U.S. patent application number 16/059597 was filed with the patent office on 2019-02-14 for aptamers for oral care applications.
The applicant listed for this patent is The Procter & Gamble Company. Invention is credited to Gregory Allen Penner, Paul Albert Sagel, Amy Violet Trejo, Juan Esteban Velasquez.
Application Number | 20190048349 16/059597 |
Document ID | / |
Family ID | 63371804 |
Filed Date | 2019-02-14 |
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United States Patent
Application |
20190048349 |
Kind Code |
A1 |
Velasquez; Juan Esteban ; et
al. |
February 14, 2019 |
APTAMERS FOR ORAL CARE APPLICATIONS
Abstract
An aptamer composition is disclosed which has one or more
oligonucleotides that include at least one of deoxyribonucleotides,
ribonucleotides, derivatives of deoxyribonucleotides, derivatives
of ribonucleotides, or mixtures thereof. The aptamer composition
has a binding affinity for a material that is at least one of
tooth, enamel, dentin, carbonated calcium-deficient hydroxyapatite,
or mixtures thereof.
Inventors: |
Velasquez; Juan Esteban;
(Cincinnati, OH) ; Trejo; Amy Violet; (Oregonia,
OH) ; Sagel; Paul Albert; (Maineville, OH) ;
Penner; Gregory Allen; (Lond, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Procter & Gamble Company |
Cincinnati |
OH |
US |
|
|
Family ID: |
63371804 |
Appl. No.: |
16/059597 |
Filed: |
August 9, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62542936 |
Aug 9, 2017 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61Q 11/00 20130101;
C12N 2310/321 20130101; C12N 2310/314 20130101; A61K 31/4706
20130101; A61K 8/606 20130101; A61K 47/549 20170801; A61K 31/7125
20130101; B82Y 5/00 20130101; C12N 2310/3341 20130101; C11D 1/90
20130101; A61K 9/1676 20130101; A61K 47/14 20130101; A61K 31/522
20130101; A61K 9/51 20130101; C12N 15/115 20130101; C12N 2310/3125
20130101; C12N 2310/313 20130101; A61K 47/26 20130101; C12N 2310/16
20130101; C12N 2310/315 20130101 |
International
Class: |
C12N 15/115 20060101
C12N015/115; A61K 9/16 20060101 A61K009/16; A61K 31/7125 20060101
A61K031/7125; A61K 47/26 20060101 A61K047/26; A61K 9/51 20060101
A61K009/51; A61K 31/522 20060101 A61K031/522; A61K 31/4706 20060101
A61K031/4706; A61K 47/14 20060101 A61K047/14 |
Claims
1. An aptamer composition comprising an oligonucleotide that is at
least one of deoxyribonucleotides, ribonucleotides, derivatives of
deoxyribonucleotides, derivatives of ribonucleotides, or mixtures
thereof; wherein the aptamer composition has a binding affinity for
at least one of tooth, enamel, dentin, carbonated calcium-deficient
hydroxyapatite, or mixtures thereof.
2. The aptamer composition of claim 1, wherein the aptamer
composition has a binding affinity for tooth.
3. The aptamer composition of claim 1, comprising at least one
oligonucleotide that has at least 50% nucleotide sequence identity
to at least one of SEQ ID NO 1 to SEQ ID NO 234.
4. The aptamer composition of claim 1, comprising at least one
oligonucleotide of SEQ ID NO 1 to SEQ ID NO 234.
5. The aptamer composition of claim 1, comprising an
oligonucleotide that is at least one of SEQ ID NO 1, SEQ ID NO 9,
SEQ ID NO 25, SEQ ID NO 112, SEQ ID NO 120, SEQ ID NO 136, or SEQ
ID NO 223 to SEQ ID NO 234.
6. The aptamer composition of claim 1, wherein the oligonucleotide
comprises one or more motifs that are at least one of SEQ ID NO
235, SEQ ID NO 236, SEQ ID NO 237, SEQ ID NO 238, SEQ ID NO 239,
SEQ ID NO 240, SEQ ID NO 241, SEQ ID NO 242, SEQ ID NO 243, and SEQ
ID NO 244.
7. The aptamer composition of claim 1, wherein the oligonucleotide
comprises natural or non-natural nucleobases.
8. The aptamer composition of claim 7, wherein the non-natural
nucleobases are at least one of hypoxanthine, xanthine,
7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine,
5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine,
6-methylisoquinoline-1-thione-2-yl, 3-methoxy-2-naphthyl,
5-propynyluracil-1-yl, 5-methylcytosin-1-yl, 2-aminoadenin-9-yl,
7-deaza-7-iodoadenin-9-yl, 7-deaza-7-propynyl-2-aminoadenin-9-yl,
phenoxazinyl, phenoxazinyl-G-clam, or mixtures thereof.
9. The aptamer composition of claim 1, wherein the nucleosides of
the oligonucleotide are linked by a chemical motif that is at least
one of natural phosphate diester, chiral phosphorothionate, chiral
methyl phosphonate, chiral phosphoramidate, chiral phosphate chiral
triester, chiral boranophosphate, chiral phosphoroselenoate,
phosphorodithioate, phosphorothionate amidate,
methylenemethylimino, 3'-amide, 3' achiral phosphoramidate, 3'
achiral methylene phosphonates, thioformacetal, thioethyl ether, or
mixtures thereof.
10. The aptamer composition of claim 1, where the derivatives of
ribonucleotides or said derivatives of deoxyribonucleotides are at
least one of locked oligonucleotides, peptide oligonucleotides,
glycol oligonucleotides, threose oligonucleotides, hexitol
oligonucleotides, altritol oligonucleotides, butyl
oligonucleotides, L-ribonucleotides, arabino oligonucleotides,
2'-fluoroarabino oligonucleotides, cyclohexene oligonucleotides,
phosphorodiamidate morpholino oligonucleotides, or mixtures
thereof.
11. The aptamer composition of claim 1, comprising at least one
polymeric material, wherein the at least one polymeric material is
covalently linked to the oligonucleotide.
12. The aptamer composition of claim 11, wherein the at least one
polymeric material is polyethylene glycol.
13. The aptamer composition of claim 1, wherein the nucleotides at
the 5'- and 3'-ends of the oligonucleotide are inverted.
14. The aptamer composition of claim 1, wherein at least one
nucleotide of the oligonucleotide is fluorinated at the 2' position
of the pentose group.
15. The aptamer composition of claim 1, wherein pyrimidine
nucleotides of the oligonucleotide are fluorinated at the 2'
position of the pentose group.
16. The aptamer composition of claim 1, wherein the oligonucleotide
is covalently or non-covalently attached to at least one oral care
active ingredient comprising whitening agents, brightening agents,
anti-stain agents, anti-cavity agents, anti-erosion agents,
anti-tartar agents, anti-calculus agents, anti-plaque agents, teeth
remineralizing agents, anti-fracture agents, strengthening agents,
abrasion resistance agents, anti-gingivitis agents, anti-microbial
agents, anti-bacterial agents, anti-fungal agents, anti-yeast
agents, anti-viral, anti-malodor agents, breath freshening agents,
flavoring agents, cooling agents, taste enhancement agents,
olfactory enhancement agents, anti-adherence agents, smoothness
agents, surface modification agents, anti-tooth pain agents,
anti-sensitivity agents, anti-inflammatory agents, gum protecting
agents, periodontal actives, tissue regeneration agents, anti-blood
coagulation agents, anti-clot stabilizer agents, salivary stimulant
agents, salivary rheology modification agents, enhanced retention
agents, soft/hard tissue targeted agents, tooth/soft tissue
cleaning agents, antioxidants, pH modifying agents, H-2
antagonists, analgesics, natural extracts and essential oils, dyes,
optical brighteners, cations, phosphates, fluoride ion sources,
peptides, nutrients, enzymes, mouth and throat products, or
mixtures thereof.
17. The aptamer composition of claim 16, wherein the oral care
active ingredient is at least one of dyes or optical
brighteners.
18. The aptamer composition of claim 16, wherein the oral care
active ingredient is 4,4'-diamino-2,2'-stilbenedisulfonic acid.
19. The aptamer composition of claim 1, wherein the oligonucleotide
is covalently or non-covalently attached to one or more
nanomaterials.
20. A method for delivering one or more oral care active
ingredients to the oral cavity comprising administering an oral
care composition comprising at least one nucleic acid aptamer and
one or more oral care active ingredients; wherein the at least one
nucleic acid aptamer and said one or more oral care active
ingredients are covalently or non-covalently attached; and wherein
the at least one nucleic acid aptamer has a binding affinity for an
oral cavity component.
Description
FIELD OF INVENTION
[0001] The present invention generally relates to nucleic acid
aptamers that have a high binding affinity and specificity for
teeth. This invention also relates to the use of such aptamers as
delivery vehicles of active ingredients to the oral cavity.
BACKGROUND OF THE INVENTION
[0002] Aptamers are short single-stranded oligonucleotides, with a
specific and complex three-dimensional shape, that bind to target
molecules. The molecular recognition of aptamers is based on
structure compatibility and intermolecular interactions, including
electrostatic forces, van der Waals interactions, hydrogen bonding,
and .pi.-.pi. stacking interactions of aromatic rings with the
target material. The targets of aptamers include, but are not
limited to, peptides, proteins, nucleotides, amino acids,
antibiotics, low molecular weight organic or inorganic compounds,
and even whole cells. The dissociation constant of aptamers
typically varies between micromolar and picomolar levels, which is
comparable to the affinity of antibodies to their antigens.
Aptamers can also be designed to have high specificity, enabling
the discrimination of target molecules from closely related
derivatives.
[0003] Aptamers are usually designed in vitro from large libraries
of random nucleic acids by Systematic Evolution of Ligands by
Exponential Enrichment (SELEX). The SELEX method was first
introduced in 1990 when single stranded RNAs were selected against
low molecular weight dyes (Ellington, A. D., Szostak, J. W., 1990.
Nature 346: 818-822). A few years later, single stranded DNA
aptamers and aptamers containing chemically modified nucleotides
were also described (Ellington, A. D., Szostak, J. W., 1992. Nature
355: 850-852; Green, L. S., et al., 1995. Chem. Biol. 2: 683-695).
Since then, aptamers for hundreds of microscopic targets, such as
cations, small molecules, proteins, cells, or tissues have been
selected. A compilation of examples from the literature is included
in the database at the website:
http://www.aptagen.com/aptamer-index/aptamer-list.aspx. However, a
need still exists for aptamers that selectively bind to surfaces in
the oral cavity, including teeth.
SUMMARY OF THE INVENTION
[0004] In this invention, we have demonstrated the use of SELEX for
the selection of aptamers against teeth and the use of such
aptamers for the delivery of active ingredients to the oral
cavity.
[0005] In certain embodiments of the present invention, an aptamer
composition is provided. The aptamer composition comprises at least
one oligonucleotide which may include: deoxyribonucleotides,
ribonucleotides, derivatives of deoxyribonucleotides, derivatives
of ribonucleotides, or mixtures thereof; wherein said aptamer
composition has a binding affinity for a material selected from the
group consisting of: tooth, enamel, dentin, carbonated
calcium-deficient hydroxyapatite, and mixtures thereof.
[0006] In another embodiment of the present invention, an aptamer
composition is provided. The aptamer composition including at least
one oligonucleotide comprising SEQ ID NO 1, SEQ ID NO 9, SEQ ID NO
25, SEQ ID NO 112, SEQ ID NO 120, SEQ ID NO 136, or SEQ ID NO 223
to SEQ ID NO 234.
[0007] In another embodiment of the present invention, an aptamer
composition includes at least one oligonucleotide having one or
more motifs comprising SEQ ID NO 235, SEQ ID NO 236, SEQ ID NO 237,
SEQ ID NO 238, SEQ ID NO 239, SEQ ID NO 240, SEQ ID NO 241, SEQ ID
NO 242, SEQ ID NO 243, or SEQ ID NO 244.
[0008] In another embodiment of the present invention, an oral care
composition is provided. The oral care composition comprises at
least one nucleic acid aptamer; wherein said at least one nucleic
acid aptamer has a binding affinity for an oral cavity component.
In another embodiment of the present invention, said oral cavity
component comprises at least one of: tooth, enamel, dentin, or any
other surfaces in the oral cavity. In yet another embodiment, said
oral cavity component is tooth.
[0009] In another embodiment of the present invention, a method for
delivering one or more oral care active ingredients to the oral
cavity is provided. The method comprises administering an oral care
composition comprising at least one nucleic acid aptamer and one or
more oral care active ingredients; wherein said at least one
nucleic acid aptamer and said one or more oral care active
ingredients are covalently or non-covalently attached; and wherein
said at least one nucleic acid aptamer has a binding affinity for
an oral cavity component.
[0010] In another embodiment of the present invention, a method for
delivering one or more oral care active ingredients to the oral
cavity is provided. The method comprises administering an oral care
composition comprising: at least one nucleic acid aptamer and one
or more nanomaterials; wherein said at least one nucleic acid
aptamer and said one or more nanomaterials are covalently or
non-covalently attached; and wherein said at least one nucleic acid
aptamer has a binding affinity for an oral cavity component.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] For a more complete understanding of the disclosure,
reference should be made to the following detailed description and
drawing FIGS.
[0012] FIG. 1 illustrates the enrichment trajectories of the top
twenty sequences in terms of copy number across different selection
rounds for Experiment A.
[0013] FIG. 2 illustrates the enrichment trajectories of the top
twenty sequences in terms of copy number across different selection
rounds for Experiment B.
[0014] FIG. 3A shows a negative control.
[0015] FIG. 3B shows the binding of the aptamer identified as
"OC1R-B1" to teeth.
[0016] FIG. 3C shows the binding of the aptamer identified as
"OC1R-B9" to teeth.
[0017] FIG. 3D shows the binding of the aptamer identified as
"OC1R-B25/OC1R-A9" to teeth.
[0018] FIG. 4 illustrates the correlation matrix ordered by
clustering (Ward.D2 method) for enrichment trajectories of the top
100 sequences in terms of copy number for Experiment B.
[0019] FIG. 5 illustrates the results of the motif analysis of
random region of aptamer OC1R-B1.
[0020] FIG. 6 illustrates the predicted secondary structure of
aptamer OC1R-B1 and its conserved motif.
[0021] FIG. 7 illustrates the results of the motif analysis of
random region of aptamer OC1R-B9.
[0022] FIG. 8 illustrates the predicted secondary structure of
aptamer OC1R-B9 and its conserved motifs.
[0023] FIG. 9 illustrates the results of the motif analysis of
random region of aptamer OC1R-A9.
[0024] FIG. 10 illustrates the predicted secondary structure of
aptamer OC1R-A9 and its conserved motif.
[0025] FIG. 11 illustrates the predicted secondary structure of
aptamer OC1D-A9.
[0026] FIG. 12 illustrates the alignment of exemplary sequences
with at least 90% nucleotide sequence identity that were identified
during the selection process.
[0027] FIG. 13 illustrates the alignment of exemplary sequences
with at least 70% nucleotide sequence identity that were identified
during the selection process.
[0028] FIG. 14 illustrates the alignment of exemplary sequences
with at least 50% nucleotide sequence identity that were identified
during the selection process.
[0029] FIG. 15 illustrates the amount of DNA Aptamers bound to
teeth.
[0030] FIG. 16 illustrates the amount of DNA Aptamers bound to
teeth after every washing.
[0031] FIG. 17 illustrates the total amount of DNA aptamers bound
(remaining), washed (eluted), and unrecovered (lost) from
teeth.
[0032] FIG. 18A illustrates the predicted secondary structures of
aptamer OC1D-B1.
[0033] FIG. 18B illustrates the predicted secondary structures of
aptamer OC1D-B1.1.
[0034] FIG. 18C illustrates the predicted secondary structures of
aptamer OC1D-B1.2.
[0035] FIG. 18D illustrates the predicted secondary structures of
aptamer OC1D-B1.3.
[0036] FIG. 19A illustrates the predicted secondary structure of
aptamer OC1D-B9.
[0037] FIG. 19B illustrates the predicted secondary structure of
aptamer OC1D-B9.1.
[0038] FIG. 19C illustrates the predicted secondary structure of
aptamer OC1D-B9.2.
[0039] FIG. 20A illustrates the predicted secondary structure of
aptamer OC1D-A9.
[0040] FIG. 20B illustrates the predicted secondary structure of
aptamer OC1D-A9.1.
[0041] FIGS. 21A-C illustrate the binding of truncated DNA aptamers
to teeth.
DETAILED DESCRIPTION OF THE INVENTION
[0042] The present invention includes oral care compositions
comprising one or more aptamers, wherein the aptamers are designed
to bind to specific targets within the oral cavity, such as tooth
surfaces or mucosal tissue. Oral care actives may be bound to the
aptamers allowing the actives to be delivered to the specific oral
cavity target, allowing for greater efficiency and affect.
Definitions
[0043] As used herein, the term "aptamer" refers to a single
stranded oligonucleotide or a peptide that has a binding affinity
for a specific target.
[0044] As used herein, the term "nucleic acid" refers to a polymer
or oligomer of nucleotides. Nucleic acids are also referred as
"ribonucleic acids" when the sugar moiety of the nucleotides is
D-ribose and as "deoxyribonucleic acids" when the sugar moiety is
2-deoxy-D-ribose.
[0045] As used herein, the term "nucleotide" usually refers to a
compound consisting of a nucleoside esterified to a monophosphate,
polyphosphate, or phosphate-derivative group via the hydroxyl group
of the 5-carbon of the sugar moiety. Nucleotides are also referred
as "ribonucleotides" when the sugar moiety is D-ribose and as
"deoxyribonucleotides" when the sugar moiety is
2-deoxy-D-ribose.
[0046] As used herein, the term "nucleoside" refers to a
glycosylamine consisting of a nucleobase, such as a purine or
pyrimidine, usually linked to a 5-carbon sugar (e.g. D-ribose or
2-deoxy-D-ribose) via a .beta.-glycosidic linkage. Nucleosides are
also referred as "ribonucleosides" when the sugar moiety is
D-ribose and as "deoxyribonucleosides" when the sugar moiety is
2-deoxy-D-ribose.
[0047] As used herein, the term "nucleobase", refers to a compound
containing a nitrogen atom that has the chemical properties of a
base. Non-limiting examples of nucleobases are compounds comprising
pyridine, purine, or pyrimidine moieties, including, but not
limited to adenine, guanine, hypoxanthine, thymine, cytosine, and
uracil.
[0048] As used herein, the term "oligonucleotide" refers to an
oligomer composed of nucleotides.
[0049] As used herein, the term "identical" or "sequence identity,"
in the context of two or more oligonucleotides, nucleic acids, or
aptamers, refers to two or more sequences that are the same or have
a specified percentage of nucleotides that are the same, when
compared and aligned for maximum correspondence, as measured using
sequence comparison algorithms or by visual inspection.
[0050] As used herein, the term "substantially homologous" or
"substantially identical" in the context of two or more
oligonucleotides, nucleic acids, or aptamers, generally refers to
two or more sequences or subsequences that have at least 40%, 60%,
80%, 90%, 95%, 96%, 97%, 98% or 99% nucleotide identity, when
compared and aligned for maximum correspondence, as measured using
sequence comparison algorithms or by visual inspection.
[0051] As used herein, the term "epitope" refers to the region of a
target that interacts with the aptamer. An epitope can be a
contiguous stretch within the target or can be represented by
multiple points that are physically proximal in a folded form of
the target.
[0052] As used herein, the term "motif" refers to the sequence of
contiguous, or series of contiguous, nucleotides occurring in a
library of aptamers with binding affinity towards a specific target
(e.g. teeth) and that exhibit a statistically significant higher
probability of occurrence than would be expected compared to a
library of random oligonucleotides. The motif sequence is
frequently the result or driver of the aptamer selection
process.
[0053] As used herein the term "binding affinity" may be calculated
using the following equation: Binding Affinity=Amount of aptamer
bound to one or more teeth/Total amount of aptamer incubated with
one or more teeth.
[0054] By "oral care composition", as used herein, is meant a
product, which in the ordinary course of usage, is not
intentionally swallowed for purposes of systemic administration of
therapeutic agents, but is rather retained in the oral cavity for a
time sufficient to contact dental surfaces or oral tissues.
Examples of oral care compositions include dentifrice, tooth gel,
subgingival gel, mouth rinse, mousse, foam, mouth spray, lozenge,
chewable tablet, chewing gum, tooth whitening strips, floss and
floss coatings, breath freshening dissolvable strips, or denture
care or adhesive product. The oral care composition may also be
incorporated onto strips or films for direct application or
attachment to oral surfaces.
[0055] The term "dentifrice", as used herein, includes tooth or
subgingival-paste, gel, or liquid formulations unless otherwise
specified. The dentifrice composition may be a single phase
composition or may be a combination of two or more separate
dentifrice compositions. The dentifrice composition may be in any
desired form, such as deep striped, surface striped, multilayered,
having a gel surrounding a paste, or any combination thereof. Each
dentifrice composition in a dentifrice comprising two or more
separate dentifrice compositions may be contained in a physically
separated compartment of a dispenser and dispensed
side-by-side.
[0056] All percentages and ratios used hereinafter are by weight of
total composition, unless otherwise indicated. All percentages,
ratios, and levels of ingredients referred to herein are based on
the actual amount of the ingredient, and do not include solvents,
fillers, or other materials with which the ingredient may be
combined as a commercially available product, unless otherwise
indicated.
[0057] All measurements referred to herein are made at 25.degree.
C. unless otherwise specified.
[0058] As used herein, the term "oral cavity" means the part of the
mouth including the teeth and gums and the cavity behind the teeth
and gums that is bounded above by the hard and soft palates and
below by the tongue and mucous membrane.
Aptamer Compositions
[0059] Nucleic acid aptamers are single-stranded oligonucleotides,
with specific secondary and tertiary structures, that can bind to
targets with high affinity and specificity. In certain embodiments
of the present invention, an aptamer composition comprises at least
one oligonucleotide comprising: deoxyribonucleotides,
ribonucleotides, derivatives of deoxyribonucleotides, derivatives
of ribonucleotides, or mixtures thereof; wherein said aptamer
composition has a binding affinity for a material that is at least
one of: tooth, enamel, dentin, hydroxyapatite, carbonated
calcium-deficient hydroxyapatite, or mixtures thereof. In another
embodiment, said aptamer composition has a binding affinity for
tooth.
[0060] In another embodiment, an aptamer composition includes at
least one oligonucleotide comprising oligonucleotides with at least
50% nucleotide sequence identity to sequences that are at least one
of SEQ ID NO 1 to SEQ ID NO 234. In another embodiment, an aptamer
composition includes at least one oligonucleotide comprising
oligonucleotides with at least 70% nucleotide sequence identity to
sequences including SEQ ID NO 1 to SEQ ID NO 234. In yet another
embodiment, an aptamer composition comprises at least one
oligonucleotide having at least 90% nucleotide sequence identity to
at least one of SEQ ID NO 1 to SEQ ID NO 234. In another
embodiment, an aptamer composition comprises at least one
oligonucleotide having at least 20 contiguous nucleotides from at
least one of SEQ ID NO 1 to SEQ ID NO 222. In another embodiment,
an aptamer composition comprises at least one oligonucleotide
having at least 40 contiguous nucleotides from at least one of SEQ
ID NO 1 to SEQ ID NO 222. In another embodiment, an aptamer
composition comprises at least one oligonucleotide having at least
60 contiguous nucleotides from at least one of SEQ ID NO 1 to SEQ
ID NO 222. In another embodiment, an aptamer composition comprises
at least one oligonucleotide having at least 70 contiguous
nucleotides from at least one of SEQ ID NO 1 to SEQ ID NO 222. In
another embodiment, an aptamer composition comprises at least one
oligonucleotide having at least 80 contiguous nucleotides from at
least one of SEQ ID NO 1 to SEQ ID NO 222.
[0061] In another embodiment, an aptamer composition comprises at
least one oligonucleotide comprising SEQ ID NO 1, SEQ ID NO 9, SEQ
ID NO 25, SEQ ID NO 112, SEQ ID NO 120, SEQ ID NO 136, or SEQ ID NO
223 to SEQ ID NO 234. In another embodiment, an aptamer composition
comprises at least one oligonucleotide having at least 50%
nucleotide sequence identity to at least one of SEQ ID NO 1, SEQ ID
NO 9, SEQ ID NO 25, SEQ ID NO 112, SEQ ID NO 120, SEQ ID NO 136, or
SEQ ID NO 223 to SEQ ID NO 234. In another embodiment, an aptamer
composition comprises at least one oligonucleotide having at least
70% nucleotide sequence identity to at least one of SEQ ID NO 1,
SEQ ID NO 9, SEQ ID NO 25, SEQ ID NO 112, SEQ ID NO 120, SEQ ID NO
136, or SEQ ID NO 223 to SEQ ID NO 234. In another embodiment, an
aptamer composition comprises at least one oligonucleotide having
at least 90% nucleotide sequence identity to at least one of SEQ ID
NO 1, SEQ ID NO 9, SEQ ID NO 25, SEQ ID NO 112, SEQ ID NO 120, SEQ
ID NO 136, or SEQ ID NO 223 to SEQ ID NO 234. Non-limiting examples
of oligonucleotides with at least 90% nucleotide sequence identity
to SEQ ID NO 1 are SEQ ID NO 49, SEQ ID NO 69, and SEQ ID NO 75. A
non-limiting example of an oligonucleotide with at least 50%
nucleotide sequence identity to SEQ ID NO 9 is SEQ ID NO 14.
[0062] In another embodiment, an oligonucleotide comprises at least
one or more motifs of SEQ ID NO 235, SEQ ID NO 236, SEQ ID NO 237,
SEQ ID NO 238, SEQ ID NO 239, SEQ ID NO 240, SEQ ID NO 241, SEQ ID
NO 242, SEQ ID NO 243, or SEQ ID NO 244. In another embodiment, an
aptamer composition comprises at least one oligonucleotide having
at least 70% nucleotide sequence identity to at least one of SEQ ID
NO 235, SEQ ID NO 236, SEQ ID NO 237, SEQ ID NO 238, SEQ ID NO 239,
SEQ ID NO 240, SEQ ID NO 241, SEQ ID NO 242, SEQ ID NO 243, or SEQ
ID NO 244. In another embodiment, an aptamer composition comprises
at least one oligonucleotide having at least 80% nucleotide
sequence identity to at least one of SEQ ID NO 235, SEQ ID NO 236,
SEQ ID NO 237, SEQ ID NO 238, SEQ ID NO 239, SEQ ID NO 240, SEQ ID
NO 241, SEQ ID NO 242, SEQ ID NO 243, or SEQ ID NO 244. In another
embodiment, an aptamer composition comprises at least one
oligonucleotide having at least 90% nucleotide sequence identity to
at least one of SEQ ID NO 235, SEQ ID NO 236, SEQ ID NO 237, SEQ ID
NO 238, SEQ ID NO 239, SEQ ID NO 240, SEQ ID NO 241, SEQ ID NO 242,
SEQ ID NO 243, or SEQ ID NO 244.
[0063] In another embodiment, the fluorinated pyrimidine
nucleotides of SEQ ID NO 1 to SEQ ID NO 111 and SEQ ID NO 223 to
228 are substituted by the corresponding natural non-fluorinated
pyrimidine nucleotides.
[0064] Chemical modifications can introduce new features into the
aptamers such as different molecular interactions with the target,
improved binding capabilities, enhanced stability of
oligonucleotide conformations, or increased resistance to
nucleases. In certain embodiments, an oligonucleotide of an aptamer
composition comprises natural or non-natural nucleobases. Natural
nucleobases are adenine, cytosine, guanine, thymine, and uracil.
