selectin-Rg, a 1:100
dilution of anti-human IgG-peroxidase conjugate (Sigma), and 0-50
nM of competitor in assay buffer. After incubation, the plate was
washed with BSA-free assay buffer to remove unbound
chimera-antibody complex and incubated for 25 min with
O-phenylenediamine dihydrochloride peroxidase substrate (Sigma) by
shaking in the dark at 22.degree. C. Absorbance was read at 450 nm
on a Bio-Kinetics Reader, Model EL312e (Bio-Tek Instruments, Laguna
Hills, Calif.). Values shown represent the mean.+-.s.e from
duplicate, or triplicate, samples from one representative
experiment.
Example 14
ssDNA Ligands to L-Selectin
A. SELEX
[0198] The starting ssDNA pool for SELEX, randomized 40BH (SEQ ID
NO: 126), contained approximately 10.sup.15 molecules (1 nmol
ssDNA). The dissociation constant of randomized ssDNA to LS-Rg is
estimated to be approximately 10 .mu.M. The SELEX protocol is
outlined in Table 11;
[0199] The initial round of SELEX was performed at 4.degree. C.
with an LS-Rg density of 16.7 pmol/.mu.l of protein A sepharose
beads. Subsequent rounds were at room temperature except as noted
in Table 11. The 2 mM EDTA elution was omitted from rounds 1-3. The
signal to noise ratio of the 50 mM EDTA elution in these three
rounds was 50, 12 and 25, respectively (Table 11). These DNAs were
amplified for the input materials of rounds 2-4. Beginning with
round 4, a 2 mM EDTA elution was added to the protocol. In this and
all subsequent rounds, the 2 mM EDTA eluted DNA was amplified for
the next round's input material.
[0200] To increase the stringency of selection, the density of
immobilized LS-Rg was reduced ten fold in round 4 with further
reductions in protein as needed to increase the stringency of
selectin (Table 11). Under these conditions a rapid increase in the
affinity of the selected pools was observed (Tables 11); at
4.degree. C., the dissociation constant of round 7 ssDNA was 60
nM.
[0201] Binding experiments with 7th round DNA revealed that the
affinity of the evolving pool for L-selectin was weakly temperature
sensitive (Kds: 60 nM, 94 nM and 230 nM at 4.degree. C., room
temperature and 37.degree. C., respectively). To enhance the
selection of ligands that bind at physiological temperature, rounds
8, 13, 16 and 17 were performed at 37.degree. C. Although
temperature sensitive, the affinity of round 15 ssDNA was optimal
at room temperature (160 pM), with 3-fold higher Kds at 4.degree.
C. and 37.degree. C.
[0202] Bulk sequencing of DNA pools indicates some non-randomness
at round 5 and dramatic non-randomness at round 13. Ligands were
cloned and sequenced from rounds 13, 15, and 17. Sequences were
aligned manually and with the aid of a NeXstar computer program
that determines consensus sequences from frequently occurring local
alignments.
B. Sequences
[0203] In Table 12, ligand sequences are shown in standard single
letter code (Cornish-Bowden, 1985 NAR 13: 3021-3030). Only the
evolved random region is shown in Table 12. Any portion of the
fixed region is shown in lower case letters. By definition, each
clone includes both the evolved sequence and the associated fixed
region, unless specifically stated otherwise A unique sequence is
operationally defined as one that differs from all others by three
or more nucleotides. Sequences that were isolated more than once
are indicated by the parenthetical number, (n), following the
ligand isolate number. These clones fall into six families and a
group of unrelated sequences or orphans (Table 12)(SEQ ID NOs:
129-180).
[0204] Family 1 is defined by ligands from 33 lineages and has a
well defined consensus sequence, TACAAGGYGYTAVACGTA (SEQ ID NO:
181). The conservation of the CAAGG and ACG and their 6 nucleotide
spacing is nearly absolute (Table 12). The consensus sequence is
flanked by variable but complementary sequences that are 3 to 5
nucleotides in length. The statistical dominance of family 1
suggests that the properties of the bulk population are a
reflection of those of family 1 ligands. Note that ssDNA family I
and 2'-NH.sub.2 family I share a common sequence, CAAGGCG and
CAAGGYG, respectively.
[0205] Family 2 is represented by a single sequence and is related
to family 1. The ligand contains the absolutely conserved CAAGG and
highly conserved ACG of family 1 although the spacing between the
two elements is strikingly different (23 compared to 6
nucleotides).
[0206] Families 4-6 are each defined by a small number of ligands
which limits confidence in their consensus sequence, while family 7
is defined by a single sequence which precludes determination of a
consensus. Family 5 appears to contain two conserved sequences,
AGGGT and RCACGAYACA, the positions of which are circularly
permuted.
C. Affinities
[0207] The dissociation constants of representative ligands from
Table 12 are shown in Table 13. These calculations assume two ssDNA
ligand binding sites per chimera. The affinity of random ssDNA
cannot be reliably determined but is estimated to be approximately
10 .mu.M.
[0208] At room temperature, the dissociation constants range from
43 pM to 1.8 nM which is at least a 5.times.10.sup.3 to
2.times.10.sup.5 fold improvement over randomized ssDNA (Table 13).
At 37.degree. C., the Kds range from 130 pM to 23 nM. The extent of
temperature sensitivity varies from insensitive (ligands LD122 and
LD127 (SEQ ID NO: 159 and 162)) to 80-fold (ligand LD112 (SEQ ID
NO: 135)). In general, among family 1 ligands the affinity of those
from round 15 is greater than that of those from round 13. For the
best ligands (LD208, LD227, LD230 and LD233 (SEQ ID NOS: 133, 134,
132, and 146)), the difference in affinity at room temperature and
37.degree. C. is about 4-fold.
[0209] The observed affinities of the evolved ssDNA ligand pools
reaffirm our proposition that it is possible to isolate
oligonucleotide ligands with affinities that are several orders of
magnitude greater than that of carbohydrate ligands.
Example 15
Specificity of ssDNA Ligands to L-Selectin
[0210] The affinity of representative cloned ligands for LS-Rg,
ES-Rg, PS-Rg, CD22.beta.-Rg and WGA was determined by
nitrocellulose partitioning and the results shown in Table 14. The
ligands are highly specific for L-selectin. The affinity for ES-Rg
is about 10.sup.3-fold lower and that for PS-Rg is about
5.times.10.sup.3-fold less than for LS-Rg. Binding above background
is not observed for CD22.beta.-Rg or for WGA at 0.7 and 1.4 .mu.M
protein, respectively, indicating that ligands neither bind the Fc
domain of the chimeric constructs nor have affinity for unrelated
sialic acid binding sites.
[0211] The specificity of oligonucleotide ligand binding contrasts
sharply with the binding of cognate carbohydrates by the selectins
and reconfirms the proposition that SELEX ligands will have greater
specificity than carbohydrate ligands.
Example 16
Cell Binding Studies
[0212] Round 15 ssDNA pool was tested for its ability to bind to
L-selectin presented in the context of a peripheral blood
mononuclear cell surface as described in Example 13, paragraph E.
The evolved pool was tested both for affinity and for specificity
by competition with an anti-L-selectin monoclonal antibody. FIG. 9
shows that the round 15 ssDNA pool binds isolated PBMCs with a
dissociation constant of approximately 1.6 nM and, as is expected
for specific binding, in a saturable fashion. FIG. 10 directly
demonstrates specificity of binding; in this experiment, binding of
2 nM .sup.32P-labeled round 15 ssDNA is completely competed by the
anti-L-selectin blocking monoclonal antibody, DREG-56, but is
unaffected by an isotype-matched irrelevant antibody. In analogous
experiments, LD201T1 (SEQ ID NO: 185) was shown to bind human PBMC
with high affinity. Binding was saturable, divalent cation
dependent, and blocked by DREG-56.
[0213] These data validate the feasibility of using immobilized,
purified protein to isolate ligands against a cell surface protein
and demonstrate the specific binding of the round 15 ssDNA pool and
of ligand LD201T1 to L-selectin in the context of a cell
surface.
[0214] The binding of LD201T1 to leukocytes in whole blood was
examined by flow cytometry. Fluorescein isothiocyanate
(FITC)-conjugated LD201T1 specifically bind human lymphocytes and
neutrophils (FIGS. 11A/B); binding is inhibited by competition with
DREG-56, unlabeled LD201, and by the addition of 4 mM EDTA (FIGS.
11A/B). These cell binding studies demonstrate that LD201T1 bind
saturably and specifically to human L-selectin on lymphocytes and
neutrophils.
Example 17
Secondary Structure of High Affinity ssDNA Ligands to
L-Selectin
[0215] In favorable instances, comparative analysis of aligned
sequences allows deduction of secondary structure and
structure-function relationships. If the nucleotides at two
positions in a sequence covary according to Watson-Crick base
pairing rules, then the nucleotides at these positions are apt to
be paired. Nonconserved sequences, especially those that vary in
length are not apt to be directly involved in function, while
highly conserved sequence are likely to be directly involved.
[0216] Comparative analysis of 24 sequences from family 1 strongly
supports a hairpin secondary structure for these ligands (FIG. 12).
In the figure, consensus nucleotides are specified, with invariant
nucleotides in bold type. To the right of the stem is a matrix
showing the number of occurrences of particular base pairs for the
positions in the stem that are on the same line. The deduced
structure consists of a GYTA tetraloop, a 3 nucleotide-pair upper
stem and a 6 to 7 nucleotide-pair lower stem. The upper and lower
stems are separated by an asymmetrical, AA internal loop or
"bulge." Two of the three base pairs in the upper stem and 6 of 7
in the lower stem are validated by covariation. The two invariant
pairs, positions 7/20 and 10/19 are both standard Watson/Crick
basepairs. This structure provides a plausible basis for the direct
involvement of invariant nucleotides (especially, A8, A9 and T15)
in binding the target protein.
[0217] The site of oligonucleotide binding on L-selectin can be
deduced from a set of competition experiments. DREG56 is an
anti-L-selectin, adhesion blocking monoclonal antibody that is
known to bind to the lectin domain. Binding of three unrelated
ligands, LD201T1 (SEQ ID NO: 185), LD174T1 (SEQ ID NO: 194) and
LD196T1 (SEQ ID NO: 195), to LS-Rg was blocked by DREG-56, but not
by an isotype-matched control. In cross-competition experiments,
LD201T1, LD174T1, or LD196T1 prevented radio-labeled LD201T1 from
binding to LS-Rg, consistent with the premise that the ligands bind
the same or overlapping sites. The blocking and competition
experiments, taken together with divalent cation-dependence of
binding, suggest that all three ligands bind to the lectin domain.
This conclusion has been verified for LD201 by photo-crosslinking
experiments.
[0218] If T15 of LD201T4 (SEQ ID NO: 187; FIG. 12) is replaced with
5-bromo-uracil, the resulting DNA photo-crosslinks at high yield
(17%) to LS-Rg following irradiation with an excimer laser as
described in Example 13, paragraph G. The high yield of
crosslinking indicates a point contact between the protein and T15.
Sequencing of the chymotryptic peptide corresponding to this point
contact revealed a peptide deriving from the lectin domain; F82 is
the crosslinking amino acid. Thus, F82 contacts T15 in a stacking
arrangement that permits high yield photo-crosslinking. By analogy
to the structure of the highly related E-selectin (Graves et al,
Nature 367, 532-538, 1994), F82 is adjacent to the proposed
carbohydrate binding site. Thus, this photo-crosslink provides
direct evidence that ligand LD201 makes contact with the lectin
domain of LS-Rg and provides an explanation for the function of the
oligonucleotides in either sterically hindering access to the
carbohydrate binding site or in altering the conformation of the
lectin domain upon DNA binding.
Example 18
L-Selectin ssDNA Ligand Truncate Data
[0219] Initial experiments to define the minimal high affinity
sequence of family 1 ligands show that more than the 26 nucleotide
hairpin (FIG. 12; Table 13) is required. Ligands corresponding to
the hairpin, LD201T4 (SEQ ID NO: 187) and LD227T1 (SEQ ID NO: 192)
derived from LD201 (SEQ ID NO: 173) and LD227 (SEQ ID NO: 134),
respectively, bind with 20-fold and 100-fold lower affinity than
their full length progenitors. The affinity of LD201T3 (SEQ ID NO:
186), a 41 nucleotide truncate of ligand LD201, is reduced about
15-fold compared to the full length ligand, while the affinity of
the 49-mer LD201T1 (SEQ ID NO: 185) is not significantly altered
(Tables 12 and 13).
[0220] Additional experiments show that truncates LD201T10 (SEQ ID
NO: 188) and LD227X1 (SEQ ID NO: 191) bind with affinities similar
to their full length counterparts. Both of these ligands have stems
that are extended at the base of the consensus stem. Alterations in
the sequence of the added stem have little, if any, effect on
binding, suggesting that it is not directly involved in binding
[0221] The added stem is separated from the consensus stem by a
single stranded bulge. The two ligands' single stranded bulges
differ in length and have unrelated sequences. Furthermore, LD201's
bulge is at the 5'-end of the original stem base while that of
LD227 is at the 3'-end. Thus, the two ligands do not present an
obvious consensus structure. Removal of the loop (LD201) or
scrambling or truncating the sequence (LD227) diminishes affinity,
suggesting that the bulged sequences may be directly involved in
binding. Note that although LD201T3 is longer than LD201T10, it is
unable to form the single stranded loop and extended stem because
of the position of the truncated ends.
Example 19
Inhibition of Binding to Sialyl Lewis.sup.X
[0222] Sialyl Lewis.sup.x is the minimal carbohydrate ligand bound
by selecting. The ability of ssDNA ligands to inhibit the binding
of L-selectin to Sialyl Lewis.sup.X was determined in competition
ELISA assays as described in Example 13, paragraph I. LD201T1 (SEQ
ID NO: 185), LD174T1 (SEQ ID NO: 194) and LD196T1 (SEQ ID NO: 195)
inhibited LS-Rg binding to immobilized SLe.sup.X in a dose
dependent manner with IC.sub.50s of approximately 3 nM. This is a
10.sup.5-10.sup.6-fold improvement over the published IC.sub.50
values for SLe.sup.X in similar plate-binding assays. A scrambled
sequence based on LD201T1 showed no activity in this assay. These
data verify that DNA ligands compete with sialyl-Lewis.sup.X for
LS-Rg binding and support the contention that low concentrations of
EDTA specifically elute ligands that bind the lectin domain's
carbohydrate binding site.
Example 20
Inhibition of Lymphocyte Trafficking by L-Selectin ssDNA
Ligands
[0223] Lymphocyte trafficking to peripheral lymph nodes is
exquisitely dependent on L-selectin. Since the ssDNA ligands binds
to human but not rodent L-selectin, a xenogeneic lymphocyte
trafficking system was established to evaluate in vivo efficacy.
Human PBMC, labeled with .sup.51Cr, were injected intravenously
into SCID mice. Cell trafficking was determined 1 hour later. In
this system, human cells traffic to peripheral and mesenteric lymph
nodes (PLN and MLN). This accumulation is inhibited by DREG-56
(FIG. 13) but not MEL-14, a monoclonal antibody that blocks murine
L-selectin-dependent trafficking. In initial experiments cells were
incubated with either DREG-56 or 3' capped and PEG-modified
oligonucleotide before injection. NX288 (SEQ ID NO: 193) inhibited
trafficking of cells to PLN (FIG. 13) and MLN in a dose-dependent
fashion but had no effect on the accumulation of cells in other
organs. At the highest dose tested (4 nmol), inhibition by the DNA
ligand was comparable to that of DREG-56 (13 pmol), while a
scrambled sequence had no significant effect (FIG. 13). The
activity of LD174T1 (SEQ ID NO: 194) was similar to that of
NX288.
[0224] To determine if the modified oligonucleotide was effective
when it was not pre-incubated with cells, DREG-56 (13 pmol/mouse)
or the modified oligonucleotide (4 nmol/mouse) was injected
intravenously into animals and 1-5 min later the radio-labeled
human cells were given intravenously. Again, both NX288 (SEQ ID NO:
193) and DREG-56 inhibited trafficking to PLN and MLN while the
scrambled sequence had no effect (FIG. 14). Therefore, the modified
oligonucleotide did not require pre-incubation with the cells to
effectively block trafficking. These experiments demonstrate, in
vivo, the efficacy of oligonucleotide ligands in inhibiting a
L-selectin dependent process.
Example 21
L-Selectin Nucleic Acid Ligand Multimers
[0225] Multivalent Complexes were made in which two nucleic acid
ligands to L-selectin were conjugated together. Multivalent
Complexes of nucleic acid ligands are described in copending U.S.
patent application Ser. No. 08/434,465, filed May 4, 1995, entitled
"Nucleic Acid Ligand Complexes" which is herein incorporated by
reference in its entirety. These multivalent Complexes were
intended to increase the binding energy to facilitate better
binding affinities through slower off-rates of the nucleic acid
ligands. These multivalent Complexes may be useful at lower doses
than their monomeric counterparts. In addition, high molecular
weight (20 kD) polyethylene gylcol (PEG) was included in some of
the Complexes to decrease the in vivo clearance rate of the
complexes. Specifically, the nucleic acid ligands incorporated into
the Complexes were LD201T1 (SEQ ID NO: 185), LD201T4 (SEQ ID NO:
187), LD201T10 (SEQ ID NO: 188) and NX303 (SEQ ID NO: 196).
Multivalent selectin nucleic acid ligand Complexes were produced as
described in Example 13, paragraph F.
[0226] A variety of monomeric nucleic acid ligands and multivalent
Complexes have been examined in flow cytometry. The multivalent
Complexes exhibited similar specificity to the monomeric forms, but
enhanced affinity as well as improved (i.e., slower) off-rate for
human lymphocytes. Titration curves, obtained from incubating
fluorescently labeled monomeric FITC-LD201T1 with peripheral blood
mononuclear cells (PBMC) purified human lymphocytes, indicated that
binding to cells is saturable. Half-saturation fluorescence
occurred at 3 nM oligonucleotide. In contrast, the branched dimeric
FITC-LD201T1 and bio-LD201T1/SA multivalent Complexes exhibited
half-saturation at approximately 0.15 nM, corresponding to an
apparent 20-fold increase in affinity. In similar experiments, half
saturation of the dumbell and fork dimers of LD201T4 was observed
at 0.1 and 0.6 nM, respectively, compared to 20 nM for monomeric
LD201T4.
[0227] Kinetic competition experiments were performed on monomeric
nucleic acid ligands and multivalent Complexes. Kinetic competition
experiments were performed with PBMC purified lymphocytes. Cells
were stained as described above but used 10 nM oligonucleotide. The
off-rate for monomeric, dimeric and multivalent Complexes was
determined by addition of 500 nM unlabeled oligonucleotide to cells
stained with fluorescently labeled ligand and measurement of the
change in the mean fluorescence intensity as a function of time.
The dissociation rate of a monomeric LD201T1 from L-selectin
expressing human lymphocytes was approximately 0.005 sec-1,
corresponding to a half-life of roughly 2.4 minutes. The LD201T1
branched dimer and biotin conjugate multivalent Complexes exhibited
apparent off-rates several times slower than that observed for the
monomeric ligand and as slow or slower than that observed for the
anti-L-selectin blocking antibody DREG56, determined under the same
conditions. A multivalent Complex containing a non-binding nucleic
acid sequence did not stain cells under identical conditions and
did not compete in the off-rate experiments. The off-rate of the
LD201T4 dumbell and fork dimers is faster than the LD201T1 branched
dimer and is better than all monomers tested. These results confirm
the proposition that dimeric and multimeric ligands bind with
higher affinities than do monomeric ligands and that the increased
affinity results from slower off-rates.
Example 22
2'-F RNA Ligands to Human L-Selectin
[0228] The experimental procedures outlined in this Example were
used to identify and characterize 2'-F RNA ligands to human
L-selectin as described in Examples 23-25.
Experimental Procedures
A) Materials
[0229] Unless otherwise indicated, all materials used in the 2'-F
RNA SELEX against the L-selectin/IgG2 chimera, LS-Rg, were
identical to those of Examples 7, paragraph A and 13, paragraph A.
SHMCK-140 buffer, used for all SELEX and binding experiments, was 1
mM CaCl.sub.2, 1 mM MgCl.sub.2, 140 mM NaCl, 5 mM KCl, and 20 mM
HEPES, pH 7.4. A soluble form of L-selectin, corresponding to the
extracellular domains, was purchased from R&D Systems and used
for some nitrocellulose filter binding experiments.