Non-limiting examples of non-natural nucleobases are hypoxanthine,
xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine,
5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine,
6-methylisoquinoline-1-thione-2-yl, 3-methoxy-2-naphthyl,
5-propynyluracil-1-yl, 5-methylcytosin-1-yl, 2-aminoadenin-9-yl,
7-deaza-7-iodoadenin-9-yl, 7-deaza-7-propynyl-2-aminoadenin-9-yl,
phenoxazinyl, phenoxazinyl-G-clam, bromouracil, 5-iodouracil, and
mixtures thereof.
[0065] Modifications of the phosphate backbone of the
oligonucleotides can also increase the resistance against nuclease
digestion. In certain embodiments, the nucleosides of
oligonucleotides are linked by a chemical motif that is at least
one of: natural phosphate diester, chiral phosphorothionate, chiral
methyl phosphonate, chiral phosphoramidate, chiral phosphate chiral
triester, chiral boranophosphate, chiral phosphoroselenoate,
phosphorodithioate, phosphorothionate amidate,
methylenemethylimino, 3'-amide, 3' achiral phosphoramidate, 3'
achiral methylene phosphonates, thioformacetal, thioethyl ether,
fluorophosphate, or mixtures thereof. In another embodiment, the
nucleosides of oligonucleotides may be linked by natural phosphate
diesters.
[0066] In another embodiment, the sugar moiety of the nucleosides
of oligonucleotides may be at least one of: ribose, deoxyribose,
2'-fluoro deoxyribose, 2'-O-methyl ribose, 2'-O-(3-amino)propyl
ribose, 2'-O-(2-methoxy)ethyl ribose,
2'-O-2-(N,N-dimethylaminooxy)ethyl ribose,
2'-O-2-[2-(N,N-dimethylamino)ethyloxy]ethyl ribose,
2'-O--N,N-dimethylacetamidyl ribose, N-morpholinophosphordiamidate,
.alpha.-deoxyribofuranosyl, other pentoses, hexoses, or mixtures
thereof.
[0067] In another embodiment, said derivatives of ribonucleotides
or derivatives of deoxyribonucleotides may be at least one of:
locked oligonucleotides, peptide oligonucleotides, glycol
oligonucleotides, threose oligonucleotides, hexitol
oligonucleotides, altritol oligonucleotides, butyl
oligonucleotides, L-ribonucleotides, arabino oligonucleotides,
2'-fluoroarabino oligonucleotides, cyclohexene oligonucleotides,
phosphorodiamidate morpholino oligonucleotides, or mixtures
thereof.
[0068] In another embodiment, the nucleotides at the 5'- and
3'-ends of an oligonucleotide are inverted. In another embodiment,
at least one nucleotide of an oligonucleotide is fluorinated at the
2' position of the pentose group. In another embodiment, the
pyrimidine nucleotides of an oligonucleotide are fluorinated at the
2' position of the pentose group. In another embodiment, the
aptamer composition further comprises at least one polymeric
material, wherein the polymeric material may be covalently linked
to an oligonucleotide; wherein the polymeric material may be
polyethylene glycol.
[0069] In another embodiment, an oligonucleotide may be between
about 10 and about 200 nucleotides in length. In another
embodiment, an oligonucleotide may be less than about 100
nucleotides in length. In yet another embodiment, an
oligonucleotide may be less than about 50 nucleotides in
length.
[0070] In another embodiment, an oligonucleotide may be covalently
or non-covalently attached to one or more oral care active
ingredients. Suitable oral care active ingredients include any
material that is generally considered as safe for use in the oral
cavity and that provides changes to the overall health of the oral
cavity; and specifically, to the condition of the oral surfaces
that such oral care active ingredients interact with. Examples of
oral conditions these actives address include, but are not limited
to, appearance and structural changes to teeth, whitening, stain
prevention and removal, stain bleaching, plaque prevention and
removal, tartar prevention and removal, cavity prevention and
treatment, inflamed and/or bleeding gums, mucosal wounds, lesions,
ulcers, aphthous ulcers, cold sores, and tooth abscesses.
[0071] In another embodiment, said one or more oral care active
ingredients are selected from the group comprising: whitening
agents, brightening agents, anti-stain agents, anti-cavity agents,
anti-erosion agents, anti-tartar agents, anti-calculus agents,
anti-plaque agents, teeth remineralizing agents, anti-fracture
agents, strengthening agents, abrasion resistance agents,
anti-gingivitis agents, anti-microbial agents, anti-bacterial
agents, anti-fungal agents, anti-yeast agents, anti-viral,
anti-malodor agents, breath freshening agents, flavoring agents,
cooling agents, taste enhancement agents, olfactory enhancement
agents, anti-adherence agents, smoothness agents, surface
modification agents, anti-tooth pain agents, anti-sensitivity
agents, anti-inflammatory agents, gum protecting agents,
periodontal actives, tissue regeneration agents, anti-blood
coagulation agents, anti-clot stabilizer agents, salivary stimulant
agents, salivary rheology modification agents, enhanced retention
agents, soft/hard tissue targeted agents, tooth/soft tissue
cleaning agents, antioxidants, pH modifying agents, H-2
antagonists, analgesics, natural extracts and essential oils, dyes,
optical brighteners, cations, phosphates, fluoride ion sources,
peptides, nutrients, enzymes, mouth and throat products, and
mixtures thereof. Non-limiting specific examples of oral care
active ingredients are listed in Section IV.
[0072] In another embodiment, an oligonucleotide is non-covalently
attached to one or more oral care active ingredients, via molecular
interactions. Examples of molecular interactions are electrostatic
forces, van der Waals interactions, hydrogen bonding, and .pi.-.pi.
stacking interactions of aromatic rings.
[0073] In another embodiment, an oligonucleotide may be covalently
attached to said one or more oral care active ingredients using one
or more linkers or spacers. Non-limiting examples of linkers are
chemically labile linkers, enzyme-labile linkers, and non-cleavable
linkers. Examples of chemically labile linkers are acid-cleavable
linkers and disulfide linkers. Acid-cleavable linkers take
advantage of low pH to trigger hydrolysis of an acid-cleavable
bond, such as a hydrazone bond, to release the active ingredient or
payload. Disulfide linkers can release the active ingredients under
reducing environments. Examples of enzyme-labile linkers are
peptide linkers that can be cleaved in the present of proteases and
.beta.-glucuronide linkers that are cleaved by glucuronidases
releasing the payload. Non-cleavable linkers can also release the
active ingredient if the aptamer is degraded by nucleases.
[0074] In another embodiment, an oligonucleotide may be covalently
or non-covalently attached to one or more nanomaterials. In another
embodiment, an oligonucleotide and one or more oral care active
ingredients may be covalently or non-covalently attached to one or
more nanomaterials. In another embodiment, one or more oral care
active ingredients are carried by one or more nanomaterials.
Non-limiting examples of nanomaterials are gold nanoparticles,
nano-scale iron oxides, carbon nanomaterials (such as single-walled
carbon nanotubes and graphene oxide), mesoporous silica
nanoparticles, quantum dots, liposomes, poly (lactide-co-glycolic
acids) nanoparticles, polymeric micelles, dendrimers, serum albumin
nanoparticles, and DNA-based nanomaterials. These nanomaterials can
serve as carriers for large volumes of oral care active
ingredients, while the aptamers can facilitate the delivery of the
nanomaterials with the actives to the expected target.
[0075] Nanomaterials can have a variety of shapes or morphologies.
Non-limiting examples of shapes or morphologies are spheres,
rectangles, polygons, disks, toroids, cones, pyramids,
rods/cylinders, and fibers. In the context of the present
invention, nanomaterials may have at least one spatial dimension
that is less than about 100 .mu.m and more preferably less than
about 10 .mu.m. Nanomaterials comprise materials in solid phase,
semi-solid phase, or liquid phase.
[0076] Aptamers can also be peptides that bind to targets with high
affinity and specificity. These peptide aptamers can be part of a
scaffold protein. Peptide aptamers can be isolated from
combinatorial libraries and improved by directed mutation or rounds
of variable region mutagenesis and selection. In certain
embodiments of the present invention, an aptamer composition may
comprise at least one peptide or protein; wherein the aptamer
composition has a binding affinity for a material selected from the
group consisting of: tooth, enamel, dentin, hydroxyapatite,
carbonated calcium-deficient hydroxyapatite, and mixtures
thereof.
Methods of Designing Aptamer Compositions
[0077] The method of designing nucleic acid aptamers known as
Systematic Evolution of Ligands by Exponential Enrichment (SELEX)
has been broadly studied and improved for the selection of aptamers
against small molecules and proteins (WO 91/19813). In brief, in
the conventional version of SELEX, the process starts with the
synthesis of a large library of oligonucleotides consisting of
randomly generated sequences of fixed length flanked by constant
5'- and 3'-ends that serve as primers. The oligonucleotides in the
library are then exposed to the target ligand and those that do not
bind the target are removed. The bound sequences are eluted and
amplified by PCR to prepare for subsequent rounds of selection in
which the stringency of the elution conditions is usually increased
to identify the tightest-binding oligonucleotides. In addition to
conventional SELEX, there are improved versions such as capillary
electrophoresis-SELEX, magnetic bead-based SELEX, cell-SELEX,
automated SELEX, complex-target SELEX, among others. A review of
aptamer screening methods is found in "Kim, Y. S. and M. B. Gu
(2014). Advances in Aptamer Screening and Small Molecule
Aptasensors. Adv. Biochem. Eng./Biotechnol. 140 (Biosensors based
on Aptamers and Enzymes): 29-67" and "Stoltenburg, R., et al.
(2007). SELEX-A (r)evolutionary method to generate high-affinity
nucleic acid ligands. Biomol. Eng. 24(4): 381-403," the contents of
which are incorporated herein by reference. Although the SELEX
method has been broadly applied, it is neither predictive nor
standardized for every target. Instead, a method must be developed
for each particular target in order for the method to lead to
viable aptamers.
[0078] Despite the large number of selected aptamers, SELEX has not
been routinely applied for the selection of aptamers with binding
affinities towards macroscopic materials and surfaces. For the
successful selection of aptamers with high binding affinity and
specificity against macroscopic materials, the epitope should be
present in sufficient amount and purity to minimize the enrichment
of unspecifically binding oligonucleotides and to increase the
specificity of the selection. Also, the presence of positively
charged groups (e.g. primary amino groups), the presence of
hydrogen bond donors and acceptors, and planarity (aromatic
compounds) facilitate the selection of aptamers. In contrast,
negatively charged molecules (e.g. containing phosphate groups)
make the selection process more difficult. Unexpectedly, in spite
of the detrimental chemical features of teeth that make aptamer
selection challenging, the inventors have found that SELEX can be
used for the design of aptamers with high binding affinity and
specificity for teeth.
Selection Library
[0079] In SELEX, the initial candidate library is generally a
mixture of chemically synthesized DNA oligonucleotides, each
comprising a long variable region of n nucleotides flanked, at the
3' and 5' ends, by conserved regions or primer recognition regions
for all the candidates of the library. These primer recognition
regions allow the central variable region to be manipulated during
SELEX, in particular by means of PCR.
[0080] The length of the variable region determines the diversity
of the library, which is equal to 4.sup.n since each position can
be occupied by one of four nucleotides A, T, G or C. For long
variable regions, huge library complexities arise. For instance,
when n=50, the theoretical diversity is 4.sup.50 or 10.sup.30,
which is an inaccessible value in practice as it corresponds to
more than 10.sup.5 tons of material for a library wherein each
sequence is represented once. The experimental limit is around
10.sup.15 different sequences, which is that of a library wherein
all candidates having a variable region of 25 nucleotides are
represented. If one chooses to manipulate a library comprising a
30-nucleotide variable region whose theoretical diversity is about
10.sup.18, only 1/1000 of the possibilities will thus be explored.
In practice, that is generally sufficient to obtain aptamers having
the desired properties. Additionally, since the polymerases used
are unreliable and introduce errors at a rate on the order of
10.sup.-4, they contribute to significantly enrich the diversity of
the sequence pool throughout the SELEX process: one candidate in
100 will be modified in each amplification cycle for a library with
a random region of 100 nucleotides in length, thus leading to the
appearance of 10.sup.13 new candidates for the overall library.
[0081] In certain embodiments of the present invention, the
starting mixture of oligonucleotides may comprise more than about
10.sup.6 different oligonucleotides or from between about 10.sup.13
to about 10.sup.15 different oligonucleotides. In another
embodiment of the present invention, the length of the variable
region may be between about 10 and about 100 nucleotides. In
another embodiment, the length of the variable region may be
between about 20 and about 60 nucleotides. In yet another
embodiment, the length of the variable region is about 40
nucleotides. Random regions shorter than 10 nucleotides may be
used, but may be constrained in their ability to form secondary or
tertiary structures and in their ability to bind to target
molecules. Random regions longer than 100 nucleotides may also be
used but may present difficulties in terms of cost of synthesis.
The randomness of the variable region is not a constraint of the
present invention. For instance, if previous knowledge exists
regarding oligonucleotides that bind to a given target, libraries
spiked with such sequences may work as well or better than
completely random ones.
[0082] In the design of primer recognition sequences care should be
taken to minimize potential annealing among sequences, fold back
regions within sequences, or annealing of the same sequence itself.
In certain embodiments of the present invention, the length of
primer recognition sequences may be between about 10 and about 40
nucleotides. In another embodiment, the length of primer
recognition sequences may be between about 12 and about 30
nucleotides. In yet another embodiment, the length of primer
recognition sequences may be between about 18 and about 26
nucleotides, i.e., about 18, 19, 20, 21, 22, 23, 24, 25 or 26
nucleotides. The length and sequence of the primer recognition
sequences determine their annealing temperature. In certain
embodiments, the primer recognition sequences of oligonucleotides
may have an annealing temperature between about 60.degree. C. and
about 72.degree. C.
[0083] Aptamers can be ribonucleotides (RNA), deoxynucleotides
(DNA), or their derivatives. When aptamers are ribonucleotides, the
first SELEX step may consist in transcribing the initial mixture of
chemically synthesized DNA oligonucleotides via the primer
recognition sequence at the 5' end. After selection, the candidates
are converted back into DNA by reverse transcription before being
amplified. RNA and DNA aptamers having comparable characteristics
have been selected against the same target and reported in the art.
Additionally, both types of aptamers can be competitive inhibitors
of one another, suggesting potential overlapping of interaction
sites.
[0084] New functionalities, such as hydrophobicity or
photoreactivity, can be incorporated into the oligonucleotides by
modifications of the nucleobases before or after selection.
Modifications at the C-5 position of pyrimidines or at the C-8 or
N-7 positions of purines are especially common and compatible with
certain enzymes used during the amplification step in SELEX. In
certain embodiments of the present invention, said oligonucleotides
comprise natural or non-natural nucleobases. Natural nucleobases
are adenine, cytosine, guanine, thymine, and uracil. Non-limiting
examples of non-natural nucleobases are hypoxanthine, xanthine,
7-methylguanine, 5,6-dihydrouracil, 5-5-methylcytosine,
5-hydroxymethylcytosine, thiouracil, 1-methylhypoxanthine,
6-methylisoquinoline-1-thione-2-yl, 3-methoxy-2-naphthyl,
5-propynyluracil-1-yl, 5-methylcytosin-1-yl, 2-aminoadenin-9-yl,
7-deaza-7-iodoadenin-9-yl, 7-deaza-7-propynyl-2-aminoadenin-9-yl,
phenoxazinyl, phenoxazinyl-G-clam, 5-bromouracil, 5-iodouracil, and
mixtures thereof. Some non-natural nucleobases, such as
5-bromouracil or 5-iodouracil, can be used to generate
photo-cross-linkable aptamers, which can be activated by UV light
to form a covalent link with the target.
[0085] In another embodiment, the nucleosides of said
oligonucleotides are linked by a chemical motif selected from the
group comprising: natural phosphate diester, chiral
phosphorothionate, chiral methyl phosphonate, chiral
phosphoramidate, chiral phosphate chiral triester, chiral
boranophosphate, chiral phosphoroselenoate, phosphorodithioate,
phosphorothionate amidate, methylenemethylimino, 3'-amide, 3'
achiral phosphoramidate, 3' achiral methylene phosphonates,
thioformacetal, thioethyl ether, fluorophosphate, and mixtures
thereof. In yet another embodiment, the nucleosides of said
oligonucleotides are linked by natural phosphate diesters.
[0086] In another embodiment, the sugar moiety of the nucleosides
of said oligonucleotides may be selected from the group comprising:
ribose, deoxyribose, 2'-fluoro deoxyribose, 2'-O-methyl ribose,
2'-O-(3-amino)propyl ribose, 2'-O-(2-methoxy)ethyl ribose,
2'-O-2-(N,N-dimethylaminooxy)ethyl ribose,
2'-O-2-[2-(N,N-dimethylamino)ethyloxy]ethyl ribose,
2'-O--N,N-dimethylacetamidyl ribose, N-morpholinophosphordiamidate,
.alpha.-deoxyribofuranosyl, other pentoses, hexoses, and mixtures
thereof.
[0087] In another embodiment, said derivatives of ribonucleotides
or said derivatives of deoxyribonucleotides are selected from the
group comprising: locked oligonucleotides, peptide
oligonucleotides, glycol oligonucleotides, threose
oligonucleotides, hexitol oligonucleotides, altritol
oligonucleotides, butyl oligonucleotides, L-ribonucleotides,
arabino oligonucleotides, 2'-fluoroarabino oligonucleotides,
cyclohexene oligonucleotides, phosphorodiamidate morpholino
oligonucleotides, and mixtures thereof.
[0088] When using modified nucleotides during the SELEX process,
they should be compatible with the enzymes used during the
amplification step. Non-limiting examples of modifications that are
compatible with commercial enzymes include modifications at the 2'
position of the sugar in RNA libraries. The ribose 2'-OH group of
pyrimidine nucleotides can be replaced with 2'-amino, 2'-fluoro,
2'-methyl, or 2'-O-methyl, which protect the RNA from degradation
by nucleases. Additional modifications in the phosphate linker,
such as phosphorothionate and boranophosphate, are also compatible
with the polymerases and confer resistance to nucleases.
[0089] In certain embodiments of the present invention, at least
one nucleotide of said oligonucleotides is fluorinated at the 2'
position of the pentose group. In another embodiment, the
pyrimidine nucleotides of said oligonucleotides are at least
partially fluorinated at the 2' position of the pentose group. In
yet another embodiment, all the pyrimidine nucleotides of said
oligonucleotides are fluorinated at the 2' position of the pentose
group. In another embodiment, at least one nucleotide of said
oligonucleotides is aminated at the 2' position of the pentose
group.
[0090] Another approach, recently described as two-dimensional
SELEX, simultaneously applies in vitro oligonucleotide selection
and dynamic combinatorial chemistry (DCC), e.g., a reversible
reaction between certain groups of the oligonucleotide (amine
groups) and a library of aldehyde compounds. The reaction produces
imine oligonucleotides which are selected on the same principles as
for conventional SELEX. It was thus possible to identify for a
target hairpin RNA modified aptamers that differ from natural
aptamers.
[0091] A very different approach relates to the use of optical
isomers. Natural oligonucleotides are D-isomers. L-analogs are
resistant to nucleases but cannot be synthesized by polymerases.
According to the laws of optical isomerism, an L-series aptamer can
form with its target (T) a complex having the same characteristics
as the complex formed by the D-series isomer and the enantiomer
(T') of the target (T). Consequently, if compound T' can be
chemically synthesized, it can be used to perform the selection of
a natural aptamer (D). Once identified, this aptamer can be
chemically synthesized in an L-series. This L-aptamer is a ligand
of the natural target (T).
Selection Step
[0092] Single stranded oligonucleotides can fold to generate
secondary and tertiary structures, resembling the formation of base
pairs. The initial sequence library is thus a library of
three-dimensional shapes, each corresponding to a distribution of
units that can trigger electrostatic interactions, create hydrogen
bonds, etc. Selection becomes a question of identifying in the
library the shape suited to the target, i.e., the shape allowing
the greatest number of interactions and the formation of the most
stable aptamer-target complex. For small targets (dyes,
antibiotics, etc.) the aptamers identified are characterized by
equilibrium dissociation constants in the micromolar range, whereas
for protein targets K.sub.d values below 10.sup.-9 M are not
rare.
[0093] Selection in each round occurs by means of physical
separation of oligonucleotides associated with the target from free
oligonucleotides. Multiple techniques may be applied
(chromatography, filter retention, electrophoresis, etc.). The
selection conditions are adjusted (relative concentration of
target/candidates, ion concentration, temperature, washing, etc.)
so that a target-binding competition occurs between the
oligonucleotides. Generally, stringency is increased as the rounds
proceed in order to promote the capture of oligonucleotides with
the highest affinity. In addition, counter-selections or negative
selections are carried out to eliminate oligonucleotides that
recognize the support or unwanted targets (e.g., filter, beads,
etc.).
[0094] The SELEX process for the selection of target-specific
aptamers is characterized by repetition of five main steps: binding
of oligonucleotides to the target, partition or removal of
oligonucleotides with low binding affinity, elution of
oligonucleotides with high binding affinity, amplification or
replication of oligonucleotides with high binding affinity, and
conditioning or preparation of the oligonucleotides for the next
cycle. This selection process is designed to identify the
oligonucleotides with the greatest affinity and specificity for the
target material.
[0095] In certain embodiments of the present invention, a method of
designing an aptamer composition comprises the step of contacting:
a) a mixture of oligonucleotides, b) a selection buffer, and c) a
target material selected from the group consisting of: tooth,
enamel, dentin, hydroxyapatite, carbonated calcium-deficient
hydroxyapatite, and mixtures thereof. In another embodiment, said
target material is tooth. In another embodiment, said tooth is at
least partially coated with saliva before said contacting step. In
another embodiment of the present invention, said mixture of
oligonucleotides comprises oligonucleotides selected from the group
consisting of deoxyribonucleotides, ribonucleotides, derivatives of
deoxyribonucleotides, derivatives of ribonucleotides, and mixtures
thereof.
[0096] SELEX cycles are usually repeated several times until
oligonucleotides with high binding affinity are identified. The
number of cycles depends on multiple variables, including target
features and concentration, design of the starting random
oligonucleotide library, selection conditions, ratio of target
binding sites to oligonucleotides, and the efficiency of the
partitioning step. In certain embodiments, said contacting step is
performed at least 5 times. In another embodiment, said contacting
step is performed between 6 and 15 times. In another embodiment,
said method further comprises the step of removing the
oligonucleotides that do not bind said target material during said
contacting step.
[0097] Oligonucleotides are oligo-anions, each unit having a charge
and hydrogen-bond donor/acceptor sites at a particular pH. Thus,
the pH and ionic strength of the selection buffer are important and
should represent the conditions of the intended aptamer
application. In certain embodiments of the present invention, the
pH of said selection buffer is between about 2 and about 9. In
another embodiment, the pH of said selection buffer is between
about 6 and about 8. In yet another embodiment, the pH of said
selection buffer is between about 2 and about 5. Selection buffers
with low pH can be important if the aptamers are expected to have
good binding affinities in acidic environments.
[0098] Cations can facilitate the proper folding of the
oligonucleotides and provide benefits in the oral cavity. In
certain embodiments of the present invention, said selection buffer
comprises cations. Non-limiting examples of cations are Ca.sup.2+,
Sn.sup.2+, Sn.sup.4+, Zn.sup.2+, Al.sup.3+, Cu.sup.2+, Fe.sup.2+,
and Fe.sup.3+. In yet another embodiment, said selection buffer
comprises divalent cations selected from the group comprising
Sn.sup.2+ and Ca.sup.2+.
[0099] In order for the aptamers to maintain their structures and
function during their application, the in vitro selection process
can be carried out under conditions similar to those for which they
are being developed. In certain embodiments of the present
invention, said selection buffer comprises a solution or suspension
of an oral care composition selected from the group comprising
dentifrices, dentifrices, toothpowders, mouthwashes, mouthrinses,
flosses, brushes, strips, sprays, patches, paint on, dissolvables,
edibles, lozenges, gums, chewables, soluble fibers, insoluble
fibers, putties, waxes, denture adhesives, denture cleansers, and
mixtures thereof. In another embodiment of the present invention,
said selection buffer comprises a solution of a dentifrice. In
another embodiment of the present invention, said selection buffer
comprises a solution of saliva.
[0100] In certain embodiments of the present invention, said
selection buffer comprises at least one surfactant. In another
embodiment, said at least one surfactant is selected from the group
comprising sodium lauryl sulfate, betaines, chlorhexidine,
sarcosinates, pluronics and triclosan. In another embodiment, said
at least one surfactant is sodium lauryl sulfate. In another
embodiment, said selection buffer comprises at least one abrasive
material selected from the group comprising aluminum hydroxide,
calcium carbonate, calcium hydrogen phosphates, calcium
pyrophosphate, calcium pyrophosphate (beta phase), silicates,
aluminosilicates, hydroxyapatite, and mixtures thereof. In yet
another embodiment, said selection buffer comprises: a) at least
one surfactant; b) at least one abrasive material selected from the
group comprising aluminum hydroxide, calcium carbonate, calcium
hydrogen phosphates, calcium pyrophosphate, calcium pyrophosphate
(beta phase), silicates, aluminosilicates, hydroxyapatite, and
mixtures thereof; c) at least one phosphate salt; and d) at least
one fluoride salt.
[0101] Negative selection or counter-selection steps can minimize
the enrichment of oligonucleotides that bind to undesired targets
or undesired epitopes within a target. For oral care applications,
binding of aptamers to teeth staining materials may not be
desirable. In certain embodiments of the present invention, said
method of designing an aptamer composition further comprises the
step of contacting: a) a mixture of oligonucleotides, b) a
selection buffer, and c) one or more teeth staining materials. In
another embodiment, said one or more teeth staining materials
comprise one or more natural or synthetic dyes or pigments selected
from the group comprising flavonoids, carotenoids, caramels,
tannins, other chromogens, and mixtures thereof. In yet another
embodiment, said one or more teeth staining materials are selected
from the group comprising wine, coffee, tea, carbonated sodas, and
mixtures thereof. During the negative selection or
counter-selection, the teeth staining materials can be either
unbound or immobilized to a support. Methods for negative selection
or counter-selection of aptamers against unbound targets have been
published in WO201735666, the content of which is incorporated
herein by reference.
[0102] In certain embodiments of the present invention, the method
of designing an aptamer composition may comprise the steps of: a)
synthesizing a mixture of oligonucleotides; b) contacting: i. said
mixture of oligonucleotides, ii. a selection buffer, and iii. a
target material selected from the group consisting of: tooth,
enamel, dentin, hydroxyapatite, carbonated calcium-deficient
hydroxyapatite, and mixtures thereof, to produce a target
suspension; c) removing the liquid phase from said target
suspension to produce a target-oligonucleotide mixture; d)
contacting said target-oligonucleotide mixture with a washing
buffer and removing the liquid phase to produce a target-aptamer
mixture; and e) contacting said target-aptamer mixture with an
elution buffer and recovering the liquid phase to produce an
aptamer mixture. In another embodiment, said steps are performed
repetitively at least 5 times. In another embodiment, said steps
are performed between 6 and 15 times.
[0103] In another embodiment, a method of designing an aptamer
composition comprising the steps of: a) synthesizing a random
mixture of deoxyribonucleotides comprising oligonucleotides
consisting of: i. a T7 promoter sequence at the 5'-end, ii. a
variable 40-nucleotide sequence in the middle, and iii. a conserved
reverse primer recognition sequence at the 3' end; b) transcribing
said random mixture of deoxyribonucleotides using pyrimidine
nucleotides fluorinated at the 2' position of the pentose group and
natural purine nucleotides and a mutant T7 polymerase to produce a
mixture of fluorinated ribonucleotides; c) contacting: i. said
mixture of fluorinated ribonucleotides, ii. a selection buffer, and
iii. a tooth, wherein said tooth is at least partially coated with
saliva, to produce a target suspension; d) removing the liquid
phase from said target suspension to produce a
tooth-oligonucleotide mixture; e) contacting said
tooth-oligonucleotide mixture with a washing buffer and removing
the liquid phase to produce a tooth-aptamer mixture; f) contacting
said tooth-aptamer mixture with an elution buffer and recovering
the liquid phase to produce an RNA aptamer mixture; g) reserve
transcribing and amplifying said RNA aptamer mixture to produce a
DNA copy of said RNA aptamer mixture; and h) sequencing said DNA
copy of said RNA aptamer mixture.
Post-Selection Modification
[0104] To enhance stability of the aptamers, chemical modifications
can be introduced in the aptamer after the selection process. For
instance, the 2'-OH groups of the ribose moieties can be replaced
by 2'-fluoro, 2'-amino, or 2'-O-methyl groups. Furthermore, the 3'-
and 5'-ends of the aptamers can be capped with different groups,
such as streptavidin-biotin, inverted thymidine, amine, phosphate,
polyethylene-glycol, cholesterol, fatty acids, proteins, enzymes,
fluorophores, among others, making the oligonucleotides resistant
to exonucleases or providing some additional benefits. Other
modifications are described in previous sections of the present
disclosure.
[0105] Unlike backbone modifications which can cause aptamer-target
interaction properties to be lost, it is possible to conjugate
various groups at one of the 3'- or 5'-ends of the oligonucleotide
in order to convert it into a delivery vehicle, tool, probe, or
sensor without disrupting its characteristics. This versatility
constitutes a significant advantage of aptamers, in particular for
their application in the current invention. In certain embodiments
of the present invention, one or more oral care active ingredients
are covalently attached to the 3'-end of an oligonucleotide. In
another embodiment, one or more oral care active ingredients are
covalently attached to the 5'-end of an oligonucleotide. In yet
another embodiment, one or more oral care active ingredients are
covalently attached to random positions of an oligonucleotide.
[0106] Incorporation of modifications to aptamers can be performed
using enzymatic or chemical methods. Non-limiting examples of
enzymes used for modification of aptamers are terminal
deoxynucleotidyl transferases (TdT), T4 RNA ligases, T4
polynucleotide kinases (PNK), DNA polymerases, RNA polymerases, and
other enzymes known by those skilled in the art. TdTs are
template-independent polymerases that can add modified
deoxynucleotides to the 3' terminus of deoxyribonucleotides. T4 RNA
ligases can be used to label ribonucleotides at the 3'-end by using
appropriately modified nucleoside 3',5'-bisphosphates. PNK can be
used to phosphorylate the 5'-end of synthetic oligonucleotides,
enabling other chemical transformations (see below). DNA and RNA
polymerases are commonly used for the random incorporation of
modified nucleotides throughout the sequence, provided such
nucleotides are compatible with the enzymes.
[0107] Non-limiting examples of chemical methods used for
modification of aptamers are periodate oxidation of
ribonucleotides, EDC activation of 5'-phosphate, random chemical
labeling methods, and other chemical methods known by those skilled
in the art, incorporated herein as embodiments of the current
invention.
[0108] During periodate oxidation, meta- and ortho-perdionates
cleave the C--C bonds between vicinal diols of 3'-ribonucleotides,
creating two aldehyde moieties that enable the conjugation of
labels or active ingredients at the 3'-end of RNA aptamers. The
resulting aldehydes can be easily reacted with hydrazide- or
primary amine-containing molecules. When amines are used, the
produced Schiff bases can be reduced to more stable secondary
amines with sodium cyanoborohydride (NaBH.sub.4).
[0109] When EDC activation of 5'-phosphate is used, the
5'-phosphate of oligonucleotides is frequently activated with EDC
(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and
imidazole to produce a reactive imidazolide intermediate, followed
by reaction with a primary amine to generate aptamers modified at
the 5'end. Because the 5' phosphate group is required for the
reaction, synthetic oligonucleotides can be first treated with a
kinase (e.g. PNK).
[0110] Random chemical labeling can be performed with different
methods. Because they allow labeling at random sites along the
aptamer, a higher degree of modification can be achieved compared
to end-labeling methods. However, since the nucleobases are
modified, binding of the aptamers to their target can be disrupted.
The most common random chemical modification methods involve the
use of photoreactive reagents, such as phenylazide-based reagents.
When the phenylazide group is exposed to UV light, it forms a
labile nitrene that reacts with double bonds and C--H and N--H
sites of the aptamers.
[0111] Additional information about methods for modification of
aptamers is summarized in "Hermanson G. T. (2008). Bioconjugate
Techniques. 2nd Edition. pp. 969-1002, Academic Press, San Diego.",
the content of which is incorporated herein by reference.
[0112] After selection, in addition to chemical modifications,
sequence truncations can be performed to remove regions that are
not essential for binding or for folding into the structure.
Moreover, aptamers can be linked together to provide different
features or better affinity. Thus, any truncations or combinations
of the aptamers described herein are incorporated as part of the
current invention.
Application of Aptamer Compositions in Oral Care Products
[0113] The aptamers of the current invention can be used in oral
care compositions to provide one or more benefits. In certain
embodiments of the present invention, an oral care composition
comprises at least one nucleic acid aptamer; wherein said at least
one nucleic acid aptamer has a binding affinity for an oral cavity
component. In another embodiment, an oral care composition
comprises at least one nucleic acid aptamer; wherein said at least
one nucleic acid aptamer has a binding affinity for an oral cavity
component selected from the group comprising: tooth, enamel,
dentin, and any other surfaces in the oral cavity. In another
embodiment, an oral care composition comprises at least one nucleic
acid aptamer; wherein said at least one nucleic acid aptamer has a
binding affinity for tooth.
[0114] The oral care compositions of the present invention can be
in different forms. Non-limiting examples of said forms are:
dentifrices (including dentifrices and toothpowders), mouthwashes,
mouthrinses, flosses, brushes, strips, sprays, patches, paint on,
dissolvables, edibles, lozenges, gums, chewables, soluble fibers,
insoluble fibers, putties, waxes, denture adhesives, denture
cleansers, liquids, pastes, Newtonian or non-Newtonian fluids,
gels, and sols.
[0115] The oral acre compositions of the present invention may
include one or more of the following:
[0116] Rheology modifiers suitable for use in the present invention
include organic and inorganic rheology modifiers, and mixtures
thereof. Inorganic rheology modifiers include hectorite and
derivatives, hydrated silicas, ternary and quaternary magnesium
silicate derivatives, bentonite and mixtures thereof. Preferred
inorganic rheology modifiers are hectorite and derivatives,
hydrated silicas and mixtures thereof. Organic rheology modifiers
include xanthan gum, carrageenan and derivatives, gellan gum,
hydroxypropyl methyl cellulose, sclerotium gum and derivatives,
pullulan, rhamsan gum, welan gum, konjac, curdlan, carbomer, algin,
alginic acid, alginates and derivatives, hydroxyethyl cellulose and
derivatives, hydroxypropyl cellulose and derivatives, starch
phosphate derivatives, guar gum and derivatives, starch and
derivatives, co-polymers of maleic acid anhydride with alkenes and
derivatives, cellulose gum and derivatives, ethylene
glycol/propylene glycol co-polymers, poloxamers and derivatives,
polyacrylates and derivatives, methyl cellulose and derivatives,
ethyl cellulose and derivatives, agar and derivatives, gum arabic
and derivatives, pectin and derivatives, chitosan and derivatives,
resinous polyethylene glycols such as PEG-XM where X is >=1,
karaya gum, locust bean gum, natto gum, co-polymers of vinyl
pyrollidone with alkenes, tragacanth gum, polyacrylamides, chitin
derivatives, gelatin, betaglucan, dextrin, dextran, cyclodextrin,
methacrylates, microcrystalline cellulose, polyquatemiums,
furcellaren gum, ghatti gum, psyllium gum, quince gum, tamarind
gum, larch gum, tara gum, and mixtures thereof. Preferred are
xanthan gum, carrageenan and derivatives, gellan gum, hydroxypropyl
methyl cellulose, sclerotium gum and derivatives, pullulan, rhamsan
gum, welan gum, konjac, curdlan, carbomer, algin, alginic acid,
alginates and derivatives, hydroxyethyl cellulose and derivatives,
hydroxypropyl cellulose and derivatives, starch phosphate
derivatives, guar gum and derivatives, starch and derivatives,
co-polymers of maleic acid anhydride with alkenes and derivatives,
cellulose gum and derivatives, ethylene glycol/propylene glycol
co-polymers, poloxamers and derivatives and mixtures thereof.
[0117] In certain embodiments amounts of rheology modifiers may
range from about 0.1% to about 15% or from about 0.5% to about 3%
by weight of the total composition, such as dentifrice.
[0118] In addition to the above components, a sweetener, a flavor,
a preservative, an effective ingredient, abrasives, fluoride ion
sources, chelating agents, antimicrobials, silicone oils and other
adjuvants such as preservatives and coloring agents, etc. may be
added as required.
[0119] As the sweetener, saccharin sodium, sucrose, maltose,
lactose, stevioside, neohesperidildigydrochalcone, glycyrrhizin,
perillartine, p-methoxycinnamic aldehyde and the like may be used,
in an amount of 0.05 to 5% by weight of the total composition.
Essential oils such as spearmint oil, peppermint oil, salvia oil,
eucalptus oil, lemon oil, lime oil, wintergreen oil and cinnamon
oil, other spices and fruit flavors as well as isolated and
synthetic flavoring materials such as 1-menthol, carvone, anethole,
eugenol and the like can be used as flavors. The flavor may be
blended in an amount of 0.1 to 5% by weight of the total
composition. Ethyl paraoxy benzonate, butyl paraoxy benzoate, etc.
may be used as the preservative. The sweetener may be added with
the abrasive. The flavor and the preservative may be added when
preparing the liquid of the slightly swollen rheology modifier or
mixed with rheology modifier after mixing with the humectant.
Enzymes such as dextranase, lytic enzyme, lysozyme, amylase and
antiplasmin agents such as EPSILON-aminocaproic acid and tranexamic
acid, fluorine compounds such as sodium monofluorophosphate sodium
fluoride and stannous fluoride, chlorhexidine salts, quaternary
ammonium salts, aluminum chlorohydroxyl allantoin, glycyrrhetinic
acid, chlorophyll, sodium chloride and phosphoric compounds may be
used as the effective ingredient. Moreover, silica gel, aluminum
silica gel, organic acids and their salts may be blended as
desired. An organic effective ingredient with low viscosity may be
added when preparing the liquid of the slightly swollen rheology
modifier.
[0120] The oral care compositions of the present invention may
comprise greater than about 0.1% by weight of a surfactant or
mixture of surfactants. Surfactant levels cited herein are on a
100% active basis, even though common raw materials such as sodium
lauryl sulphate may be supplied as aqueous solutions of lower
activity.
[0121] Suitable surfactant levels are from about 0.1% to about 15%,
from about 0.25% to about 10%, or from about 0.5% to about 5% by
weight of the total composition. Suitable surfactants for use
herein include anionic, amphoteric, non-ionic, zwitterionic and
cationic surfactants, though anionic, amphoteric, non-ionic and
zwitterionic surfactants (and mixtures thereof) are preferred.
[0122] Useful anionic surfactants herein include the water-soluble
salts of alkyl sulphates and alkyl ether sulphates having from 10
to 18 carbon atoms in the alkyl radical and the water-soluble salts
of sulphonated monoglycerides of fatty acids having from 10 to 18
carbon atoms. Sodium lauryl sulphate and sodium coconut
monoglyceride sulphonates are examples of anionic surfactants o
this type.
[0123] Suitable cationic surfactants useful in the present
invention can be broadly defined as derivatives of aliphatic
quaternary ammonium compounds having one long alkyl chain
containing from about 8 to 18 carbon atoms such as lauryl
trimethylammonium chloride; cetyl pyridinium chloride; benzalkonium
chloride; cetyl trimethylammonium bromide;
di-isobutylphenoxyethyl-dimethylbenzylammonium chloride; coconut
alkyltrimethyl-ammonium nitrite; cetyl pyridinium fluoride; etc.
Certain cationic surfactants can also act as germicides in the
compositions disclosed herein.
[0124] Suitable nonionic surfactants that can be used in the
compositions of the present invention can be broadly defined as
compounds produced by the condensation of alkylene oxide groups
(hydrophilic in nature) with an organic hydrophobic compound which
may be aliphatic and/or aromatic in nature. Examples of suitable
nonionic surfactants include the poloxamers; sorbitan derivatives,
such as sorbitan di-isostearate; ethylene oxide condensates of
hydrogenated castor oil, such as PEG-30 hydrogenated castor oil;
ethylene oxide condensates of aliphatic alcohols or alkyl phenols;
products derived from the condensation of ethylene oxide with the
reaction product of propylene oxide and ethylene diamine; long
chain tertiary amine oxides; long chain tertiary phosphine oxides;
long chain dialkyl sulphoxides and mixtures of such materials.
These materials are useful for stabilising foams without
contributing to excess viscosity build for the oral care
composition.
[0125] Zwitterionic surfactants can be broadly described as
derivatives of aliphatic quaternary ammonium, phosphonium, and
sulphonium compounds, in which the aliphatic radicals can be
straight chain or branched, and wherein one of the aliphatic
substituents contains from about 8 to 18 carbon atoms and one
contains an anionic water-solubilising group, e.g., carboxy,
sulphonate, sulphate, phosphate or phosphonate.
[0126] The oral care compositions of the present invention may
comprise greater than about 50% liquid carrier materials. Water may
comprise from about 20% to about 70% or from about 30% to about 50%
by weight of the total composition. These amounts of water include
the free water which is added plus that which is introduced with
other materials such as with sorbitol and with surfactant
solutions.
[0127] Generally, the liquid carrier may further include one or
more humectants. Suitable humectants include glycerin, sorbitol,
and other edible polyhydric alcohols, such as low molecular weight
polyethylene glycols at levels of from about 15% to about 50%. To
provide the best balance of foaming properties and resistance to
drying out, the ratio of total water to total humectant may be from
about 0.65:1 to about 1.5:1, or from about 0.85:1 to about
1.3:1.
[0128] The viscosities of the oral care compositions herein may be
affected by the viscosity of Newtonian liquids present in the
composition. These may be either pure liquids such as glycerin or
water, or a solution of a solute in a solvent such as a sorbitol
solution in water. The level of contribution of the Newtonian
liquid to the viscosity of the non-Newtonian oral care composition
will depend upon the level at which the Newtonian liquid is
incorporated. Water may be present in a significant amount in an
oral care composition, and has a Newtonian viscosity of
approximately 1 mPas at 25 deg. C. Humectants such as glycerin and
sorbitol solutions typically have a significantly higher Newtonian
viscosity than water. As a result, the total level of humectant,
the ratio of water to humectant, and the choice of humectants,
helps to determine the high shear rate viscosity of the oral care
compositions.
[0129] Common humectants such as sorbitol, glycerin,
polyethyleneglycols, propylene glycols and mixtures thereof may be
used, but the specific levels and ratios used will differ depending
on the choice of humectant. Sorbitol may be used, but due to its
relatively high Newtonian viscosity, in certain embodiments cannot
be incorporated at levels above 45% by weight of the composition,
as it contributes significantly to the high shear rate viscosity of
the oral care composition. Conversely, propylene glycol may be
employed at higher levels as it has a lower Newtonian viscosity
than sorbitol, and hence does not contribute as much to the high
shear rate viscosity of the oral care composition. Glycerin has an
intermediate Newtonian viscosity in between that of sorbitol and
polyethylene glycol.
[0130] Ethanol may also be present in the oral care compositions.
These amounts may range from about 0.5 to about 5%, or from about
1.5 to about 3.5% by weight of the total composition. Ethanol can
be a useful solvent and can also serve to enhance the impact of a
flavour, though in this latter respect only low levels are usually
employed. Non-ethanolic solvents such as propylene glycol may also
be employed. Also useful herein are low molecular weight
polyethylene glycols.
[0131] The oral care compositions of the present invention may
comprise a dental abrasive, such as those used in dentifrices.
Abrasives serve to polish the teeth, remove surface deposits, or
both. The abrasive material contemplated for use herein can be any
material which does not excessively abrade dentine. Suitable
abrasives include insoluble phosphate polishing agents, such as,
for example, dicalcium phosphate, tricalcium phosphate, calcium
pyrophosphate, beta-phase calcium pyrophosphate, dicalcium
phosphate dihydrate, anhydrous calcium phosphate, insoluble sodium
metaphosphate, and the like. Also suitable are chalk-type abrasives
such as calcium and magnesium carbonates, silicas including
xerogels, hydrogels, aerogels and precipitates, alumina and
hydrates thereof such as alpha alumina trihydrate, aluminosilicates
such as calcined aluminium silicate and aluminium silicate,
magnesium and zirconium silicates such as magnesium trisilicate and
thermosetting polymerised resins such as particulate condensation
products of urea and formaldehyde, polymethylmethacrylate, powdered
polyethylene and others such as disclosed in U.S. Pat. No.
3,070,510. Mixtures of abrasives can also be used. The abrasive
polishing materials generally have an average particle size of from
about 0.1 to about 30 .mu.m, or from about 1 to about 15 .mu.m.
[0132] Silica dental abrasives of various types offer exceptional
dental cleaning and polishing performance without unduly abrading
tooth enamel or dentin. The silica abrasive can be precipitated
silica or silica gels such as the silica xerogels described in U.S.
Pat. No. 3,538,230, U.S. Pat. No. 3,862,307. Silicas may be used
that have an oil absorption from 30 g per 100 g to 100 g per 100 g
of silica. It has been found that silicas with low oil absorption
levels are less structuring, and therefore do not build the
viscosity of the oral care composition to the same degree as those
silicas that are more highly structuring, and therefore have higher
oil absorption levels. As used herein, oil absorption is measured
by measuring the maximum amount of linseed oil the silica can
absorb at 25 deg. C.
[0133] Suitable abrasive levels may be from about 0% to about 20%
by weight of the total composition, in certain embodiments less
than 10%, such as from 1% to 10%. In certain embodiments abrasive
levels from 3% to 5% by weight of the total composition can be
used.
[0134] For anticaries protection, a source of fluoride ion will
normally be present in the oral care composition. Fluoride sources
include sodium fluoride, potassium fluoride, calcium fluoride,
stannous fluoride, stannous monofluorophosphate and sodium
monofluoro-phosphate. Suitable levels provide from 25 to 2500 ppm
of available fluoride ion by weight of the oral care
composition.
[0135] Suitable chelating agents include organic acids and their
salts, such as tartaric acid and pharmaceutically-acceptable salts
thereof, citric acid and alkali metal citrates and mixtures
thereof. Chelating agents are able to complex calcium found in the
cell walls of the bacteria. Chelating agents can also disrupt
plaque by removing calcium from the calcium bridges which help hold
this biomass intact. However, it is possible to use a chelating
agent which has an affinity for calcium that is too high, resulting
in tooth demineralisation. In certain embodiments the chelating
agents have a calcium binding constant of about 101 to about 105 to
provide improved cleaning with reduced plaque and calculus
formation. The amounts of chelating that may be used in the
formulations of the present invention are about 0.1% to about 2.5%,
from about 0.5% to about 2.5% or from about 1.0% to about 2.5%. The
tartaric acid salt chelating agent can be used alone or in
combination with other optional chelating agents.
[0136] Another group of agents particularly suitable for use as
chelating agents in the present invention are the water soluble
polyphosphates, polyphosphonates, and pyro-phosphates which are
useful as anticalculus agents. The pyrophosphate salts used in the
present compositions can be any of the alkali metal pyrophosphate
salts. An effective amount of pyrophosphate salt useful in the
present composition is generally enough to provide at least 1.0%
pyrophosphate ion or from about 1.5% to about 6% of such ions. The
pyrophosphate salts are described in more detail in Kirk &
Othmer, Encyclopedia of Chemical Technology, Second Edition, Volume
15, Interscience Publishers (1968).
[0137] Water soluble polyphosphates such as sodium
tripolyphosphate, potassium tripolyphosphate and sodium
hexametaphosphate may be used. Other long chain anticalculus agents
of this type are described in WO98/22079. Also preferred are the
water soluble diphosphonates. Suitable soluble diphosphonates
include ethane-1-hydroxy-1,1,-diphosphonate (EHDP) and
aza-cycloheptane-diphosphonate (AHP). The tripolyphosphates and
diphosphonates are particularly effective as they provide both
anti-tartar activity and stain removal activity without building
viscosity as much as much as less water soluble chemical stain
removal agents and are stable with respect to hydrolysis in water.
The soluble polyphosphates and diphosphonates are beneficial as
destaining actives. Without wishing to be bound by theory, it is
believed that these ingredients remove stain by desorbing stained
pellicle from the enamel surface of the tooth. Suitable levels of
water soluble polyphosphates and diphosphonates are from about 0.1%
to about 10%, from about 1% to about 5%, or from about 1.5% to
about 3% by weight of the oral care composition.
[0138] Still another possible group of chelating agents suitable
for use in the present invention are the anionic polymeric
polycarboxylates. Such materials are well known in the art, being
employed in the form of their free acids or partially or preferably
fully neutralised water-soluble alkali metal (e.g. potassium and
preferably sodium) or ammonium salts. Additional polymeric
polycarboxylates are disclosed in U.S. Pat. No. 4,138,477 and U.S.
Pat. No. 4,183,914, and include copolymers of maleic anhydride with
styrene, isobutylene or ethyl vinyl ether, polyacrylic,
polyitaconic and polymaleic acids, and sulphoacrylic oligomers of
MW as low as 1,000 available as Uniroyal ND-2.
[0139] Also useful for the present invention are antimicrobial
agents. A wide variety of antimicrobial agents can be used,
including stannous salts such as stannous pyrophosphate and
stannous gluconate; zinc salt, such as zinc lactate and zinc
citrate; copper salts, such as copper bisglycinate; quaternary
ammonium salts, such as cetyl pyridinium chloride and
tetradecylethyl pyridinium chloride; bis-biguanide salts; and
nonionic antimicrobial agents such as triclosan. Certain flavour
oils, such as thymol, may also have antimicrobial activity. Such
agents are disclosed in U.S. Pat. No. 2,946,725 and U.S. Pat. No.
4,051,234. Also useful is sodium chlorite, described in WO
99/43290.
[0140] Antimicrobial agents, if present, are typically included at
levels of from about 0.01% to about 10%. Levels of stannous and
cationic antimicrobial agents can be kept to less than about 5% or
less than about 1% to avoid staining problems.
[0141] In certain embodiments antimicrobial agents are non-cationic
antimicrobial agent, such as those described in U.S. Pat. No.
5,037,637. A particularly effective antimicrobial agent is
2',4,4'-trichloro-2-hydroxy-diphenyl ether (triclosan).
[0142] An optional ingredient in the present compositions is a
silicone oil. Silicone oils can be useful as plaque barriers, as
disclosed in WO 96/19191. Suitable classes of silicone oils
include, but are not limited to, dimethicones, dimethiconols,
dimethicone copolyols and aminoalkylsilicones. Silicone oils are
generally present in a level of from about 0.1% to about 15%, from
about 0.5% to about 5%, or from about 0.5% to about 3% by
weight.
[0143] Sweetening agents such as sodium saccharin, sodium
cyclamate, Acesulfame K, aspartame, sucrose and the like may be
included at levels from about 0.1 to 5% by weight. Other additives
may also be incorporated including flavours, preservatives,
opacifiers and colorants. Typical colorants are D&C Yellow No.
10, FD&C Blue No. 1, FD&C Red No. 40, D&C Red No. 33
and combinations thereof. Levels of the colorant may range from
about 0.0001 to about 0.1%.
[0144] The oral care composition preferably comprises at least one
nucleic acid aptamer at a level where upon directed use, promotes
one or more benefits without detriment to the oral cavity component
it is applied to. In certain embodiments of the present invention,
said oral care composition comprises between about 0.00001% to
about 10% of at least one nucleic acid aptamer. In another
embodiment, said oral care composition comprises between about
0.00005% to about 5% of at least one nucleic acid aptamer. In
another embodiment, said oral care composition comprises between
about 0.0001% to about 1% of at least one nucleic acid aptamer.
[0145] In another embodiment, an oral care composition comprises at
least one peptide aptamer; wherein said at least one peptide
aptamer has a binding affinity for an oral cavity component
selected from the group consisting of: tooth, enamel, dentin, and
any other surfaces in the oral cavity. In another embodiment, said
at least one peptide aptamer has a binding affinity for tooth.
[0146] The aptamers of the present invention could provide several
benefits when bound to an oral cavity component, including, but not
limited to, teeth remineralization (e.g. by improving calcium
deposition on teeth), teeth acid resistance, appearance and
structural changes to teeth, stain prevention (e.g. by repelling
teeth staining materials such as dyes or pigments), plaque
prevention, tartar prevention, and cavity prevention and treatment.
As an example, if aptamers comprising fluorinated nucleotides are
degraded or decomposed after binding, they could effectively
deliver fluoride ions to teeth, which can provide cavity prevention
benefits. Non-limiting examples of fluorinated nucleotides include
fluorophosphate nucleotides, 2'-fluoro deoxyribonucleotides, and
nucleotides with fluorinated nucleobases.
[0147] The combined use of aptamers that bind to different epitopes
of a particular target (e.g. tooth) could provide a greater overall
target coverage and/or efficacy across different individuals.
Identification of aptamers binding to different epitopes can be
achieved by performing a covariance analysis for the change in
oligonucleotide frequency during the rounds of SELEX selection as
described in Example 3. In certain embodiments of the present
invention, an oral care composition comprises at least two
different nucleic acid aptamers; wherein said at least two
different nucleic acid aptamers have binding affinities for
different epitopes of tooth. In another embodiment, said at least
two different nucleic acid aptamers are selected from the group
consisting of SEQ ID NO 1, SEQ ID NO 9, SEQ ID NO 25, SEQ ID NO
112, SEQ ID NO 120, SEQ ID NO 136, and SEQ ID NO 223 to SEQ ID NO
234.
[0148] The aptamers of the current invention can also be formulated
in oral care products to effectively deliver active ingredients to
oral cavity components. In certain embodiments of the present
invention, a method for delivering one or more oral care active
ingredients to the oral cavity comprises administering an oral care
composition comprising at least one nucleic acid aptamer and one or
more oral care active ingredients; wherein said at least one
nucleic acid aptamer and said one or more oral care active
ingredients are covalently or non-covalently attached; and wherein
said at least one nucleic acid aptamer has a binding affinity for
an oral cavity component. Examples of the oral conditions these
oral care active ingredients address include, but are not limited
to, appearance and structural changes to teeth, whitening, stain
prevention and removal, stain bleaching, plaque prevention and
removal, tartar prevention and removal, cavity prevention and
treatment, inflamed and/or bleeding gums, mucosal wounds, lesions,
ulcers, aphthous ulcers, cold sores, and tooth abscesses.
[0149] In another embodiment, said oral cavity component in said
method of delivering one or more oral care active ingredients is
selected from the group comprising: tooth, enamel, dentin, and any
other surfaces in the oral cavity. In another embodiment, said oral
cavity component is tooth.
[0150] In another embodiment, a method for delivering one or more
oral care active ingredients to the oral cavity comprises
administering an oral care composition comprising at least one
peptide aptamer and one or more oral care active ingredients;
wherein said at least one peptide aptamer and said one or more oral
care active ingredients are covalently or non-covalently attached;
and wherein said at least one peptide aptamer has a binding
affinity for an oral cavity component.