B) SELEX
[0230] The SELEX procedure is described in detail in U.S. Pat. No.
5,270,163 and elsewhere. Procedures are essentially identical to
those in Examples 7 and 13 except as noted. The variable regions of
synthetic DNA templates were randomized at either 30 or 40
positions and were flanked by N7 5' and 3' fixed regions producing
transcripts 30N7 (SEQ ID NO: 292) and 40N7 (SEQ ID NO: 389). The
primers for the PCR were the following:
[0231] N7 5' Primer 5' taatacgactcactatagggaggacgatgcgg 3' (SEQ ID
NO: 65)
[0232] N7 3' Primer 5' tcgggcgagtcgtcctg 3 ' (SEQ ID NO: 66)
[0233] The initial RNA pool was made by first Klenow extending 3
nmol of synthetic single stranded DNA and then transcribing the
resulting double stranded molecules with T7 RNA polymerase. Klenow
extension conditions: 6 nmols primer 5N7, 3 nmols 30N7 or 40n7,
1.times. Klenow Buffer, 1.8 mM each of DATP, dCTP, dGTP and dTTP in
a reaction volume of 0.5 ml.
[0234] For subsequent rounds, eluted RNA was the template for AMV
reverse transcriptase mediated synthesis of single-stranded cDNA.
These single-stranded DNA molecules were converted into
double-stranded transcription templates by PCR amplification. PCR
conditions were 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 7.5 mM
MgCl.sub.2, 0.2 mM of each dATP, DCTP, dGTP, and dTTP, and 100 U/ml
of Taq DNA polymerase. Transcription reactions contained one third
of the purified PCR reaction, 200 nM T7 RNA polymerase, 80 mM HEPES
(pH 8.0), 12 mM MgCl.sub.2, 5 mM DTT, 2 mM spermidine, 1 mM each of
2'-OH ATP, 2'-OH GTP, 3 mM each of 2'-F CTP, 2'-F UTP, and 250 nM
.alpha.-.sup.32P 2'-OH ATP. Note that in all transcription
reactions 2'-F CTP and 2'-F UTP replaced CTP and UTP.
[0235] The strategy for partitioning LS-Rg/RNA complexes from
unbound RNA is outlined in Table 15 and is essentially identical to
that of Example 7, paragraph B. In the initial SELEX rounds, which
were performed at 37.degree. C., the density of immobilized LS-Rg
was 10 pmols/.mu.l of Protein A Sepharose 4 Fast Flow beads. LS-Rg
was coupled to protein A sepharose beads according to the
manufacturer's instructions (Pharmacia Biotech). In later rounds,
the density of LS-Rg was reduced (Table 15), as needed, to increase
the stringency of selection. At the seventh round, both SELEXes
were branched. One branch was continued as previously described
(Example 7, paragraph B). In the second branch of both SELEXes, the
RNA pool was pre-annealed to oligonucleotides that are
complementary to the 5' and 3' fixed sequences. These rounds are
termed "counter-selected" rounds. Before each round, RNA was batch
adsorbed to 100 .mu.l of protein A sepharose beads for 15 minutes
in a 2 ml siliconized column. Unbound RNA and RNA eluted with
minimal washing (two volumes) were combined and used for SELEX
input material. For SELEX, extensively washed, immobilized LS-Rg
was batch incubated with pre-adsorbed RNA for 1 to 2 hours in a 2
ml column with constant rocking. Unbound RNA was removed by
extensive batch washing (500 .mu.l SHMCK 140/wash). In addition,
the counter selected rounds were extensively washed with buffer
containing 200 nM of both complementary oligos. Bound RNA was
eluted as two fractions; first, bound RNA was eluted by incubating
and washing columns with 100 .mu.L 5 mM EDTA in SHMCK 140 without
divalent cations; second, the remaining elutable RNA was removed by
incubating and/or washing with 500 .mu.L 50 mM EDTA in SHMCK 140
without divalents. The percentage of input RNA that was eluted is
recorded in Table 22. In every round, an equal volume of protein A
sepharose beads without LS-Rg was treated identically to the SELEX
beads to determine background binding. All unadsorbed, wash and
eluted fractions were counted in a Beckman LS6500 scintillation
counter in order to monitor each round of SELEX.
[0236] The 5 mM EDTA eluates were processed for use in the
following round (Table 15). After precipitating with
isopropanol/ethanol (1:1, v/v), the RNA was reverse transcribed
into cDNA by AMV reverse transcriptase either at 48.degree. C. for
15 minutes and then 65.degree. C. for 15 minutes in 50 mM Tris-Cl
pH (8.3), 60 mM NaCl, 6 mM Mg(OAc).sub.2, 10 mM DTT, 200 pmol DNA
primer, 0.5 mM each of dNTPs, and 0.4 unit/.mu.L AMV RT.
Transcripts of the PCR product were used to initiate the next round
of SELEX.
C) Nitrocellulose Filter Binding Assay
[0237] As described in SELEX Patent Applications, a nitrocellulose
filter partitioning method was used to determine the affinity of
RNA ligands for LS-Rg and for other proteins. Filter discs
(nitrocellulose/cellulose acetate mixed matrix, 0.45 .mu.m pore
size, Millipore) were placed on a vacuum manifold and washed with 3
ml of SHMCK 140 buffer under vacuum. Reaction mixtures, containing
.sup.32P labeled RNA pools and unlabeled LS-Rg, were incubated in
SHMCK 140 for 10-20 min at 37.degree. C., and then immediately
washed with 3 ml SHMCK 140. The filters were air-dried and counted
in a Beckman LS6500 liquid scintillation counter without fluor.
Alternatively, binding studies employed 96 well micro-titer
manifolds essentially as described in Example 13, paragraph E.
D) Cloning and Sequencing
[0238] 12th round PCR products were re-amplified with primers which
contain either a BamHI or a HinDIII restriction endonuclease
recognition site. Using these restriction sites, the DNA sequences
were inserted directionally into the pUC9 vector. These recombinant
plasmids were transformed into E. coli strain DH5a (Life
Technologies, Gaithersburg, Md.). Plasmid DNA was prepared
according to the alkaline lysis method (Quiagen, QIAwell,
Chattsworth Calif.). Approximately 300 clones were sequenced using
the ABI Prism protocol (Perkin Elmer, Foster City, Calif.).
Sequences are shown in Table 16.
E) Cell Binding Studies
[0239] Binding of evolved ligands to L-selectin presented in the
context of a cell surface was tested by flow cytometry experiments
with human lymphocytes. Briefly, peripheral blood mononuclear cells
(PBMC) were purified on histoplaque by standard techniques. To
evaluate leukocyte binding by unlabeled 2'-F ligands, cells (500
cells/mL) were incubated with fluorescein labeled FITC-LD201T1 (SEQ
ID NO: 185) in the presence of increasing concentrations of
individual, unlabeled 2'-F ligands in 0.25 mL SMHCK buffer (140 mM
NaCl, 1 mM MgCl.sub.2, 1 mM CaCl.sub.2, 5 mM, KCl, 20 mM HEPES pH
7.4, 8.9 mM NaOH, 0.1% (w/v) BSA, 0.1% (w/v) sodium azide) at room
temperature for 15 minutes. Fluorescent staining of cells was
quantified on a FACSCaliber fluorescent activated cell sorter
(Becton Dickinson, San Jose, Calif.). The affinity of the 2'-F
competitor was calculated from the flurorescence inhibition
curves.
Example 23
2'-F RNA Ligands to L-Selectin
A. SELEX
[0240] The starting RNA pools for SELEX, randomized 30N7 (SEQ ID
NO: 292) or 40N7 (SEQ ID NO: 389) contained approximately 10.sup.14
molecules (0.7 nmol RNA). The SELEX protocol is outlined in Table
15 and Example 22. All rounds were selected at 37.degree. C. The
dissociation constant of randomized RNA to LS-Rg is estimated to be
approximately 10 .mu.M. After six rounds the pool affinities had
improved to approximately 300 nM. An aliquot of the RNA recovered
from the seventh round was used as the starting material for the
first counter-selected rounds. Five rounds of counter-selection and
five additional standard rounds were performed in parallel. Thus, a
total of twelve rounds were performed in both branches of both
SELEXes: 30N7, counter-selected 30N7, 40N7 and counter-selected
40N7. The affinities of each of the 12th round pools ranged from 60
to 400 pM. Ligands were cloned from these pools.
B. Sequences of 2'-F RNA Ligands to L-Selectin
[0241] In Table 16, ligand sequences are shown in standard single
letter code (Cornish-Bowden, 1985 NAR 13: 3021-3030). Fixed region
sequence is shown in lower case letters. By definition, each clone
includes both the evolved sequence and the associated fixed region,
unless specifically stated otherwise. A unique sequence is
operationally defined as one that differs from all others by three
or more nucleotides. Sequences that were isolated more than once
are indicated by the parenthetical number, (n), following the
ligand isolate number.
[0242] The 30N7 and 40N7 SELEX final pools shared a common major
sequence family, even though identical sequences from the two
SELEXes are rare (Table 16). Most ligands (72 of the 92 unique
sequences) from the 30N7 and 40N7 SELEXes contain one of two
related sequence motifs, RYGYGUUUUCRAGY or RYGYGUUWWUCRAGY. These
motifs define family 1. Within the family there are three
subfamilies. Subfamily la ligands (53/66) contain an additional
sequence motif, CUYARRY, one nucleotide 5' to the family 1
consensus motifs. Subfamily 1b (9/66 unique sequences) lacks the
CUYARRY motif. Subfamily 1c (5/66) is also missing the CUYARRY
motif, has an A inserted between the Y and G of consensus YGUU and
lacks the consensus GA base pair. The significance of the sequence
subfamilies is reflected in the postulated secondary structure of
the ligands (Example 25).
[0243] A second family, composed of 5 sequences, has a relatively
well defined consensus: UACUAN.sub.0-1UGURCG . . .
UYCACUAAGN.sub.1-2CCC (Table 16). Family 3 has a short, unreliable
consensus motif (Table 16). In addition, there are approximately 12
orphans or apparently unrelated sequences. Three of the orphan
sequences were recovered at least twice (Table 16).
C. Affinities
[0244] The dissociation constants of representative ligands from
Table 16 are shown in Table 17. These calculations assume two
ligand binding sites per chimera. The affinity of random 2'-F RNA
cannot be reliably determined but is estimated to be approximately
10 .mu.M.
[0245] The dissociation constants range from 34 pM to 315 nM at
37.degree. C. Binding affinity is not expected to be temperature
sensitive since selection was at 37.degree. C. and 2'-F RNA forms
thermal stable structures, but binding has not been tested at lower
temperatures. For the most part, the extreme differences in
affinity may be related to predicted secondary structure (Example
25).
[0246] The observed affinities of the evolved 2'-F RNA ligands
reaffirm our proposition that it is possible to isolate
oligonucleotide ligands with affinities that are several orders of
magnitude greater than that of carbohydrate ligands.
Example 24
Cell Binding Studies
[0247] The ability of full length 2'-F ligands to bind to
L-selectin presented in the context of a cell surface was tested by
competition-flow cytometry experiments with human peripheral blood
lymphocytes. Lymphocytes were stained with 10 nM FITC-conjugated
DNA ligand FITC-LD201T1 (SEQ ID NO: 185) in the presence of
increasing concentrations of unlabeled 2'-F ligands as described in
Example 22, paragraph E. Ligands LF1513 (SEQ ID NO: 321), LF1514
(SEQ ID NO: 297), LF1613 (SEQ ID NO: 331) and LF1618 (SEQ ID NO:
351) inhibited the binding of FITC-LD201T1 in a concentration
dependent manner, with complete inhibition observed at competitor
concentrations of 10 to 300 nM. These results demonstrate that the
2'-F ligands are capable of binding cell surface L-selectin and
suggest that the 2'-F ligands and LD201T1 bind the same or
overlapping sites. The affinities of the fluoro ligands, calculated
from the competition curves, range from 0.2 to 25 nM. The affinity
of two of the ligands for L-selectin on human lymphocytes, LF1613
(Kd=0.2 nM) and LF1514 (Kd=0.8 nM), is significantly better than
that of the DNA ligand LD201T1 (Kd=3 nM). The reasonable agreement
between the affinities for purified protein and lymphocyte
L-selectin suggests that binding to lymphocytes is specific for
L-selectin. These data validate the feasibility of using
immobilized, purified protein to isolate ligands against a cell
surface protein.
Example 25
Secondary Structure of High Affinity 2'-F RNA Ligands to
L-Selectin
[0248] In favorable instances, comparative analysis of aligned
sequences allows deduction of secondary structure and
structure-function relationships. If the nucleotides at two
positions in a sequence covary according to Watson-Crick base
pairing rules, then the nucleotides at these positions are apt to
be paired. Nonconserved sequences, especially those that vary in
length are not apt to be directly involved in function, while
highly conserved sequence are likely to be directly involved.
[0249] The deduced secondary structure of family la ligands from
comparative analysis of 21 unique sequences is a hairpin motif
(FIG. 15) consisting of a 4 to 7 nucleotide terminal loop, a 6 base
upper stem and a lower stem of 4 or more base pairs. The consensus
terminal loops are either a UUUU tetraloop or a UUWWU pentaloop.
Hexa- and heptaloops are relatively rare. The upper and lower stems
are delineated by a 7 nucleotide bulge in the 5'-half of the stem.
Four of the six base pairs in the upper stem and all base pairs in
the lower stem are supported by Watson-Crick covariation. Of the
two invariant base pairs in the upper stem, one is the loop closing
GC, while the other is a non-standard GA. The lower stem is most
often 4 or 5 base pairs long but can be extended. While the
sequence of the upper stem is strongly conserved, that of the lower
stem is not, with the possible exception of the YR' base pair
adjacent to the internal bulge. This base pair appears to covary
with the 3' position of the 7 nucleotide bulge in a manner which
minimizes the likelihood of extending the upper stem. Both the
sequence (CUYARRY) and length (7 nt) of the bulge are highly
conserved.
[0250] In terms of comparative analysis, the 7 nucleotide bulge,
the upper stem and the 5' and 3' positions of the terminal loop are
most apt to be directly involved in L-selectin binding.
Specifically, the 5' U and 3' U of the terminal loop, the invariant
GC and GA base pairs of the upper stem and the conserved C, U and A
of the bulge are the mostly likely candidates. The lower stem,
because of its variability in length and sequence, is less likely
to be directly involved. The importance of the bulge for binding is
supported by the poor affinity of ligand LF1512 (SEQ ID NO: 357;
Kd=315 nM); the simplest structure for this ligand is a UUUU
tetraloop and a ten base pair, nearly perfect, consensus stem which
is missing only the 7 nucleotide bulge.
[0251] The deduced secondary structure of family 1b is similar to
that of family 1a, except that the upper stem is usually 7 base
pairs in length and that the single stranded bulge which does not
have a highly conserved consensus is only 4 nucleotide long. This
structure may be an acceptable variation of the 1a secondary
structure with the upper stem's increased length allowing a shorter
bulge; the affinity of ligand LF1511 (SEQ ID NO: 332) is 300
pM.
[0252] Although family 1c has a consensus sequence, GUUUUCNR that
is related to 1a and 1b, a convincing consensus secondary structure
is not evident, perhaps due to insufficient data. The most highly
structured member of the family, LF1618 (SEQ ID NO: 351), permits a
UUUU tetraloop and "upper" stem of 7 base pairs but has neither a
lower stem nor the consensus 7 nucleotide bulge sequence of 1a. The
upper stem differs from those of 1a and 1b in that it has an
unpaired A adjacent to the loop closing G and does not have the
invariant GA base pair of 1a and 1b. The affinity of LF1618 is a
modest 10 nM which suggests that family 1c forms a less successful
structure.
[0253] Predictions of minimal high affinity sequences for family 1
ligands can be made and serve as a partial test of the postulated
secondary structure. Truncates which include only the upper stem
and terminal loop, LF1514T1 (SEQ ID NO: 385) or these two elements
plus the 7 nucleotide bulge sequence, LF1514T2 (SEQ ID NO: 386),
are not expected to bind with high affinity. On the other hand,
there is a reasonable, but not rigorous, expectation that ligands
truncated at the base of the lower consensus stem, LF1514T4 (SEQ ID
NO: 387) and LF1807T4 (SEQ ID NO: 388), will bind with high
affinity. In side by side comparisons, the affinities of LF1514T1
and LF1514T2 for LS-Rg were reduced at least 100-fold in comparison
to full length LD1514 (SEQ ID NO: 297), while the affinity of
LF1514T4 was reduced less than two fold and that of LF1807T4
approximately three-fold. The correspondence between the predicted
and observed truncate affinities supports the postulated secondary
structure.
[0254] Since the ssDNA ligand LD201T1 (SEQ ID NO: 185) and the
adhesion blocking anti-human L-selectin antibody DREG56 are known
to bind to the lectin domain of L-selectin, competition between
radio-labeled LF1807 (SEQ ID NO: 309) and either unlabeled DREG56
or unlabeled LD201T1 can serve to determine if the 2'-F ligands
also bind the lectin domain of purified LS-Rg. In these
experiments, both DREG56 and LD201T1 gave concentration dependent
inhibition of LF1807 binding. Complete inhibition was attained with
300 nM Mab and 1 .mu.M LD201T1. The competitors' affinities of
LS-Rg, calculated from the competition curves, were in good
agreement with their known affinities. These results are consistent
with the premise that LF1807, NX280 and DREG56 have the same or
overlapping binding sites and consequently it is expected that 2'-F
ligands will be antagonists of L-selectin mediated adhesion. These
results also reaffirm the proposition that the SELEX protocol, with
5 mM elution of bound oligonucleotides, preferentially elutes
ligands bound at or near the lectin domain's bound calcium.
Example 26
ssDNA Ligands to Human P-Selectin
[0255] PS-Rg is a chimeric protein in which the lectin, EGF, and
the first two CRD domains of human P-selectin are joined to the Fc
domain of a human G1 immunoglobulin (R. M. Nelson et al., 1993,
supra). Purified chimera is provided by A. Varki. Soluble
P-selectin is purchased from R&D Systems. Unless otherwise
indicated, all materials used in the ssDNA SELEX against the
P-selectin/IgG.sub.1 chimera, PS-Rg, are identical to those of
Examples 7 and 13.
[0256] The SELEX procedure is described in detail in U.S. Pat. No.
5,270,163. The specific strategies and procedures for evolving high
affinity ssDNA antagonists to P-selectin are described in Examples
7 and 13.
Example 27
2'-F RNA Ligands to Human P-Selectin
[0257] The Experimental procedures outlined in this Example were
used to identify 2'-F RNA ligands to human P-selectin as described
in Examples 28-34.
Experimental Procedures
A) Materials
[0258] PS-Rg is a chimeric protein in which the extracellular
domain of human P-selectin is joined to the Fc domain of a human G2
immunoglobulin (Norgard et al., 1993, PNAS 90:1068-1072). ES-Rg and
CD22.beta.-Rg are analogous constructs of E-selectin and CD220
joined to a human G1 immunoglobulin Fc domain (R. M. Nelson et al.,
1993, supra; I. Stamenkovic et al., 1991, Cell 66, 1133-1144) while
LS-Rg has L-selectin joined to an IgG2 Fc domain. Purified chimera
were provided by A. Varki. Soluble P-selectin was purchased from
R&D Systems. Protein A Sepharose 4 Fast Flow beads were
purchased from Pharmacia Biotech. Anti-P-selectin monoclonal
antibodies: G1 was obtained from Centocor. The 2'-F modified CTP
and UTP were prepared according to Pieken et. al. (1991, Science
253:314-317). DNA oligonucleotides were synthesized by Operon. All
other reagents and chemicals were purchased from commercial
sources. Unless otherwise indicated, experiments utilized HSMC
buffer (1 mM CaCl.sub.2, 1 mM MgCl.sub.2, 150 mM NaCl, 20.0 mM
HEPES, pH 7.4).
B) SELEX
[0259] The SELEX procedure is described in detail in U.S. Pat. No.
5,270,163 and elsewhere. The nucleotide sequence of the synthetic
DNA template for the PS-Rg SELEX was randomized at 50 positions.