[0151] In another embodiment, said one or more oral care active
ingredients are selected from the group comprising: whitening
agents, brightening agents, anti-stain agents, anti-cavity agents,
anti-erosion agents, anti-tartar agents, anti-calculus agents,
anti-plaque agents, teeth remineralizing agents, anti-fracture
agents, strengthening agents, abrasion resistance agents,
anti-gingivitis agents, anti-microbial agents, anti-bacterial
agents, anti-fungal agents, anti-yeast agents, anti-viral,
anti-malodor agents, breath freshening agents, flavoring agents,
cooling agents, taste enhancement agents, olfactory enhancement
agents, anti-adherence agents, smoothness agents, surface
modification agents, anti-tooth pain agents, anti-sensitivity
agents, anti-inflammatory agents, gum protecting agents,
periodontal actives, tissue regeneration agents, anti-blood
coagulation agents, anti-clot stabilizer agents, salivary stimulant
agents, salivary rheology modification agents, enhanced retention
agents, soft/hard tissue targeted agents, tooth/soft tissue
cleaning agents, antioxidants, pH modifying agents, H-2
antagonists, analgesics, natural extracts and essential oils, dyes,
optical brighteners, cations, phosphates, fluoride ion sources,
peptides, nutrients, enzymes, mouth and throat products, and
mixtures thereof. The present invention also includes other oral
care active ingredients previously disclosed in the art. In another
embodiment, said oral care active ingredient is selected from the
group consisting of dyes and optical brighteners. In another
embodiment, said oral care active ingredient is selected from the
group consisting of dyes and optical brighteners and said at least
one nucleic acid aptamer has a binding affinity for tooth.
[0152] Non-limiting examples of whitening agents are dyes, optical
brighteners, peroxides, metal chlorites, perborates, percarbonates,
peroxyacids, and mixtures thereof. Suitable peroxide compounds
include hydrogen peroxide, calcium peroxide, carbamide peroxide,
and mixtures thereof. Most preferred is carbamide peroxide.
Suitable metal chlorites include calcium chlorite, barium chlorite,
magnesium chlorite, lithium chlorite, sodium chlorite, and
potassium chlorite. Additional whitening actives may be
hypochlorite and chlorine dioxide. The preferred chlorite is sodium
chlorite.
[0153] Dyes and optical brighteners can provide desirable whitening
and other cosmetic effects on teeth. Non-limiting examples of dyes
are triarylmethane dyes, including brilliant blue FCF (FD&C
blue 1 or D&C blue 4), fast green FCF (FD&C green 3), and
patent blue V; indigoid dyes, including indigo carmine (FD&C
blue 2); anthraquinone dyes, including sunset violet 13 (D&C
violet 2); azoic dyes; xanthene dyes; natural dyes, including
chlorophylls, spirulina, and anthocyanins; their derivatives; and
mixtures thereof.
[0154] Optical brighteners, also known as fluorescent whitening
agents, are organic compounds that are colorless to weakly colored
in solution, absorb ultraviolet light (e.g. from daylight, ca.
300-430 nm), and reemit most of the absorbed energy as blue
fluorescent light (400-500 nm). Thus, in daylight, optical
brighteners can compensate for the often undesirable yellowish tone
found in teeth and other materials. Furthermore, since day UV light
(not perceived by the eye) is converted to visible light, the
brightness of the teeth can be enhanced to produce a luminous
white. Non-limiting examples of optical brighteners are derivatives
of carbocyles, stilbene and 4,4'-diaminostilbene, including
4,4'-diamino-2,2'-stilbenedisulfonic acid; derivatives of
distyrylbenzenes, distyrylbiphenyls, and divinylstilbenes;
derivatives of triazinylaminostilbenes; derivatives of
stilbenyl-2H-triazoles; derivatives of benzoxazoles,
stilbenylbenzoxazoles, and bis(benzoxazoles); derivatives of
furans, benzo[b]furans, benzimidazoles,
bix(benzo[b]furan-2-yl)biphenyls, and cationic benzimidazoles;
derivatives of 1,3-diphenyl-2-pyrazolines; derivatives of
coumarins; derivatives of napthalimides; derivatives of
1,3,5-triazin-2-yl; derivatives of bis(benzoxazol-2-yl); and
mixtures thereof. A review of commonly used optical brighteners is
found in "Optical Brighteners" by Siegrist, A. E., Eckhardt, C.,
Kaschig, J. and Schmidt, E.; Ullmann's Encyclopedia of Industrial
Chemistry, Wiley and Sons, 2003, the contents of which are
incorporated herein by reference. In certain embodiments of the
present invention, said oral care active ingredient is
4,4'-diamino-2,2'-stilbenedisulfonic acid.
[0155] Non-limiting examples of anti-cavity agents are: a)
phosphorus-containing agents, including polyphosphates such as
pyrophosphate, tripolyphosphate, trimetaphosphate, and
hexametaphosphate; organic phosphates such as glycerophosphate,
phytate, 1,6-fructose diphosphate, calcium lactophosphate,
casein-phosphopeptide amorphous calcium phosphate (CPP-ACP), and
sodium caseinate; phosphoproteins; phosphonates such as ethane
hydroxy diphosphonate; and phosphosilicates; b) calcium-containing
agents, including calcium lactate; c) anti-microbial agents; d)
metals and their cations, including zinc, tin, aluminum, copper,
iron, and calcium; e) other organic agents including citrate; and
f) fluoride-ion sources agents, including sodium fluoride, stannous
fluoride, amine fluorides such as olaflur (amine fluoride 297) and
dectaflur, sodium monofluorophosphate, fluorosilicates,
fluorozirconates, fluorostannites, fluoroborates, fluorotitanates,
and fluorogermanates. Non-limiting examples of cations are
Ca.sup.2+, Sn.sup.2+, Sn.sup.4+, Zn.sup.2+, Al.sup.3+, Cu.sup.2+,
Fe.sup.2+, and Fe.sup.3+.
[0156] Anti-tartar agents known for use in dental care products
also include phosphates, such as pyrophosphates, polyphosphates,
polyphosphonates and mixtures thereof. Pyrophosphates are among the
best known for use in dental care products. Pyrophosphate ions
delivered to the teeth derive from pyrophosphate salts. The
pyrophosphate salts useful in the present compositions include the
dialkali metal pyrophosphate salts, tetra-alkali metal
pyrophosphate salts, and mixtures thereof. Disodium dihydrogen
pyrophosphate (Na.sub.2H.sub.2P.sub.2O.sub.7), tetrasodium
pyrophosphate (Na.sub.4P.sub.2O.sub.7), and tetrapotassium
pyrophosphate (K.sub.4P.sub.207) in their unhydrated as well as
hydrated forms are the preferred species. While any of the
above-mentioned pyrophosphate salts may be used, tetrasodium
pyrophosphate salt is preferred.
[0157] The pyrophosphate salts are described in more detail in Kirk
& Othmer, Encyclopedia of Chemical Technology, Third Edition,
Volume 17, Wiley-Interscience Publishers (1982). Additional
anti-calculus agents include pyrophosphates or polyphosphates
disclosed in U.S. Pat. No. 4,590,066 issued to Parran & Sakkab
on May 20, 1986; polyacrylates and other polycarboxylates, such as
those disclosed in U.S. Pat. No. 3,429,963 issued to Shedlovsky on
Feb. 25, 1969, U.S. Pat. No. 4,304,766 issued to Chang on Dec. 8,
1981 and U.S. Pat. No. 4,661,341 issued to Benedict & Sunberg
on Apr. 28, 1987; polyepoxysuccinates such as those disclosed in
U.S. Pat. No. 4,846,650 issued to Benedict, Bush & Sunberg on
Jul. 11, 1989; ethylenediaminetetraacetic acid as disclosed in
British Patent No. 490,384 dated Feb. 15, 1937; nitrilotriacetic
acid and related compounds as disclosed in U.S. Pat. No. 3,678,154
issued to Widder & Briner on Jul. 18, 1972; polyphosphonates as
disclosed in U.S. Pat. No. 3,737,533 issued to Francis on Jun. 5,
1973, U.S. Pat. No. 3,988,443 issued to Ploger, Schmidt-Dunker
& Gloxhuber on Oct. 26, 1976 and U.S. Pat. No. 4,877,603 issued
to Degenhardt & Kozikowski on Oct. 31, 1989. Anti-calculus
phosphates include potassium and sodium pyrophosphates; sodium
tripolyphosphate; diphosphonates, such as
ethane-1-hydroxy-1,1-diphosphonate,
1-azacycloheptane-1,1-diphosphonate, and linear alkyl
diphosphonates; linear carboxylic acids; and sodium zinc
citrate.
[0158] Agents that may be used in place of or in combination with
the pyrophosphate salt include such known materials as synthetic
anionic polymers including polyacrylates and copolymers of maleic
anhydride or acid and methyl vinyl ether (e.g., Gantrez), as
described, for example, in U.S. Pat. No. 4,627,977, to Gaffar et
al., as well as, e.g., polyamino propoane sulfonic acid (AMPS),
zinc citrate trihydrate, polyphosphates (e.g., tripolyphosphate;
hexametaphosphate), diphosphonates (e.g., EHDP; AHP), polypeptides
(such as polyaspartic and polyglutamic acids), and mixtures
thereof.
[0159] Fluoride ion sources are well known for use in oral care
compositions as anti-cavity agents. Fluoride ions are contained in
a number of oral care compositions for this purpose, particularly
dentifrices. Patents disclosing such dentifrices include U.S. Pat.
No. 3,538,230 to Pader et al; U.S. Pat. No. 3,689,637 to Pader;
U.S. Pat. No. 3,711,604 to Colodney et al; U.S. Pat. No. 3,911,104
to Harrison; U.S. Pat. No. 3,935,306 to Roberts et al; and U.S.
Pat. No. 4,040,858 to Wason.
[0160] Application of fluoride ions to dental enamel serves to
protect teeth against decay. A wide variety of fluoride
ion-yielding materials can be employed as sources of soluble
fluoride in the instant compositions. Examples of suitable fluoride
ion-yielding materials are found in Briner et al; U.S. Pat. No.
3,535,421; issued Oct. 20, 1970 and Widder et al; U.S. Pat. No.
3,678,154; issued Jul. 18, 1972. Preferred fluoride ion sources for
use herein include sodium fluoride, potassium fluoride and ammonium
fluoride. Sodium fluoride is particularly preferred.
[0161] Anti-microbial agents can also be present in the oral care
compositions or substances of the present invention. Such agents
may include, but are not limited to, triclosan,
5-chloro-2-(2,4-dichlorophenoxy)-phenol, as described in The Merck
Index, 11th ed. (1989), pp. 1529 (entry no. 9573) in U.S. Pat. No.
3,506,720, and in European Patent Application No. 0,251,591 of
Beecham Group, PLC, published Jan. 7, 1988; chlorhexidine (Merck
Index, no. 2090), alexidine (Merck Index, no. 222); hexetidine
(Merck Index, no. 4624); sanguinarine (Merck Index, no. 8320);
benzalkonium chloride (Merck Index, no. 1066); salicylanilide
(Merck Index, no. 8299); domiphen bromide (Merck Index, no. 3411);
cetylpyridinium chloride (CPC) (Merck Index, no. 2024;
tetradecylpyridinium chloride (TPC); N-tetradecyl-4-ethylpyridinium
chloride (TDEPC); octenidine; delmopinol, octapinol, and other
piperidino derivatives; nisin preparations; zinc/stannous ion
agents; bacteriocins; antibiotics such as augmentin, amoxicillin,
tetracycline, doxycycline, minocycline, and metronidazole; and
analogs and salts of the above anti-microbial anti-plaque agents;
essential oils inclyding thymol, geraniol, carvacrol, citral,
hinokitiol, eucalyptol, catechol (particularly 4-allyl catechol)
and mixtures thereof; methyl salicylate; hydrogen peroxide; metal
salts of chlorite, and mixtures thereof.
[0162] Non-limiting examples of flavoring and cooling agents are
menthol, menthone, methyl acetate, menthofuran, 1,8-cineol,
R-(-)-carvone, limonene, dihydrocarvone, methyl salicylate, sugar
alcohols or polyols (e.g. xylitol, sorbitol, and erythritol), and
their derivatives. A non-limiting example of teeth remineralizing
agents is hydroxyapatite nanocrystals.
[0163] Anti-inflammatory agents can also be present in the oral
care compositions or substances of the present invention. Such
agents may include, but are not limited to, non-steroidal
anti-inflammatory agents or NSAIDs such as ketorolac, flurbiprofen,
ibuprofen, naproxen, indomethacin, aspirin, ketoprofen, piroxicam
and meclofenamic acid. Use of NSAIDs such as ketorolac is claimed
in U.S. Pat. No. 5,626,838, issued May 6, 1997. Disclosed therein
are methods of preventing and/or treating primary and reoccurring
squamous cell carcinoma of the oral cavity or oropharynx by topical
administration to the oral cavity or oropharynx an effective amount
of an NSAID.
[0164] Nutrients may improve the condition of the oral cavity.
Nutrients include minerals, vitamins, oral nutritional supplements,
enteral nutritional supplements, and mixtures thereof. Minerals
that can be included with the compositions of the present invention
include calcium, phosphorus, fluoride, zinc, manganese, potassium
and mixtures thereof. These minerals are disclosed in Drug Facts
and Comparisons (loose leaf drug information service), Wolters
Kluer Company, St. Louis, Mo., (c)1997, pp 10-17. Vitamins can be
included with minerals or used separately. Vitamins include
Vitamins C and D, thiamine, riboflavin, calcium pantothenate,
niacin, folic acid, nicotinamide, pyridoxine, cyanocobalamin,
para-aminobenzoic acid, bioflavonoids, and mixtures thereof. Such
vitamins are disclosed in Drug Facts and Comparisons (loose leaf
drug information service), Wolters Kluer Company, St. Louis, Mo.,
(c)1997, pp. 3-10. Oral nutritional supplements include amino
acids, lipotropics, fish oil, and mixtures thereof, as disclosed in
Drug Facts and Comparisons (loose leaf drug information service),
Wolters Kluer Company, St. Louis, Mo., (c)1997, pp. 54-54e. Amino
acids include L-tryptophan, L-lysine, methionine, threonine,
levocarnitine or L-carnitine and mixtures thereof. Lipotropics
include, but, are not limited to choline, inositol, betaine,
linoleic acid, linolenic acid, and mixtures thereof. Fish oil
contains large amounts of omega-3 (N-3) polyunsaturated fatty
acids, eicosapentaenoic acid and docosahexaenoic acid. Enteral
nutritional supplements include, but, are not limited to protein
products, glucose polymers, corn oil, safflower oil, medium chain
triglycerides as disclosed in Drug Facts and Comparisons (loose
leaf drug information service), Wolters Kluer Company, St. Louis,
Mo., (c) 1997, pp. 55-57.
[0165] Enzymes provide several benefits when used for cleansing of
the oral cavity. Proteases break down salivary proteins which are
absorbed onto the tooth surface and form the pellicle; the first
layer of resulting plaque. Proteases along with lipases destroy
bacteria by lysing proteins and lipids which form the structural
component of bacterial cell walls and membranes. Dextranases break
down the organic skeletal structure produced by bacteria that forms
a matrix for bacterial adhesion. Proteases and amylases not only
prevent plaque formation but also prevent the development of
calculus by breaking-up the carbohydrate-protein complex that binds
calcium, preventing mineralization. Enzymes useful in the present
invention include any of the commercially available proteases,
glucanohydrolases, endoglycosidases, amylases, mutanases, lipases
and mucinases or compatible mixtures thereof. Preferred are the
proteases, dextranases, endoglycosidases and mutanases, most
preferred being papain, endoglycosidase or a mixture of dextranase
and mutanase. Additional enzymes suitable for use in the present
invention are disclosed in U.S. Pat. No. 5,000,939 to Dring et al.;
U.S. Pat. No. 4,992,420 to Neeser; U.S. Pat. No. 4,355,022 to
Rabussay; U.S. Pat. No. 4,154,815 to Pader; U.S. Pat. No. 4,058,595
to Colodney; U.S. Pat. No. 3,991,177 to Virda et al. and 3,696,191
to Weeks.
[0166] Other materials that can be used with the present invention
include commonly known mouth and throat products. Such products are
disclosed in Drug Facts and Comparisons (loose leaf drug
information service), Wolters Kluer Company, St. Louis, Mo.,
(c)1997, pp. 520b-527. These products include anti-fungal,
antibiotic and analgesic agents.
[0167] Antioxidants are generally recognized as useful in
compositions such as those of the present invention. Antioxidants
are disclosed in texts such as Cadenas and Packer, The Handbook of
Antioxidants, (c) 1996 by Marcel Dekker, Inc. Antioxidants that may
be included in the oral care composition or substance of the
present invention include, but are not limited to vitamin E,
ascorbic acid, uric acid, carotenoids, Vitamin A, flavonoids and
polyphenols, herbal antioxidants, melatonin, aminoindoles, lipoic
acids and mixtures thereof.
[0168] In another embodiment, a method for delivering one or more
oral care active ingredients to the oral cavity comprises
administering an oral care composition comprising: at least one
nucleic acid aptamer and one or more nanomaterials; wherein said at
least one nucleic acid aptamer and said one or more nanomaterials
are covalently or non-covalently attached; and wherein said at
least one nucleic acid aptamer has a binding affinity for an oral
cavity component.
[0169] In another embodiment, a method for delivering one or more
oral care active ingredients to the oral cavity comprises
administering an oral care composition comprising: a) at least one
nucleic acid aptamer; b) one or more nanomaterials; and c) and one
or more oral care active ingredients; wherein said at least one
nucleic acid aptamer and said one or more nanomaterials are
covalently or non-covalently attached; and wherein said at least
one nucleic acid aptamer has a binding affinity for an oral cavity
component. In another embodiment, said one or more oral care active
ingredients are covalently or non-covalently attached to said one
or more nanomaterials. In yet another embodiment, said one or more
oral care active ingredients are carried by said one or more
nanomaterials.
[0170] Non-limiting examples of nanomaterials are gold
nanoparticles, nano-scale iron oxides, carbon nanomaterials (such
as single-walled carbon nanotubes and graphene oxide), mesoporous
silica nanoparticles, quantum dots, liposomes, poly
(lactide-co-glycolic acids) nanoparticles, polymeric micelles,
dendrimers, serum albumin nanoparticles, and DNA-based
nanomaterials.
[0171] In addition to at least one nucleic acid aptamer, the oral
care compositions of the present invention may also include
surfactants and/or detergents, polishing agents, abrasive
materials, binders, carriers, thickening agents, humectants, salts,
and other ingredients. Surfactants or detergents can provide a
desirable foaming quality. Suitable surfactants are those which are
reasonably stable and foam throughout a wide pH range. The
surfactant may be anionic, nonionic, amphoteric, zwitterionic,
cationic, or mixtures thereof. Anionic surfactants useful herein
include the water-soluble salts of alkyl sulfates having from 8 to
20 carbon atoms in the alkyl radical (e.g., sodium alkyl sulfate)
and the water-soluble salts of sulfonated monoglycerides of fatty
acids having from 8 to 20 carbon atoms. Sodium lauryl sulfate and
sodium coconut monoglyceride sulfonates are examples of anionic
surfactants of this type. Other suitable anionic surfactants are
sarcosinates, such as sodium lauroyl sarcosinate, taurates, sodium
lauryl sulfoacetate, sodium lauroyl isethionate, sodium laureth
carboxylate, and sodium dodecyl benzenesulfonate. Mixtures of
anionic surfactants can also be employed. Many suitable anionic
surfactants are disclosed by Agricola et al., U.S. Pat. No.
3,959,458, issued May 25, 1976, incorporated herein in its entirety
by reference. Nonionic surfactants which can be used in the
compositions of the present invention can be broadly defined as
compounds produced by the condensation of alkylene oxide groups
(hydrophilic in nature) with an organic hydrophobic compound which
may be aliphatic or alkyl-aromatic in nature. Examples of suitable
nonionic surfactants include poloxamers (sold under trade name
Pluronic), polyoxyethylene, polyoxyethylene sorbitan esters (sold
under trade name Tweens), fatty alcohol ethoxylates, polyethylene
oxide condensates of alkyl phenols, products derived from the
condensation of ethylene oxide with the reaction product of
propylene oxide and ethylene diamine, ethylene oxide condensates of
aliphatic alcohols, long chain tertiary amine oxides, long chain
tertiary phosphine oxides, long chain dialkyl sulfoxides, and
mixtures of such materials. The amphoteric surfactants useful in
the present invention can be broadly described as derivatives of
aliphatic secondary and tertiary amines in which the aliphatic
radical can be a straight chain or branched and wherein one of the
aliphatic substituents contains from about 8 to about 18 carbon
atoms and one contains an anionic water-solubilizing group, e.g.,
carboxylate, sulfonate, sulfate, phosphate, or phosphonate. Other
suitable amphoteric surfactants are betaines, specifically
cocamidopropyl betaine. Mixtures of amphoteric surfactants can also
be employed. Many of these suitable nonionic and amphoteric
surfactants are disclosed by Gieske et al., U.S. Pat. No.
4,051,234, issued Sep. 27, 1977, incorporated herein by reference
in its entirety. The present composition typically comprises one or
more surfactants each at a level of from about 0.25% to about 12%,
preferably from about 0.5% to about 8%, and most preferably from
about 1% to about 6%, by weight of the composition.
[0172] The binder system, generally, is a primary factor that
determines the rheological characteristics of the oral care
composition. The binder also acts to keep any solid phase of an
oral care component suspended, thus preventing separation of the
solid phase portion of the oral care component from the liquid
phase portion. Additionally, the binder can provide body or
thickness to the oral care composition. Thus, in some instances, a
binder can also provide a thickening function to an oral care
composition. Examples of binders include sodium
carboxymethyl-cellulose, cellulose ether, xanthan gum, carrageenan,
sodium alginate, carbopol, or silicates such as hydrous sodium
lithium magnesium silicate. Other examples of suitable binders
include polymers such as hydroxypropyl methylcellulose,
hydroxyethyl cellulose, guar gum, tragacanth gum, karaya gum,
arabic gum, Irish moss, starch, and alginate. Alternatively, the
binder can include a clay, for example, a synthetic clay such as a
hectorite, or a natural clay. Each of the binders can be used alone
or in combination with other binders.
[0173] Non-limiting examples of thickening agents include
thickening silica, polymers, clays, and combinations thereof.
Thickening silica, for example, SILODENT 15 hydrated silica, in the
amount between about 4% to about 8% by weight (e.g., about 6%)
provide desirable in-mouth characteristics. The phrase "in-mouth
characteristics" as described herein relates to the body and
thickness of the dentifrice as it foams in the mouth of a user.
[0174] Non-limiting examples of polishing agents include abrasive
materials, such as carbonates (e.g., sodium bicarbonate, calcium
carbonate) water-colloidal silica, precipitated silicas (e.g.,
hydrated silica), sodium aluminosilicates, silica grades containing
alumina, hydrated alumina, dicalcium phosphates, calcium hydrogen
phosphates, calcium pyrophosphate, calcium pyrophosphate (beta
phase), hydroxyapatite, insoluble sodium metaphosphate, and
magnesiums (e.g., trimagnesium phosphate). A suitable amount of
polishing agent is an amount that safely provides good polishing
and cleaning and which, when combined with other ingredients gives
a smooth, flowable, and not excessively gritty composition. In
general, when polishing agents are included, they are present in an
amount from about 5% to about 50% by weight (e.g., from about 5% to
about 35%, or from about 7% to about 25%).
[0175] Examples of carriers include water, polyethylene glycol,
glycerin, polypropylene glycol, starches, sucrose, alcohols (e.g.,
methanol, ethanol, isopropanol, etc.), or combinations thereof.
Examples of combinations include various water and alcohol
combinations and various polyethylene glycol and polypropylene
glycol combinations. In general, the amount of carrier included is
determined based on the concentration of the binder system along
with the amount of dissolved salts, surfactants, and dispersed
phase.
[0176] Generally, humectants are polyols. Examples of humectants
include glycerin, sorbitol propyleneglycol, xylitol, lactitol,
polypropylene glycol, polyethylene glycol, hydrogenated corn syrup
and mixtures thereof. In general, when humectants are included they
can be present in an amount from about 10% to about 60% by
weight.
[0177] Examples of buffers and salts include primary, secondary, or
tertiary alkali metal phosphates, citric acid, sodium citrate,
sodium saccharin, tetrasodium pyrophosphate, sodium hydroxide, and
the like. The oral care compositions of the present invention may
also include active ingredients, for example, to prevent cavities,
to whiten teeth, to freshen breath, to deliver oral medication, and
to provide other therapeutic and cosmetic benefits such as those
described above. Examples of active ingredients include the
following: anti-caries agents (e.g., water soluble fluoride salts,
fluorosilicates, fluorozirconates, fluorostannites, fluoroborates,
fluorotitanates, fluorogermanates, mixed halides and casine);
anti-tartar agents; anti-calculus agents (e.g. alkali-metal
pyrophosphates, hypophosphite-containing polymers, organic
phosphocitrates, phosphocitrates, polyphosphates); anti-bacterial
agents (e.g., bacteriocins, antibodies, enzymes); anti-bacterial
enhancing agents; anti-microbial agents (e.g., Triclosan,
chlorhexidine, copper-, zinc- and stannous salts such as zinc
citrate, zinc sulfate, zinc glycinate, sanguinarine extract,
metronidazole, quaternary ammonium compounds, such as
cetylpyridinium chloride; bis-guanides, such as chlorhexidine
digluconate, hexetidine, octenidine, alexidine; and halogenated
bisphenolic compounds, such as 2,2'
methylenbis-(4-chloro-6-bromophenol)); desensitizing agents (e.g.,
potassium citrate, potassium chloride, potassium tartrate,
potassium bicarbonate, potassium oxalate, potassium nitrate and
strontium salts); whitening agents (e.g., bleaching agents such as
peroxy compounds, e.g. potassium peroxydiphosphate); anti-plaque
agents; gum protecting agents (e.g., vegetable oils such as
sunflower oil, rape seed oil, soybean oil and safflower oil, and
other oils such as silicone oils and hydrocarbon oils). The gum
protection agent may be an agent capable of improving the
permeability barrier of the gums. Other active ingredients include
wound healing agents (e.g., urea, allantoin, panthenol, alkali
metal thiocyanates, chamomile-based actives and acetylsalicylic
acid derivatives, ibuprofen, flurbiprofen, aspirin, indomethacin
etc.); tooth buffering agents; demineralization agents;
anti-inflammatory agents; anti-malodor agent; breath freshing
agents; and agents for the treatment of oral conditions such as
gingivitis or periodontitis.
[0178] The oral care compositions of the present invention may also
include one or more of other ingredients, comprising: phenolic
compounds (e.g., phenol and its homologues, including
2-methyl-phenol, 3-methyl-phenol. 4-methyl-phenol, 4-ethyl-phenol,
2,4-dimethol-phenol, and 3,4-dimethol-phenol); sweetening agents
(e.g., sodium saccharin, sodium cyclamate, sucrose, lactose,
maltose, and fructose); flavors (e.g., peppermint oil, spearmint
oil, eucalyptus oil, aniseed oil, fennel oil, caraway oil, methyl
acetate, cinnamaldehyde, anethol, vanillin, thymol and other
natural or nature-identical essential oils or synthetic flavors);
preservatives (e.g., p-hydroxybenzoic acid methyl, ethyl, or propyl
ester, sodium sorbate, sodium benzoate, bromochlorophene,
triclosan, hexetidine, phenyl silicylate, biguanides, and
peroxides); opacifying and coloring agents such as titanium dioxide
or FD&C dyes; and vitamins such as retinol, tocopherol or
ascorbic acid.