This variable region was flanked by N8 5' and 3' fixed regions. The
transcript 50N8 has the sequence 5'
gggagacaagaauaaacgcucaa-50N-uucgacaggaggcucacaac- aggc 3' (SEQ ID
NO: 390). All C and U have 2'-F substituted for 2'-OH on the
ribose. The primers for the PCR were the following:
[0260] N8 5' Primer 5' taatacgactcactatagggagacaagaataaacgctcaa 3'
(SEQ ID NO: 197)
[0261] N8 3' Primer 5' gcctgttgtgagcctcctgtcgaa 3' (SEQ ID NO: 198)
The fixed regions include primer annealing sites for PCR and cDNA
synthesis as well as a consensus T7 promoter to allow in vitro
transcription. The initial RNA pool was made by first Klenow
extending 1 nmol of synthetic single stranded DNA and then
transcribing the resulting double stranded molecules with T7 RNA
polymerase. Klenow extension conditions: 3.5 nmols primer 5N8, 1.4
nmols 40N8, 1.times. Klenow Buffer, 0.4 mM each of DATP, dCTP, dGTP
and dTTP in a reaction volume of 1 ml.
[0262] For subsequent rounds, eluted RNA was the template for AMV
reverse transcriptase mediated synthesis of single stranded cDNA.
These single-stranded DNA molecules were converted into
double-stranded transcription templates by PCR amplification. PCR
conditions were 50 mM KCl, 10 mM Tris-Cl, pH 8.3, 7.5 mM
MgCl.sub.2, 1 mM of each dATP, dCTP, dGTP, and dTTP, and 25 U/ml of
Taq DNA polymerase. Transcription reactions contained 0.5 mM DNA
template, 200 nM T7 RNA polymerase, 40 mM Tris-HCl (pH 8.0), 12 mM
MgCl.sub.2, 5 mM DTT, 1 mM spermidine, 4% PEG 8000, 1 mM each of
2'-OH ATP and 2'-OH GTP, 3.3 mM each of 2'-F CTP and 2'-F UTP, and
250 nM .alpha.-.sup.32P 2'-OH ATP.
[0263] The strategy for partitioning PS-Rg/RNA complexes from
unbound RNA is essentially identical to the strategy detailed in
Example 7 for ligands to L-selectin (Table 18).
[0264] In the initial SELEX rounds, which were performed at
37.degree. C., the density of immobilized PS-Rg was 20 pmols/.mu.l
of Protein A Sepharose 4 Fast Flow beads. In later rounds, the
density of PS-Rg was reduced (Table 18), as needed, to increase the
stringency of selection. Beginning with the second round, SELEX was
often done at more than one PS-Rg density. At each round, the
eluted material from only one PS-Rg density was carried
forward.
[0265] Before each round, RNA was batch adsorbed to 100 .mu.l of
protein A sepharose beads for 1 hour in a 2 ml siliconized column.
Unbound RNA and RNA eluted with minimal washing (two volumes) were
combined and used for SELEX input material. For SELEX, extensively
washed, immobilized PS-Rg was batch incubated with pre-adsorbed RNA
for 0.5 to 1 hours in a 2 ml siliconized column with frequent
mixing. Unbound RNA was removed by extensive batch washing (500
.mu.l HSMC/wash). Bound RNA was eluted as two fractions; first,
bound RNA was eluted by incubating and washing columns with 5 mM
EDTA in HSMC without divalent cations; second, the remaining
elutable RNA was removed by incubating and/or washing with 50 mM
EDTA in HSMC without divalents. The percentage of input RNA that
was eluted is recorded in Table 18. In every round, an equal volume
of protein A sepharose beads without PS-Rg was treated identically
to the SELEX beads to determine background binding. All unadsorbed,
wash and eluted fractions were counted in a Beckman LS6500
scintillation counter in order to monitor each round of SELEX.
[0266] The eluted fractions were processed for use in the following
round (Table 18). After precipitating with 300 mM Sodium Acetate pH
7 in ethanol (2.5 volumes), the RNA was resuspended in 80 .mu.l of
H.sub.2O and 40 .mu.l were reverse transcribed into cDNA by AMV
reverse transcriptase at 48.degree. C. for 30 minutes, in 50 mM
Tris-Cl pH (8.3), 60 mM NaCl, 6 mM Mg(OAc).sub.2, 10 mM DTT, 200
pmol DNA primer, 0.4 mM each of dNTPs, and 0.4 unit/.mu.l AMV RT.
Transcripts of the PCR product were used to initiate the next round
of SELEX.
C) Nitrocellulose Filter Binding Assay
[0267] As described in SELEX Patent Applications, a nitrocellulose
filter partitioning method was used to determine the affinity of
RNA ligands for PS-Rg and for other proteins. Filter discs
(nitrocellulose/cellulose acetate mixed matrix, 0.45 .mu.m pore
size, Millipore) were placed on a vacuum manifold and washed with 2
ml of HSMC buffer under vacuum. Reaction mixtures, containing
.sup.32P labeled RNA pools and unlabeled PS-Rg, were incubated in
HSMC for 10-20 min at 4.degree. C., room temperature or 37.degree.
C., filtered, and then immediately washed with 4 ml HSMC at the
same temperature. The filters were air-dried and counted in a
Beckman LS6500 liquid scintillation counter without fluor.
[0268] PS-Rg is a dimeric protein that is the expression product of
a recombinant gene constructed by fusing the DNA sequence that
encodes the extracellular domains of human P-selectin to the DNA
that encodes a human IgG.sub.1 Fc region. For affinity
calculations, one ligand binding site per PS-Rg monomer (two per
dimer) were assumed. The monomer concentration is defined as 2
times the PS-Rg dimer concentration. The equilibrium dissociation
constant, K.sub.d, for an RNA pool or specific ligand is calculated
as described in Example 7, paragraph C.
D) Cloning and Sequencing
[0269] Twelfth round PCR products were re-amplified with primers
which contain either a BamHI or a HinDIII restriction endonuclease
recognition site. Using these restriction sites, the DNA sequences
were inserted directionally into the pUC9 vector. These recombinant
plasmids were transformed into E. coli strain JM109 (Life
Technologies, Gaithersburg, Md.). Plasmid DNA was prepared
according to the alkaline hydrolysis method (PERFECTprep, 5'-3',
Boulder, Colo.). Approximately 50 clones were sequenced using the
Sequenase protocol (Amersham, Arlington Heights, Ill.). The
resulting ligand sequences are shown in Table 19.
E) Boundary Experiments
[0270] The minimal high affinity sequence of individual ligands was
determined by boundary experiments (Tuerk et. al. 1990, J. Mol.
Biol. 213: 749). Individual RNA ligands, .sup.32P-labeled at the
5'-end for the 3' boundary and .sup.32P-labeled at the 3'-end for
the 5' boundary, are hydrolyzed in 50 mM Na2CO3 pH 9 for 8 minutes
at 95.degree. C. The resulting partial hydrolysate contains a
population of end-labeled molecules whose hydrolyzed ends
correspond to each of the purine positions in the full length
molecule. The hydrolysate is incubated with PS-Rg (at
concentrations 5-fold above, below and at the measured Kd for the
ligand). The RNA concentration is significantly lower than the Kd.
The reaction is incubated at room temperature for 30 minutes,
filtered, and then immediately washed with 5 ml HSMC at the same
temperature. The bound RNA is extracted from the filter and then
electrophoresed on an 8% denaturing gel adjacent to hydrolyzed RNA
which has not been incubated with PS-Rg. Analysis is as described
in Tuerk et. al. 1990, J. Mol. Biol. 213: 749.
F) 2'-O-Methyl Substitution Experiments
[0271] In order to decrease the susceptibility of the 2'-F
pyrimidine RNA ligands to nuclease digestion, post-SELEX
modification experiments were performed to identify 2'-OH purines
that are replaceable with 2'-OMe purines without loss of affinity
as described in Green et. al. (1995, J. Mol. Biol. 247: 60-68).
Briefly, seven oligonucleotides were synthesized, each with three
mixed positions. A mixed position is defined as a 2'-OH purine
nucleotide within the RNA which has been synthesized with 2:1 ratio
of 2'-OH:2'-OMe. Since the coupling efficiency of 2'-OH
phosphoramidites is lower than that of 2'-OMes, the resulting RNA
has 25-50% 2'-OH at each mixed position. .sup.32P end-labeled RNA
ligands are then incubated with concentrations of PS-Rg 2-fold
above and 2.5-fold below the Kd of the unmodified ligand at room
temperature for 30 minutes, filtered, and then immediately washed
with 5 ml HSMC at the same temperature. The bound RNA (Selected
RNA) is extracted from the filter and then hydrolyzed with 50 mM
Na.sub.2CO.sub.3 pH 9 for 8 minutes at 95.degree. C. in parallel
with RNA which has not been exposed to binding and filtration
(Unselected RNA). The Selected RNA is then electrophoresed on a 20%
denaturing gel adjacent to Unselected RNA.
[0272] To determine the affect on binding affinity of 2'-OMe
substitution at a particular position, the ratio of intensities of
the Unselected:Selected bands that correspond to the position in
question are calculated. The Unselected:Selected ratio when the
position is mixed is compared to the mean ratio for that position
from experiments in which the position is not mixed. If the
Unselected:Selected ratio of the mixed position is significantly
greater than that when the position is not mixed, 2'-OMe may
increase affinity. Conversely, if the ratio is significantly less,
2'-OMe may decrease affinity. If the ratios are not significantly
different, 2'-OMe substitution has no affect.
G) Cell Binding Studies
[0273] The ability of evolved ligand pools and cloned ligands to
bind to P-selectin presented in the context of a cell surface was
tested in experiments with human platelet suspensions. Whole blood
from normal volunteers was collected in Vacutainer 6457 tubes.
Within 5 minutes of collection, 485 g of blood was stimulated with
15 .mu.l Bio/Data THROMBINEX for 5 minutes at room temperature. A
100 .mu.l aliquot of stimulated blood was transferred to 1 ml of
BB- (140 MM NaCl, 20 mM HEPES pH 7.35, 5 mM KCl, 0.01% NaN.sub.3)
at 4.degree. C. and spun at 735.times.g for 5 minutes. This step
was repeated and the resulting pellet was re-suspended in 1 ml of
BB+ (140 mM NaCl, 20 mM HEPES pH 7.35, 5 mM KCl. 0.01% NaN.sub.3, 1
mM CaCl.sub.2, 1 mM MgCl.sub.2) at 4.degree. C.
[0274] To detect antigen expression, 15 .mu.l BB+ containing FITC
conjugated anti-CD61 or PE conjugated anti-CD62 antibody (Becton
Dickinson) was incubated for 20-30 minutes at 4.degree. C. with 10
.mu.l of platelet suspension. This was diluted to 200 .mu.l with
4.degree. C. BB+ and analyzed on a Becton Dickinson FACSCaliber
using 488 nm excitation and FL1 (530 nm emission) or FL2 (580 rum
emission) with the machine live gated on platelets. Between 1000
and 5000 events in this gate were recorded.
[0275] To detect oligonucleotide ligand binding, 15 .mu.l BB+
containing ligand conjugated to either FITC or biotin was incubated
20-30 minutes at 4.degree. C. with 10 .mu.l platelet suspension.
The FITC-ligand incubations were diluted to 200 .mu.l with BB+ and
analyzed on a FACSCaliber flow cytometer. The biotinylated-ligand
reactions were incubated with streptavidin-phycoerythrin (SA-PE)
(Becton Dickinson) for 20 minutes at 4.degree. C., before dilution
and analysis. Wash steps with 500 .mu.l BB+ and 700.times.g spins
have been used without compromising the quality of the results.
[0276] The specificity of binding to P-selectin (CD62P) expressed
on platelets was tested by competition with the P-selectin specific
blocking monoclonal antibody, G1. Saturability of binding was
tested by self-competition with unlabeled RNA.
H) Inhibition of Selectin Binding to Sialyl-Lewis.sup.X
[0277] The ability of evolved RNA pools or cloned ligands to
inhibit the binding of PS-Rg to sialyl-Lewis.sup.X was tested in
competitive ELISA assays (C. Foxall et al., 1992, supra). For these
assays, the wells of Corning (25801) 96 well microtiter plates were
coated with 100 ng of a sialyl-Lewis.sup.X/BSA conjugate, air dried
overnight, washed with 300 .mu.l of PBS(-) and then blocked with 1%
BSA in HSMC for 60 min at room temperature. RNA ligands were
incubated with PS-Rg in HSMC/1% BSA at room temperature for 15 min.
After removal of the blocking solution, 50 .mu.l of PS-Rg (10 nM)
or a PS-Rg (10 nM)/RNA ligand mix was added to the coated, blocked
wells and incubated at room temperature for 60 minutes. The binding
solution was removed, wells were washed with 300 .mu.l of PBS(-)
and then probed with HRP conjugated anti-human IgG, at room
temperature to quantitate PS-Rg binding. After a 30 minute
incubation at room temperature in the dark with OPD peroxidase
substrate (Sigma P9187), the extent of PS-Rg binding and percent
inhibition was determined from the OD.sub.450.
Example 28
2'-F RNA Ligands to Human P-selectin
A. SELEX
[0278] The starting RNA pool for SELEX, randomized 50N8 (SEQ ID NO:
390), contained approximately 10.sup.15 molecules (1 nmol RNA). The
SELEX protocol is outlined in Table 18. The dissociation constant
of randomized RNA to PS-Rg is estimated to be approximately 2.5
.mu.M. An eight-fold difference was observed in the RNA elution
profiles with 5 mM EDTA from SELEX and background beads for rounds
1 and 2, while the 50 mM elution produced a 30-40 fold excess over
background Table 18. For rounds 1 through 3, the 5 mM and 50 mM
eluted RNAs were pooled and processed for the next round. Beginning
with round 4, only the 5 mM eluate was processed for the following
round. To increase the stringency of selection, the density of
immobilized PS-Rg was reduced five fold in round 2 and again in
round three without greatly reducing the fraction eluted from the
column. The density of immobilized PS-Rg was further reduced
1.6-fold in round 4 and remained at this density until round 8,
with further reductions in protein density at later rounds. The
affinity of the selected pools rapidly increased and the pools
gradually evolved biphasic binding characteristics.
[0279] Binding experiments with 12th round RNA revealed that the
affinity of the evolving pool for P-selectin was not temperature
sensitive. Bulk sequencing of 2nd, 6th, 11th and 12th RNA pools
revealed noticeable non-randomness by round twelve. The 6th round
RNA bound monophasically at 37.degree. C. with a dissociation
constant of approximately 85 nM, while the 11th and 12th round RNAs
bound biphasically with high affinity Kds of approximately 100 and
20 pM, respectively. The binding of all tested pools required
divalent cations. In the absence of divalent cations, the Kds of
the 12th round pools increased to >10 nM. (HSMC, minus
Ca.sup.++/Mg.sup.++, plus 2 mM EDTA). The 12th round pool showed
high specificity for PS-Rg with measured Kd's of 1.2 .mu.M and 4.9
.mu.M for ES-Rg and LS-Rg, respectively.
B. RNA Sequences
[0280] In Table 19, ligand sequences are shown in standard single
letter code (Cornish-Bowden, 1985 NAR 13: 3021-3030). Fixed region
sequence is shown in lower case letters. By definition, each clone
includes both the evolved sequence and the associated fixed region,
unless specifically stated otherwise. From the twelfth round, 21 of
44 sequenced ligands were unique. A unique sequence is
operationally defined as one that differs from all others by three
or more nucleotides. Sequences that were isolated more than once,
are indicated by the parenthetical number, (n), following the
ligand isolate number. These clones fall into five sequence
families (1-5) and a group of two unrelated sequences (Orphans)(SEQ
ID NOs: 199-219).
[0281] Family 1 is defined by 23 ligands from 13 independent
lineages. The consensus sequence is composed of two variably spaced
sequences, CUCAACGAMC and CGCGAG (Table 19). In 11 of 13 ligands
the CUCAA of the consensus is from 5' fixed sequence which
consequently minimizes variability and in turn reduces confidence
in interpreting the importance of CUCAA or the paired GAG (see
Example 27).
[0282] Families 2-5 are each represented by multiple isolates of a
single sequence which precludes determination of consensus
sequences.
D. Affinities
[0283] The dissociation constants for representative ligands,
including all orphans, were determined by nitrocellulose filter
binding experiments and are listed in Table 20. These calculations
assume two binding sites per chimera. The affinity of random RNA is
estimated to be approximately 2.5 .mu.M.
[0284] In general, ligands bind monophasically with dissociation
constants ranging from 15 pM to 450 pM at 37.degree. C. Some of the
highest affinity ligands bind biphasically. Full length ligands of
families 14 show no temperature dependence. The observed affinities
substantiate the proposition that it is possible to isolate
oligonucleotide ligands with affinities that are several orders of
magnitude greater than that of carbohydrate ligands.
Example 29
Specificity of 2'-F RNA Ligands
[0285] The affinity of P-selectin ligands to ES-Rg, LS-Rg and
CD22.beta.-Rg were determined by nitrocellulose partitioning. As
indicated in Table 20, the ligands are highly specific for
P-selectin. In general, a ligand's affinity for ES-Rg and LS-Rg is
at least 10.sup.4-fold lower than for PS-Rg. Binding above
background is not observed for CD22.beta.-Rg at the highest protein
concentration tested (660 nM), indicating that ligands do not bind
the Fc domain of the chimeric constructs nor do they have affinity
for the sialic acid binding site of this unrelated lectin. The
specificity of oligonucleotide ligand binding contrasts sharply
with the binding of cognate carbohydrates by the selectins and
confirms the proposition that SELEX ligands will have greater
specificity than carbohydrate ligands.
Example 30
Inhibition of Binding to sialyl-Lewis.sup.X
[0286] Oligonucleotide ligands, eluted by 2-5 mM EDTA, are expected
to derive part of their binding energy from contacts with the
lectin domain's bound Ca.sup.++ and consequently, are expected to
compete with sialyl-Lewis.sup.X for binding. In competition assays,
the selected oligonucleotide ligands competitively inhibit PS-Rg
binding to immobilized sialyl-Lewis.sup.X with IC50s ranging from 1
to 4 nM (Table 20). Specifically, ligand PF377 (SEQ ID NO: 206) has
an IC50 of approximately 2 nM. Complete inhibition is attained at
10 nM ligand. This result is typical of high affinity ligands and
is reasonable under the experimental conditions. The IC50s of
ligands whose Kds are much lower than the PS-Rg concentration (10
nM) are limited by the protein concentration and are expected to be
approximately one half the PS-Rg concentration. The specificity of
competition is demonstrated by the inability of round 2 RNA
(Kd.about.1 .mu.M) to inhibit PS-Rg binding to immobilized
sialyl-Lewis.sup.X. These data verify that 2'-F RNA ligands are
functional antagonists of PS-Rg.
Example 31
Secondary Structure of High Affinity Ligands
[0287] In favorable instances, comparative analysis of aligned
sequences allows deduction of secondary structure and
structure-function relationships. If the nucleotides at two
positions in a sequence covary according to Watson-Crick base
pairing rules, then the nucleotides at these positions are apt to
be paired. Nonconserved sequences, especially those that vary in
length are not apt to be directly involved in function, while
highly conserved sequences are likely to be directly involved.
[0288] Comparative analysis of the family 1 alignment suggests a
hairpin motif, the stem of which contains three asymmetrical
internal loops (FIG. 16). In the figure, consensus positions are
specified, with invariant nucleotides in bold type. To the right of
the stem is a matrix showing the number of occurrences of
particular base pairs for the positions in the stem that are on the
same line. The matrix shows that 6 of the stem's 9 base pairs are
supported by Watson-Crick covariation. Portions of the two
consensus motifs, CUC and GAG, form the terminus of the stem.
Conclusions regarding a direct role of the terminus in binding are
tempered by the use of fixed sequence (11 of 13 ligands) which
limits variability. The variability of the loop's sequence and
length suggests that it is not directly involved in binding. This
conclusion is reenforced by ligand PF422 (SEQ ID NO: 202) which is
a circular permutation of the consensus motif. Although the loop
that connects the stem's two halves is at the opposite end relative
to other ligands, PF422 binds with high (Kd=172 pM; Table 21)
affinity.