[0179] An example of synthesizing aptamers that can be used in oral
care compositions, such as dentifrice, is shown below.
Aptamer Preparation:
[0180] Aptamers SEQ ID NO 1, SEQ ID NO 9, and SEQ ID NO 25 are
synthesized by enzymatic transcription from the corresponding
double stranded DNA templates using a mixture of 15 mM 2'-fluoro
CTP, 15 mM 2'-fluoro UTP, 5 mM ATP, 5 mM GTP, a mutant T7
polymerase (T7 R&DNA), and other standard reagents are used.
The aptamers are then cleaned up with a Zymo RNA cleanup column,
following manufacturer's instructions, and eluted on the reaction
buffer (e.g. phosphate buffered saline (PBS) with EDTA: 10 mM
sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2).
Conjugation Reaction:
[0181] First, a solution of 4,4'-diamino-2,2'-stilbenedisulfonic
acid (0.25 M) and imidazole (0.1 M) in water (pH 6) is prepared.
Then, EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide
hydrochloride) is weighed in a reaction vial and mixed with an
aliquot of an aptamer solution prepared as above. An aliquot of the
amine/imidazole solution is added immediately to the reaction vial
and vortexed until all the components are dissolved. An additional
aliquot of imidazole solution (0.1 M, pH 6) is added to the
reaction vial and the reaction mixture is incubated at room
temperature for at least 2 hours. Following incubation, the
unreacted EDC and its by-products and imidazole are separated from
the modified aptamer by dialysis or by using a spin desalting
column and a suitable buffer (e.g. 10 mM sodium phosphate, 0.15 M
NaCl, 10 mM EDTA, pH 7.2). Additional details about the conjugation
protocols are described in "Hermanson G. T. (2008). Bioconjugate
Techniques. 2nd Edition. pp. 969-1002, Academic Press, San Diego.",
the content of which is incorporated herein by reference.
[0182] The produced modified aptamer is conjugated with
4,4'-diamino-2,2'-stilbenedisulfonic acid at the 5'-end and can be
formulated in an oral care composition (e.g. dentifrice) to provide
teeth whitening benefits when contacted with teeth.
[0183] An example of a potential dentifrice formulation comprising
one or more aptamers of the present invention is shown below in
TABLE 1. Sample dentifrice formulations can be prepared using
standard methods known in the art using the components listed in
TABLE 1.
TABLE-US-00001 TABLE 1 Weight % of Components Composition Sorbitol
solution (70%) 32.577 Sodium hydroxide (50% soln.) 1.740 Water QS
Saccharin sodium 0.450 Xanthan gum 0.300 Sodium fluoride 0.243
Carboxymethylcellulose 1.050 Sodium acid pyrophosphate 3.190
Carbomer 0.300 Flavor 1.4 Sodium lauryl sulfate (28% soln.) 6.000
Mica titanium dioxide 0.400 Aptamer 0.1-0.01 Silica 22 Total
100
EXAMPLES
Example 1. Aptamers Design
A. Preparation of the Immobilization Field
[0184] The immobilization field was prepared by synthesizing a
random library of eight nucleotide oligonucleotides with a
disulfide group on the 5'-end (immobilization field library) as
described elsewhere (PLoS One. 2018 Jan. 5; 13(1):e0190212). In
brief, the 8-mer thiolated random oligonucleotide library was
dissolved in 50 .mu.L of 1.times.PBS buffer (pH 7.4) at a final
concentration of 10 .mu.M. The surface of a gold coated glass slide
with dimensions of 7 mm.times.10 mm.times.0.3 mm (Xantec, Germany).
was treated with five sequential 10 .mu.L drops of the
immobilization field library. The slide was then allowed to
incubate for 1 hour in the dark at room temperature in order to
facilitate conjugation of the immobilization field library onto the
gold surface.
[0185] After this incubation period, the immobilization field
library was considered to have been conjugated onto the gold
surface. The remaining solution was removed, and the surface was
allowed to dry at room temperature.
[0186] The remaining surface was then blocked with short thiol
terminated polyethylene glycol (PEG-SH) with molecular formula:
CH.sub.3O--(CH.sub.2CH.sub.2O).sub.n--CH.sub.2CH.sub.2SH and an
average molecular weight of 550 daltons. An aliquot of 50 .mu.L of
the PEG-SH solution in 1.times.PBS buffer at a concentration of 286
.mu.M was applied to the chip and allowed to incubate overnight at
room temperature with gentle shaking. This process was repeated in
a second blocking step, with an incubation period of 30 minutes at
room temperature with gentle shaking.
[0187] Following blocking of the chip, the latter was washed with
600 .mu.L of 1.times.HEPES buffer (10 mM HEPES, pH 7.4, 120 mM
NaCl, 5 mM KCl, 5 mM MgCl.sub.2) for 5 minutes with shaking at room
temperature.
B. Library Preparation
[0188] A DNA library of about 10.sup.15 different sequences,
containing a random region of 40 nucleotides flanked by two
conserved regions, i.e. T7 promoter sequence at the 5'-end
(5'-GGGAAGAGAAGGACATATGAT-3') and a 3' reverse primer recognition
sequence (5'-TTGACTAGTACATGACCACTT-3'), was transcribed to RNA
using a mixture of 3:1 2'-fluoro pyrimidines nucleotides and
natural purine nucleotides and a mutant T7 polymerase (T7
R&DNA).
[0189] An aliquot of the transcribed selection library comprising
about 10.sup.15 RNA sequences was diluted in 50 .mu.L of 1.times.
selection buffer (10 mM cacodylate buffer, 120 mM NaCl, 5 mM KCl,
50 .mu.M SnF.sub.2). An equimolar number of oligonucleotides
complementary to the conserved regions of the library sequences (T7
promoter primer and 3' reverse primer) or blockers was added and
incubated with the selection library in a total volume of 100
.mu.L. This solution was heated for 10 minutes at 45.degree. C. to
ensure removal of any secondary or tertiary structures which could
interfere with the proper annealing of the blockers to the
selection library. The blockers were then allowed to anneal to the
selection library by allowing the mixture to equilibrate to room
temperature for 15 minutes.
[0190] This blocked selection library was then exposed to the
immobilization field in five sequential 10 .mu.L drops. The blocked
selection library was incubated on the immobilization field for 30
minutes with slow shaking in an incubator at room temperature. The
solution remaining on top after this time period was removed and
discarded. The chip was washed twice with the addition of 50 .mu.L
of selection buffer. The buffer was pipetted over the chip and then
discarded.
[0191] The blocked selection library sequences which were bound to
the immobilization field were recovered from the chip by applying
50 .mu.L of 60% DMSO and incubating for 10 min at room temperature.
The solution was removed to a fresh tube, and the process was
repeated two more times. The three elution solutions were combined
(150 .mu.L in total). The RNA sequences were then cleaned up with a
Zymo RNA cleanup column (Zymo Research, Irvine, Calif.), following
manufacturer's instructions. The purified selection library was
eluted with 35 .mu.L of water and combined with 10 .mu.L of
5.times. selection buffer and 5 .mu.L of 500 .mu.M SnF.sub.2.
C. Aptamer Selection
[0192] A clean unerupted third molar tooth was washed with three
successive applications of 1 mL water and dipped into a sample of
human saliva collected from several individuals. Saliva was
pipetted over regions of the tooth that appeared to not be coated.
The 50 .mu.L library solution prepared as described above (section
B) was pipetted into a depression on a microscope slide (concavity
slides, 3.2 mm thick; United Scientific) and the tooth was placed
in this depression. The slide was then placed in a shaking
incubator at 50 rpm, 37.degree. C., for 1 hour. The tooth was
removed and washed twice with 1 mL each of selection buffer. Then
the tooth was placed on a fresh depression slide and 50 .mu.L of
60% DMSO was added to elute bound sequences. This elution process
was repeated and the two elution solutions were combined (100 .mu.L
in total). The eluted RNA library was cleaned up with a Zymo RNA
clean up column, following manufacturer's instructions. The library
was reverse transcribed into DNA with Protoscript RT II enzyme and
PCR amplified in a two-step process. First, four separate PCR
reactions were performed with different numbers of sequential PCR
cycles (e.g. 4, 6, 8, and 10 cycles). Then, the products of each of
these PCR reactions were analyzed by gel electrophoresis to
determine the optimum number of cycles required for amplification,
i.e. as high a yield as possible without the appearance of any
concatemers of the PCR product. Then, this number of PCR cycles was
applied for library amplification to complete the selection
round.
[0193] The library was split into two aliquots to perform two
experiments under the same conditions (Experiment A and Experiment
B). The selection process was repeated eleven more times.
Dentifrice (Crest Cavity Protection) was added at a concentration
of 0.322% in the selection buffer during selection rounds 6, 9, and
12.
[0194] Negative selections against coffee and wine were also
performed. During selection rounds 7 and 10, an aliquot of 5 .mu.L
of instant coffee was added to the library solution (for a final
1:10 dilution) and the mixture was incubated with the
immobilization field for 30 minutes with shaking in an incubator at
room temperature. Oligonucleotides with specificity for molecules
present in the coffee are not expected to bind the immobilization
field. Thus, the solution remaining on top after this time period
was removed and discarded. The chip was washed twice with the
addition of 50 .mu.L of selection buffer. The buffer was pipetted
onto the surface and then discarded. The library sequences which
were bound to the immobilization field were recovered from the chip
by applying 50 .mu.L of 60% DMSO and incubating for 10 min at room
temperature. The solution was removed to a fresh tube, and the
process was repeated two more times. The three elution solutions
were combined (150 .mu.L in total). The RNA sequences were then
cleaned up with a Zymo RNA cleanup column, following manufacturer's
instructions. The library was reverse transcribed into DNA with
Protoscript RT II enzyme and PCR amplified in a two-step process,
as described above, to complete the selection cycle. The same
process was performed with wine during selection rounds 8 and
11.
D. Aptamers Sequencing
[0195] Aliquots of selection rounds 7 to 12 for both experiments
were prepared for next generation sequencing (NGS) analysis. A
total of more than 23 million sequences were analyzed. The number
of sequences captured was much lower for selection rounds 11 and 12
as a function of the increased stringency of selection. One
indication that a selection was successful is the observation that
the copy number of certain sequences increased over selection
rounds (see FIG. 1 and FIG. 2). In the graphs shown in FIGS. 1 and
2, the top 20 sequences based on the frequency on round 12 of the
selection process were graphed. For instance, for FIG. 1, the
sequences are OC1R-A1 to OC1R-A20 in order from the top line to the
bottom line (based on round 12). FIG. 1 shows the enrichment
trajectories of the top twenty sequences in terms of copy number
across different selection rounds for Experiment A. FIG. 2 shows
the enrichment trajectories of the top twenty sequences in terms of
copy number across different selection rounds for Experiment B. The
top sequences in terms of copy number for every selection
experiment are listed in TABLE 2. Interestingly, the top 15
sequences, based on copy number, in selection experiment A were
also identified in the top 40 sequences of selection experiment B.
Furthermore, the top 2 sequences in both experiments were
identical, but in the reverse ranking.
Example 2. RNA Aptamers Binding
[0196] DNA oligonucleotides encoding for selected aptamers
(OC1R-B1/OC1R-A2,OC1R-B9, and OC1R-B25/OC1R-A9) and one encoding
for a negative control aptamer (Neg) were transcribed to RNA using
a mixture of 1 mM biotinylated UTP, 15 mM 2'-fluoro CTP, 14 mM
2'-fluoro UTP, 5 mM ATP, 5 mM GTP, a mutant T7 polymerase (T7
R&DNA), and other standard reagents. The modified RNA
oligonucleotides were then cleaned up with a Zymo RNA cleanup
column, following manufacturer's instructions. An aliquot of 250
.mu.L of 1 .mu.M modified RNA in 1.times. binding buffer (10 mM
cacodylate buffer, 120 mM NaCl, 5 mM KCl, 50 .mu.M SnF.sub.2, and
0.322% dentifrice) was placed in the depression of a microscope
slide (concavity slides, 3.2 mm thick; United Scientific, Waukegan,
Ill.). Separately, a clean unerupted third molar tooth was washed
with water, dried, and coated with human saliva collected fresh
from several. The tooth was then placed into the depression of the
slide containing the modified RNA and incubated for 30 minutes at
room temperature. The tooth was removed from the slide and washed
twice with 250 .mu.L of binding buffer.
[0197] A solution of streptavidin-horse radish peroxidase (HRP) in
binding buffer was prepared and an aliquot of 250 .mu.L was placed
into the depression of a clean slide (concavity slides, 3.2 mm
thick; United Scientific). The tooth was also placed into the same
depression and incubated for 30 minutes at room temperature. After
incubation, the tooth was washed with 2 mL of binding buffer.
Finally, to detect aptamer binding, the tooth was immersed into a
solution of 10.times. LumiGLO.RTM. (Cell Signaling Technology,
Danvers, Mass.) and 10.times. hydrogen peroxide (50:50 mixture of
20.times. stocks). Only aptamers that bind to the tooth generated
chemoluminescence in darkness (see FIGS. 3A-3D). FIGS. 3A-3D show
the binding of different aptamers to teeth as demonstrated by the
chemoluminescence of the teeth in darkness. FIG. 3A shows a
negative control. FIG. 3B shows the binding of the aptamer
identified as "OC1R-B1" to teeth. FIG. 3C shows the binding of the
aptamer identified as "OC1R-B9" to teeth. FIG. 3D shows the binding
of the aptamer identified as "OC1R-B25/OC1R-A9" to teeth.
Example 3. Covariance Analysis of Sequences
[0198] A covariance analysis for the change in sequence frequency
was performed on the top 100 aptamers of Experiment B. First, for
each selection round, the frequency data was normalized by dividing
the observed frequency of each aptamer by the average of the
frequencies of the top 100 aptamers. This normalization allowed
eliminating potential differences caused by PCR amplification prior
to NGS analysis among different selection rounds. Then, the
normalized values of each aptamer in selection round 7 were
subtracted from the normalized values of the corresponding aptamer
in selection rounds 8 to 12. The resulting matrix was used for the
correlation analysis.
[0199] A Pearson correlation across the selection rounds was
performed. Since a different tooth was used in each selection
round, it is reasonable to assume that the covariance among aptamer
frequencies would be due to covariance in the abundance of the
epitope within the tooth that they bind to. Thus, each cluster of
covarying aptamers corresponds to a group of aptamers that bind to
a different epitope within the tooth. An Euclidean distance matrix
from the correlation matrix was generated and used as the basis for
clustering with a Ward.D2 algorithm (see FIG. 4). FIG. 4 shows a
correlation matrix ordered by clustering (Ward.D2 method) for
enrichment trajectories of top 100 aptamers of Experiment B. These
analyses were performed with the software R.
[0200] Based on FIG. 4, five different epitopes are likely the
binding sites of the selected aptamers. The sequences numbers (SEQ
ID NOs) of such aptamers for each cluster/epitope are listed
below:
Cluster A: 29, 66, 4, 89, 3, 31, 1, 49, 69, 75, 81, 5, 41, 37, 6,
8, 14, 21, 61
Cluster B: 16, 24, 33, 76, 45, 36, 52, 51, 15, 93
Cluster C: 87, 9, 62, 77, 79, 35, 73, 7, 78, 98, 55, 59, 71, 18,
95, 53, 13, 20, 23, 30, 84, 22, 34
Cluster D: 12, 17, 11, 39, 82, 27, 10, 26, 38, 92, 65, 56, 88, 42,
47, 28, 63, 91, 32, 96, 46, 60
Cluster E: 99, 43, 80, 100, 44, 97, 58, 67, 74, 85, 86, 19, 90, 50,
25, 83, 54, 40, 48, 72, 64, 57, 68, 94, 2, 70
[0201] The covariance analysis suggests that aptamers OC1R-B1 (SEQ
ID NO 1), OC1R-B9 (SEQ ID NO 9), and OC1R-B25/OC1R-A9 (SEQ ID NO
25) bind to different epitopes within a tooth. Furthermore,
aptamers within cluster A likely bind to the same epitope as
OC1R-B1, aptamers within cluster C likely bind to the same epitope
as OC1R-B9, and aptamers within cluster E likely bind to the same
epitope as OC1R-B25 (or OC1R-A9). Aptamers in clusters B and D
probably bind to epitopes different than the aptamers described
above.
[0202] It should be noted that a significant negative correlation
among clusters was observed, which could be caused by the variation
in the chemical composition of the enamel of the teeth used in this
study. This variation could be due to either natural variation
among individuals from whom the teeth were extracted or differences
on post-extraction treatment of the teeth.
[0203] The combined use of aptamers from different clusters could
provide a greater overall tooth coverage and/or efficacy across
different individuals and it is included as one of the embodiment
of the present invention. Aptamers binding to different teeth
epitopes can be selected using the information from this example.
Furthermore, the use of these aptamers could be used to diagnose
the chemical composition of teeth enamel in vivo.
Example 4. Motif and Predicted Structure Analysis
[0204] Aptamers bind to target molecules on the basis of the lowest
free-energy shape that they form. The lowest free energy shape is a
function of homology between regions within the single stranded
sequence. These regions of homology fold back onto each other and
thus create the secondary and tertiary shape of the aptamer that is
crucial to enable binding. We characterized the core
characteristics of these aptamers through a combined analysis of
conserved motif sequences and their effect on the predicted
structure of the whole aptamer. A motif in this context is defined
as a contiguous sequence of nucleotides of a defined length. For
this example, we considered each possible overlapping six
nucleotide motif within the random region of each aptamer
characterized.
[0205] The frequency of motifs of six or more nucleotides from the
random regions of aptamers OC1R-B1, OC1R-B9, and OC1R-A9 within a
subset of the selection library (top 1,000 oligonucleotides in
terms of copy number) was determined. Since these aptamers were all
selected for binding to the same target (teeth), it stands to
reason that motifs within this library that were present at higher
frequencies than a random distribution would represent key
sequences within the aptamers that were selected for. Moreover,
predicted structures containing these sequences should be
reasonably expected to represent structures that have been selected
for. As such, an analysis of the selection of motif sequences and
the predicted structures that they form provides a basis for a more
general understanding of the necessary requirements for aptamers
that have the capacity to bind to teeth.
[0206] The prediction of the secondary structures of the aptamers
was performed with RNAstructure
(https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/Predictl/Predictl-
.html).
[0207] A. Analysis of the Role of Conserved Motifs on Structure
within the Aptamer OC1R-B1:
[0208] The results of motif analysis are presented in FIG. 5. The
overlapping six nucleotide motifs comprising the random region of
the aptamer are provided consecutively along the x axis in this
figure. The y axis provides a statistical significance (Z value)
for each motif in the library. The Z value was computed as the
observed frequency of this motif in the library minus the average
of the frequency for all motifs in the library and this subtractant
was divided by the standard deviation of all motifs in the library
to provide the Z value. Thus, a Z value of 2 represents a frequency
of this motif in the library that is two standard deviations
greater than the average value for all motifs.
[0209] In FIG. 5, it is clear that the sequence UUCCUA and the
sequence UUUAUCUU were conserved at a level that represented more
than two standard deviations from the average. These two conserved
sequences were separated by a G and U consecutively that were not
conserved. These two consensus sequences can be joined by
considering these non-conserved nucleotides as N's.
TABLE-US-00002 SEQ ID NO 235: 5' UUCCUANNUUUAUCUU 3'.
[0210] The lowest free energy predicted structure of the OC1R-B1
aptamer is shown in FIG. 6. The numbers in FIGS. 6, 8, 10, 11, 12,
18A-18D, 19A-19C, 20A and 20B indicate the position along the
sequence from the 5'-end to the 3'-end; and are useful to track and
align oligos. It appears that the consensus motif is key to the
creation of a small loop between stems, as well as the joining of
the two ends of the aptamer. The sequence of the stem (double
stranded regions) itself is of importance as alteration of this
sequence while maintaining double stranded structure can destroy
binding capacity. It is thought that the sequence nature of the
double stranded region is important in terms of the stacking energy
of the bases and that this stacking energy is affected by events
such as binding elsewhere in the aptamer. The conserved motif noted
as SEQ ID NO 235 is highlighted in a box. SEQ ID NO's 235 to 244
are shown in TABLE 5.
[0211] Given that we have shown that the DNA version of this
aptamer also binds effectively to teeth (see Example 7), it stands
to reason that the conclusions arrived at within this example
regarding conserved motifs in the RNA sequence would apply to the
DNA sequence as well. Thus, any sequences containing the
corresponding deoxyribonucleotide motif
TABLE-US-00003 SEQ ID NO 236: 5'-TTCCTANNTTTATCTT-3'
are also expected to bind to teeth and are included as embodiments
of the present invention.
[0212] B. Analysis of the Role of Conserved Motifs on Structure
within the Aptamer OC1R-B9:
[0213] The analysis of the role of conserved motifs on structure
within aptamer OC1R-B9 was performed in a manner identical to that
described for OC1R-B1. FIG. 7 provides a summary of the motif
analysis for aptamer OC1R-B9. There is a ten nucleotide motif near
the 5' end of the aptamer that was present at a frequency that was
more than two standard deviations from the overall average motif
frequency in the selected libraries,
TABLE-US-00004 SEQ ID NO 237: 5'-UUCGCANAAG-3'.
This sequence has a single degenerate nucleotide within it. There
is an additional highly conserved 14 nucleotide motif near the 3'
end of the random sequence,
TABLE-US-00005 SEQ ID NO 238: 5'-UGCGGCANGCGCGU-3'.
[0214] This sequence also contains one degenerate nucleotide. The
predicted secondary structure of OC1R-B9 and its conserved motifs
is illustrated in FIG. 8. It is clear in this figure that the two
consensus motifs are related to each other, each comprising a
different side of the same double stranded secondary structure.
Sequences containing any of these motifs are also expected to bind
to teeth and are included as embodiments of the present invention.
Given that we demonstrated that the DNA version of this aptamer
also bound to teeth, it stands to reason that the same consensus
motifs within the DNA version of this aptamer would be necessary
core elements for the binding function. Thus, any sequences
containing any of the corresponding deoxyribonucleotide motifs:
TABLE-US-00006 SEQ ID NO 239: 5'-TTCGCANAAG-3' SEQ ID NO 240:
5'-TGCGGCANGCGCGT-3'
are also expected to bind to teeth and are included as embodiments
of the present invention.
[0215] C. Analysis of the Role of Conserved Motifs on Structure
within Aptamer OC1R-A9:
The motif analysis and predicted secondary structure for the
aptamer OC1R-A9 were performed in a manner identical to that
described for aptamer OC1R-B1 and OC1R-B9. The motif analysis for
the aptamer OC1R-A9 is provided in FIG. 9. In FIG. 9, as described
in Section A above, the y axis provides a statistical significance
(Z value) for each motif in the library. The Z value was computed
as the observed frequency of this motif in the library minus the
average of the frequency for all motifs in the library and this
subtractant was divided by the standard deviation of all motifs in
the library to provide the Z value." The ten nucleotide motif,
TABLE-US-00007 SEQ ID NO 241: 5'-CUUUUCUUCC-3'
was present at a frequency that was more than two standard
deviations from the overall average frequency of motifs within this
selected library. The role of this motif on the structure of the
aptamer is provided in FIG. 10. It is clear that this motif is
responsible for the formation of a double stranded region within
the aptamer. Therefore, it stands to reason that this double
stranded region within the aptamer is necessary for binding to
teeth. This motif within the DNA version of this aptamer, OC1D-A9
is also responsible for the formation of a double stranded region
(FIG. 11). Therefore, sequences containing this motif are also
expected to bind to teeth and are included as embodiments of the
present invention. Therefore, it stands to reason that any DNA
sequences containing the corresponding deoxyribonucleotide
motif
TABLE-US-00008 SEQ ID NO 242: 5'-CTTTTCTTCC-3'
are also expected to bind to teeth and are included as embodiments
of the present invention.
[0216] D. Analysis of Common Motifs within Aptamer Library:
[0217] A search for common motifs within the top 10,000 sequences
in terms of frequency from Experiment B was performed. The lead
motif identified in terms of significant deviation from random
distribution was SEQ ID NO 243.
TABLE-US-00009 SEQ ID NO 243: 5'-GCGCGCGC-3'
This motif was found in each of the following sequences, in which
the 5'- and 3'-primer recognition sequences were eliminated for
simplicity. Oligonucleotides comprising the motif SEQ ID NO 243 are
included as an embodiment of the current invention.
TABLE-US-00010 OC1R-B71 AAGCCGGCCCGGGAACAUGUCACGCGCGCGCGCAAAGUAG
OC1R-B125 GAAUUAUGAUAGACUAUGAGUCAAUAGCGCGCGCUGGAGG OC1R-B181
CACCGGGUGACGAAACGGCGCGCGCGCUAGCCUGCUAGCA OC1R-B190
ACACCGAGGGACUGAAUGGGGAGGCCGCGCGCGCUGAGGG OC1R-B302
AAGUCAGGACGCGAUGGGUGCGCGCGCGCGUGCGACAUAA OC1R-B306
ACGGCGCGCGCAGGCUACCACUACUGACUCGGCAAUGAUA OC1R-B316
CAACACUGCGCGCGCCGGACAACAAGCGAUUCCUGAGUAC OC1R-B317
CAACUCGACCUGCGCGCGCCUACCCCCAAACUCAACUACC OC1R-B338
CGAGGGUUAGAUCGGGGCGCGCGCACUUGUGGUUGUCCAA OC1R-B348
GAACGAGAGGCGCGCGCUGGUGGACCGGGGGAUAGGGGAU OC1R-B390
AACCGGUCAACACGAUCCUGAGCGCGCGCACGGAUGAAUU OC1R-B394
AACGGGCAUGACAGCCUCACGCCAAGGCGCGCGCGGGUAA OC1R-B395
AACGUCAAAAAACACUGCGCGCGCUGGAUAGAUGAACGUA OC1R-B404
ACAAAGCAGCCAGCGCGCGCUUGACCCGGAGAGUAGCACA OC1R-B463
CAUCCAUGCGCGCGCAGGGGUUAAGCGAGGGUCCUCCAUG OC1R-B497
CUCCGCGCGCGCACUUACCAUCUCUUCGUAAGGAAGUCGA OC1R-B606
AACCUGCAUUAGGCGCGCGCGCGUAGUUGGCACAGCUGUG OC1R-B690
AGCCAGCAGAAUGCCAACCAACGUAGCCGAGGCGCGCGCA OC1R-B875
CUGCUCUCCGGGCGCGCGCACCAACCUCUAGGCAGCUUGG OC1R-B952
GGCAGGGUAAUCUGGGCUGCGCGCGCACCUACAAUGGCUA OC1R-B973
GUGCAAGUUAUGAGAUUGGACGCACCGCGCGCGCAGCCUU OC1R-B986
UAAAGUUGGGUUCGGGGGCGCGCGCACUUCAUCACGACUA OC1R-B1115
UUUCACGCUUCCUGCGCGCGCUAGUGGCAACUCUACCUCC
[0218] Any sequences containing the corresponding
deoxyribonucleotide motif
TABLE-US-00011 SEQ ID NO 244: 5'-GCGCGCGC-3'
are also expected to bind to teeth and are included as embodiments
of the present invention.
Example 5. Analysis of Sequences Similarity
[0219] Alignment of SEQ ID NO 1 to SEQ ID NO 111 was performed
using the software Align X, a component of Vector NTI Advanced
11.5.4 by Themo Fisher Scientific. Several groups of sequences have
at least 90%, at least 70%, or at least 50% nucleotide sequence
identity as illustrated in the alignments of FIGS. 12, 13, and 14.
In these alignments, only the central variable region of the
aptamers is included for simplicity. Thus, oligonucleotides with at
least 50%, at least 70%, or at least 90% nucleotide sequence
identity to sequences selected from the group consisting of SEQ ID
NO 1 to SEQ ID NO 111 are included as embodiments of the current
invention. FIG. 12 shows the alignment of exemplary sequences with
at least 90% nucleotide sequence identity that were identified
during the selection process. FIG. 13 shows the alignment of
exemplary sequences with at least 70% nucleotide sequence identity
that were identified during the selection process. FIG. 14 shows
the alignment of exemplary sequences with at least 50% nucleotide
sequence identity that were identified during the selection
process.
Example 7. DNA Aptamers Binding
[0220] Selected DNA aptamers (OC1D-B1/OC1D-A2, OC1D-B9, and
OC1D-B25/OC1D-A9) were chemically synthesized with a FAM
fluorophore on the 5'end (Eurofins). An aliquot of 250 .mu.L of 1
.mu.M DNA aptamer in 1.times. binding buffer (10 mM cacodylate
buffer, 120 mM NaCl, 5 mM KCl, 50 .mu.M SnF.sub.2, and 0.322%
toothpaste; pH 7.2) was placed in the depression of a microscope
slide (concavity slides, 3.2 mm thick; United Scientific).
Separately, a clean unerupted third molar tooth was washed with
water, dried, and coated with human saliva collected fresh from
several individuals. The tooth was then placed into the depression
of the slide containing the DNA aptamer and incubated for 20
minutes at room temperature. The amount of aptamer bound was
determined by measuring the fluorescence remaining in the solution
after the tooth was removed (see FIG. 15). The tooth was removed
from the slide and washed several times with 250 .mu.L of neutral
binding buffer. The fluorescence of each wash solution was measured
(see FIG. 16). Bound aptamers were recovered by washing the teeth
with two aliquots of 250 mM NaOH. The fluorescence of each elution
solution was also measured (see FIG. 17). Not all the aptamer
incubated with the teeth was recovered probably due to very strong
binding or adsorption inside the teeth.
[0221] Given that we have shown that the DNA version of aptamers
OC1R-B1/OC1R-A2, OC1R-B9, and OC1R-B25/OC1R-A9 also bind
effectively to teeth, it stands to reason that the conclusions
arrived at within this example would apply to the DNA versions of
the remaining selected aptamers (SEQ ID NO: 112 to SEQ ID NO: 222),
included herein as part of the invention and listed in Table 3.
Example 8. Truncation of Aptamers
[0222] Starting from the predicted secondary structure of the
selected aptamers (OC1D-B1, OC1D-B9, and OC1D-A9), smaller
oligonucleotides comprising some of the secondary structure
elements were designed. Consideration of consensus motifs within
these predicted structures was included in the design of the
truncated aptamers (FIGS. 5 to 10). The truncated aptamers all
contained highly selected motif sequences that were involved in
stem/loop structures within the original aptamers. These selected
structures were conserved in the truncated aptamers.
[0223] For instance, aptamers OC1D-B1.1, OC1D-B1.2, and OC1D-B1.3
were derived from aptamer OC1D-B1 (see FIGS. 18A-18D). OC1D-B1.1
comprises completely the bent stem/loop structure of the structure,
OC1D-B1.2 comprises just the top of the bent stem/loop structure,
while OC1D-B1.3 comprises the tight stem/loop structure. Aptamers
OC1D-B9.1 and OC1D-B9.2 were derived from aptamer OC1D-B9 (see
FIGS. 19A-19C) and correspond to its two-bottom stem/loop
substructures. Aptamer OC1D-A9.1 was derived from aptamer OC1D-A9
(see FIGS. 20A and 20B) with a view to maintain the conserved motif
element in a minimal aptamer.
[0224] FIGS. 21A-21C illustrate the binding results for each of
these truncated aptamers to teeth. These binding assays were
performed and analyzed in a manner identical to that described
previously for the full-length aptamers (see Example 7). For
aptamers from OC1D-B1, the truncated aptamer containing the entire
bent stem/loop structure (OC1D-B1.1) performed better on teeth
compared to the other two. This provides confirmation that the
conserved motif identified in the RNA aptamer is conserved and key
to the function of this aptamer as well. For aptamers from OC1D-B9,
only the truncated aptamer OC1D-B9.2 containing the bottom bent
stem/loop structure binds well on teeth. This corresponds well to
the higher level of statistical significance associated with the
conserved motif within OC1D-B9.2 compared to the conserved motif
within OC1D-B9.1. Finally, truncated aptamer OC1D-A9.1 has a higher
binding affinity than the parent aptamer, but not as high as other
truncated aptamers. The list of sequences of truncated aptamers is
included in TABLE 4.
TABLE-US-00012 TABLE 2 List of top sequences from selection
experiments A and B. All the pyrimidine nucleotides are fluorinated
at the 2' position of the pentose group. Copy SEQ ID NO Name Total
Sequence Number 1 OC1R-B1 GGGAAGAGAAGGACAUAUGAUUCAUGUGAGAUGA 16160
or OC1R- UGUGUGUUCCUAGUUUUAUCUUGCUCUUUGACUA A2 GUACAUGACCACUU 2
OC1R-B2 GGGAAGAGAAGGACAUAUGAUUAGGCUAACUGUU 7945 or OC1R-
CAGGGAUUUGAUAUGCAUGAGGAGCACUUGACUA A1 GUACAUGACCACUU 3 OC1R-B3
GGGAAGAGAAGGACAUAUGAUCCGCUCUAAAGUA 7939 or OC1R-
CCAACCGCGGGAGCUAAAUGCAAGCCGUUGACUAG A19 UACAUGACCACUU 4 OC1R-B4
GGGAAGAGAAGGACAUAUGAUUGUGUCAGGCUCU 4041
AGAGUCUAGACGGCCGGGGUCCCGGAUUUGACUA GUACAUGACCACUU 5 OC1R-B5
GGGAAGAGAAGGACAUAUGAUCCUUAUGUCUAGC 2867
GGCCUUACGCGAUUAGUGGCGUUUUGUUUGACUA GUACAUGACCACUU 6 OC1R-B6
GGGAAGAGAAGGACAUAUGAUCUUUAUGUAUUAU 1841
CAGUCAUACCGGACGCAGCCCGCUGGAUUGACUAG UACAUGACCACUU 7 OC1R-B7
GGGAAGAGAAGGACAUAUGAUUGUGUUAUUACAC 1464 or OC1R-
UUCGUGAUUUUCCUUGCUUUUCUAUUUUUGACUA A3 GUACAUGACCACUU 8 OC1R-B8
GGGAAGAGAAGGACAUAUGAUCCAACAUCUAAAG 1373
UACUGGUCGCCUAGGGAGACUGUUCGGUUGACUA GUACAUGACCACUU 9 OC1R-B9
GGGAAGAGAAGGACAUAUGAUGCUAUAUUCGCAA 851
AAGCAGGCUGAGUGCGGCAGGCGCGUGUUGACUA GUACAUGACCACUU 10 OC1R-
GGGAAGAGAAGGACAUAUGAUUCAUUCAUUCGCA 759 B10
ACACAAUUGUAUUCGCAUCUGCGAUUUUUGACUA GUACAUGACCACUU 11 OC1R-
GGGAAGAGAAGGACAUAUGAUCUUUCUCUUUUCU 561 B11 or
AAUAUUUAAUUUAUUGGGUACCAAUUUUUGACUA OC1R- GUACAUGACCACUU A11 12
OC1R- GGGAAGAGAAGGACAUAUGAUCUUUGUUUCGCAU 425 B12 or
ACGUUUUCUUUUUCUCUCUUCUUAUUUUUGACUA OC1R-A7 GUACAUGACCACUU 13 OC1R-
GGGAAGAGAAGGACAUAUGAUUAUUCUGUUCUUC 402 B13 or
AAAAAUCUUUUAGCGUAUACGCUAUUUUUGACUA OC1R-A5 GUACAUGACCACUU 14 OC1R-
GGGAAGAGAAGGACAUAUGAUUUCCUUAUGUUCG 396 B14
GUCAACAGGGACUGCUGCAGCACCGGCUUGACUAG UACAUGACCACUU 15 OC1R-
GGGAAGAGAAGGACAUAUGAUUAAGCGCACUCAA 371 B15
CAGGGUCUAUGAUCCGCGCCGAUCAUGUUGACUAG UACAUGACCACUU 16 OC1R-
GGGAAGAGAAGGACAUAUGAUCCGCUUUCCAUUG 357 B16 or
AGAUUAUAAGCUGUUAGAGACUUAUUUUUGACUA OC1R- GUACAUGACCACUU A15 17
OC1R- GGGAAGAGAAGGACAUAUGAUUUUCGAAACGUUU 353 B17 or
CUUUCAAGUUCUUAAUCAUUCCCAUUUUUGACUA OC1R-A8 GUACAUGACCACUU 18 OC1R-
GGGAAGAGAAGGACAUAUGAUCAUUAGAUGCGCA 297 B18
GUUCGAAGCCGGUACAGCUGGCGCGCGUUGACUAG UACAUGACCACUU 19 OC1R-
GGGAAGAGAAGGACAUAUGAUAAAGAAUAACCUU 290 B19
AAAAUAACACCACCGCCUCACAGCAUAUUGACUAG UACAUGACCACUU 20 OC1R-
GGGAAGAGAAGGACAUAUGAUAAAUUGAUCUAUU 282 B20 or
CUUUUCGGUGCUAUUUAUCUUCCAUUUUUGACUA OC1R-A6 GUACAUGACCACUU 21 OC1R-
GGGAAGAGAAGGACAUAUGAUCUACUCGCGCGGC 282 B21
GGACAAAAGCGCAACCCAGCACCCAUGUUGACUAG UACAUGACCACUU 22 OC1R-
GGGAAGAGAAGGACAUAUGAUUCUUAGUUUGUAA 255 B22 or
UUACUUUUCCUUCCUUUUAUUCUAUUUUUGACUA OC1R- GUACAUGACCACUU A10 23
OC1R- GGGAAGAGAAGGACAUAUGAUAACCCGCGCAGAC 227 B23
UUACAAGCGCGCAAAAAAAGGGUACGUUUGACUA GUACAUGACCACUU 24 OC1R-
GGGAAGAGAAGGACAUAUGAUAUUCCUUUAUGCC 209 B24 or
GCAUCAUUUUAUUGUUUAUGACAAUUUUUGACUA OC1R- GUACAUGACCACUU A23 25
OC1R- GGGAAGAGAAGGACAUAUGAUAUUUCGUACUACU 209 B25 or
UUUCUUCCAAGCUUCAAUCGCCCAUUUUUGACUAG OC1R-A9 UACAUGACCACUU 26 OC1R-
GGGAAGAGAAGGACAUAUGAUUCACUCAUUCGCA 198 B26 or
ACACAAUUGUAUUCGCAUCUGCGAUUUUUGACUA OC1R- GUACAUGACCACUU A24 27
OC1R- GGGAAGAGAAGGACAUAUGAUAUUAUUUCCACAG 190 B27 or
UUCCUUUAUCCACACAUCUUCUCAUUUUUGACUAG OC1R- UACAUGACCACUU A12 28
OC1R- GGGAAGAGAAGGACAUAUGAUAAACUCGUUAUCU 187 B28
AUUCGUUUAUUUGCAUCUCUUUCAUUUUUGACUA GUACAUGACCACUU 29 OC1R-
GGGAAGAGAAGGACAUAUGAUCCAACCUCUAAAG 185 B29
UACUGGUCGCCUAGGGAGACUGUUCGGUUGACUA GUACAUGACCACUU 30 OC1R-
GGGAAGAGAAGGACAUAUGAUUUCCUUUUUGCUA 179 B30
UUUCCGUUAAUGUAAACUCUCCUAUUUUUGACUA OC1R- GUACAUGACCACUU A13 31
OC1R- GGGAAGAGAAGGACAUAUGAUCCUUAUGGCCUAG 167 B31
UAGGGAUCCGGGCGCCGACCAGCGCGAUUGACUAG UACAUGACCACUU 32 OC1R-
GGGAAGAGAAGGACAUAUGAUCGUCUGUCUUCUU 153 B32
CGAAUACGUUUUGGGCUAAGCCCAUUUUUGACUA OC1R- GUACAUGACCACUU A18 33
OC1R- GGGAAGAGAAGGACAUAUGAUUCAACCAAACUGC 143 B33
CGACGACCGAGGUAUGUCCUUAUGUACUUGACUA GUACAUGACCACUU 34 OC1R-
GGGAAGAGAAGGACAUAUGAUUACGGGUCUGAGC 142 B34
AAAAGCGAAGGAAGCAGGCGCAGGGAUUUGACUA GUACAUGACCACUU 35 OC1R-
GGGAAGAGAAGGACAUAUGAUUCUCUCAUUCGCA 134 B35 or
ACACAAUUGUAUUCGCAUCUGCGAUUUUUGACUA OC1R-A4 GUACAUGACCACUU 36 OC1R-
GGGAAGAGAAGGACAUAUGAUGCUCUAAAGUACU 127 B36
AAGCGUUUGCGCCGAUGCCCGGACCGCUUGACUAG UACAUGACCACUU 37 OC1R-
GGGAAGAGAAGGACAUAUGAUACUUCAUUAAUGU 126 B37
GAGGCCGUCAGGGGGCAACCUUCGAGCUUGACUAG UACAUGACCACUU 38 OC1R-
GGGAAGAGAAGGACAUAUGAUUCCUUAUUCUUGU 126 B38
UACUACUUUCUUUUCCUAUUUUUUUCUUUGACUA GUACAUGACCACUU 39 OC1R-
GGGAAGAGAAGGACAUAUGAUCGUUAUUUUCAUU 125 B39 or
UUCUUGUUCCCCAUAUGCCCAGGCGCAUUGACUAG OC1R- UACAUGACCACUU A14 40
OC1R- GGGAAGAGAAGGACAUAUGAUACCAGCGGCGUAG 120 B40
AAACGUACAGCUCGCCUGUAACGCCUGUUGACUAG UACAUGACCACUU 41 OC1R-
GGGAAGAGAAGGACAUAUGAUCGAUAUGGGUGCG 107 B41
GGAAUGUACGUUCACCGAAUAUGCUCCUUGACUA GUACAUGACCACUU 42 OC1R-
GGGAAGAGAAGGACAUAUGAUUAACAGUGCGUAG 95 B42
UCAUAUCGAAUGUUUAUCUUCCUAUUUUUGACUA GUACAUGACCACUU 43 OC1R-
GGGAAGAGAAGGACAUAUGAUCAGACUCUCGCCC 94 B43
AAUUCGCAAGGCGUUGCAUUGCGAUUUUUGACUA GUACAUGACCACUU 44 OC1R-
GGGAAGAGAAGGACAUAUGAUUUCCAACUCUCCA 88 B44
CGAGAGCAUGGGUCGAAUGACUCAUUUUUGACUA GUACAUGACCACUU 45 OC1R-
GGGAAGAGAAGGACAUAUGAUGCAUCGCGCGUCA 86 B45
CUCAACUCGUGAUUACCGAGGGCGCCGUUGACUAG UACAUGACCACUU 46 OC1R-
GGGAAGAGAAGGACAUAUGAUCUGAAUCUUUCCG 82 B46
CAGCCCUGUCCUUUUAAAGACAGGUUUUUGACUA GUACAUGACCACUU 47 OC1R-
GGGAAGAGAAGGACAUAUGAUUUUGUUACUUACU 70 B47
UCGUCUAUCUUCUGUUGCACACAGUUUUUGACUA GUACAUGACCACUU 48 OC1R-
GGGAAGAGAAGGACAUAUGAUUCAAAUCUUCAGC 69 B48
GAUAAUGGCACAAUUUCCGCGCCAUUUUUGACUA GUACAUGACCACUU 49 OC1R-
GGGAAGAGAAGGACAUAUGAUUUAUGUGAGAUGA 67 B49
UGUGUGUUCCUAGUUUUAUCUUGCUCUUUGACUA GUACAUGACCACUU 50 OC1R-
GGGAAGAGAAGGACAUAUGAUCCACUUUUCCAUU 62 B50
AACUGUUGCGGGCAAGUAGCACCGUUUUUGACUA GUACAUGACCACUU 51 OC1R-
GGGAAGAGAAGGACAUAUGAUAGAGAAGACCAUU 59 B51
CGGAAAGAGCUGCGUGUCCUUAUGUACUUGACUA GUACAUGACCACUU 52 OC1R-
GGGAAGAGAAGGACAUAUGAUUCUUAUGUAGCAA 58 B52
GCAAAAUGUGCCGCCGAGCCGACGCCAUUGACUAG UACAUGACCACUU 53 OC1R-
GGGAAGAGAAGGACAUAUGAUAAGCGCAUAAUAA 56 B53
GCCAGCCAGUUCUUGGCGCGCGGGGUAUUGACUAG UACAUGACCACUU 54 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGUCCGCAUUUC 56 B54
UAUUUUCUAUAUGGCUUACUGCCAUUUUUGACUA GUACAUGACCACUU 55 OC1R-
GGGAAGAGAAGGACAUAUGAUAUAAAGAACACGC 44 B55
AAAACCACCCGGACACCCGGUGCCGUGUUGACUAG UACAUGACCACUU 56 OC1R-
GGGAAGAGAAGGACAUAUGAUACACAGGCGGUGG 42 B56
AGCCGAAGGGCACCGGGACAAACCGACUUGACUAG UACAUGACCACUU 57 OC1R-
GGGAAGAGAAGGACAUAUGAUAGUUCCGGCGCAG 39 B57
CAGCGUCCUCACGUUUUACGUGCCCCAUUGACUAG UACAUGACCACUU 58 OC1R-
GGGAAGAGAAGGACAUAUGAUGACCGUCGCGAUC 39 B58
GUUUAUAAUGUUCUGGAUCUUUCAUUUUUGACUA GUACAUGACCACUU 59 OC1R-
GGGAAGAGAAGGACAUAUGAUAAGUGGGGCCCCG 37
B59 ACGACUUUUCCUUCCUCUCUUCCGGCAUUGACUAG UACAUGACCACUU 60 OC1R-
GGGAAGAGAAGGACAUAUGAUAUCAACAUACCAA 37 B60
AAUGUCAUUUCCAAUCUUUUCCCAUUUUUGACUA GUACAUGACCACUU 61 OC1R-
GGGAAGAGAAGGACAUAUGAUAGCGAACAAACAA 36 B61
GGGUGCCCAGGCCCCCUUCGCACAUCGUUGACUAG UACAUGACCACUU 62 OC1R-
GGGAAGAGAAGGACAUAUGAUCCUCUGUAACGCA 35 B62
AAGUCAAGUCGCGCAAGGCCGCCCGCGUUGACUAG UACAUGACCACUU 63 OC1R-
GGGAAGAGAAGGACAUAUGAUCUUCAUCUGCGAU 35 B63
UACGGUACACUUUAGUGUAUCGUUUUUUUGACUA GUACAUGACCACUU 64 OC1R-
GGGAAGAGAAGGACAUAUGAUGCCUAUGUGCUAG 35 B64
AUGCAGCAGCAACCGCCGGCGACUGGAUUGACUAG UACAUGACCACUU 65 OC1R-
GGGAAGAGAAGGACAUAUGAUCCGCGCCCUAACCU 33 B65
UCUGACCAAGCUUCCCUGGCACUUGGUUGACUAGU ACAUGACCACUU 66 OC1R-
GGGAAGAGAAGGACAUAUGAUCCUUAUGUAUUAU 33 B66
CAGUCAUACCGGACGCAGCCCGCUGGAUUGACUAG UACAUGACCACUU 67 OC1R-
GGGAAGAGAAGGACAUAUGAUCUAAUCUAUACUG 33 B67
GCUGCUAACGCUUUUUCUUUUCCAUUUUUGACUA GUACAUGACCACUU 68 OC1R-
GGGAAGAGAAGGACAUAUGAUCAGUUUACGCGGA 32 B68
GUCGUUUGUGUCCAUUUCUUCUCAUUUUUGACUA GUACAUGACCACUU 69 OC1R-
GGGAAGAGAAGGACAUAUGAUUCACGUGAGAUGA 32 B69
UGUGUGUUCCUAGUUUUAUCUUGCUCUUUGACUA GUACAUGACCACUU 70 OC1R-
GGGAAGAGAAGGACAUAUGAUUCCUUGUGUACCG 32 B70
CUCCGAAUGUGCUCCAGCGCGCCUCGGUUGACUAG UACAUGACCACUU 71 OC1R-
GGGAAGAGAAGGACAUAUGAUAAGCCGGCCCGGG 31 B71
AACAUGUCACGCGCGCGCGCAAAGUAGUUGACUAG UACAUGACCACUU 72 OC1R-
GGGAAGAGAAGGACAUAUGAUCCUGGAUUUCCGA 31 B72
AAUUAGAGUGCCGUUUCGUUACGGUUUUUGACUA GUACAUGACCACUU 73 OC1R-
GGGAAGAGAAGGACAUAUGAUCGUGUCAUCCGCA 31 B73
CAAGGAGGCCUGCAUGGCAGGGACACGUUGACUA GUACAUGACCACUU 74 OC1R-
GGGAAGAGAAGGACAUAUGAUGAGUAGACUUUUU 31 B74
GUAUCAUUUUUUUAUCGUAAGAUAUUUUUGACUA GUACAUGACCACUU 75 OC1R-
GGGAAGAGAAGGACAUAUGAUCCAUGUGAGAUGA 30 B75
UGUGUGUUCCUAGUUUUAUCUUGCUCUUUGACUA GUACAUGACCACUU 76 OC1R-
GGGAAGAGAAGGACAUAUGAUCUUUGCUCUAGAG 29 B76
UGUAGUCUAUGAGGGACAAGGUAGCCAUUGACUA GUACAUGACCACUU 77 OC1R-
GGGAAGAGAAGGACAUAUGAUGUUGGUUUUCUUU 29 B77
CUCUUUCUUUUCUUUCUCUUUCUAUUUUUGACUA GUACAUGACCACUU 78 OC1R-
GGGAAGAGAAGGACAUAUGAUCAAUCGGGCGGGG 28 B78
GUAAGAGGCGUGCGCAGCGUGGAGGUGUUGACUA GUACAUGACCACUU 79 OC1R-
GGGAAGAGAAGGACAUAUGAUCACCGUGGUGCGC 27 B79
AAAGCCGCAACGAGAACUGCGGAAUCGUUGACUA GUACAUGACCACUU 80 OC1R-
GGGAAGAGAAGGACAUAUGAUUGCUUUAAGUCUU 26 B80
UUUAUCAUUUUGUUUCCUUCAUUUUUUUUGACUA GUACAUGACCACUU 81 OC1R-
GGGAAGAGAAGGACAUAUGAUCGACUAGUUAUAC 25 B81
UGCAAAGGCUAUAAGCGCGAGCGCGCGUUGACUA GUACAUGACCACUU 82 OC1R-
GGGAAGAGAAGGACAUAUGAUGAGUAAUAGAUGG 25 B82
CGUACACAAAUCGGAUACGACGAGCGCUUGACUAG UACAUGACCACUU 83 OC1R-
GGGAAGAGAAGGACAUAUGAUUUUCGCUUCAAGA 25 B83
UUCCCAACGCCUUGUAAGUCAAGGUUUUUGACUA GUACAUGACCACUU 84 OC1R-
GGGAAGAGAAGGACAUAUGAUGUGUGAGAUGAGC 24 B84
CCCUGGACCAGACGCACGCUCGCACUGUUGACUAG UACAUGACCACUU 85 OC1R-
GGGAAGAGAAGGACAUAUGAUCAGGAUGCGGCGC 23 B85
CGGUAAUUGACUUCCCCCUACGUAGGAUUGACUAG UACAUGACCACUU 86 OC1R-
GGGAAGAGAAGGACAUAUGAUCAGGGACCCGGCC 22 B86
GGUGCAUCUCCUUCUUUAGCGUACGCCUUGACUAG UACAUGACCACUU 87 OC1R-
GGGAAGAGAAGGACAUAUGAUCUGCUCUAAAGUA 22 B87
CCAACCGCGGGAGCUAAAUGCAAGCCGUUGACUAG UACAUGACCACUU 88 OC1R-
GGGAAGAGAAGGACAUAUGAUGAUUGCCAUGCAU 22 B88
UAGGGGGGGACGCGCGCGAAAGGGAGAUUGACUA GUACAUGACCACUU 89 OC1R-
GGGAAGAGAAGGACAUAUGAUUCGCUCUAAAGUA 22 B89
CCAACCGCGGGAGCUAAAUGCAAGCCGUUGACUAG UACAUGACCACUU 90 OC1R-
GGGAAGAGAAGGACAUAUGAUAAAAAACCGGGGU 21 B90
UCUUAAUUUUCAUUGUUCGUCGUACUUUUGACUA GUACAUGACCACUU 91 OC1R-
GGGAAGAGAAGGACAUAUGAUAACCCAUUGGUGA 21 B91
AUCGCAACCACAGCCAGCCCGGCGCGAUUGACUAG UACAUGACCACUU 92 OC1R-
GGGAAGAGAAGGACAUAUGAUCGAAGUGAGGGGA 21 B92
UCGCGCGGGGUGCACCUAAAUAUGGGAUUGACUA GUACAUGACCACUU 93 OC1R-
GGGAAGAGAAGGACAUAUGAUAGCCUUAUGUACU 20 B93
AUAGAAGUCAGCUAUCCGCCGCACAAUUUGACUAG UACAUGACCACUU 94 OC1R-
GGGAAGAGAAGGACAUAUGAUCGUUGUUUUUCCC 20 B94
AAAGCUCGUUAGCAUUCAUUCCUAUUUUUGACUA GUACAUGACCACUU 95 OC1R-
GGGAAGAGAAGGACAUAUGAUGAUCAUCAGCGGA 20 B95
AAGCACGAAACGCCACGGGCCGCGGCAUUGACUAG UACAUGACCACUU 96 OC1R-
GGGAAGAGAAGGACAUAUGAUUCCUUCCUAUUGA 20 B96
CAAUGCGCCCGGGCCUCUUCAAUUGUAUUGACUAG UACAUGACCACUU 97 OC1R-
GGGAAGAGAAGGACAUAUGAUAGUUGCCGCGCGG 18 B97
CGCAAGAUUGGAGAGUCCCGGGCUGUAUUGACUA GUACAUGACCACUU 98 OC1R-
GGGAAGAGAAGGACAUAUGAUCAUAAGUUCGUUC 18 B98
AUUCCGUUAACACGCGUAUGGCGUUUUUUGACUA GUACAUGACCACUU 99 OC1R-
GGGAAGAGAAGGACAUAUGAUCCUUUGUCUCCAA 18 B99
AUCUUAGGACUGAAUGAGUGCCUAUUUUUGACUA GUACAUGACCACUU 100 OC1R-
GGGAAGAGAAGGACAUAUGAUCUUCUUUGAGAAU 18 B100
UCUCUUUUUACAAUUCCGGCGCCGUGAUUGACUAG UACAUGACCACUU 101 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGCUAACUGUU 3130 A16
UAGGGAUUUGAUAUGCAUGAGGAGCACUUGACUA GUACAUGACCACUU 102 OC1R-
GGGAAGAGAAGGACAUAUGAUCGUCUGUCUUCUU 2970 A17
CGAAUACGUUUUGGGCUAAGCCCAUUUUUGACUA GUACAUGACCACUU 103 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGCUAACUGCU 2753 A20
CAGGGAUUUGAUAUGCAUGAGGAGCACUUGACUA GUACAUGACCACUU 104 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGCUAACUGUU 2642 A21
CAGGGACUUGAUAUGCAUGAGGAGCACUUGACUA GUACAUGACCACUU 105 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGCUCACUGUU 2627 A22
CAGGGAUUUGAUAUGCAUGAGGAGCACUUGACUA GUACAUGACCACUU 106 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGCUAACUGUU 2250 A25
CAGGGAUUUGAUAUGCAUGGGGAGCACUUGACUA GUACAUGACCACUU 107 OC1R-
GGGAAGAGAAGGACAUAUGAUUUCUUCCUAUUGA 2195 A26
CGAUGCGCCCGGGCCUCUUCAAUUGUAUUGACUAG UACAUGACCACUU 108 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGCUAACUGUU 2156 A27
CGGGGAUUUGAUAUGCAUGAGGAGCACUUGACUA GUACAUGACCACUU 109 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGCUAACUGUU 2074 A28
CAGGGAUUUGAUAUGCACGAGGAGCACUUGACUA GUACAUGACCACUU 110 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGUUAACUGUU 2042 A29