Example 32
Boundary Experiments
[0289] Boundary experiments were performed on a number of
P-selectin ligands as described in Example 27 and the results are
shown in Table 21. The results for family 1 ligands are consistent
with their proposed secondary structure. The composite boundary
species vary in size from 38-90 nucleotides, but are 40-45
nucleotides in family 1. Affinities of these truncated ligands are
shown in Table 22. In general, the truncates lose no more than
10-fold in affinity in comparison to the full length, effectively
inhibit the binding of PS-Rg to sialyl-Lewis.sup.x and maintain
binding specificity for PS-Rg (Table 22). These data validate the
boundary method for identifying the minimal high affinity binding
element of the RNA ligands.
Example 33
Binding of 2'-F RNA Ligands to Human Platelets
[0290] Since the P-selectin ligands were isolated against purified
protein, their ability to bind P-selectin presented in the context
of a cell surface was determined in flow cytometry experiments with
activated human platelets. Platelets were gated by side scatter and
CD61 expression. CD61 is a constitutively expressed antigen on the
surface of both resting and activated platelets. The expression of
P-selectin was monitored with anti-CD62P monoclonal antibody
(Becton Dickinson). The mean fluorescence intensity of activated
platelets, stained with biotintylated-PF377s1 (SEQ ID NO:
223)/SA-PE (Example 27, paragraph G), is 5 times greater than that
of similarly stained resting platelets. In titration experiments,
half maximal fluorescence occurs at approximately 50 pM PF377s1
(EC50) which is consistent with its equilibrium dissociation
constant, 60 pM, for PS-Rg. Binding to platelets is specific by the
criterion that it is saturable. Saturability has been demonstrated
not only by titration but also by competition with unlabeled
PF377s1.
[0291] Binding to platelets is P-selectin specific by the criteria
that 1) oligonucleotides that do not bind PS-Rg do not bind
platelets; 2) that binding of PF377s1 to platelets is divalent
cation dependent; and most importantly 3) that binding is inhibited
by the anti-P-selectin adhesion blocking monoclonal antibody G1,
but not by an isotype control antibody. These data validate the
feasibility of using immobilized, purified protein to isolate
highly specific ligands against a cell surface P-selectin.
Example 34
2'-O-Methyl Substitution Experiments
[0292] 2'-OMe purine substitutions were performed on ligand PF377s1
(SEQ ID NO: 223) as described in Example 27 paragraph F and the
results are shown in Table 23. The data indicate that 2'-OMe
purines at positions 7-9, 15, 27, 28 and 31 enhance binding while
substitutions at positions 13, 14, 16, 18, 21 22, 24, and 30 have
little or no affect on affinity. Thus it appears that up to 15
positions may be substituted with only slight losses in affinity.
In partial confirmation of this expectation, the affinity of 377s1
simultaneously substituted with 2'-OMe purines at 11 positions
(PF377M6, SEQ ID NO: 235) is 250 pM (Table 22).
Example 35
2'-NH.sub.2 RNA Ligands to Human P-Selectin
[0293] The experimental procedures described in this Example are
used in Examples 36-38 to isolate and characterize 2'-NH.sub.2 RNA
ligands to human P-selectin.
Experimental Procedures
A) Materials
[0294] Unless otherwise indicated, all materials used in the
2'-NH.sub.2 RNA SELEX against the P-selectin/IgG.sub.1 chimera,
PS-Rg, were identical to those of Example 27. The 2'-NH.sub.2
modified CTP and UTP were prepared according to Pieken et. al.
(1991, Science 253:314-317). The buffer for SELEX experiments was 1
mM CaCl.sub.2, 1 mM MgCl.sub.2, 150 mM NaCl, 10.0 mM HEPES, pH
7.4.
B) SELEX
[0295] The SELEX procedure is described in detail in U.S. Pat. No.
5,270,163 and elsewhere. The nucleotide sequence of the synthetic
DNA template for the PS-Rg SELEX was randomized at 50 positions.
This variable region was flanked by N8 5' and 3' fixed regions. The
transcript 50N8 has the sequence 5' gggagacaagaauaaac
gcucaa-50N-uucgacaggaggcucacaa- caggc 3' (SEQ ID NO: 248). All C
and U have 2'-NH.sub.2 substituted for 2'-OH on the ribose. The
primers for the PCR were the following:
[0296] N8 5' Primer 5' taatacgactcactatagggagacaagaataaacgctcaa 3'
(SEQ ID NO: 249)
[0297] N8 3' Primer 5' gcctgttgtgagcctcctgtcgaa 3' (SEQ ID NO:
250). The procedures used to isolate 2'-NH.sub.2 oligonucleotide
ligands to P-selectin are identical to those described 2'-F ligands
in Example 27, except that transcription reactions utilized 1 mM
each, 2'-NH.sub.2-CTP and 2'-NH.sub.2-UTP, in place of 3.3 mM each
2'-F-CTP and 2'-F-UTP.
C) Nitrocellulose Filter Binding Assay
[0298] As described in SELEX Patent Applications and in Example 27,
paragraph C, a nitrocellulose filter partitioning method was used
to determine the affinity of RNA ligands for PS-Rg and for other
proteins. Either a Gibco BRL 96 well manifold, as described in
Example 23 or a 12 well Millipore manifold (Example 7C) was used
for these experiments. Binding data were analyzed as described in
Example 7, paragraph C.
D) Cloning and Sequencing
[0299] Twelfth round PCR products were re-amplified with primers
which contain either a BamHI or a HinDIII restriction endonuclease
recognition site. Approximately 75 ligands were cloned and
sequenced using the procedures described in Example 7, paragraph D.
The resulting sequences are shown in Table 25.
E) Cell Binding Studies
[0300] The ability of evolved ligand pools to bind to P-selectin
presented in the context of a cell surface was tested in flow
cytometry experiments with human platelet suspensions as described
in Example 7, paragraph E.
Example 36
2'-NH.sub.2 RNA Ligands to Human P-Selectin
A. SELEX
[0301] The starting 2'-NH.sub.2 RNA pool for SELEX, randomized 50N8
(SEQ ID NO: 248), contained approximately 1015 molecules (1 nmol
2'-NH.sub.2 RNA). The dissociation constant of randomized RNA to
PS-Rg is estimated to be approximately 6.4 .mu.M. The SELEX
protocol is outlined in Table 24.
[0302] The initial round of SELEX was performed at 37.degree. C.
with an PS-Rg density of 20 pmol/.mu.l of protein A sepharose
beads. Subsequent rounds were all at 37.degree. C. In the first
round there was no signal above background for the 5 mM EDTA
elution, whereas the 50 mM EDTA elution had a signal 7 fold above
background, consequently, the two elutions were combined and
processed for the next round. This scheme was continued through
round 6. Starting with round seven only the 5 mM eluate was
processed for the next round. To increase the stringency of
selection, the density of immobilized PS-Rg was reduced ten fold in
round 6 with further reductions in protein density at later rounds.
Under these conditions a rapid increase in the affinity of the
selected pools was observed.
[0303] Binding experiments with 12th round RNA revealed that the
affinity of the evolving pool for P-selectin was temperature
sensitive despite performing the selection at 37.degree. C., (Kds:
13 pM, 91 pM and 390 pM at 4.degree. C., room temperature and
37.degree. C., respectively). Bulk sequencing of RNA pools
indicated dramatic non-randomness at round 10 with not many visible
changes in round 12. Ligands were cloned and sequenced from round
12.
B. 2'-NH.sub.2 RNA Sequences
[0304] In Table 25, the 2'-NH.sub.2 RNA ligand sequences are shown
in standard single letter code (Cornish-Bowden, 1985 NAR 13:
3021-3030)(SEQ ID NOS: 251-290). The evolved random region is shown
in upper case letters in Table 25. Any portion of the fixed region
is shown in lower case letters. By definition, each clone includes
both the evolved sequence and the associated fixed region, unless
specifically stated otherwise. From the twelfth round, 40/61
sequenced ligands were unique. A unique sequence is operationally
defined as one that differs from all others by three or more
nucleotides. Sequences that were isolated more than once are
indicated by the parenthetical number, (n), following the ligand
isolate number. Ligands from family 1 dominate the final pool
containing 16/61 sequences, which are derived from multiple
lineages. Families 2 and 3 are represented by slight mutational
variations of a single sequence. Sequences labeled as "others" do
not have any obvious similarities. Family 1 is characterized by the
consensus sequence GGGAAGAAGAC (SEQ ID NO: 291).
C. Affinities
[0305] The dissociation constants of representative ligands are
shown in Table 26. These calculations assume two RNA ligand binding
sites per chimera. The affinity of random 2'-NH.sub.2 RNA is
estimated to be approximately 10 .mu.M.
[0306] At 37.degree. C., the dissociation constants range from 60
pM to 50 nM which is at least a l.times.10.sup.3 to l>10.sup.5
fold improvement over randomized 2'-NH.sub.2 RNA (Table 26). There
is a marked temperature sensitivity for Clone PA350 (SEQ ID NO:
252) with an increase in affinity of 6 fold at 4.degree. C. (Table
26). The observed affinities of the evolved 2'-NH.sub.2 ligand
pools reaffirm our proposition that it is possible to isolate
oligonucleotide ligands with affinities that are several orders of
magnitude greater than that of carbohydrate ligands.
Example 37
Specificity of 2'-NH.sub.2 RNA Ligands to P-Selectin
[0307] The affinity of clone PA350 (SEQ ID NO: 252) for LS-Rg and
ES-Rg was determined by nitrocellulose partitioning and the results
shown in Table 26. The ligands are highly specific for P-selectin.
The affinity for ES-Rg is about 600-fold lower and that for LS-Rg
is about 5.times.10.sup.5-fold less than for PS-Rg. Binding above
background is not observed for CD22.beta.-Rg indicating that
ligands neither bind the Fc domain of the chimeric constructs nor
have affinity for unrelated sialic acid binding sites.
[0308] The specificity of oligonucleotide ligand binding contrasts
sharply with the binding of cognate carbohydrates by the selectins
and reconfirms the proposition that SELEX ligands will have greater
specificity than carbohydrate ligands.
Example 38
Cell Binding Studies
[0309] FITC-labeled ligand PA350 (FITC-350) (SEQ ID NO: 252) was
tested for its ability to bind to P-selectin presented in the
context of a platelet cell surface by flow cytometry experiments as
described in Example 23, paragraph G.
[0310] The specificity of FITC-PA350 for binding to P-selectin was
tested by competition experiments in which FITC-PA350 and unlabeled
blocking monoclonal antibody G1 were simultaneously added to
stimulated platelets. G1 effectively competes with FITC-PA350 for
binding to platelets, while an isotype matched control has little
or no effect which demonstrates that FITC-PA350 specifically binds
to P-selectin. The specificity of binding is further verified by
the observation that oligonucleotide binding is saturable; binding
of 10 nM FITC-PA350 is inhibited by 200 nM unlabeled PA350. In
addition, the binding of FITC-PA350 is dependent on divalent
cations; at 10 nM FITC-PA350 activated platelets are not stained in
excess of autofluorescence in the presence of 5 mM EDTA.
[0311] These data validate the feasibility of using immobilized,
purified protein to isolate ligands against a cell surface protein
and the binding specificity of 2'-NH.sub.2 ligands to P-selectin in
the context of a cell surface.
Example 39
Inhibition of P-selectin Binding to Sialyl Lewis.sup.x
[0312] In competition assays, ligands PA341 (SEQ ID NO: 251) and
PA350 (SEQ ID NO: 252) competitively inhibit PS-Rg binding to
immobilized sialyl-Lewis.sup.x with IC50s ranging from 2 to 5 nM
(Table 26). This result is typical of high affinity ligands and is
reasonable under the experimental conditions. The IC50s of ligands
whose Kds are much lower than the PS-Rg concentration (10 nM) are
limited by the protein concentration and are expected to be
approximately one half the PS-Rg concentration. The specificity of
competition is demonstrated by the inability of round 2 RNA
(Kd.about.1 .mu.M) to inhibit PS-Rg binding to immobilized
sialyl-Lewis.sup.x. These data verify that 2-NH.sub.2 RNA ligands
are functional antagonists of P-selectin.
Example 40
2'-NH.sub.2 RNA Ligands to Human E-Selectin
[0313] ES-Rg is a chimeric protein in which the extracellular
domain of human E-selectin is joined to the Fc domain of a human G1
immunoglobulin (R. M. Nelson et al., 1993, supra). Purified chimera
were provided by A. Varki. Unless otherwise indicated, all
materials used in this SELEX are similar to those of Examples 7 and
13.
[0314] The SELEX procedure is described in detail in U.S. Pat. No.
5,270,163 and elsewhere. The rationale and experimental procedures
are the same as those described in Examples 7 and 13.
1TABLE 1 Wheat Germ Agglutinin Selex Gel Total Total Protein Total
RNA Volume Volume % RNA % RNA Kd Round (pmole) (pmole) (.mu.l)
(.mu.l) Eluted Amplified (nM) 1 5,800 2,020 50 276 0.05 0.05
6,000,000 2 5,800 1,070 50 276 0.12 0.12 3 5,800 1,770 50 280 0.21
0.21 4 5,800 900 50 263 3 3 5 5,800 500 50 271 28.5 28.5 600 6a
5,800 1,000 50 282 28.8 6b 580 1,000 5 237 5.7 0.18 400 7 580 940 5
245 12.8 0.87 320 8 580 192 5 265 21.4 0.64 260 9 58 170 0.5 215
3.8 0.06 130 10 58 184 0.5 210 5.2 0.12 94 11 58 180 0.5 210 2.3
0.07 68 Wheat Germ Lectin Sepharose 6 MB, WGA density,
approximately 5 mg/ml of gel or 116 .mu.M. RNA Loading Conditions:
Rounds 1-5, 2 hrs @ room temperature on roller; incubation time
reduced to 1 hr. for Rounds 6-11. RNA Elution Conditions: Rounds
1-5, 200 .mu.l of 2 mM (GlcNAc)3, 15 min. @ room temperature on
roller; 2x 200 .mu.l wash with same buffer. Rounds 6: 200 .mu.l of
0.2 mM (GlcNAc)3, incubated as above; washed sequentially with 200
.mu.l of 0.5, 1, 1.5, 2 and 10 mM (GlcNAc)3. #Rounds 7-8: 200 .mu.l
of 0.2 mM (GlcNAc)3, incubated as in round 6; wash twice with sane
buffer; washed sequentially with 3x 200 .mu.l each, of 0.5, 1.0,
1.5, 2.0 and 10 mM (G1cNAc)3. Rounds 9-11: incubated 15 @ room
temperature in 200 .mu.l of 1 mM (GlcNAc); #washed 2x with 200
.mu.l of same buffer; incubation and washes repeated with 1.5, 2.0
and 10 mM (GlcNAc). % RNA Eluted: percentage of input RNA eluted
with (GlcNAc)3 % RNA Amplified: percentage of input RNA amplified;
Rounds 1-5: entire eluted RNA sample amplified. Rounds 6-11: pooled
2 mM and 10 mM RNA, amplified for subsequent round. Rounds 9-11:
1.5 mM RNA amplified separately.
[0315]
2TABLE 2 Wheat Germ Agglutinin 2' NH.sub.2 RNA Ligands SEQ ID
Ligand NO. SEQUENCE FAMILY 1 11.8 4
AUGGUUGGCCUGGGCGCAGGCUUCGAAGACUCGGCGGGAA CGGGAAUGgcuccgcc 11.4(3) 5
CAGGCACUG AAAACUCGGCGGGAA CG AAAG UAGUGCCGACUCAGACGCGU 11.10 6
AGUCUGGCCAAAGACUCGGCGGGAA CGUAAAACGGCCAGAAUU 11.35 7
GUAGGAGGUUCCAUCACC AGGACUCGGCGGGAA CG GAA, GGUGAUGS 11.5 8
ACAAGGAUCGAUGGCGAGCCGGGGAGG GCUCGGCGGGAA CG AAA UCUgcuccgcc 11.26 9
UUGGGCAGGCAGAGCGAGACCGGGGGCUCGGCGGGAA CG GAACAGGAAUcgcuccgcc 11.19
10 AAGGGAUGGGAUUGGGACGAGCGGCC AAGACUCGGCGGGAA CG AAG GGUcgcuccgcc
11.15 11 aaucauacac aagaCUCCGCGGGAA CG AAA
GUGUCAUGGUAGCAAGUCCAAUGGUGGACUCUc 11.34 12 aaucauacac
aagaCUCGGCGGGAA CGUGAA GUGGGUAGGUAGCUGAAGACGGUCUGGGCGCCA 6.8 13
AAGGGAUGGGAUUGGGACGAGCGGCC AAGACUCGGCGGGAA CG AAG GGUCCgcuccgcc 6.9
14 aaucauacaca agaCUCGGCGGGAA CG AAG
UGUGUGAGUAACGAUCACUUGGUACUAAAGCCC 6.23 15 aaucauacac
aagaCUCGGCGGGAAUCG AAA GUGUACUGAAUUAGAACGGUGGGCCUGCUCAUCGU 6.26 16
aaucauacaca agaCUCGGCGGGAAUCGUAA
UGUGGAUGAUAGCACGAUGGCAGYAGUAGUCGGACCGC 6.14 17
aaucauacacaagaCAGCGGCGG AGUC A GUGAAAGCGUGGGGGGYGCGGGAGGUCUACCCUGAC
CON- 56 AAGACUCGGCGGGAA CG AAA SENSUS: FAMILY 2 11.12 18
CGGCUGUGUGUGGU AGCGUCAUAGUAGGAGUCGUCACGAACCAA GGCgcuccgcc 11.24(2)
19 CGGCUGU GUGGUGUUGGAGCGUCAUAGUAGGAGUCGUCACGAACCAA GGCgcuccgcc
11.27(2) 20 CGAUGCGAGGCAAGAA AUGGAGUCGUUACGAACCC UCUUGCAGUGCGCGc
11.32 21 CGUGCGGAGCAAAUAGGGGAUC AUCGAGUCGU ACGAACCGUUAUCGCcgcuccgcc
11.6 22 CUGGGGAGCAGGAUAUGAGAUGUGCGGGGCA AUGGAGUCGUGACGAACC gcuccgcc
CON- 57 GGAGUCGUGACGAACC SENSUS: FAMILY 3 11.13 23
GUCCGCCCCCAGGGAUGCAACGGGGUGGCUCUAAAA- GGCUUGGCUAA 11.23 24
GAGAAUGAGCAUGGCCGGGGCAGGAAGUGGGUGGC- AACGGAGGCCA 6.3 25
GAUACAGCGCGGGUCUAAAGACCUUGCCCCUAGG AUGCAACGGGGUGCGUCCGCC 6.7 26
UGAAGGGUGGUAAGAGAGAGUCUGAGCUCGUC- CUAGGGAUGCAACGGCACGUCCGCC 6.20 27
CAAACCUGCAGUCGCGCGGUGAAACCUAGGGUUGCAACGGUACAUCGCUGUCGUCCGCC 6.34 28
GUGGACUGGAAUCUUCGAGGACAGGAACGUUCCUAGGGAUGCAACOGACCGUCCGCC 6.35 29
GUGUACCAAUGGAGGCAAUGCUGCGGGAAUGGAGGCCUAGGGAUGCAAC 6.5 30
GUCCCUAGGGAUGCAACGGGCAGCAUUCGCAUAGGAGUAAUCG- GAGGUC 6.16 31
GCCUAGGGAUGCAACGGCGAAUGGAUAGCGAUGUCGUGGACAGCCAGGU 6.19 32
AUCGAACCUAGGGAUOCAACGGUGAAGGUUGUGAGGAUUCGCCAUUAGGC 6.21 33
GCUAGGGAUGCCGCAGAAUGGUCGCGGAUGU- AAUAGGUGAAGAUUGUUGC 6.25 34
GGACCUAGGGAUGCAACGGUCCGACCUUGAUGCGCGGGUGUCCAAGCUAC 6.33 35
AAGGGAGGAGCUAGAGAGGGAAAGGUUACUACGCGCCAGAAUAGGAUGU CON- 58
CCUAGGGAUGCAACGG SENSUS: FAMILY 4 11.2 36 CCAACGUA CAUCGCGAGCUGGUG
GAGAGUUCAUGA GGGUGUUACGGGGU 11.33 37 CCCAACGUGUCAUCGCGAGCUGGCG
GAGAGUUCAUG GGGU UACGGGU 11.28 38 GUUGCUGCGAGCUGGGGGCGGCGA
GAAGGUAGGCGGUCCGAGUGUU CGAAU 11.7(4) 39 aCUGGCAAGRAGUGCGUGAGGGUAC-
GUUAG GGGUGUU UGGGCCGAUCGCAU CON- 59 RCUGG GAGRGU GGGUGUU SENSUS:
FAMILY 5 11.20(5) 40 UUGGUCGUACUGGACAGAGCCGUGGUAGAGGGAUUGGGACAAAG-
UGUCA FAMILY 6 6.15 41
UGUGAGAAAGUGGCCAACUUUAGGACGUCGGUGGACUGYGCGGGUAGGCUC 6.28 42
CAGGCAGAUGUGUCUGAGUUCGUCGGAGUA GACGUCGGUGGAC GCGGAAC CON- 60
UGUGNNNNAGUNNNNNNNNNUA GACGUCGGUGGACNNNGCGG SENSUS: FAMILY 7 6.24
43 UGUGAUUAGGCAGUUGCAGCCGCC GU GCGGAGACGU GA CUCGAG GAUUC 6.27 44
UGCCGGUGGAAAGGCGGGUAGGU GA CCCGAG GAUUCCUACCAAGCCAU 11.3 45
GAGGUGRA UGGGAGAGUGGAGCCCGGGUGACUCGAGGAUUCCCGU CON- 61 GGGNNNGU GA
CYCGRG GAYUC SENSUS: FAMILY 8 6.2 46
GUCAUGCUGUGGCUGAACAUACUGGUGAAAGUUCAGUAGGGUGGAUACAgcuccgcc 6.6(2) 47
CCGGGGAUGGUGAGUCGGGCAGUGUGACCGAACUGGUGCCCGCUGAGAgcucc CON- 62
UGANCNNACUGGUGNNNGNGNAG SENSUS: FAMILY 9 6.11 48 ACACUAACCAGGUCUCU
GAACGCGGGAC GGAGGUG UGGGCGAGGUGGAA 6.13 49
CCGUCUCCCGAGAACCAGGCAGAGGACGUGCUG- AAGGAGCUG CAUCUAGAA 6.17 50
CCGUCUCC GAGAACCAGGCAGAGGAGGUGCUGAAGGRGCUGGCAUCUACAA CON- 63 GUCUCY
GAACNNGGNA GGANGUGNUG GAGNUG SENSUS: ORPHANS 6.1 51
CCCGCACAUAAUGUAGGGAACAAUGUUAUGGCGGAAUUGAUAAC- CGGU 6.4 52
CGAUGUUAGCGCCUCCGGGAGAGGUUAGGGUCGUGCGGNAAGAGUGAG- GU 6.18 53
GGUACGGGCGAGACGAGAUGGACUUAUAGGUCGAUGAACGGGUAGCAGC- UC 11.30 54
CGGUUGCUGAACAGAACGUGAGUCUUGGUGAGUCGCACAGAUUGUCCU 11.29 55
ACUGAGUAAGGUCUGGCGUGGCAUUAGGUUAGUGGGAGGCUUGGAGUAGc
[0316]
3TABLE 3 Dissociation Constants of RNA Ligands to WGA Ligand SEQ ID
NO: Kd Family 1 11.8 4 9.2 nM 11.4 5 32 nM 11.35 7 90 nM 11.5 8 44
nM 11.26 9 38 nM 11.19 10 22 nM 11.15 11 54 nM 11.34 12 92 nM 6.8
13 11 nM 6.9 14 396 nM 6.23 15 824 nM 6.14 17 <5% Family 2 11.12
18 15.2 nM 11.24 19 19.4 nM 11.27 20 30 nM 11.32 21 274 nM 11.6 22
702 nM Family 3 11.13 23 <5% 11.23 24 <5% 6.3 25 120 nM 6.2
27 <5% 6.34 28 <5% 6.35 29 <5% 6.5 30 678 nM 6.16 31
<5% 6.19 32 74 nM Family 4 11.2 36 62 nM 11.33 37 <5% 11.28
38 9.2 nM 11.7 39 16 nM Family 5 11.2 40 1.4 nM Family 7 6.27 44 56
nM 11.3 45 410 nM Family 8 6.6 47 <5% Family 9 6.11 48 <5%
Orphans 11.3 54 56 nM 11.29 55 32 nM The Kds of ligands that show
<5% binding at 1 .mu.M WGA is estimated to be >20 .mu.M.
[0317]
4TABLE 4 Specificity of RNA Ligands to WGA Kds for
N-acetyl-glucosamine Binding Lectins Ligand 6.8 Ligand 11.20 Ligand
11.24 LECTIN (SEQ ID NO: 13) (SEQ ID NO: 40) (SEQ ID NO: 19)
Triticum 11.4 nM 1.4 nM 19.2 nM vulgare (WGA) Canavalia <5%*
<5%* <5%* ensiformis (Con A)** Datura <5%* 11.2 .mu.M
<5%* stramonium Ulex europaeus 4.4 .mu.M 2.2 .mu.M <5%*
(UEA-II) *Less than 5% binding at 1 .mu.M protein; estimated Kd
>20 .mu.M **succinylated Con A
[0318]
5TABLE 5 INHIBITION OF RNA LIGAND BINDING TO WHEAT GERM AGGULTININ
Ligand SEQ ID NO: Competitor IC.sub.50 (.mu.M) Max Inhib K.sub.c
(.mu.M) 6.8 13 (GlcNAc).sub.3 95 >95% 10.9 11.20 40
(GlcNAc).sub.3 120 >95% 8.4 11.24 19 (GlcNAc).sub.3 120 >95%
19.4 K.sub.c is the dissociation constant of (GlcNAc).sub.3
calculated from these data, assuming competitive inhibition and two
RNA ligand binding sites per dimer.
[0319]
6TABLE 6 INHIBITION OF WGA MEDIATED AGGLUTINATION OF SHEEP
ERYTHROCYTES Inhibitory Concentration (.mu.M) Inhibitor SEQ ID NO:
Complete Partial 6.8 13 0.5 0.12 11.20 40 0.5 0.12 11.24 19 * 2
(GlcNAc).sub.3 8 2 GlcNAc 780 200 * Complete inhibition of
agglutination by ligand 11.24 was not observed in this
experiment.
[0320]
7TABLE 7a L-Selectin 2'NH.sub.2-RNA SELEX at 4.degree. C. % 5 mM %
50 mM Total Total EDTA EDTA SELEX RNA Protein RNA:LS- Bead Total
Eluted Eluted Round # pmoles pmoles Rg Ratio Volume Volume RNA RNA
Kd(nM) Rnd 0 10,000 Rnd 1 1060 167.0 6.3 10 .mu.L .about.100 .mu.L
0.498 0.301 Rnd 2 962 167.0 5.8 10 .mu.L .about.100 .mu.L 0.306
0.114 Rnd 3 509 167.0 3.0 10 .mu.L .about.100 .mu.L 1.480 0.713 Rnd
4 407 167.0 2.4 10 .mu.L .about.100 .mu.L 5.010 1.596 434 Rnd 5 429
167.0 2.6 10 .mu.L .about.100 .mu.L 8.357 7.047 439 16.7 26.3 10
.mu.L .about.100 .mu.L 0.984 0.492 133 Rnd 6 452 167.0 2.7 10 .mu.L
.about.100 .mu.L 7.409 6.579 46 16.7 2.8 10 .mu.L .about.100 .mu.L
3.468 1.312 37 Rnd 7 43 16.7 2.6 10 .mu.L .about.100 .mu.L 8.679
2.430 44 16.7 2.6 10 .mu.L .about.100 .mu.L 7.539 2.358 22 4.2 5.2
10 .mu.L .about.100 .mu.L 2.748 1.298 Rnd 8 43 16.7 2.6 10 .mu.L
.about.100 .mu.L 8.139 1.393 33 23 4.2 5.5 10 .mu.L .about.100
.mu.L 2.754 0.516 Rnd 9 23 4.2 5.5 10 .mu.L .about.100 .mu.L 4.352
0.761 Rnd 10 21 4.2 5.0 10 .mu.L .about.100 .mu.L 6.820 1.123 13 23
8.4 2.7 50 .mu.L .about.150 .mu.L 14.756 1.934 Rnd 11 30 10.5 2.9
250 .mu.L .about.500 .mu.L 0.707 0.033 Rnd 12 12 10.5 1.1 250 .mu.L
.about.500 .mu.L 3.283 0.137 Rnd 13 7 1 7 250 .mu.L .about.500
.mu.L 4.188 0.136 0.3 Rnd 14 9 1 9 250 .mu.L .about.500 .mu.L 4.817
0.438 0.7 L-Selectin Rg was immobilized on Protein A Sepharose 4
Fast Flow. Protein A density is approximately 6 mg/ml drained gel
(143 .mu.M). RNA Loading Conditions: All selections were carried
out in the cold room. The RNA used in each selection was first
incubated for 30 minutes with 100 .mu.L Protein A Sepharose in the
cold room on a roller. Only RNA which flowed through this column
was used on the LS-Rg selection column. The RNA was incubated on
the selection column for 90 minutes on a roller before being washed
extensively with binding buffer (20 mM HEPES pH 7.4 150 mM NaCl, 1
mM MgCl.sub.2, 1 mM CaCl.sub.2.) RNA Elution Conditions: RNA was
eluted by incubating the extensively-washed columns in 100 .mu.L of
HEPES buffered EDTA (pH 7.4) for 30 minutes on a roller followed by
three 100 .mu.L HEPES buffered EDTA washes.
[0321]
8TABLE 7b L-Selectin 2'NH.sub.2-RNA SELEX at Room Temperature % 5
mM % 50 mM Total Total EDTA EDTA SELEX RNA Protein RNA:LS- Bead
Total Eluted Eluted Round # pmoles pmoles Rg Ratio Volume Volume
RNA RNA Kd (nM) Rnd 7 43 10.0 4.3 10 .mu.L .about.100 .mu.L 1.205
0.463 Rnd 8 35 10 3.5 10 .mu.L .about.100 .mu.L 6.642 0.401 35 10
3.5 10 .mu.L .about.100 .mu.L 5.540 0.391 Rnd 9 24 2.5 9.6 10 .mu.L
.about.100 .mu.L 1.473 0.383 13 Rnd 10 30 6.3 4.9 250 .mu.L
.about.500 .mu.L 0.707 0.033 Rnd 11 12 6.3 1.9 250 .mu.L .about.500
.mu.L 3.283 0.134 Rnd 12 6 0.6 9.4 250 .mu.L .about.500 .mu.L 0.877
0.109 0.3 Rnd 13 1 0.6 1.4 250 .mu.L .about.500 .mu.L 5.496 0.739
0.7 L-Selectin Rg was immobilized on Protein A Sepharose 4 Fast
Flow. Protein A density is approximately 6 mg/ml drained gel (143
.mu.M). RNA Loading Conditions: Selections were carried out at room
temperature. The RNA used in each selection was first incubated for
30 minutes with 100 .mu.L Protein A Sepharose at room temp. Only
RNA which flowed through this column was used on the LS-Rg
selection column. The RNA was incubated on the selection column for
90 minutes on a roller before being washed extensively with binding
buffer (20 mM HEPES pH 7.4 150 mM NaCl, 1 mM MgCl.sub.2, 1 mM
CaCl.sub.2.) RNA Elution Conditions: RNA was eluted by incubating
the extensively-washed columns in 100 .mu.L of HEPES buffered EDTA
(pH 7.4) for 30 minutes on a roller followed by three 100 .mu.L
HEPES buffered EDTA washes.
[0322]
9TABLE 8 L-Selectin 2' NH.sub.2 RNA LIGANDS Ligand SEQ ID NO.
Sequences Family I F13.32(5) 67
CGCGUAUGUGUGAAAGCGUGUGCACGGAGGCGU-CUACAAU 6.60(2) 68
GGCAUUGUGUGAAUAGCUGAUCCCACAGGUAA- CAACAGCA 6.50(3) 69
UAAUGUGUGAAUCAAGCAGUCUGAAUAGAUUAGACAAAAU 6.79 70
AUGUGUGAGUAGCUGAGCCCCCGAGUAUGAWACCUGACUA F14.9 71
AAACCUUGAUGUGUGAUAGAGCAUCCCCCAGGCGACGUAC F14.21 72
UUGAGAUGUGUGAGUACAAGCUCAAAAUCCCGUUGG- AGG F14.25 73
UAGAGGUAGUAUGUGUGGGAGAUGAAAAUAC- UGUGGAAAG F13.48(2) 74
AAAGUUAUGAGUCCGUAUAUCAAGGUCGACAUGUGUGAAU 6.71 75
CACGAAAAACCCGAAUUGGGUCGCCCAUAAGGAUGUGUGA 6.28 76
GUAAAGAGAUCCUAAUGGCUCGCUAGAUGUGAUGUGAAAC CONSENSUS: 118 AUGUGUGA
Family II F14.20(26) 77 UAACAA CAAUCAAGGCGGGUUCACCGCCCCAGUAUGAGUG
F14.12(22) 78 UAACAA CAAUCAAGGCGGGUUYACCGCCCCAGUAUGAGUA F14.11(12)
79 UAACAA CAAUCAAGGCGGGUUYACCGCUCCAGUAUGAGUA F13.45(9) 80 UAACAA
CAAUCAAGGCGGGUUCACCGCCCCAGUAUGAGUG 6.80 81 ACCAAGCAAUCUA
GGUCGAACGCUACA CAUGAAUGACGUc CONSENSUS: 119 CAA CAAUC AUGAGUR
Family III 6.17 82 GAACAUGAAGUAAUCAAAGUCGUACC AAUAUACAGGAAGC 6.49
83 GAACAUGAAGUAAGAC CGUCAC AAUUCGAAUGAUUGAAUA 6.16 84
GAACAUGAAGUAAAA AGUCGACG AAUUAGCUGUAACCAAAA 6.37 85 GAACAUGAAGUAAA
AGUCUG AGUUAGUAAAUUACAGUGAU 6.78 86 GAACUUGAAGUUGA ANUCGCUAA
GGUUAUGGAUUCAAGAUU 6.26 87 AACAUGAAGUAAUA AGUC
GACGUAAUUAGCUGUAACUAAA 6.40 88 AACAUGAAGUAAA AGUCUG
AGUUAGAAAUUACAAGUGAU- F13.57 89 UAACAUAAAGUAGCG
CGUCUGUGAGAGGAAGUGCCUGGAU CONSENSUS: 120 AACAUGAAGUA AGUC ARUUAG
Family IV 6.58 90 AUAGAACCGCAAGGAUAACCUCGACCGUGGUCAACUGAGA 6.69 91
UAAGAACCGCUAGCGCACGAUCAAACAAAGAGAAACAAA- CONSENSUS: 121 AGAACCGCWAG
Family V 6.56 92 UUCUCUCCAAGAACYGAGCGAAUAAACSACCGGASUCACA F13.55 93
UGUCUCUCCUGACUUUUAUUCUUAGUUCGAGCUGUCCUGG CONSENSUS: 122 UCUCUCC
Family VI F14.27 94 CCGUACAUGGUAARCCU CGAAGGAUUCCCGGGAUGAUCCC
F14.53 95 UCCCAGAGUCCCGUGAUGCGAAGAAUCCAUUAGUACCAGA CONSENSUS: 123
CGAAGAAUYC Family VII F13.42 96 GAUGUAAAUGACAAAUGAACCUCGAAAGAUUG-
CACACUC F13.51 97 AUGUAAAUCUAGGCAGAAACGUAGGGCAUCCACCG- CAACGA
CONSENSUS: 124 AUGUAAAU Family VIII 6.33(11) 98
AUAACCCAAGCAGCNUCGAGAAAGAGCUCCAUAGAUGAU- 6.41 99
CAAAGCACGCGUAUGGCAUGAAACUGGCANCCCAAGUAAG CONSENSUS: 125 AACCCAAG
Family IX F13.46(4) 100 CAAAAGGUUGACGUAGCGAAGCUCUCAAAAUGGUCAUGAC
Family X F14.2 101 AAGUGAAGCUAAAGCGGAGGG CCAUUCAGUUUCNCACCA
F14.13(2) 102 AAGUGAAGCUAAAGSGGAGGG CCACUCAGAAACGCACCA Family XI
6.72(2) 103 CACCGCUAAOCAGUGGCAUAGCCCAGUAACCUGUAAGAGA 6.42 104
CAC-GCUAAGCAGUGGCAUAGC---GWAACCUGUAAGAGA Family XII 6.30(5) 105
AGAUUACCAUAACCGCGUAGUCGAAGACAUAUAGUAGCGA Family XIII 6.52(2) 106
ACUCGGGUAGAACGCGACUUGCCACCACUCCCAUAAAGAC Orphans 6.14 107
UCAGAACUCUGCCGCUGUAGACAAAGAGGAGCUUAGCGAA 6.36 108
AAUGAGCAUCGAGAGAGCGCGAACUCAUCGAGCGUACUAA 6.41 119
CAAAGCACGCGUAUGGCAUGAAACUGGCANCCCAAGUAAG 6.44 110
GAUGCAGCAACCUGAAAACGGCGUCCAC- AGGUAAUAACAG 6.70 111
AAACUCGCUACAAACACCCAAUCCUAGAACGUUAUGGAGA 6.76 112
CUAGCAUAGCCACCGGAACAGACAGAUACGAGCACGAUCA 6.89 113
GAUUCGGAGUACUGAAAAACAACCCUCAAAAGUGCAUAGG 6.81 114
GUCCAGGACGGACCGCAGCUGUGAUACA- AUCGACUUACAC 6.70 115
AAACUCGCUACAAACACCCAAUCCUAGAACGUUAUGGAGA F13.59 116
CGGCCCUUAUCGGAGGUCUGCGCCACUAAUUACAUCCAC F14.70 117
UCCAGAGCGUGAAGAUCAACGUCCCGGNGUCGAAGA
[0323]
10TABLE 9 Dissociation Constants of 2' NH.sub.2 RNA Ligands to
L-Selectin* Ligand SEQ ID NO: 4.degree. C. Rm Temp Family I F13.32
67 15.7 nM 14.9 nM F13.48 74 15.9 nM 9.2 nM F14.9 71 8.2 nM 15.4 nM
F14.21 72 2.3 nM 15.9 nM F14.25 73 1300 nM Family II F14.12 78 5.8
pM 1.7 nM (0.68) (0.62) 16.2 nM 94 nM F14.20 77 58 pM 1.0 nM (0.68)
(0.28) 60 nM 48 nM Family III F13.57 89 3.0 nM 75 nM Family V
F13.55 93 62 pM 1.5 nM Family VI F14.53 95 97 pM 142 nM (0.65) 14.5
nM F14.27 94 145 nM Family VII F13.42 96 2.0 nM 5.5 nM F13.51 97
8.8 nM 18 nM Family X F14.2 101 1.8 nM 7.2 nM F14.13 102 1.3 nM
(0.74) 270 nM Orphans F13.59 116 <5% <5% F14.70 117 2.0 nM
7.8 nM (0.75) (0.58) 254 nM 265 nM *Kds of monophasic binding
ligands are indicated by a single number; the high affinity Kd
(ie., K.sub.d1), the mole fraction binding with K.sub.d1, and the
low affinity K.sub.d (ie., K.sub.d2) are presented for biphasic
binding ligands.
[0324]
11TABLE 10 Specificity of 2' NH.sub.2 RNA Ligands to L-Selectin*
Ligand SEQ ID NO: LS-Rg ES-Rg PS-Rg CD22-Rg Family I F13.32 67 15.7
nM <5% 17 .mu.M <5% F13.48 74 15.9 nM <5% 720 nM <5%
F14.9 71 8.2 nM <5% <5% F14.21 72 2.3 nM 2.6 .mu.M <5%
F14.25 73 1300 nM Family II F14.12 78 60 pM 47 nM 910 nM <5%
F14.20 77 58 pM 70 nM <5% (0.68) 60 nM Family III F13.57 89 3.0
nM 2.7 .mu.M <5% Family V F13.55 93 62 pM 49 nM 5.8 .mu.M <5%
Family VI F14.53 95 97 pM 355 nM 5.2 .mu.M <5% (0.65) 14.5 nM
Family VII F13.42 96 2.0 nM 4.4 .mu.M <5% F13.51 97 8.8 nM 2.0
.mu.M Family X F14.2 101 1.8 nM 1.9 .mu.M 450 nM <5% Orphans
F13.59 116 <5% <5% <5% F14.70 117 2.0 nM 5.9 .mu.M <5%
(0.75) 254 nM *Dissociation constants were determined at 4.degree.