CAGGGAUUUGAUAUGCAUGAGGAGCACUUGACUA GUACAUGACCACUU 111 OC1R-
GGGAAGAGAAGGACAUAUGAUUAGGCUAACUGUU 2031 A30
CAGGGAUUUGAUGUGCAUGAGGAGCACUUGACUA GUACAUGACCACUU
TABLE-US-00013 TABLE 3 List of deoxyribonucleotides aptamers based
on the top sequences from selection experiments A and B. SEQ ID NO
Name Total Sequence 112 OC1D-
GGGAAGAGAAGGACATATGATTCATGTGAGATGATGTGTGTTCCTAG B1 or
TTTTATCTTGCTCTTTGACTAGTACATGACCACTT OC1D- A2 113 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGTTCAGGGATTTGATA B2 or
TGCATGAGGAGCACTTGACTAGTACATGACCACTT OC1D- A1 114 OC1D-
GGGAAGAGAAGGACATATGATCCGCTCTAAAGTACCAACCGCGGGA B3 or
GCTAAATGCAAGCCGTTGACTAGTACATGACCACTT OC1D- A19 115 OC1D-
GGGAAGAGAAGGACATATGATTGTGTCAGGCTCTAGAGTCTAGACGG B4
CCGGGGTCCCGGATTTGACTAGTACATGACCACTT 116 OC1D-
GGGAAGAGAAGGACATATGATCCTTATGTCTAGCGGCCTTACGCGAT B5
TAGTGGCGTTTTGTTTGACTAGTACATGACCACTT 117 OC1D-
GGGAAGAGAAGGACATATGATCTTTATGTATTATCAGTCATACCGGA B6
CGCAGCCCGCTGGATTGACTAGTACATGACCACTT 118 OC1D-
GGGAAGAGAAGGACATATGATTGTGTTATTACACTTCGTGATTTTCCT B7 or
TGCTTTTCTATTTTTGACTAGTACATGACCACTT OC1D- A3 119 OC1D-
GGGAAGAGAAGGACATATGATCCAACATCTAAAGTACTGGTCGCCTA B8
GGGAGACTGTTCGGTTGACTAGTACATGACCACTT 120 OC1D-
GGGAAGAGAAGGACATATGATGCTATATTCGCAAAAGCAGGCTGAG B9
TGCGGCAGGCGCGTGTTGACTAGTACATGACCACTT 121 OC1D-
GGGAAGAGAAGGACATATGATTCATTCATTCGCAACACAATTGTATT B10
CGCATCTGCGATTTTTGACTAGTACATGACCACTT 122 OC1D-
GGGAAGAGAAGGACATATGATCTTTCTCTTTTCTAATATTTAATTTAT B11 or
TGGGTACCAATTTTTGACTAGTACATGACCACTT OC1D- A11 123 OC1D-
GGGAAGAGAAGGACATATGATCTTTGTTTCGCATACGTTTTCTTTTTC B12 or
TCTCTTCTTATTTTTGACTAGTACATGACCACTT OC1D- A7 124 OC1D-
GGGAAGAGAAGGACATATGATTATTCTGTTCTTCAAAAATCTTTTAG B13 or
CGTATACGCTATTTTTGACTAGTACATGACCACTT OC1D- A5 125 OC1D-
GGGAAGAGAAGGACATATGATTTCCTTATGTTCGGTCAACAGGGACT B14
GCTGCAGCACCGGCTTGACTAGTACATGACCACTT 126 OC1D-
GGGAAGAGAAGGACATATGATTAAGCGCACTCAACAGGGTCTATGA B15
TCCGCGCCGATCATGTTGACTAGTACATGACCACTT 127 OC1D-
GGGAAGAGAAGGACATATGATCCGCTTTCCATTGAGATTATAAGCTG B16 or
TTAGAGACTTATTTTTGACTAGTACATGACCACTT OC1D- A15 128 OC1D-
GGGAAGAGAAGGACATATGATTTTCGAAACGTTTCTTTCAAGTTCTT B17 or
AATCATTCCCATTTTTGACTAGTACATGACCACTT OC1D- A8 129 OC1D-
GGGAAGAGAAGGACATATGATCATTAGATGCGCAGTTCGAAGCCGG B18
TACAGCTGGCGCGCGTTGACTAGTACATGACCACTT 130 OC1D-
GGGAAGAGAAGGACATATGATAAAGAATAACCTTAAAATAACACCA B19
CCGCCTCACAGCATATTGACTAGTACATGACCACTT 131 OC1D-
GGGAAGAGAAGGACATATGATAAATTGATCTATTCTTTTCGGTGCTA B20 or
TTTATCTTCCATTTTTGACTAGTACATGACCACTT OC1D- A6 132 OC1D-
GGGAAGAGAAGGACATATGATCTACTCGCGCGGCGGACAAAAGCGC B21
AACCCAGCACCCATGTTGACTAGTACATGACCACTT 133 OC1D-
GGGAAGAGAAGGACATATGATTCTTAGTTTGTAATTACTTTTCCTTCC B22 or
TTTTATTCTATTTTTGACTAGTACATGACCACTT OC1D- A10 134 OC1D-
GGGAAGAGAAGGACATATGATAACCCGCGCAGACTTACAAGCGCGC B23
AAAAAAAGGGTACGTTTGACTAGTACATGACCACTT 135 OC1D-
GGGAAGAGAAGGACATATGATATTCCTTTATGCCGCATCATTTTATTG B24 or
TTTATGACAATTTTTGACTAGTACATGACCACTT OC1D- A23 136 OC1D-
GGGAAGAGAAGGACATATGATATTTCGTACTACTTTTCTTCCAAGCTT B25 or
CAATCGCCCATTTTTGACTAGTACATGACCACTT OC1D- A9 137 OC1D-
GGGAAGAGAAGGACATATGATTCACTCATTCGCAACACAATTGTATT B26 or
CGCATCTGCGATTTTTGACTAGTACATGACCACTT OC1D- A24 138 OC1D-
GGGAAGAGAAGGACATATGATATTATTTCCACAGTTCCTTTATCCAC B27 or
ACATCTTCTCATTTTTGACTAGTACATGACCACTT OC1D- A12 139 OC1D-
GGGAAGAGAAGGACATATGATAAACTCGTTATCTATTCGTTTATTTG B28
CATCTCTTTCATTTTTGACTAGTACATGACCACTT 140 OC1D-
GGGAAGAGAAGGACATATGATCCAACCTCTAAAGTACTGGTCGCCTA B29
GGGAGACTGTTCGGTTGACTAGTACATGACCACTT 141 OC1D-
GGGAAGAGAAGGACATATGATTTCCTTTTTGCTATTTCCGTTAATGTA B30
AACTCTCCTATTTTTGACTAGTACATGACCACTT OC1D- A13 142 OC1D-
GGGAAGAGAAGGACATATGATCCTTATGGCCTAGTAGGGATCCGGGC B31
GCCGACCAGCGCGATTGACTAGTACATGACCACTT 143 OC1D-
GGGAAGAGAAGGACATATGATCGTCTGTCTTCTTCGAATACGTTTTG B32
GGCTAAGCCCATTTTTGACTAGTACATGACCACTT OC1D- A18 144 OC1D-
GGGAAGAGAAGGACATATGATTCAACCAAACTGCCGACGACCGAGG B33
TATGTCCTTATGTACTTGACTAGTACATGACCACTT 145 OC1D-
GGGAAGAGAAGGACATATGATTACGGGTCTGAGCAAAAGCGAAGGA B34
AGCAGGCGCAGGGATTTGACTAGTACATGACCACTT 146 OC1D-
GGGAAGAGAAGGACATATGATTCTCTCATTCGCAACACAATTGTATT B35 or
CGCATCTGCGATTTTTGACTAGTACATGACCACTT OC1D- A4 147 OC1D-
GGGAAGAGAAGGACATATGATGCTCTAAAGTACTAAGCGTTTGCGCC B36
GATGCCCGGACCGCTTGACTAGTACATGACCACTT 148 OC1D-
GGGAAGAGAAGGACATATGATACTTCATTAATGTGAGGCCGTCAGGG B37
GGCAACCTTCGAGCTTGACTAGTACATGACCACTT 149 OC1D-
GGGAAGAGAAGGACATATGATTCCTTATTCTTGTTACTACTTTCTTTT B38
CCTATTTTTTTCTTTGACTAGTACATGACCACTT 150 OC1D-
GGGAAGAGAAGGACATATGATCGTTATTTTCATTTTCTTGTTCCCCAT B39 or
ATGCCCAGGCGCATTGACTAGTACATGACCACTT OC1D- A14 151 OC1D-
GGGAAGAGAAGGACATATGATACCAGCGGCGTAGAAACGTACAGCT B40
CGCCTGTAACGCCTGTTGACTAGTACATGACCACTT 152 OC1D-
GGGAAGAGAAGGACATATGATCGATATGGGTGCGGGAATGTACGTT B41
CACCGAATATGCTCCTTGACTAGTACATGACCACTT 153 OC1D-
GGGAAGAGAAGGACATATGATTAACAGTGCGTAGTCATATCGAATGT B42
TTATCTTCCTATTTTTGACTAGTACATGACCACTT 154 OC1D-
GGGAAGAGAAGGACATATGATCAGACTCTCGCCCAATTCGCAAGGC B43
GTTGCATTGCGATTTTTGACTAGTACATGACCACTT 155 OC1D-
GGGAAGAGAAGGACATATGATTTCCAACTCTCCACGAGAGCATGGGT B44
CGAATGACTCATTTTTGACTAGTACATGACCACTT 156 OC1D-
GGGAAGAGAAGGACATATGATGCATCGCGCGTCACTCAACTCGTGAT B45
TACCGAGGGCGCCGTTGACTAGTACATGACCACTT 157 OC1D-
GGGAAGAGAAGGACATATGATCTGAATCTTTCCGCAGCCCTGTCCTT B46
TTAAAGACAGGTTTTTGACTAGTACATGACCACTT 158 OC1D-
GGGAAGAGAAGGACATATGATTTTGTTACTTACTTCGTCTATCTTCTG B47
TTGCACACAGTTTTTGACTAGTACATGACCACTT 159 OC1D-
GGGAAGAGAAGGACATATGATTCAAATCTTCAGCGATAATGGCACA B48
ATTTCCGCGCCATTTTTGACTAGTACATGACCACTT 160 OC1D-
GGGAAGAGAAGGACATATGATTTATGTGAGATGATGTGTGTTCCTAG B49
TTTTATCTTGCTCTTTGACTAGTACATGACCACTT 161 OC1D-
GGGAAGAGAAGGACATATGATCCACTTTTCCATTAACTGTTGCGGGC B50
AAGTAGCACCGTTTTTGACTAGTACATGACCACTT 162 OC1D-
GGGAAGAGAAGGACATATGATAGAGAAGACCATTCGGAAAGAGCTG B51
CGTGTCCTTATGTACTTGACTAGTACATGACCACTT 163 OC1D-
GGGAAGAGAAGGACATATGATTCTTATGTAGCAAGCAAAATGTGCCG B52
CCGAGCCGACGCCATTGACTAGTACATGACCACTT 164 OC1D-
GGGAAGAGAAGGACATATGATAAGCGCATAATAAGCCAGCCAGTTC B53
TTGGCGCGCGGGGTATTGACTAGTACATGACCACTT 165 OC1D-
GGGAAGAGAAGGACATATGATTAGTCCGCATTTCTATTTTCTATATG B54
GCTTACTGCCATTTTTGACTAGTACATGACCACTT 166 OC1D-
GGGAAGAGAAGGACATATGATATAAAGAACACGCAAAACCACCCGG B55
ACACCCGGTGCCGTGTTGACTAGTACATGACCACTT 167 OC1D-
GGGAAGAGAAGGACATATGATACACAGGCGGTGGAGCCGAAGGGCA B56
CCGGGACAAACCGACTTGACTAGTACATGACCACTT 168 OC1D-
GGGAAGAGAAGGACATATGATAGTTCCGGCGCAGCAGCGTCCTCAC B57
GTTTTACGTGCCCCATTGACTAGTACATGACCACTT 169 OC1D-
GGGAAGAGAAGGACATATGATGACCGTCGCGATCGTTTATAATGTTC B58
TGGATCTTTCATTTTTGACTAGTACATGACCACTT 170 OC1D-
GGGAAGAGAAGGACATATGATAAGTGGGGCCCCGACGACTTTTCCTT B59
CCTCTCTTCCGGCATTGACTAGTACATGACCACTT 171 OC1D-
GGGAAGAGAAGGACATATGATATCAACATACCAAAATGTCATTTCCA B60
ATCTTTTCCCATTTTTGACTAGTACATGACCACTT 172 OC1D-
GGGAAGAGAAGGACATATGATAGCGAACAAACAAGGGTGCCCAGGC B61
CCCCTTCGCACATCGTTGACTAGTACATGACCACTT 173 OC1D-
GGGAAGAGAAGGACATATGATCCTCTGTAACGCAAAGTCAAGTCGC B62
GCAAGGCCGCCCGCGTTGACTAGTACATGACCACTT 174 OC1D-
GGGAAGAGAAGGACATATGATCTTCATCTGCGATTACGGTACACTTT B63
AGTGTATCGTTTTTTTGACTAGTACATGACCACTT 175 OC1D-
GGGAAGAGAAGGACATATGATGCCTATGTGCTAGATGCAGCAGCAA B64
CCGCCGGCGACTGGATTGACTAGTACATGACCACTT 176 OC1D-
GGGAAGAGAAGGACATATGATCCGCGCCCTAACCTTCTGACCAAGCT B65
TCCCTGGCACTTGGTTGACTAGTACATGACCACTT 177 OC1D-
GGGAAGAGAAGGACATATGATCCTTATGTATTATCAGTCATACCGGA B66
CGCAGCCCGCTGGATTGACTAGTACATGACCACTT 178 OC1D-
GGGAAGAGAAGGACATATGATCTAATCTATACTGGCTGCTAACGCTT B67
TTTCTTTTCCATTTTTGACTAGTACATGACCACTT 179 OC1D-
GGGAAGAGAAGGACATATGATCAGTTTACGCGGAGTCGTTTGTGTCC B68
ATTTCTTCTCATTTTTGACTAGTACATGACCACTT 180 OC1D-
GGGAAGAGAAGGACATATGATTCACGTGAGATGATGTGTGTTCCTAG
B69 TTTTATCTTGCTCTTTGACTAGTACATGACCACTT 181 OC1D-
GGGAAGAGAAGGACATATGATTCCTTGTGTACCGCTCCGAATGTGCT B70
CCAGCGCGCCTCGGTTGACTAGTACATGACCACTT 182 OC1D-
GGGAAGAGAAGGACATATGATAAGCCGGCCCGGGAACATGTCACGC B71
GCGCGCGCAAAGTAGTTGACTAGTACATGACCACTT 183 OC1D-
GGGAAGAGAAGGACATATGATCCTGGATTTCCGAAATTAGAGTGCCG B72
TTTCGTTACGGTTTTTGACTAGTACATGACCACTT 184 OC1D-
GGGAAGAGAAGGACATATGATCGTGTCATCCGCACAAGGAGGCCTG B73
CATGGCAGGGACACGTTGACTAGTACATGACCACTT 185 OC1D-
GGGAAGAGAAGGACATATGATGAGTAGACTTTTTGTATCATTTTTTTA B74
TCGTAAGATATTTTTGACTAGTACATGACCACTT 186 OC1D-
GGGAAGAGAAGGACATATGATCCATGTGAGATGATGTGTGTTCCTAG B75
TTTTATCTTGCTCTTTGACTAGTACATGACCACTT 187 OC1D-
GGGAAGAGAAGGACATATGATCTTTGCTCTAGAGTGTAGTCTATGAG B76
GGACAAGGTAGCCATTGACTAGTACATGACCACTT 188 OC1D-
GGGAAGAGAAGGACATATGATGTTGGTTTTCTTTCTCTTTCTTTTCTTT B77
CTCTTTCTATTTTTGACTAGTACATGACCACTT 189 OC1D-
GGGAAGAGAAGGACATATGATCAATCGGGCGGGGGTAAGAGGCGTG B78
CGCAGCGTGGAGGTGTTGACTAGTACATGACCACTT 190 OC1D-
GGGAAGAGAAGGACATATGATCACCGTGGTGCGCAAAGCCGCAACG B79
AGAACTGCGGAATCGTTGACTAGTACATGACCACTT 191 OC1D-
GGGAAGAGAAGGACATATGATTGCTTTAAGTCTTTTTATCATTTTGTT B80
TCCTTCATTTTTTTTGACTAGTACATGACCACTT 192 OC1D-
GGGAAGAGAAGGACATATGATCGACTAGTTATACTGCAAAGGCTATA B81
AGCGCGAGCGCGCGTTGACTAGTACATGACCACTT 193 OC1D-
GGGAAGAGAAGGACATATGATGAGTAATAGATGGCGTACACAAATC B82
GGATACGACGAGCGCTTGACTAGTACATGACCACTT 194 OC1D-
GGGAAGAGAAGGACATATGATTTTCGCTTCAAGATTCCCAACGCCTT B83
GTAAGTCAAGGTTTTTGACTAGTACATGACCACTT 195 OC1D-
GGGAAGAGAAGGACATATGATGTGTGAGATGAGCCCCTGGACCAGA B84
CGCACGCTCGCACTGTTGACTAGTACATGACCACTT 196 OC1D-
GGGAAGAGAAGGACATATGATCAGGATGCGGCGCCGGTAATTGACT B85
TCCCCCTACGTAGGATTGACTAGTACATGACCACTT 197 OC1D-
GGGAAGAGAAGGACATATGATCAGGGACCCGGCCGGTGCATCTCCTT B86
CTTTAGCGTACGCCTTGACTAGTACATGACCACTT 198 OC1D-
GGGAAGAGAAGGACATATGATCTGCTCTAAAGTACCAACCGCGGGA B87
GCTAAATGCAAGCCGTTGACTAGTACATGACCACTT 199 OC1D-
GGGAAGAGAAGGACATATGATGATTGCCATGCATTAGGGGGGGACG B88
CGCGCGAAAGGGAGATTGACTAGTACATGACCACTT 200 OC1D-
GGGAAGAGAAGGACATATGATTCGCTCTAAAGTACCAACCGCGGGA B89
GCTAAATGCAAGCCGTTGACTAGTACATGACCACTT 201 OC1D-
GGGAAGAGAAGGACATATGATAAAAAACCGGGGTTCTTAATTTTCAT B90
TGTTCGTCGTACTTTTGACTAGTACATGACCACTT 202 OC1D-
GGGAAGAGAAGGACATATGATAACCCATTGGTGAATCGCAACCACA B91
GCCAGCCCGGCGCGATTGACTAGTACATGACCACTT 203 OC1D-
GGGAAGAGAAGGACATATGATCGAAGTGAGGGGATCGCGCGGGGTG B92
CACCTAAATATGGGATTGACTAGTACATGACCACTT 204 OC1D-
GGGAAGAGAAGGACATATGATAGCCTTATGTACTATAGAAGTCAGCT B93
ATCCGCCGCACAATTTGACTAGTACATGACCACTT 205 OC1D-
GGGAAGAGAAGGACATATGATCGTTGTTTTTCCCAAAGCTCGTTAGC B94
ATTCATTCCTATTTTTGACTAGTACATGACCACTT 206 OC1D-
GGGAAGAGAAGGACATATGATGATCATCAGCGGAAAGCACGAAACG B95
CCACGGGCCGCGGCATTGACTAGTACATGACCACTT 207 OC1D-
GGGAAGAGAAGGACATATGATTCCTTCCTATTGACAATGCGCCCGGG B96
CCTCTTCAATTGTATTGACTAGTACATGACCACTT 208 OC1D-
GGGAAGAGAAGGACATATGATAGTTGCCGCGCGGCGCAAGATTGGA B97
GAGTCCCGGGCTGTATTGACTAGTACATGACCACTT 209 OC1D-
GGGAAGAGAAGGACATATGATCATAAGTTCGTTCATTCCGTTAACAC B98
GCGTATGGCGTTTTTTGACTAGTACATGACCACTT 210 OC1D-
GGGAAGAGAAGGACATATGATCCTTTGTCTCCAAATCTTAGGACTGA B99
ATGAGTGCCTATTTTTGACTAGTACATGACCACTT 211 OC1D-
GGGAAGAGAAGGACATATGATCTTCTTTGAGAATTCTCTTTTTACAAT B100
TCCGGCGCCGTGATTGACTAGTACATGACCACTT 212 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGTTTAGGGATTTGATA A16
TGCATGAGGAGCACTTGACTAGTACATGACCACTT 213 OC1D-
GGGAAGAGAAGGACATATGATCGTCTGTCTTCTTCGAATACGTTTTG A17
GGCTAAGCCCATTTTTGACTAGTACATGACCACTT 214 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGCTCAGGGATTTGATA A20
TGCATGAGGAGCACTTGACTAGTACATGACCACTT 215 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGTTCAGGGACTTGATA A21
TGCATGAGGAGCACTTGACTAGTACATGACCACTT 216 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTCACTGTTCAGGGATTTGATA A22
TGCATGAGGAGCACTTGACTAGTACATGACCACTT 217 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGTTCAGGGATTTGATA A25
TGCATGGGGAGCACTTGACTAGTACATGACCACTT 218 OC1D-
GGGAAGAGAAGGACATATGATTTCTTCCTATTGACGATGCGCCCGGG A26
CCTCTTCAATTGTATTGACTAGTACATGACCACTT 219 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGTTCGGGGATTTGATA A27
TGCATGAGGAGCACTTGACTAGTACATGACCACTT 220 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGTTCAGGGATTTGATA A28
TGCACGAGGAGCACTTGACTAGTACATGACCACTT 221 OC1D-
GGGAAGAGAAGGACATATGATTAGGTTAACTGTTCAGGGATTTGATA A29
TGCATGAGGAGCACTTGACTAGTACATGACCACTT 222 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGTTCAGGGATTTGATG A30
TGCATGAGGAGCACTTGACTAGTACATGACCACTT 112 OC1D-
GGGAAGAGAAGGACATATGATTCATGTGAGATGATGTGTGTTCCTAG B1 or
TTTTATCTTGCTCTTTGACTAGTACATGACCACTT OC1D- A2 113 OC1D-
GGGAAGAGAAGGACATATGATTAGGCTAACTGTTCAGGGATTTGATA B2 or
TGCATGAGGAGCACTTGACTAGTACATGACCACTT OC1D- A1 114 OC1D-
GGGAAGAGAAGGACATATGATCCGCTCTAAAGTACCAACCGCGGGA B3 or
GCTAAATGCAAGCCGTTGACTAGTACATGACCACTT OC1D- A19 115 OC1D-
GGGAAGAGAAGGACATATGATTGTGTCAGGCTCTAGAGTCTAGACGG B4
CCGGGGTCCCGGATTTGACTAGTACATGACCACTT 116 OC1D-
GGGAAGAGAAGGACATATGATCCTTATGTCTAGCGGCCTTACGCGAT B5
TAGTGGCGTTTTGTTTGACTAGTACATGACCACTT 117 OC1D-
GGGAAGAGAAGGACATATGATCTTTATGTATTATCAGTCATACCGGA B6
CGCAGCCCGCTGGATTGACTAGTACATGACCACTT 118 OC1D-
GGGAAGAGAAGGACATATGATTGTGTTATTACACTTCGTGATTTTCCT B7 or
TGCTTTTCTATTTTTGACTAGTACATGACCACTT OC1D- A3 119 OC1D-
GGGAAGAGAAGGACATATGATCCAACATCTAAAGTACTGGTCGCCTA B8
GGGAGACTGTTCGGTTGACTAGTACATGACCACTT 120 OC1D-
GGGAAGAGAAGGACATATGATGCTATATTCGCAAAAGCAGGCTGAG B9
TGCGGCAGGCGCGTGTTGACTAGTACATGACCACTT 121 OC1D-
GGGAAGAGAAGGACATATGATTCATTCATTCGCAACACAATTGTATT B10
CGCATCTGCGATTTTTGACTAGTACATGACCACTT 122 OC1D-
GGGAAGAGAAGGACATATGATCTTTCTCTTTTCTAATATTTAATTTAT B11 or
TGGGTACCAATTTTTGACTAGTACATGACCACTT OC1D- A11 123 OC1D-
GGGAAGAGAAGGACATATGATCTTTGTTTCGCATACGTTTTCTTTTTC B12 or
TCTCTTCTTATTTTTGACTAGTACATGACCACTT OC1D- A7 124 OC1D-
GGGAAGAGAAGGACATATGATTATTCTGTTCTTCAAAAATCTTTTAG B13 or
CGTATACGCTATTTTTGACTAGTACATGACCACTT OC1D- A5 125 OC1D-
GGGAAGAGAAGGACATATGATTTCCTTATGTTCGGTCAACAGGGACT B14
GCTGCAGCACCGGCTTGACTAGTACATGACCACTT 126 OC1D-
GGGAAGAGAAGGACATATGATTAAGCGCACTCAACAGGGTCTATGA B15
TCCGCGCCGATCATGTTGACTAGTACATGACCACTT 127 OC1D-
GGGAAGAGAAGGACATATGATCCGCTTTCCATTGAGATTATAAGCTG B16 or
TTAGAGACTTATTTTTGACTAGTACATGACCACTT OC1D- A15 128 OC1D-
GGGAAGAGAAGGACATATGATTTTCGAAACGTTTCTTTCAAGTTCTT B17 or
AATCATTCCCATTTTTGACTAGTACATGACCACTT OC1D- A8 129 OC1D-
GGGAAGAGAAGGACATATGATCATTAGATGCGCAGTTCGAAGCCGG B18
TACAGCTGGCGCGCGTTGACTAGTACATGACCACTT 130 OC1D-
GGGAAGAGAAGGACATATGATAAAGAATAACCTTAAAATAACACCA B19
CCGCCTCACAGCATATTGACTAGTACATGACCACTT
TABLE-US-00014 TABLE 4 List of truncated aptamers. SEQ ID NO Name
Sequence 223 OC1D-B1.1 ATGTGTGTTCCTAGTTTTATCTTGCTCTTTGACTA
GTACATGACCACTTG 224 OC1D-B1.2 TTCCTAGTTTTATCTTGCTCTTTGACTAGTA 225
OC1D-B1.3 GGACATATGATTCATGTGAGA 226 OC1D-B9.1
TTCGCAAAAGCAGGCTGAGTGCGGC 227 OC1D-B9.2
CAGGCGCGTGTTGACTAGTACATGACCAC 228 OC1D-A9.1
GGGAAGAGAAGGACATATGATATTTCGTACTACTT TTCTTCCAA 229 OC1R-B1.1
AUGUGUGUUCCUAGUUUUAUCUUGCUCUUUGACUA GUACAUGACCACUUG 230 OC1R-B1.2
UUCCUAGUUUUAUCUUGCUCUUUGACUAGUA 231 OC1R-B1.3 GGACAUAUGAUUCAUGUGAGA
232 OC1R-B9.1 UUCGCAAAAGCAGGCUGAGUGCGGC 233 OC1R-B9.2
CAGGCGCGUGUUGACUAGUACAUGACCAC 234 OC1R-A9.1
GGGAAGAGAAGGACAUAUGAUAUUUCGUACUACUU UUCUUCCAA
TABLE-US-00015 TABLE 5 List of Conserved Motif Sequences SEQ ID NO
Sequence 235 UUCCUANNUUUAUCUU 236 TTCCTANNTTTATCTT 237 UUCGCANAAG
238 UGCGGCANGCGCGU 239 TTCGCANAAG 240 TGCGGCANGCGCGT 241 CUUUUCUUCC
242 CTTTTCTTCC 243 NGCGCGCGCN 244 NGCGCGCGCN
Sequence CWU 1
1
244182RNAArtificial SequenceSynthetic aptamer sequence 1gggaagagaa
ggacauauga uucaugugag augaugugug uuccuaguuu uaucuugcuc 60uuugacuagu
acaugaccac uu 82282RNAArtificial SequenceSynthetic aptamer sequence
2gggaagagaa ggacauauga uuaggcuaac uguucaggga uuugauaugc augaggagca
60cuugacuagu acaugaccac uu 82382RNAArtificial SequenceSynthetic
aptamer sequence 3gggaagagaa ggacauauga uccgcucuaa aguaccaacc
gcgggagcua aaugcaagcc 60guugacuagu acaugaccac uu 82482RNAArtificial
SequenceSynthetic aptamer sequence 4gggaagagaa ggacauauga
uugugucagg cucuagaguc uagacggccg gggucccgga 60uuugacuagu acaugaccac
uu 82582RNAArtificial SequenceSynthetic aptamer sequence
5gggaagagaa ggacauauga uccuuauguc uagcggccuu acgcgauuag uggcguuuug
60uuugacuagu acaugaccac uu 82682RNAArtificial SequenceSynthetic
aptamer sequence 6gggaagagaa ggacauauga ucuuuaugua uuaucaguca
uaccggacgc agcccgcugg 60auugacuagu acaugaccac uu 82782RNAArtificial
SequenceSynthetic aptamer sequence 7gggaagagaa ggacauauga
uuguguuauu acacuucgug auuuuccuug cuuuucuauu 60uuugacuagu acaugaccac
uu 82882RNAArtificial SequenceSynthetic aptamer sequence
8gggaagagaa ggacauauga uccaacaucu aaaguacugg ucgccuaggg agacuguucg
60guugacuagu acaugaccac uu 82982RNAArtificial SequenceSynthetic
aptamer sequence 9gggaagagaa ggacauauga ugcuauauuc gcaaaagcag
gcugagugcg gcaggcgcgu 60guugacuagu acaugaccac uu
821082RNAArtificial SequenceSynthetic aptamer sequence 10gggaagagaa
ggacauauga uucauucauu cgcaacacaa uuguauucgc aucugcgauu 60uuugacuagu
acaugaccac uu 821182RNAArtificial SequenceSynthetic aptamer
sequence 11gggaagagaa ggacauauga ucuuucucuu uucuaauauu uaauuuauug
gguaccaauu 60uuugacuagu acaugaccac uu 821282RNAArtificial
SequenceSynthetic aptamer sequence 12gggaagagaa ggacauauga
ucuuuguuuc gcauacguuu ucuuuuucuc ucuucuuauu 60uuugacuagu acaugaccac
uu 821382RNAArtificial SequenceSynthetic aptamer sequence
13gggaagagaa ggacauauga uuauucuguu cuucaaaaau cuuuuagcgu auacgcuauu
60uuugacuagu acaugaccac uu 821482RNAArtificial SequenceSynthetic