C. in HSMC buffer. When <5% binding was observed at the highest
protein concentration, the Kd is estimated to be >20 .mu.M.
[0325]
12TABLE 11 L-SELECTIN ssDNA SELEX Total Total DNA Prot. DNA: Bead %
Eluted % Eluted Kd, nM signal:bkgd Round Temp. pmol pmol Protein
Vol. Total Vol. 2 mM EDTA 50 mM EDTA 4 degrees 2 mM Rnd 0 10,000
Rnd 1 4 930 167 5.6 10 .mu.L .about.100 .mu.L n/a 5.5 50 Rnd 2 25
400 167 2.4 10 .mu.L .about.100 .mu.L n/a 2.19 12 Rnd 3 25 460 167
2.8 10 .mu.L .about.100 .mu.L n/a 2.55 25 Rnd 4 25 100 16.7 6 10
.mu.L .about.100 .mu.L 0.35 0.29 1.3 Rnd 5 25 100 16.7 6 10 .mu.L
.about.100 .mu.L 0.23 0.08 967 3 Rnd 6 25 1000 16.7 60 10 .mu.L
.about.100 .mu.L 1.42 0.38 4 Rnd 7 25 100 16.7 6 10 .mu.L
.about.100 .mu.L 6.9 0.93 60 18 Rnd 8 37 100 16.7 6 10 .mu.L
.about.100 .mu.L 1.9 0.31 9 Rnd 9 25 10 1.67 6 10 .mu.L .about.100
.mu.L 0.5 0.16 2.1 1.6 Rnd 10 25 10 1.67 6 10 .mu.L .about.100
.mu.L 2.2 0.57 5 Rnd 11 25 2.5 0.42 6 10 .mu.L .about.100 .mu.L
0.37 0.07 1.3 @ 25.degree. C. 8 Rnd 12 25 2.5 0.42 6 10 .mu.L
.about.100 .mu.L 0.86 0.13 11 Rnd 13 37 2.5 0.42 6 10 .mu.L
.about.100 .mu.L 0.7 0.35 0.44 @ 25.degree. C. 5 Rnd 14 25 5 0.84 6
50 .mu.L .about.100 .mu.L 2.8 0.76 4 Rnd 15 25 1.25 0.21 6 50 .mu.L
.about.100 .mu.L 1.7 0.5 0.16 @ 25.degree. C. 7 Binding Buffer,
Rounds 1-9 10 mM HEPES, pH at room temp w/NaOH to 7.4 100 mM NaCl 1
mM MgCl2 1 mM CaCl2 5 mM KCl Elution Buffers: replace divalent
cations with EDTA
[0326]
13TABLE 12 L-Selectin ssDNA Ligands Ligand SEQ ID NO SEQUENCE
Family 1 LD204(3) 129 GGAACACGTGAGGTTTAC AAGGCACTCGAC GTAAACACTT
LD145 130 CCCCGAAGAACATTTTAC AAGGTGCTAAAC GTAAAATCAG LD183(2) 131
GGCATCCCTGAGTCATTAC AAGGTTCTTAAC GTAATGTAC LD230(2) 132
TGCACACCTGAGGGTTAC AAGGCGCTAGAC GTAACCTCTC LD208(7) 133 CACGTTTC
AAGGGGTTACAC GAAACGATTCACTCCTTGGC LD227(5) 134 CGGACATGAGCGTTAC
AAGGTGCTAAAC GTAACGTACTT LD112 135 CGCATCCACATAGTTC AAGGGGCPACAC
GAAATATGCA LD137 136 TACCCCTTGgGCCTCATAGAC AAGGTCTTAAAC GTTAGC
LD179(2) 137 CACATGCCTGACGCGGTAC AAGGCCTGG AC GTAACGTTG LD182 138
TAGTGCTCCACGTATTC AAGGTGCTAAAC GAAGACGGCCT LD190 139 AGCGATGC
AAGGGGCTACAC GCAACGATTTAGATGCTCT LD193(2) 140 CCAGGAGCACAGTAC
AAGGTGTTAAAC GTAATGTCTGGT LD199 141 ACCACACCTGGGCGGTAC AAGGAGTTATCC
GTAACGTGT LD201(2) 142 CAAGGTAACCAGTAC AAGGTGCTAAAC GTAATGGCTTCG
LD203 143 ACCCCCGACCCGAGTAC AAGGCATTCGAC GTAATCTGGT LD207 144
CAGTAC AAGGTGTTAAAC GTAATGCCGATCGAGTTGTAT LD216 145 ACAACGAGTAC
AAGGAGATAGAC GTAATCGGCGCAGGTATC LD233(5) 146 CACGACAGAGAAC
AAGGCGTTAGAC GTTATCCGACCACG LD191 147 AGGGAGAAC AAGGTGCTAAAC
GTTTATCTACACTTCACCT LD128(3) 148 AGGACC AAGGTGTTAAAC
GGCTCCCCTGGCTATGCCTCTT LD111(2) 149 gcTACAC AAGGTGCTAAAC
GTAGAGCCAGATCCGATCTGAGC LD139 150 GGAC AAGGCACTCGAC
GTAGTTTATAACTCCCTCCGGgCC LD237 151 gcTACAC AAGGGGCCAAAC
GGAAGCCAGACGCGGATCTGACA LD173 152 CGGCTATAC NNGGTGCTAAAC
GCAGAGACTCGATCAACA LD209 153 GAGTAGCC AAGGCGTTAGAC
GGAGGGGGAATGGAAGCTTG LD221 154 GAGTAGCC AAGGCGTTAGAC GGAGGGGGAATGG
LD108 155 GAGTAGCC AAGGCGTTAGAC GGAGGGGGAATGTGAGCACA LD141 156
TAGCTCCACACAC AASSCGCRGCAC ATAGGGGATATCTGG LD539 175
CGGCAGGGCACTAAC AAGGTGTTAAAC GTTACGGATGCC LD547 176
TGCACACCGGCCCACCCGGAC AAGGCGCTAGAC GAAATGACTCTGTTCTG LD516 177
GACGAAGAGGCC AAGGTGATAACC GGAGTTTCCGTCCGC LD543 178
AAGGACTTAGCTATCC AAGGCACTCGAC GAAGAGCCCGA LD545 179 ATGCCCAGTTC
AAGGTTCTGACC GAAATGACTCTGTTCTG Truncates LD201T1 185
tagCCAAGGTAACCAGTAC AAGGTGCTAAAC GTAATGGCTTCGgcttac LD201T3 186
GTAACCAGTAC AAGGTGCTAAAC GTAATGGCTTCGgcttac LD201T4 187 CCAGTAC
AAGGTCCTAAAC GTAATGG LD201T10 188 CGCGGTAACCAGTAC AAGGTGCTAAAC
GTAATGGCGCG LD201T12 189 GCGGTAACCAGTAC AAGGTGCTAAAC GTAATGGCGC
LD227t5 190 ACATGAGCGTTAC AAGGTGCTAAAC GTAACGTACTTgcttactctcatgt
LD227x1 191 cgcGCGTTAC AAGGTGCTAAAC GTAACGTACTTgcttactcgcg LD227t1
192 GCGTTAC AAGGTGCTAAAC GTAACGT NX288 193
dt.sub.atagCCAAGGTAACCAGTAC AAGGTGCTAAAC
GTAATGGCTTTCGgcttact[3'3']t NX303 196 dt.sub.aCCAGTAc AAGGTGCTAAAC
GTAATGGt[3'3']t Consensus: 181 TAC AAGCYGYTAVAC GTA Family 2
LD181(3) 157 CAT CAAGGACTTTGCCCGAAACCCTAGGTTCACG TGTGGG Family 4
LD174(2) 158 CATTCACCATGGCCCCTTCCTACGTATGTTCTGCGGGTG LD122 159
GCAACGTGGCCCCGTT TAGCTCATTTGACCGTTCCATCCG LD239 160
CCACAGACAATCGCAGTCCCCGTG TAGCTCTGGGTGTCT LD533 180 GCAGCGTGGCCCTGTT
TAGCTCATTTGACCGTTCCATCCG Truncates LD174t1 194
tagCCATTCACCATGGCCCCTTCCTACGTATGTTCTGCGGGTGgctta Consensus: 182
GGCCCCGT Family 5 LD109 161 CCACCGTGATGCACGATACATGAGGGTGTGTCAGCGCAT
LD127 162 CGAGGTAGTCGTTATAGGGTGCGCACCACACACAGCGGTRG Consensus: 183
RCACGAYACA Family 6 LD196 163
TGGCCGTACGGGCCGTGCACCCACTTACCTGGGAAGTGA LD229 164
CTCTGCTTACCTCATGTAGTTCCAAGCTTGGCGTAATCATG Truncate LD196t1 195
agcTGGCGGTACGGGCCGTGCACCCACTTACCTGGGAAGTGAgctta Consensus: 184
CTTACCT Family 7 LD206(2) 165 AGCCTTGT ACGGGGTTACAC
ACAACGATTTAGATCCTCT Orphans LD214 166 TGATGCGACTTTAGTCGAACGTTA-
CTGGGGCTCAGAGGACA LD102 167 CGAGGATCTGATACTTATTGAACATAM-
CCGCACNCAGGCTT LD530 168 CGATCGTGTGTCATGCTACCTACGATCTGA- CTA LD504
169 GCACACAAGTCAAGCATGCGACCTTCAACCATCGACCCGA LD509 170
ATGCCAGTGCAGGCTTCCATCCATCAGTCTGACANNNNNN LD523 171
CACTTCGGCTCTACTCCACCTCGGTCCTCCACTCCACAG- LD527 172
CGCTAACTGACCCTCGATCCCCCCAAGCCATCCTCATCGC LD541 173
ATCTGACTAGCTCGGCGAGAGTACCCGCTCATGGCTTCGGCGAATGCCCT LD548 174
TCCTGAGACCTTACAATAGGCTGCGGTACTGCAACGTGGA
[0327]
14TABLE 13 Dissociation Constants of ssDNA Ligands to L-Selectin
Room Ligand SEQ ID NO: Temperature 37.degree. C. Family 1 LD111 149
330 pM 11.8 nM LD128 148 310 pM 1.8 nM LD108 155 160 pM 8.5 nM
LD112 135 300 pM 23.2 nM LD137 136 520 pM 0.65 nM LD139 150 210 pM
6.8 nM LD145 130 920 pM 8.8 nM LD179 137 180 pM 590 pM LD182 138
130 pM 2.0 nM LD183 131 170 pM 1.0 nM LD193 140 88 pM 970 pM LD201
142 110 pM 1.2 nM LD204 129 100 pM 3.7 nM LD208 155 110 pM 380 pM
LD227 134 43 pM 160 pM LD230 132 57 pM 260 pM LD233 146 110 nM 380
pM Family 2 LD181 157 84 pM 1.8 nM Family 4 LD122 159 1.8 nM 2.1 nM
LD174 158 43 pM 370 pM LD239 160 170 pM 1.6 nM Family 5 LD109 161
190 pM 9.6 nM LD127 162 1.0 nM 890 pM Family 6 LD196 163 130 pM 3.4
nM Family 7 LD206 165 330 pM 6.0 nM Orphans LD102 167 not
determined 7.9 nM LD214 166 660 pM 8.4 nM Round 15 160 pM 660 pM
Pool LD201T1* 4.8 nM LD201T3* 43 nM *LD201T1 and LD201T3 were made
by solid state synthesis; the Kd of the synthetic full length LD201
control was 3.8 nM while that of enzymatically synthesized LD201
was 1.8 nM.
[0328]
15TABLE 14 Specificities of ssDNA Ligands to L-Selectin* Ligand SEQ
ID NO: LS-Rg ES-Rg PS-Rg Family 1 LD111 149 1.1 nM 1.2 .mu.M 840 nM
LD201 142 110 nM 37 nM 1.0 .mu.M LD204 129 450 pM 1.5 .mu.M 2.9
.mu.M LD227 134 64 pM 33 nM 560 nM LD230 132 44 pM 19 nM 600 nM
LD233 146 120 pM 39 nM 420 nM Family 2 LD181 157 200 pM 37 nM 1.6
.mu.M Family 4 LD122 159 340 pM 400 nM 420 nM LD174 158 46 pM 28 nM
380 nM Family 5 LD127 162 250 pM 1.3 .mu.M 780 nM Family 6 LD196
163 220 pM 50 nM 3.4 .mu.M Family 7 LD206 165 120 pM 100 nM 600 nM
*Kds were determined at room temperature. In assays with 700 nM
CD22 .beta.-Rg and 1.4 .mu.M WGA less than 1% and 3% binding,
respectively, was observed for all ligands suggesting that the
dissociation constants are greater than 100 .mu.M for these
proteins.
[0329]
16TABLE 15 Summary of Selection Conditions and Results from 2'F RNA
Human L-selectin SELEXes Total Total Temp, % Bound % 5 mM EDTA
SELEX RNA Protein Time, LS-Rg EDTA Signal/ Round pmoles pmoles Vol.
Sites Eluted Bkgnd Kd(nM) 30n7 2'Fluro SELEX 1 630 100 37.degree.
C. 15' 10 .mu.l 0.7 0.1 20 2 656 100 37.degree. C. 15' 10 .mu.l 2.8
0.4 24 3 608 100 37.degree. C. 15' 10 .mu.l 11.6 1.9 68 10000 4 193
20 37.degree. C. 15' 10 .mu.l 7.4 0.8 24 5 193 20 37.degree. C. 15'
10 .mu.l 19.7 2.1 17 850 6 86 10 37.degree. C. 15' 10 .mu.l 15.7
1.9 8 360 7 17 2 37.degree. C. 15' 10 .mu.l 12.1 1.4 3 8 17 2
37.degree. C. 15' 10 .mu.l 55.1 6.6 2 9 19 2 37.degree. C. 15' 10
.mu.l 40.1 4.2 4 10 18 2 37.degree. C. 15' 10 .mu.l 28.4 3.3 3 3 11
103 12.5 37.degree. C. 15' 50 .mu.l 647.7 8.3 65 11 27 2.5
37.degree. C. 15' 50 .mu.l 63.1 5.9 3 0.5 12 89 5 37.degree. C. 15'
50 .mu.l 53.2 3.0 7 12 79 5 37.degree. C. 15' 50 .mu.l 54.8 3.5 65
0.4 40n7 2'Fluro SELEX 1 677 100 37.degree. C. 15' 10 .mu.l 1.8 0.3
31 2 659 100 37.degree. C. 15' 10 .mu.l 5.8 0.9 19 3 499 100
37.degree. C. 15' 10 .mu.l 9.6 1.9 25 10000 4 187 20 37.degree. C.
15' 10 .mu.l 4.3 0.5 7 5 179 20 37.degree. C. 15' 10 .mu.l 19.7 2.2
8 1024 6 89 10 37.degree. C. 15' 10 .mu.l 17.7 2.0 12 240 7 19 2
37.degree. C. 15' 10 .mu.l 17.3 1.8 2 8 17 2 37.degree. C. 15' 10
.mu.l 78.9 10.4 5 9 19 2 37.degree. C. 15' 10 .mu.l 36.5 4.1 3 10
18 2 37.degree. C. 15' 10 .mu.l 14.1 2.3 2 0.9 11 99 12.5
37.degree. C. 15' 50 .mu.l 60.3 7.7 16 11 22 2.5 37.degree. C. 15'
50 .mu.l 90.1 10.4 18 0.3 12 89 5 37.degree. C. 15' 50 .mu.l 53.2
3.0 7 12 92 5 37.degree. C. 15' 50 .mu.l 92.2 5.0 80 0.1 30n7
Primer Competition Counter-SELEX 1 168 20 37.degree. C.15' 100
.mu.l 2.1 0.25 6 2 189 20 37.degree. C.15' 100 .mu.l 15.4 1.62 119
3 185 20 37.degree. C.15' 100 .mu.l 9.2 0.99 66 2 4 95 5 37.degree.
C.15' 100 .mu.l 44.0 2.33 6 0.3 5 100 5 37.degree. C.15' 100 .mu.l
29.0 1.43 43 5 104 5 37.degree. C.15' 100 .mu.l 36.0 1.70 24 0.4
40n7 Primer Competition Counter-SELEX 1 155 20 37.degree. C.15' 100
.mu.l 1.9 0.25 5 2 184 20 37.degree. C.15' 100 .mu.l 26.8 2.92 172
3 117 20 37.degree. C.15' 100 .mu.l 12.9 2.21 78 2 4 93 5
37.degree. C.15' 100 .mu.l 46.0 2.43 3 0.2 5 93 5 37.degree. C.15'
100 .mu.l 37.0 2.00 52 5 94 5 37.degree. C.15' 100 .mu.l 42.0 2.25
15 0.06
[0330]
17TABLE 16 L-Selectin 2' F Ligands Sequences SEQ ID Ligand Sequence
NO. Family 1a LF1518 gggaggacgau gcggG CAAAUUG CAUGCG UU-UU--
CGAGUG CUUGC UcagacGacucgcccga 293 LF1817 gggaggacgaugc ggUG
CUUAAAC AACGCG UGAAU-- CGAGUU CAUC CACUCCUCCU cagacgacucgcccg 294 a
LF1813 gggaggacgaugcggUUAAU UCAGU CUCAAAC GGUGCG UUUAU-- CGAGCC
ACUGA UcwgacgacucgcccgaA 295 LF1822 gggaggacgaugcggCU UAGAG CUCAAAC
GGUGUG ACUUU-- CAAGCC CUCUA UGCCcagacgacucgcccga 296 LF1514
gggaggacgaugc ggUAC CUCAAAU UGCGUG UU-UU-- CAAGCA GUAUc
agacgacucgcccga 297 LF1529 gggaggacgaugcg gACC CUCAAAU AACGUG
UCUUU-- CAAGUU GGUc agacgacucgcccga 298 LF1527(2) gggaggacgaugcg
gACC CUCAAAU AGCGUG CAUUU-- CAAGCU GGUc agacgacucgcccga 299
LF1536(2) gggaAgacgaugc ggCG CUCAAAU AAUGCG UUAAU-- CGAAUU CGCC
cagacgacucgcccga 300 LF1614 gggaggacgaugcggCA AACAAG CUCAAAU GACGUG
UUUUU-- CAAGUC CUUGUU GUcagacgacucgcccga 301 LF1625
gggaggacgaugcggUA GUAAGU CUCAAAU GUUGCG UUUUU-- CGAAAC ACUUAC
AUcaGacgacucgcccga 302 LF1728 gggaggacgaugc ggAGA CUCAAAU GGUGUG
UU-UU-- CAAGCC UCUCC cagUcgacucgcccga 303 LF1729 gggaggacgaugc ggUG
CUCAAAU GAUGCG UUUCU-- CGAAUC CACC cAgacgacucgcccga GG 304 LF1815
gggaggacgaugc ggCCAUCGGU CUUGGGC AACGCG UU-UU-- CGAGUU ACCUAUGGUc
agacgacucgcccga 305 LF1834 gggaggacgaugcggCCAUC GGU CUUGGGC AACGCG
UU-UU-- CGAGUU aCC UACAUcagacgacucgcccga 306 LF1508 gggaggacgaugcg
gGACC CUUAGGC AACGUG UU-UU-- CAAGUU GGUc agacgacucgcccga 307 LF1828
gggaggacgaugcgg ACGUAGCU CUUAGGC AAUGCG UAUUU-- CGAAUU AGCUGUGU
cagacgacucgcccga 308 LF1807 gggaggacgaugc ggAGU CUUAGGC AGCGCG
UU-UU-- CGAGCU ACUCC AUCGCCAGUcagacgacucgcccga 309 LF1825
gggaAgacgaugcgg AAUGCU CUUAGGC AGCGCG UUAAU-- CGAGCU
AGCACAUCCUcagacgacucgcccga 310 LF1855 gggaggacgaugG ggAGU CUUAGGC
AGCGCG UU-UU-- CGAGCU ACUCC AUCGCCAGUcagacgacucgcccga 311 LF1811
gggaggacgaugcgg UAAUCU CUUAGGC AUCGCG UUAAU-- CGAGAU AGAUCACCGU
cagacgacucgcccga 312 LF1626 gggaggacgaugcgg CAAUGUCh CUUAGGC CACGCG
UUAAU-- CGAGCG UGACUGU cagacgacucgcccgag 313 LF1808(3)
gggaggacgaugc ggCAUGGU CUUAGGC GACGCG UUUAUAU CGAGUC ACCAUGCU
cagacgacucgcccga 314 LF1719(2)* gggaggacgaugcgg GAUG CUUAGGC GCCGUG
UU-UU-- CAAGGC CAUc agacgacucgcccga 315 LF1619 gggaggacgaugcggU
AAUUGU CUUAGGC GCCGUG UU-AU-- CAAGGC ACAAUU UCCCUcagacgacucgcccga
316 LF1620 gggaagacgaugcggCUACUA GUGU CUUAGGC GGAGUG UUUAU-- CAAUCC
ACAC aUcagacgacucgcccga 317 LF1756 gggaggacgaugcggA CUGA CUUAGGC
UGCGCG CACUU-- CGAGCA UcaG acgacucgcccga 318 LF1629(2)
gggaggacgaugcgg UGGUGUGU CUUUGGC ACCGCG UAUUUU- CGAGGU ACACAUca
gacgacucgcccga 319 LF1821 gggaggacgaugcggUG GUGUGU CUUUGGC ACCGCG
UA-UU-- CUCGAG GUACAC AUcagacgacucgcccga 320 LF1513 gggaggacgaugcg
gGCU CUUCAGC AACGUG UU-AU-- CAAGUU AGCCc agacgacucgcccga 321 LF1615
gggaggacgaugc ggCGUAA CUUCAGC GGUGUG UUAAU-- CAAGCC UUACGCC
AUCUcagacgacucgcccga 322 LF1521(2) gaggacgaugc ggGCU CUUAAGC AACGUG
UU-AU-- CAAGUU AGCCc agacgacucgcccga 323 LF1651 gggaggacga ugcggU
CUCAAGC aAUGCG UUUAU-- CGAAUU ACCGUA CGCCUCCGUcagacgacucgccc 324 ga
LF1830 gggaggacgaugcggAA AUCU CUUAAGC AGCGUG UAAAU-- CAAGCU AGAU
CUUCGUcagacgacucgcccga 325 LF1523(2)* gggaggacgaugc ggUU CUUAAGC
AGCGCG UCAAU-- CGAGCU AACC cagacgacucgcccga 326 LF1708**
gggaggacgaugc ggAU CUUAAGC AGCGCG UCAAU-- CGAGCU AACC
cagacgacucgcccgag 327 LF1851 ACAGCUGAUGACCAUGAUUACGCCAAG CUUAAGC
AGCGCG UU-UU-- CGAGCU CAUGUUGGUcagacgacucgcccga 328 LF1610(3)**
gggaggac gaugcggAGGGU CUUAAGC AGUGUG AUAAU-- CAAACU ACUCUCCGUGUc
agacgacucgcccga 329 LF1712 gggaggacgaugc ggGAU CUUAAGC AGUGCG
UUAUU-- CGAACU AUCCc agacgacucgcccga 330 LF1613(3)
gggaggacgaugcggUGC UAUU CUUAAGC GGCGUG UUUUU-- CAAGCC AAUA
UCAUcagacgacucgcccga 331 LF1735 gggaggac gaugcggU CUUAAGC GGCGCG
AUUUU-- CGAGCC ACCGCAUCCUC CGUGcaGacgacucgccc 332 ga LF1731
gggaggacgaugcg gCCU CUUAAGC GUCGUG UUUUU-- CAAGCU GGUc
agacgacucgcccga 333 LF1853 ggga ggacgaugcggAUACCACCU CUUAAGC GACGUG
CAUUU-- CAAGUC AGAUGGucagacgacUcgcccga 334 LF1816
gggaggacgAugcggUGCUA UU CUUAAGC GGCGUG UAAAU-- CAAGCU AG
AUCAUCGUcagacgacucgcccga 335 LF1622(3)* gggaggacgaugcggA ACGACU
CUUAAGC UGUGCG UU-UU-- CGAACA AGUCGU AACUcagacgacucgcccga 336
LF1725 gggaggacgaugc ggCU CUCAUUU wGCGCG UAAAU-- CGAGCU AGCC
cagacgacucgcccga 337 LF1632 gggaggacgaugcggAG UCwCU CUCcacC AkCGUG
UkUUAAU CAAGCU AnUG CCUcagacGacucgcccga 338 LF1856
gggaggacGaugcggUCUAC GGUCU CUCUGGC GGUGCG UAAAU-- CkAACC AGAUCG
cagacgacucgcccga 339 LF1631 gggaggacgaugc ggUdAUUU CyUAAUC hGAGCG
UUUAU-- CUAUCU mAAUkAUC CUcagacgacucgcccga 340 LF1730 gggaggacgaugc
ggaU CgCAAUmU GUwGCG UU-CU-- CkAAAC AGCC Ucagacgacucgcccga 341
LF1852 gggaggacgaugc ggAACUU CUUAGGC AGCGUG CUAGU-- CAAGCU AAGUUCC
ACCUcagacgacucgcccga 371 LF1653 gggaggacgaugcggC ACAAU CUUCGGC
AGCGUG CAAGAU- CAAGCU AUUGU UGUcagacgacucgcccga 372 LF1554
gggaggacgaugc ggCGGU CUUAAGC AGUGUG UCAAU-- CAAACU AUCGUc
agacgacucgcccga 366 LF1722 gggaggacgaugc ggUU CUUAAGC AGCGCG UCAAU-
CGAGCU AACC cagacgacucgcccga 367 Truncates LF1514T1 UGCGUG UU-UU--
CAAGCA 385 LF1514T2 CUCAAAU UGCGUG UU-UU-- CAAGCA 386 LF1514T4
ggUAC CUCAAAU UGCGUG UU-UU-- CAAGCA GUAUc 387 LF1807T5 ggAGU
CUUAGGC AGCGCG UU-UU-- CGAGCU ACUCC 388 Family 1b LF1511(4)
gggaggacgaugcgg UGGUU CUAG GCACGUG UU-UU-- CAAGUGU AAUca
gacgacucgcccga 342 LF1753 gggaggac gaugc ggAA ACAUGUG UU-UU--
CGAAUGU gCUC UCCUCCCCAAACAACyCCCCCAA 343 LF1524 gggaggacg augc ggAA
GGCCGUG UUAAU-- CAAGGCU GCAAU AAAUCAUCCUCCC cagacgac 344 ucgcccga
LF1810 g ggaggacgaugc ggAG GAUGGUG UUCAU-- CAAGAUU GCUCGUUCUUU
ACUGCGUUcagacgac 345 ucgcccga LF1621(2)* gggaggacgaugcggUCAA
AGUGAAG AAUG GACaGCG UU-UU-- CGAGUU GCUUCACU cagacGacucgcccga 346
LF1826(2)* gggaggacgaugcgg GGAG AAUG GCCAGCG UUUAU-- CGAGGU
GCUCCGUUAACCGG cAgacgacucgccc 347 ga LF1713 gggaggacgaugcgg GAGG
AAUG GACwGCG UAUAU-- CGAGUUG CCUc agacgacucgcccga 348 LF1520
gggaggacgaugcg gAUCG AUU UCAUGCG UUUUU-- CGAGUGA CGAUc
agacgacucgcccga 349 LF1552 gggaggacgaugcggA GACc CUA AGmGsG UksUUUU
CAAsCU GGUc wgacgacuzgcccga 350 Family 1c LF1618(2) gggaggacgaugcgg
UUAGCCUACACUCUAGGUUCAG UU-UU-- CGAAUCUUCCACCG cWgacgacucgcccga 351
LF1528(3) gggaggacgaugcgg UUAGGUCAAUGAUCUUAG UU-UU-- CGAUUCGU
cagacgacucgcccga 352 LF1718 gggaggacga ugcggA CGUGUG UAUCrAr
UU-UU-- CCGCUG UUUGUG cagacgacucgcccga 353 LF1623 gggaggacGaugcgg
ACAGGGUUCUUAG GCGGAG UG-UU-- CAUCAA UCCAACCAUGU cagacgacucgcccga
354 LF1557 gggaggacgaugcgg CGAUUUCCAC AGUUUG UCUUAUU CCGCAU AU
cagacgacucgcccga 355 Family 1 (Unclassified) LF1707 gggaggacgaugcgg
AUAyUCAgCUyGUGUk UU-UU-- CdAUCUUCCC cagacgacucgcccga 356 LF1512
gggaggacgaugc ggCACACGUG UU-UU-- CAAGUGUGCU CCUGGGAU
cagacgacucgcccga 357 LF1535(2) gggaggacgaugc ggCAAUGUG UUUCU---
CAAAUUGCU UUCUCCCUU cagacgacucgcccga 358 LF1711 gggaAgacg augcggUG
UUGAU-- CAAUG AAUGUCCUCCUCCUACCC cagacgacucgc 364 ccga LF1517
gggaggacgau gcgguG UUUGU-- CAAUGU CAUGAUUAGUUUUCCCA cagacgacucgc
365 ccga Family 2 LF1627(2) gggaggacgaugc ggAUACUACCGUGCG AACaCUAAG
UCCCGUCUGUCCACUCCU cagacgacucgcccga 359 LF1724(2)* gggaggacgaugc
ggAUaCUA-UGUGCG UUCACUAAG UCCCGUC-GUCCCCU cagacgacucgcccga 360
LF1652(2) gggaggacgaugc ggGUACUA UGUACG AUCaCUAAG
CCCCAUCACCCUUCUCACU cagacnacucgcccga 361 LF1519 gggaggacgaugc
ggUUACUA UGUACA UUUACUAAG ACCCAACGU cagacgacucgcccga 362 LF1608
gggaggacgaugc ggUUwCUA UGUwCGCCUUACUAAGUACCCGUCGACUGUCCCAU
cagacgacucgcccga 363 Family 3 LF1710 gggaggacgaugcgg
AAUGrCCCGUUACCAwCAAUGCGCCUCdUUGmCCCCAAACAACyCCCCCAA 368 LF1829
gacgaugcgg AAUyUCGUGyUAcGCGUyyYCUAUCCAAUCUACCCCmUCUCCAAU
cagacgacyc----- 369 LF1509 gggaggacgaugcgg
CGCUUACAAUAAUUCUCCCUGAGUACAGCucag acgacucgcccga 370 Orphans LF1507
gggaggacgaugcgg UCAUUAACCAAGAUAUGCGAAUCACCUCCU cagacgacucgcccga 373
LF1516(2) gggaggacgaugcgg UCAUUCUCUAAAAAAGUAUUCCGUACCUCCa
cagacgacucgcccga 374 LF1530(2)* gggaggacgaugcgg
GUGAUCUUUUAUGCUCCUCUUGUUUCCUGU cagacgacucgcccga 375 LF1835(4*)
gggaggacnaugcgg UCUAGGCaUCGCUAUUCUUUACUGAUAUAAUUACUCCCCU
cagacgacucgcccga 376 monster gggaggacgaugcgg
AGUwwGCNCGGUCCAGUCACAUCCwAUCCC cagacGacucgcccga 377 LF1522
gggaggacgAugcgg CUCUCAUAUkGwGUrUUyUUCmUUCsrGGCUCAAACAAyyCCCCCAA 378
LF1727 gggaggacgaugcgg CUUGUUAGUUAAACUCGAGUCUCCACCCCU
cagacgacucgcccga 379 LF1510 gggaggacgaugcgg
UCUCUwCUvACvUGUrUUCACAUUUUCGCyUCAAACAACyCCCCCAA 380 LF1715
gggaggacgaugcgg UUrACAAUGrssCUCrCCUUCCCwGGUCCU cagacgacucgcccga 381
LF1809 AggaggacGaugcgg UUAUCUGAArCwUGCGUAAmCUArUGUsAAAsUGCAACrA
cRaacaacYcScccaa 382 LF1533 Aggaagacgaugcgg
UUCGAUUUAUUUGUGUCAUUGUUCUUCCAU cagacgacucgcccga 383 LF1720
--------------- -----------GUGAUGACAUGGAUUACGC cagacgacucgcccga
384
[0331]
18TABLE 17 2' Fluoro L-selectin SELEXes: Full Length Transcribed
Ligands: Protein and Lymphocyte Binding Affinity L-selectin#
Lymphocytes## LIGAND SEQ ID NO Kd (nM) Kd (nM) LF1508 307 0.5
LF1511 342 0.48 LF1512 357 315 LF1513 321 0.16 4 LF1514 297 0.13
0.8 LF1516 374 1.3* LF1518 293 0.42 LF1520 339 0.5* LF1521 323
0.25* LF1523 326 0.25 LF1524 344 2.1* LF1527 299 0.32 LF1528 352
--* LF1529 298 0.6 LF1535 358 --* LF1536 300 0.22* LF1610 329 0.53
LF1613 331 0.034 0.2 LF1614 301 0.17 LF1615 322 0.32 LF1618 351 9.6
25 LF1707 356 0.16* LF1708 327 70 LF1712 330 0.065* LF1713 338
0.22* LF1718 353 6.4* LF1807 309 0.034 LF1808 314 0.6 LF1810 345
8.1* LF1811 312 0.19 LF1815 305 0.18* LF1816 335 --* LF1817 294
2.3* 40N7 -- NX280 1.6 3 #Nitrocellulose filter partitioning @
37.degree. C.; *designate soluble L-selectin, others LS-Rg;
--indicates binding was undetectable ##Flow cytometry competition @
room temperature;
[0332]
19TABLE 18 P-SELECTIN 2'F RNA SELEX % RNA Signal to % RNA Signal to
eluted Noise- eluted Noise- SELEX RNA Load PS-Rg Bead Total 5 mM 5
mM 50 mM 50 mM % Retained Round # (pmol) (pmol) Volume Volume EDTA
EDTA EDTA EDTA on column Kd(nM) Rnd 1 320 200 10 .mu.l 125 .mu.l
1.4 8 8.3 40 0.7 2500 Rnd 2 510 100 10 .mu.l 125 .mu.l 1.8 9 3.5 30
0.6 200 40 10 .mu.l 125 .mu.l 1.7 5 2.6 12 0.3 Rnd 3 200 40 10
.mu.l 125 .mu.l 2.3 15 3.0 13 0.1 40 8 10 .mu.l 125 .mu.l 1.3 4 0.8
8 0.3 1200 Rnd 4 25 5 10 .mu.l 125 .mu.l 1.2 3 0.6 3 0.7 Rnd 5 25 5
10 .mu.l 125 .mu.l 0.9 3 0.15 1.5 0.3 280-900 Rnd 6 25 5 10 .mu.l
125 .mu.l 0.8 2 0.0 1 0.4 85 Rnd 7 50 5 10 .mu.l 125 .mu.l 4.0 8
1.0 4.3 0.5 13 Rnd 8 50 5 10 .mu.l 125 .mu.l 4.6 16 0.4 6.7 0.3 5
10 1 10 .mu.l 125 .mu.l 4.5 6 0.2 2.3 1.4 5 Rnd 9 10 1 10 .mu.l 125
.mu.l 5.3 28 0.05 1.5 1.2 10 1 100 .mu.l 250 .mu.l 2.8 6 0.3 2 0.8
Rnd 10 5 0.5 10 .mu.l 500 .mu.l 5.6 20 0.2 5 1.2 Rnd 11 5 1 250
.mu.l 500 .mu.l 10 11 0.4 2 2.5 0.1-2 1 0.2 10 .mu.l 500 .mu.l 14.2
15 0.6 3 13 Rnd 12 1 0.1 250 .mu.l 500 .mu.l 4.5 4 0.8 2 4.7
0.02-20 Rnd 13 0.1 0.01 250 .mu.l 500 .mu.l 2.6 2 ND ND 3.6
[0333]
20TABLE 19 P-Selectin 2'-F RNA Ligands SEQ ID Ligand Sequence NO.
Family 1 PF373(6)
gggagacaagaauaaacgcucaaCGAAUCAGUAAACAUAACACCAUGAAACAUAAAUAGCACGC-
GAGACGUCuucgacaggaggcucacaa 199 caggc PF424
gggagacaagaauaaacgcucaaCGAGUUCACAUGGGAGCAAUCUCCGAAUAAACAACACGCKAKCGCAAAuu-
cgacaggaggcucacaac 200 aggc PF412 gggagacaagaauaaacgcucaaC-
GACCACAAUACAAACUCGUAUGGAACACGCGAGCGACAGUGACGCAUUuucgacaggaggcucacaa
201 caggc PF422 gggagacaagaauaaacgcucaaCGUCAAGCCAGAAUCCGGAACACGC-
GAGAAAACAAAUCAACGACCAAUCGAuucgacaggaggcucac 202 aacaggc PF426
gggagacaagaauaaacgcucaaCGACCACAAUAACCGGAAAUCCCCGCGGUUACGGAACACGCGAA-
CAUGAAuucgacaggaggcucaca 203 acaggc PF398
gggagacaagaauaaacgcucaaCGAACCACGGGGAAAUCCACCAGUAACACGCGAGGCAAACAGACCCUCuu-
cgacaggaggcucacaac 204 aggc PF380(2)
gggagacaagaauaaacgcucaacCGAGCAAAAGUACUCA
CGGGACCAGGAGAUCAGCAACACGCGAGACGA- AAuucgacaggaggcuca 205 caacaggc
PF377(2)
gggagacaagaauaaacgcucaaCGAGCCAGGAACAUCGACGUCAGCAAACGCGAGCGCAACCAGUAACACCu-
ucgacaggaggcucacaa 206 caggc PF387(2)
gggagacaagaauaaacgcucaaCGCACCAGGAACAACGAGAACCAUCAGUAAACGCGAGCGAUUGCAUGuuc-
gacaggaggcucacaaca 207 ggc PF383 gggagacaagaauaaacgcucaaCG-
CACCAGGAACAACAAGAACCAUCAGUAAGCGCGAGCGAUUGCAUAuucgacaggaggcucacaaca
208 ggc PF395 gggagacaagaauaaacgcucaaCGAGCAAGGAACGAAUACAAACCAGGAA-
ACUCAGCAACACGCGAGCAGUAAGAAuucgacaggaggcu 209 cacaacaggc PF416(2)
gggagacaagaauaaacgcucaaCAGUUCACUCAACCGGCACCAGACUACGAUCAGCAUUGGCG-
AGUGAACACuucgacaggaggcucaca 210 acaggc PF388(2)
gggagacaagaauaaacgcucaaCUGGCAACGGGAUAACAACAAAUGU
CACCAGCACUAGCGAGACGGAAGG- uucgacaggaggcucaca 211 acaggc Family 1
Truncates PF373s1 CUCAACGAAUCAGUAAACAUAACACCAUGAAACAUAA-
AUAGCACGCGAG 220 PF424s1
CUCAACGAGUUCACAUGGGAGCAAUCUCCGAAUAAACAACACGCGAG 221 PF3981
CUCAACGAACCACGGGGAAAUCCACCAGUAACACGCGAG 222 PF377s1
CUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAG 223 PF377s2
CGCUCAACGAGCCAGGAACAUCGACGACAGCAAACGCGAGCG 224 PF377L1
CUCAACGAGCCAGGACUACGAUCAGCCAAACGCGAG 225 PF387s1
CUCAACGCACCAGGAACAACGAGAACCAUCAGUA- AACGCGAG 226 PF383s1
CUCAACGCACCAGGAACAACAAGAAC- CAUCAGUAAGCGCGAG 227 PF416s2
CACUCAACCGGCACCAGACUACGAUCAGCAUUGGCGAGUG 228 PF422s1
GAAUCCGGAACACGCGAGAAAACAAAUCAACGACCAAUCGAUUCG 229 2'-O-Methyl
Substituted Truncates PF377M1 CUCAACGAGCCAGGACGUCAGCAAACGCGAG 230
PF3772 CUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAG 231 PF377M3
CUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAG 232 PF377M4
CUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAG 233 PF377M5
CUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAG 234 PF377M6
CUCAACGAGCCAGGAACAUCGACGUCAGCAAACGCGAG 235 Family 2 PF378(8)
gggagacaagaauaaacgcucaaCGAUGAGCGUGACCGAAGCUAUAAUCAGGUCGAUUCACCA-
AGCAAUCUUAuucgacaggaggcucaca 212 acaggc Family 3 PF381(5)
gggagacaagaauaaacgcucaaAGGAUCACACAAACAUCGGUCAAUAAAUAAGUAU-
UGAUAGCGGGGAUAuucgacaggaggcucacaac 213 aggc Family 4 PF411(2)
gggagacaagaauaaacgcucaaCAACCCAACCAUCUAGAGCUUCGAA-
CCAUGGUAUACAAGGGAACACAAAAuucgacaggaggcucaca 214 acaggc Family 5
PF396(2) gggagacaagaauaaacgcucaaGCGGUCAGAAACAAUAGCUG-
GAUACAUACCGCGCAUCCGCUGGGCGAUAuucgacaggaggcucacaa 215 caggc Orphans
PF386 gggagacaagaauaaacgcucaaACAAGAGAGUCAAACCAAGU-
GAGAUCAGAGCGUUUAGCGCGGAAAGCACAuucgacaggaggcucaca 216 acaggc PF382
gggagacaagaauaaacgcucaaACUCGACUAGUAAUCACCCUAGCAUAAAUCUCCUCGAGCACAG-
ACGAUAuucgacaggaggcucacaa 217 caggc PF404
gggagacaagaauaaacgcucaaUCAGCAGUAAGCGAUCCUAUAAAGAUCAACUAGCCAAAGAUGACUUAuuc-
gacaggaggcucacaaca 218 ggc PF417 gggagacaagaauaaacgcucaaAA-
AGACGUAUUCGAUUCGAAACGAGAAAGACUUCAAGUGAGCCCGCAGuucgacaggaggcucacaac
219 aggc
[0334]
21TABLE 20 Dissociation Constants and Specificity of 2'F RNA
Ligands to P-Selectin SEQ Kd S LeX Kd Kd ID Ligand (PS-Rg) (IC50)
(ES-Rg) (LS-Rg) Tm(.degree. C.) NO. PF373 49.5 pM >3 .mu.M >3
.mu.M 199 PF377 18.5 pM 3 nM 2.3 .mu.M >3 .mu.M 53.degree. C.
206 PF378 51.5 pM 212 PF380 74.5 pM 4 nM 205 PF381 16.5 pM 1 nM 213
PF386 45.5 pM 216 PF387 16 pM 207 PF388 90 pM 211 PF395 26 pM 209
PF396 24 pM 215 PF398 46 pM 204 PF404 47.5 pM 218 PF411 13 pM 2 nM
214 PF412 450 pM 201 PF416 63 pM 210 PF417 69 pM 219 PF422 172 pM 3
nM 202 PF424 36.5 pM 200
[0335]
22TABLE 21 Boundary Results for 2' F RNA Ligands to P-Selectin SEQ
ID Kd(pM) Clone# NO. FAMILY 1 56 PF373s1 cucaaCGAAUCAG UA
AACAUAACACCAUGAAACA UAAAUAGCACGCGAG 220 178 PF424s1
cucaaCGAGUUCACAUG GGAGCAAUCUCCGAA UAAACAACACGCGAG 221 63 PF398s1
cucaaCGAACCAC GG GGAAAUCCA CCAGUAACACGCGAG 222 ND PF380s1
cucaaCGAGCAAAAGUACUCACGGGACCAGGAGA UCAGCAACACGCGAG ACGAAA- 236 uucg
50 PF377s1 cucaaCGAGCCAG GA ACAUCGACG UCAGCAAA CGCGAGCG 223 50
PF377s2 cg cucaaCGAGCCAG GA ACAUCGACG UCAGCAAA CGCGAG CG 234 PF412
cg cucaaCGACCACAA UA CAAACUCG UAUGGAACACGCGAG CG 237 63 PF387s1 cg
cucaaCGCACCAG GA ACAACGAGAACCA UCAGUAAA CGCGAG CG 226 10000 PF383s1
acg cucaaCGCACCAG GA ACAACAAGAACCA UCAGUAAG CGCGAG CG 227 PF388 cg
cucaaCuGGCAAC GG GAUAACAACAAAUGUCA CCAGCACU AGCGAG ACG 238 150
PF416s1 UCA CUCAACCGGCACCA GA CUACGA UCAGCAUU GGCGAG UG 239 PF395
gggagacaagaauaaacg cucaaCGAGCAAG GA ACGAAUACAAACCAGGAAACUCAGCAACAC-
GCGAG CA 240 PF426 cucaaCGACCACAA UA ACCGGAAAUCCCCGCGGU
UACCGGAACACGCGAA CA 241 1000 PF422s1 AUCAACGACCAAUC GA uucg3'
5'GAAUCCGGAACACGCGAG - 229 AAAACAA FAMILY 2 PF378
agaauaaacgcucaaCGAUGAGCGUGACCGAAGCUAUAAUCAGGUCGAUUCACCAAGCAAUCUUAuucg
242 FAMILY 3 PF381
acgcucaaAGGAUCACACAAACAUCGGUCAAUAAAUAAGUAUUGAUAGCG 243 FAMILY 4
PF396 gcucaaGCGGUCAGAAACAAUAGCUGGAUACAU- ACCGCGCAUCCGCUGGGCG 244
FAMILY 5 PF411
ACCAUCUAGAGCUUCGAACCAUGGUAUACAAGGGAACACAAAAuucgcggaggcucca 245
ORPHANS PF386 gggagacaaga- 246
uaaacgcucaaACAAGAGAGUCAAACCAAGUGAGAUCAGAGCGUUUAGCGCGGAAAGCACA-
uucgacaggaggcucacaacaggc PF417 gggagacaagaauaaacgcucaaAAAGACGUAUU-
CGAUUCGAAACGAGAAAGAC UUCAAGUGAGCCCGCAG- 247 uucgacaggaggcuca
[0336]
23TABLE 22 Dissociation Constants and Specificity of Truncated 2'F
RNA Ligands to P-Selectin Kd S LeX Kd Kd Tm # Ligand (PS-Rg) (IC50)
(ES-Rg) (LS-Rg) (.degree. C.) Bases SEQ ID NO. PF373s1 56 pM 3 nM
>3 .mu.M >3 .mu.M 220 PF377s1 60 pM 2 nM >3 .mu.M >3
.mu.M 59.degree. C. 38 223 PF377s2 45 pM 4 nM 42 224 PF383s1 10000
pM 25 nM 46 227 PF387s1 63 pM 2 nM >3 .mu.M >3 .mu.M 46 226
PF398s1 178 pM 2 nM >3 .mu.M >3 .mu.M 39 222 PF416s2 150 pM 3
nM 42 228 PF422s1 1000 pM 8 nM >3 .mu.M >3 .mu.M 44 229
PF377s1B 65 pM 3 nM >3 .mu.M >3 .mu.M 38 223 PF377s1B:SA 30
pM 38 223 PF377s1F 60 pM 3 nM 38 223 PF377s1- 125 pM 2 nM 41 223
5'NH2 PF377L1 220 pM 4 nM >3 .mu.M >3 .mu.M 35 225 PF377t3'
30 pM 2 nM 59 223 PF377M1 120 pM >3 .mu.M 38 230 PF377M2 1700 pM
38 231 PF377M3 900 pM 10 nM >3 .mu.M 38 232 PF377M4 1700 pM 38
233 PF377M5 60 pM 2 nM >3 .mu.M 38 234 PF377M6 250 pM 38 235
[0337]
24TABLE 23 2'OMe Substitution of 2'F RNA Ligands to P-Selectin
Purine Unmixed Std. Mixed Mixed Predicted Actual Position Ratio
Dev. 40 pM 200 pM Pref. Pref. 4 1.07 0.12 0.3 0.4 2'-OH untested 5
1.00 1.00 0.4 0.7 2'-OH untested 7 1.00 0.13 1.2 1.5 2'-O--Me
2'-O--Me 8 1.03 0.20 2.3 1.3 2'-O--Me 2'-O--Me 12 0.83 0.12 0.4 0.5
2'-OH untested 13 0.90 0.17 0.8 0.8 neutral 2'-O--Me 14 0.73 0.15
0.8 0.9 neutral 2'-O--Me 15 0.63 0.15 0.8 1.3 2'-0--Me 2'O--Me 16
0.67 0.10 0.5 0.7 neutral untested 18 0.60 0.10 0.7 0.7 neutral
2'-O--Me 21 0.87 0.30 0.5 0.7 neutral 2'-O--Me 22 0.72 0.16 0.7 0.8
neutral 2'-O--Me 24 0.70 0.16 0.6 0.8 neutral 2'-O--Me 27 0.83 0.12
1.3 1.5 2'-O--Me 2'-O-Me 28 0.69 0.09 0.6 1.0 2'-O--Me ? 30 0.90
0.00 0.8 1.0 neutral ? 31 0.92 0.16 1.2 1.5 2'-O--Me 2'O--Me 32
1.10 0.06 0.5 0.8 2'-OH untested 34 0.93 0.06 0.7 0.9 2'-OH
untested
[0338]
25TABLE 24 % RNA Signal to % RNA Signal to eluted Noise- eluted
Noise- % SELEX RNA Load PS-Rg Bead Total 5 mM 5 mM 50 mM 50 mM
Retained Round # (pmol) (pmol) Volume Volume EDTA EDTA EDTA EDTA on
column Kd (nM) Rnd 1 330 200 10 .mu.l 125 .mu.l 0.0 1 1.3 6.5 0.2
6350 Rnd 2 300 100 10 .mu.l 100 .mu.l 0.8 8 0.3 2.7 0.6 Rnd 3 550
100 10 .mu.l 125 .mu.l 0.6 21 0.2 8 0.1 1900 Rnd 4 500 100 10 .mu.l
125 .mu.l 1.0 33 0.8 10 0.4 Rnd 5 365 100 10 .mu.l 125 .mu.l 1.5 30
1.6 32 0.4 470 Rnd 6 500 50 10 .mu.l 125 .mu.l 1.9 22 0.9 17 0.3 50
5 10 .mu.l 125 .mu.l 1.1 5 0.4 2.3 1.2 103 Rnd 7 50 5 10 .mu.l 125
.mu.l 1.8 7 0.05 1.8 0.6 31 Rnd 8 50 5 10 .mu.l 125 .mu.l 3.6 7 0.0
<1 0.6 Rnd 9 10 1 10 .mu.l 125 .mu.l 3.3 5 0.1 2 1.2 Rnd 10 1
0.2 10 .mu.l 500 .mu.l 2.5 3 0.0 <1 0.3 0.2-6 Rnd 11 1 0.1 10
.mu.l 500 .mu.l 2.0 2 0.0 <1 5.0 1 0.1 250 .mu.l 500 .mu.l 1.5 2
0.0 <1 12.0 Rnd 12 1 0.1 10 .mu.l 500 .mu.l 4.1 5 0.2 2 3.2 1
0.1 250 .mu.l 500 .mu.l 3.1 2 0.2 1 14.0
[0339]
26TABLE 25 P-Selectin 2' NH2 RNA Ligands SEQ ID Ligand Sequence NO.
family 1 PA341(7)
gggagacaagaauaaacgcucaaGCCCCAAACGCAAGCGAGCAUCCGCAACAGGGAAGAAGACA-
GACGAAUGAuucgacaggaggcucaca 251 acaggc PA350
gggagacaagaauaaacgcucaaGCCCCAAACGCAAGUGAGCAUCCGCAACAGGGAAGAAGACAGACGAUUGA-
uucgacaggaggcucaca 252 acaggc PA466
gggagacaagaaauaaacncucaaGCCCCAAACGCAAGUGAGCAUCCGCAACAGGGAAGAAGACAGAUGAAUG-
Auucgacaggaggcucac 253 aacaggc PA473 gggagacaagaauaaacncucaaGCCCCAA
GCAAGUGAGCAUCCGCAACAGGGAAGAAGACAGACGAGUG- Auucgacaggaggcucac 254
aacaggc PA477(3) gggagacaagaauaaacncucaaGCCCCAAaCGCAAGUG
AGCAUCCGCAACAGGGAAGAAGACAGACGAAUG- Auucgacaggaggcucac 255 aacaggc
PA328(3) gggagacaagaauaaacgcucaaGCAAAAGGCGUAAAUACACC
UCCGCAACUGGGAAGAAGACGCAGGGACG- GuucgacaggNggcucac 256 aacaggc
family 2 PA337(6)
gggagacaagaauaaacgcucaaACAGCUACAAGUGGGACAACAGGGUACAGCGGAGAGAAACA-
UCCAAACAAGuucgacaggaggcucac 257 aacaggc family 3 PA448(7)
gggagacaagaauaaacgcucaaAUCAACUAAACAACGCAGUCACGAGAACGACCGG-
KCUGACUCCGAAAG uucgacaggaggcucac 258 aacaggc others PA325
gggagacaagaauaaacgcucaaACGAGAGCACCAAGGCAACAGAUGCAGAAG-
AAGUGUGCGCGCGCGAAA uucgacaggaggcucac 259 aacaggc PA327
gggagacaagaauaaacgcucaaUAAGACAACGAACAGACAGAAGCGAAAAAGGGGCGCCGCAGCAACAACAA-
Auucgacaggaggcucac 260 aacaggc PA446
gggagacaagaauaaacgcucaaCGUGUACCACAACAGUUCCACG
GAAGCUGGAAUAGGACGCAGAGGAA uucgacaggaggcucac 261 aacaggc PA313
gggagacaagaauaaacgcucaaACAAAAUUWUGGUGGGCCCCGcAACMGGGRGGRAGRCCGUUGAAGGC
uucgacaggaggcucac 262 aacaggc PA336
gggagacaagaauaaacgcucaaGAUCAUAACGAGAGGAGAGGGAGAACUACACGCGCGCGAGGAAAGAG
uucgacaggaggcucac 263 aacaggc PA301
gggagacaagaauaaacgcucaaACACAAAUCGGGCAGGGACUGGGUUGGGCACGGCAGGGCGCC
uucgacaggaggcucac 264 aacaggc PA305
gggagacaagaauaaacgcucaaGUGGGCUCGGGCCGGAUGUCUACGGGUGUGAAGAAACCCCUAGGGCAGGG
uucgacaggaggcucac 265 aacaggc PA309
gggagacaagaauaaacgcucaaGAUCAGCGGAACUAAGAAAUGGAAGGCUAAGCACCGGGAUCGGGAGAA
uucgacaggaggcucac 266 aacaggc PA315
gggagacaagaauaaacgcucaaUAACAAAGCAGCAAAGUACCAGAGGAGAGUUGGCAGGGUUUAGGCAGC
uucgacaggaggcucac 267 aacaggc PA318
gggagaca-gaauaaacgcucaaAGACCAAGGGACAGCAGCGGGGAAAAACAGAUCACAGCUGUAAGAGGGC
uucgacaggaggcucac 268 aacaggc PA319
gggagacaagaauaaacgcucaaAGUCGGGGAUAGAAACACACUAAGAAGUGCAUCAGGUAGGAGAUAA
uucgacaggaggcucac 269 aacaggc PA320
gggagacaagaauaaacgcucaaGAGUAUCACACAAACCGGCACGGACUAAGCAGAAGGAGGUACGGAAGA
uucgacaggaggcucac 270 aacaggc PA321
gggagacaagaauaaacNcucaaCGAAAUAGAAGGAACAGAAGAAUGGBGAWGNGGGAAAUgGCAACGAA
uucgacaggaggcucac 271 aacaggc PA324
gggagacaagaauaaacgcucaaACGAGACCCUGGAUACGAGGCUGAGGGAAAGGGAGMMMRRAMCUARRCKC
uucgacaggaggcucac 272 aacaggc PA329
gggagacaagaauaaacgcucaaGAAGGAUACUUAGGACUACGUGGGAUGGGAUGAAAUGGGAGAACGGGAG
uucgacaggaggcucac 273 aacaggc PA330
gggagacaagaauaaacgcucaaAACGCACAAAGUAAGGGACGGGAUGGAUCGCCCUAGGCUGGAAGGGAAC
uucgacaggaggcucac 274 aacaggc PA332
gggagacaagaauaaacgcucaaGGUGAACGGCAGCAAGGCCCAAAACGUAAGGCCGGAAACNGGAGAGGGA
uucgacaggnggcucac 275 aacaggc PA335
gggagacaagaauaaacgcucaaUGAUAUACACGUAAGCACUGAACCAGGCUGAGAUCCAUCAGUGCCCAGG
uucgacaggaggcucac 276 aacaggc PA336
gggagacaagaauaaacgcucaaGAUCAUAACGAGAGGAGAGGGAGAACUACACGCGCGCGAGGAAAGAG
uucgacaggaggcucac 277 aacaggc PA338
gggagacaagaauaaacgcucaaUCAAGUAAGGAGGAAGGGUCGUGACAGAAAAACGAGCAAAAAACGCGAG
uucgacaggaggcucac 278 aacaggc PA339
gggagacaagaauaaacgcucaaAAGGUGCCGGGUUGGAGGGGUAGCAAGAAAUGGCUAGGGCGCASGA
uucgacaggnggcucac 279 aacaggc PA342
gggagacaagaauaaacgcucaaCCAACGCGCACCCCGCAGCAAACGAAAUUGGGGAGACAGGUGCAAGACAG
uucgacaggaggcucac 280 aacaggc PA349
gggagacaagaauaaackcucaaCAAACAAUAUCGGCGCAGGAAAACGUAGAAACGAAAMGGAGCUGCGYGGA
uucgacaggaggcucac 281 aacaggc PA351
gggagacaagaauaaacgcucaaUGAUAGCACAGUGUAUAAGAAAACGCAACACCGCGCGCGGAAGAG
uucgacaggaggcucac 282 aacaggc PA352
gggagacaagaauaaacgcucaaGAUCAUCGCAGUAUCGGAAUCGACCCUCAGUGGGUGACAUGCGGACAAG
uucgacaggaggcucac 283 aacaggc PA353
gggagacaagaauaaacgcucaaGUACCGGGAAGGGAUGAACUGGGAUAUGGGAACGGAGGUCAGAGGCACGA
uucgacaggaggcucac 284 aacaggc PA354
gggagacaagaauaaacgcucaaGCAAUGGAACGCUAGGAGGGAACAUAAGCAGGGCGAGCGGAGUCGAUAGC
uucgacaggaggcucac 285 aacaggc PA447
gggagacaagaauaaacgcucaaAACAGAACUGAUCGGCGCAGGUUGAUAAAGGGGCAGCGGGAAGAUCACAA
uucgacaggaggcucac 286 aacaggc PA463
gggagacaagaauaaacgcucaaGGGAAACGGAAAGGGACAAGGCGAACAGACGAGAAGUAGACGGAGUAGGA
uucgacaggaggcucac 287 aacaggc PA465
gggagacaagaauaaacgcucaaNNNGAGGAAGGGCACGCAAGGAAACAAAACACAAAGCAGAAGUAGUAAGA
uucgacaggaggcucac 288 aacaggc PA467
gggagacaagaauaaacgcucaaGUACRCAGUGAGCAGAAGCAGAGAGACUUGGGAUGGGAUGAAAUGGKC
uucgacaggaggcucac 289 aacaggc PA479
gggagacaagaauaaacNcucaaCCGACGUGGACDCGCAUCGGCAUCCAGACCAGGCUGNBCNGCACCASACG
uucgacaggaggcucac 290 aacaggc
[0340]
27TABLE 26 Dissociation Constants and Specificity of 2'NH2 RNA
Ligands to P-Selectin Kd Kd SLeX Kd Kd SEQ ID Ligand (PS-Rg) (4oC)
(IC50) (ES-Rg) (LS-Rg) NO. PA 301 2.5 nM 264 PA 305 0.21 pM 265 PA
309 0.656 pM 266 PA 315 5 nM 267 PA 318 2 nM 268 PA 319 11 nM 269
PA 320 4.5 nM 270 PA 321 8 nM 271 PA 325 >10 nM 259 PA 327 13.5
nM 260 PA 328 3 nM 256 PA 329 4 nM 273 PA 330 0.237 nM 274 PA 335
10.5 nM 276 PA 336 15 nM 277 PA 337 4.5 nM 257 PA 338 57 nM 278 PA
339 13.5 nM 279 PA 341 0.44 nM 3 nM 251 PA 342 4 nM 280 PA 350 0.06
nM 0.01 nM 2 nM 375 nM >3 .mu.M 252 PA 351 2 nM 282 PA 352 6 nM
283 PA 353 9 nM 284 PA 354 5 nM 285 PA 447 50 nM 286 PA 448 5 nM
258 PA 463 8 nM 287 PA 465 >50 nM 288 PA 466 0.43 nM 253 PA 467
24 nM 289 PA 473 0.36 nM 254 PA 477 0.57 nM 255
[0341]
Sequence CWU 1
1
* * * * *