aptamer sequence 14gggaagagaa ggacauauga uuuccuuaug uucggucaac
agggacugcu gcagcaccgg 60cuugacuagu acaugaccac uu
821582RNAArtificial SequenceSynthetic aptamer sequence 15gggaagagaa
ggacauauga uuaagcgcac ucaacagggu cuaugauccg cgccgaucau 60guugacuagu
acaugaccac uu 821682RNAArtificial SequenceSynthetic aptamer
sequence 16gggaagagaa ggacauauga uccgcuuucc auugagauua uaagcuguua
gagacuuauu 60uuugacuagu acaugaccac uu 821782RNAArtificial
SequenceSynthetic aptamer sequence 17gggaagagaa ggacauauga
uuuucgaaac guuucuuuca aguucuuaau cauucccauu 60uuugacuagu acaugaccac
uu 821882RNAArtificial SequenceSynthetic aptamer sequence
18gggaagagaa ggacauauga ucauuagaug cgcaguucga agccgguaca gcuggcgcgc
60guugacuagu acaugaccac uu 821982RNAArtificial SequenceSynthetic
aptamer sequence 19gggaagagaa ggacauauga uaaagaauaa ccuuaaaaua
acaccaccgc cucacagcau 60auugacuagu acaugaccac uu
822082RNAArtificial SequenceSynthetic aptamer sequence 20gggaagagaa
ggacauauga uaaauugauc uauucuuuuc ggugcuauuu aucuuccauu 60uuugacuagu
acaugaccac uu 822182RNAArtificial SequenceSynthetic aptamer
sequence 21gggaagagaa ggacauauga ucuacucgcg cggcggacaa aagcgcaacc
cagcacccau 60guugacuagu acaugaccac uu 822282RNAArtificial
SequenceSynthetic aptamer sequence 22gggaagagaa ggacauauga
uucuuaguuu guaauuacuu uuccuuccuu uuauucuauu 60uuugacuagu acaugaccac
uu 822382RNAArtificial SequenceSynthetic aptamer sequence
23gggaagagaa ggacauauga uaacccgcgc agacuuacaa gcgcgcaaaa aaaggguacg
60uuugacuagu acaugaccac uu 822482RNAArtificial SequenceSynthetic
aptamer sequence 24gggaagagaa ggacauauga uauuccuuua ugccgcauca
uuuuauuguu uaugacaauu 60uuugacuagu acaugaccac uu
822582RNAArtificial SequenceSynthetic aptamer sequence 25gggaagagaa
ggacauauga uauuucguac uacuuuucuu ccaagcuuca aucgcccauu 60uuugacuagu
acaugaccac uu 822682RNAArtificial SequenceSynthetic aptamer
sequence 26gggaagagaa ggacauauga uucacucauu cgcaacacaa uuguauucgc
aucugcgauu 60uuugacuagu acaugaccac uu 822782RNAArtificial
SequenceSynthetic aptamer sequence 27gggaagagaa ggacauauga
uauuauuucc acaguuccuu uauccacaca ucuucucauu 60uuugacuagu acaugaccac
uu 822882RNAArtificial SequenceSynthetic aptamer sequence
28gggaagagaa ggacauauga uaaacucguu aucuauucgu uuauuugcau cucuuucauu
60uuugacuagu acaugaccac uu 822982RNAArtificial SequenceSynthetic
aptamer sequence 29gggaagagaa ggacauauga uccaaccucu aaaguacugg
ucgccuaggg agacuguucg 60guugacuagu acaugaccac uu
823082RNAArtificial SequenceSynthetic aptamer sequence 30gggaagagaa
ggacauauga uuuccuuuuu gcuauuuccg uuaauguaaa cucuccuauu 60uuugacuagu
acaugaccac uu 823182RNAArtificial SequenceSynthetic aptamer
sequence 31gggaagagaa ggacauauga uccuuauggc cuaguaggga uccgggcgcc
gaccagcgcg 60auugacuagu acaugaccac uu 823282RNAArtificial
SequenceSynthetic aptamer sequence 32gggaagagaa ggacauauga
ucgucugucu ucuucgaaua cguuuugggc uaagcccauu 60uuugacuagu acaugaccac
uu 823382RNAArtificial SequenceSynthetic aptamer sequence
33gggaagagaa ggacauauga uucaaccaaa cugccgacga ccgagguaug uccuuaugua
60cuugacuagu acaugaccac uu 823482RNAArtificial SequenceSynthetic
aptamer sequence 34gggaagagaa ggacauauga uuacgggucu gagcaaaagc
gaaggaagca ggcgcaggga 60uuugacuagu acaugaccac uu
823582RNAArtificial SequenceSynthetic aptamer sequence 35gggaagagaa
ggacauauga uucucucauu cgcaacacaa uuguauucgc aucugcgauu 60uuugacuagu
acaugaccac uu 823682RNAArtificial SequenceSynthetic aptamer
sequence 36gggaagagaa ggacauauga ugcucuaaag uacuaagcgu uugcgccgau
gcccggaccg 60cuugacuagu acaugaccac uu 823782RNAArtificial
SequenceSynthetic aptamer sequence 37gggaagagaa ggacauauga
uacuucauua augugaggcc gucagggggc aaccuucgag 60cuugacuagu acaugaccac
uu 823882RNAArtificial SequenceSynthetic aptamer sequence
38gggaagagaa ggacauauga uuccuuauuc uuguuacuac uuucuuuucc uauuuuuuuc
60uuugacuagu acaugaccac uu 823982RNAArtificial SequenceSynthetic
aptamer sequence 39gggaagagaa ggacauauga ucguuauuuu cauuuucuug
uuccccauau gcccaggcgc 60auugacuagu acaugaccac uu
824082RNAArtificial SequenceSynthetic aptamer sequence 40gggaagagaa
ggacauauga uaccagcggc guagaaacgu acagcucgcc uguaacgccu 60guugacuagu
acaugaccac uu 824182RNAArtificial SequenceSynthetic aptamer
sequence 41gggaagagaa ggacauauga ucgauauggg ugcgggaaug uacguucacc
gaauaugcuc 60cuugacuagu acaugaccac uu 824282RNAArtificial
SequenceSynthetic aptamer sequence 42gggaagagaa ggacauauga
uuaacagugc guagucauau cgaauguuua ucuuccuauu 60uuugacuagu acaugaccac
uu 824382RNAArtificial SequenceSynthetic aptamer sequence
43gggaagagaa ggacauauga ucagacucuc gcccaauucg caaggcguug cauugcgauu
60uuugacuagu acaugaccac uu 824482RNAArtificial SequenceSynthetic
aptamer sequence 44gggaagagaa ggacauauga uuuccaacuc uccacgagag
caugggucga augacucauu 60uuugacuagu acaugaccac uu
824582RNAArtificial SequenceSynthetic aptamer sequence 45gggaagagaa
ggacauauga ugcaucgcgc gucacucaac ucgugauuac cgagggcgcc 60guugacuagu
acaugaccac uu 824682RNAArtificial SequenceSynthetic aptamer
sequence 46gggaagagaa ggacauauga ucugaaucuu uccgcagccc uguccuuuua
aagacagguu 60uuugacuagu acaugaccac uu 824782RNAArtificial
SequenceSynthetic aptamer sequence 47gggaagagaa ggacauauga
uuuuguuacu uacuucgucu aucuucuguu gcacacaguu 60uuugacuagu acaugaccac
uu 824882RNAArtificial SequenceSynthetic aptamer sequence
48gggaagagaa ggacauauga uucaaaucuu cagcgauaau ggcacaauuu ccgcgccauu
60uuugacuagu acaugaccac uu 824982RNAArtificial SequenceSynthetic
aptamer sequence 49gggaagagaa ggacauauga uuuaugugag augaugugug
uuccuaguuu uaucuugcuc 60uuugacuagu acaugaccac uu
825082RNAArtificial SequenceSynthetic aptamer sequence 50gggaagagaa
ggacauauga uccacuuuuc cauuaacugu ugcgggcaag uagcaccguu 60uuugacuagu
acaugaccac uu 825182RNAArtificial SequenceSynthetic aptamer
sequence 51gggaagagaa ggacauauga uagagaagac cauucggaaa gagcugcgug
uccuuaugua 60cuugacuagu acaugaccac uu 825282RNAArtificial
SequenceSynthetic aptamer sequence 52gggaagagaa ggacauauga
uucuuaugua gcaagcaaaa ugugccgccg agccgacgcc 60auugacuagu acaugaccac
uu 825382RNAArtificial SequenceSynthetic aptamer sequence
53gggaagagaa ggacauauga uaagcgcaua auaagccagc caguucuugg cgcgcggggu
60auugacuagu acaugaccac uu 825482RNAArtificial SequenceSynthetic
aptamer sequence 54gggaagagaa ggacauauga uuaguccgca uuucuauuuu
cuauauggcu uacugccauu 60uuugacuagu acaugaccac uu
825582RNAArtificial SequenceSynthetic aptamer sequence 55gggaagagaa
ggacauauga uauaaagaac acgcaaaacc acccggacac ccggugccgu 60guugacuagu
acaugaccac uu 825682RNAArtificial SequenceSynthetic aptamer
sequence 56gggaagagaa ggacauauga uacacaggcg guggagccga agggcaccgg
gacaaaccga 60cuugacuagu acaugaccac uu 825782RNAArtificial
SequenceSynthetic aptamer sequence 57gggaagagaa ggacauauga
uaguuccggc gcagcagcgu ccucacguuu uacgugcccc 60auugacuagu acaugaccac
uu 825882RNAArtificial SequenceSynthetic aptamer sequence
58gggaagagaa ggacauauga ugaccgucgc gaucguuuau aauguucugg aucuuucauu
60uuugacuagu acaugaccac uu 825982RNAArtificial SequenceSynthetic
aptamer sequence 59gggaagagaa ggacauauga uaaguggggc cccgacgacu
uuuccuuccu cucuuccggc 60auugacuagu acaugaccac uu
826082RNAArtificial SequenceSynthetic aptamer sequence 60gggaagagaa
ggacauauga uaucaacaua ccaaaauguc auuuccaauc uuuucccauu 60uuugacuagu
acaugaccac uu 826182RNAArtificial SequenceSynthetic aptamer
sequence 61gggaagagaa ggacauauga uagcgaacaa acaagggugc ccaggccccc
uucgcacauc 60guugacuagu acaugaccac uu 826282RNAArtificial
SequenceSynthetic aptamer sequence 62gggaagagaa ggacauauga
uccucuguaa cgcaaaguca agucgcgcaa ggccgcccgc 60guugacuagu acaugaccac
uu 826382RNAArtificial SequenceSynthetic aptamer sequence
63gggaagagaa ggacauauga ucuucaucug cgauuacggu acacuuuagu guaucguuuu
60uuugacuagu acaugaccac uu 826482RNAArtificial SequenceSynthetic
aptamer sequence 64gggaagagaa ggacauauga ugccuaugug cuagaugcag
cagcaaccgc cggcgacugg 60auugacuagu acaugaccac uu
826582RNAArtificial SequenceSynthetic aptamer sequence 65gggaagagaa
ggacauauga uccgcgcccu aaccuucuga ccaagcuucc cuggcacuug 60guugacuagu
acaugaccac uu 826682RNAArtificial SequenceSynthetic aptamer
sequence 66gggaagagaa ggacauauga uccuuaugua uuaucaguca uaccggacgc
agcccgcugg 60auugacuagu acaugaccac uu 826782RNAArtificial
SequenceSynthetic aptamer sequence 67gggaagagaa ggacauauga
ucuaaucuau acuggcugcu aacgcuuuuu cuuuuccauu 60uuugacuagu acaugaccac
uu 826882RNAArtificial SequenceSynthetic aptamer sequence
68gggaagagaa ggacauauga ucaguuuacg cggagucguu uguguccauu ucuucucauu
60uuugacuagu acaugaccac uu 826982RNAArtificial SequenceSynthetic
aptamer sequence 69gggaagagaa ggacauauga uucacgugag augaugugug
uuccuaguuu uaucuugcuc 60uuugacuagu acaugaccac uu
827082RNAArtificial SequenceSynthetic aptamer sequence 70gggaagagaa
ggacauauga uuccuugugu accgcuccga augugcucca gcgcgccucg 60guugacuagu
acaugaccac uu 827182RNAArtificial SequenceSynthetic aptamer
sequence 71gggaagagaa ggacauauga uaagccggcc cgggaacaug ucacgcgcgc
gcgcaaagua 60guugacuagu acaugaccac uu 827282RNAArtificial
SequenceSynthetic aptamer sequence 72gggaagagaa ggacauauga
uccuggauuu ccgaaauuag agugccguuu cguuacgguu 60uuugacuagu acaugaccac
uu 827382RNAArtificial SequenceSynthetic aptamer sequence
73gggaagagaa ggacauauga ucgugucauc cgcacaagga ggccugcaug gcagggacac
60guugacuagu acaugaccac uu 827482RNAArtificial SequenceSynthetic
aptamer sequence 74gggaagagaa ggacauauga ugaguagacu uuuuguauca
uuuuuuuauc guaagauauu 60uuugacuagu acaugaccac uu
827582RNAArtificial SequenceSynthetic aptamer sequence 75gggaagagaa
ggacauauga uccaugugag augaugugug uuccuaguuu uaucuugcuc 60uuugacuagu
acaugaccac uu 827682RNAArtificial SequenceSynthetic aptamer
sequence 76gggaagagaa ggacauauga ucuuugcucu agaguguagu cuaugaggga
caagguagcc 60auugacuagu acaugaccac uu 827782RNAArtificial
SequenceSynthetic aptamer sequence 77gggaagagaa ggacauauga
uguugguuuu cuuucucuuu cuuuucuuuc ucuuucuauu 60uuugacuagu acaugaccac
uu 827882RNAArtificial SequenceSynthetic aptamer sequence
78gggaagagaa ggacauauga ucaaucgggc ggggguaaga ggcgugcgca gcguggaggu
60guugacuagu acaugaccac uu 827982RNAArtificial SequenceSynthetic
aptamer sequence 79gggaagagaa ggacauauga ucaccguggu gcgcaaagcc
gcaacgagaa cugcggaauc 60guugacuagu acaugaccac uu
828082RNAArtificial SequenceSynthetic aptamer sequence 80gggaagagaa
ggacauauga uugcuuuaag ucuuuuuauc auuuuguuuc cuucauuuuu 60uuugacuagu
acaugaccac uu 828182RNAArtificial SequenceSynthetic aptamer
sequence 81gggaagagaa ggacauauga ucgacuaguu auacugcaaa ggcuauaagc
gcgagcgcgc 60guugacuagu acaugaccac uu 828282RNAArtificial
SequenceSynthetic aptamer sequence 82gggaagagaa ggacauauga
ugaguaauag auggcguaca caaaucggau acgacgagcg 60cuugacuagu acaugaccac
uu 828382RNAArtificial SequenceSynthetic aptamer sequence
83gggaagagaa ggacauauga uuuucgcuuc aagauuccca acgccuugua agucaagguu
60uuugacuagu acaugaccac uu 828482RNAArtificial SequenceSynthetic
aptamer sequence 84gggaagagaa ggacauauga ugugugagau gagccccugg
accagacgca cgcucgcacu 60guugacuagu
acaugaccac uu 828582RNAArtificial SequenceSynthetic aptamer
sequence 85gggaagagaa ggacauauga ucaggaugcg gcgccgguaa uugacuuccc
ccuacguagg 60auugacuagu acaugaccac uu 828682RNAArtificial
SequenceSynthetic aptamer sequence 86gggaagagaa ggacauauga
ucagggaccc ggccggugca ucuccuucuu uagcguacgc 60cuugacuagu acaugaccac
uu 828782RNAArtificial SequenceSynthetic aptamer sequence
87gggaagagaa ggacauauga ucugcucuaa aguaccaacc gcgggagcua aaugcaagcc
60guugacuagu acaugaccac uu 828882RNAArtificial SequenceSynthetic
aptamer sequence 88gggaagagaa ggacauauga ugauugccau gcauuagggg
gggacgcgcg cgaaagggag 60auugacuagu acaugaccac uu
828982RNAArtificial SequenceSynthetic aptamer sequence 89gggaagagaa
ggacauauga uucgcucuaa aguaccaacc gcgggagcua aaugcaagcc 60guugacuagu
acaugaccac uu 829082RNAArtificial SequenceSynthetic aptamer
sequence 90gggaagagaa ggacauauga uaaaaaaccg ggguucuuaa uuuucauugu
ucgucguacu 60uuugacuagu acaugaccac uu 829182RNAArtificial
SequenceSynthetic aptamer sequence 91gggaagagaa ggacauauga
uaacccauug gugaaucgca accacagcca gcccggcgcg 60auugacuagu acaugaccac
uu 829282RNAArtificial SequenceSynthetic aptamer sequence
92gggaagagaa ggacauauga ucgaagugag gggaucgcgc ggggugcacc uaaauauggg
60auugacuagu acaugaccac uu 829382RNAArtificial SequenceSynthetic
aptamer sequence 93gggaagagaa ggacauauga uagccuuaug uacuauagaa
gucagcuauc cgccgcacaa 60uuugacuagu acaugaccac uu
829482RNAArtificial SequenceSynthetic aptamer sequence 94gggaagagaa
ggacauauga ucguuguuuu ucccaaagcu cguuagcauu cauuccuauu 60uuugacuagu
acaugaccac uu 829582RNAArtificial SequenceSynthetic aptamer
sequence 95gggaagagaa ggacauauga ugaucaucag cggaaagcac gaaacgccac
gggccgcggc 60auugacuagu acaugaccac uu 829682RNAArtificial
SequenceSynthetic aptamer sequence 96gggaagagaa ggacauauga
uuccuuccua uugacaaugc gcccgggccu cuucaauugu 60auugacuagu acaugaccac
uu 829782RNAArtificial SequenceSynthetic aptamer sequence
97gggaagagaa ggacauauga uaguugccgc gcggcgcaag auuggagagu cccgggcugu
60auugacuagu acaugaccac uu 829882RNAArtificial SequenceSynthetic
aptamer sequence 98gggaagagaa ggacauauga ucauaaguuc guucauuccg
uuaacacgcg uauggcguuu 60uuugacuagu acaugaccac uu
829982RNAArtificial SequenceSynthetic aptamer sequence 99gggaagagaa
ggacauauga uccuuugucu ccaaaucuua ggacugaaug agugccuauu 60uuugacuagu
acaugaccac uu 8210082RNAArtificial SequenceSynthetic aptamer
sequence 100gggaagagaa ggacauauga ucuucuuuga gaauucucuu uuuacaauuc
cggcgccgug 60auugacuagu acaugaccac uu 8210182RNAArtificial
SequenceSynthetic aptamer sequence 101gggaagagaa ggacauauga
uuaggcuaac uguuuaggga uuugauaugc augaggagca 60cuugacuagu acaugaccac
uu 8210282RNAArtificial SequenceSynthetic aptamer sequence
102gggaagagaa ggacauauga uuaggcuaac uguucaggga guugauaugc
augaggagca 60cuugacuagu acaugaccac uu 8210382RNAArtificial
SequenceSynthetic aptamer sequence 103gggaagagaa ggacauauga
uuaggcuaac ugcucaggga uuugauaugc augaggagca 60cuugacuagu acaugaccac
uu 8210482RNAArtificial SequenceSynthetic aptamer sequence
104gggaagagaa ggacauauga uuaggcuaac uguucaggga cuugauaugc
augaggagca 60cuugacuagu acaugaccac uu 8210582RNAArtificial
SequenceSynthetic aptamer sequence 105gggaagagaa ggacauauga
uuaggcucac uguucaggga uuugauaugc augaggagca 60cuugacuagu acaugaccac
uu 8210682RNAArtificial SequenceSynthetic aptamer sequence
106gggaagagaa ggacauauga uuaggcuaac uguucaggga uuugauaugc
auggggagca 60cuugacuagu acaugaccac uu 8210782RNAArtificial
SequenceSynthetic aptamer sequence 107gggaagagaa ggacauauga
uuucuuccua uugacgaugc gcccgggccu cuucaauugu 60auugacuagu acaugaccac
uu 8210882RNAArtificial SequenceSynthetic aptamer sequence
108gggaagagaa ggacauauga uuaggcuaac uguucgggga uuugauaugc
augaggagca 60cuugacuagu acaugaccac uu 8210982RNAArtificial
SequenceSynthetic aptamer sequence 109gggaagagaa ggacauauga
uuaggcuaac uguucaggga uuugauaugc acgaggagca 60cuugacuagu acaugaccac
uu 8211082RNAArtificial SequenceSynthetic aptamer sequence
110gggaagagaa ggacauauga uuagguuaac uguucaggga uuugauaugc
augaggagca 60cuugacuagu acaugaccac uu 8211182RNAArtificial
SequenceSynthetic aptamer sequence 111gggaagagaa ggacauauga
uuaggcuaac uguucaggga uuugaugugc augaggagca 60cuugacuagu acaugaccac
uu 8211282DNAArtificial SequenceSynthetic aptamer sequence
112gggaagagaa ggacatatga ttcatgtgag atgatgtgtg ttcctagttt
tatcttgctc 60tttgactagt acatgaccac tt 8211382DNAArtificial
SequenceSynthetic aptamer sequence 113gggaagagaa ggacatatga
ttaggctaac tgttcaggga tttgatatgc atgaggagca 60cttgactagt acatgaccac
tt 8211482DNAArtificial SequenceSynthetic aptamer sequence
114gggaagagaa ggacatatga tccgctctaa agtaccaacc gcgggagcta
aatgcaagcc 60gttgactagt acatgaccac tt 8211582DNAArtificial
SequenceSynthetic aptamer sequence 115gggaagagaa ggacatatga
ttgtgtcagg ctctagagtc tagacggccg gggtcccgga 60tttgactagt acatgaccac
tt 8211682DNAArtificial SequenceSynthetic aptamer sequence
116gggaagagaa ggacatatga tccttatgtc tagcggcctt acgcgattag
tggcgttttg 60tttgactagt acatgaccac tt 8211782DNAArtificial
SequenceSynthetic aptamer sequence 117gggaagagaa ggacatatga
tctttatgta ttatcagtca taccggacgc agcccgctgg 60attgactagt acatgaccac
tt 8211882DNAArtificial SequenceSynthetic aptamer sequence
118gggaagagaa ggacatatga ttgtgttatt acacttcgtg attttccttg
cttttctatt 60tttgactagt acatgaccac tt 8211982DNAArtificial
SequenceSynthetic aptamer sequence 119gggaagagaa ggacatatga
tccaacatct aaagtactgg tcgcctaggg agactgttcg 60gttgactagt acatgaccac
tt 8212082DNAArtificial SequenceSynthetic aptamer sequence
120gggaagagaa ggacatatga tgctatattc gcaaaagcag gctgagtgcg
gcaggcgcgt 60gttgactagt acatgaccac tt 8212182DNAArtificial
SequenceSynthetic aptamer sequence 121gggaagagaa ggacatatga
ttcattcatt cgcaacacaa ttgtattcgc atctgcgatt 60tttgactagt acatgaccac
tt 8212282DNAArtificial SequenceSynthetic aptamer sequence
122gggaagagaa ggacatatga tctttctctt ttctaatatt taatttattg
ggtaccaatt 60tttgactagt acatgaccac tt 8212382DNAArtificial
SequenceSynthetic aptamer sequence 123gggaagagaa ggacatatga
tctttgtttc gcatacgttt tctttttctc tcttcttatt 60tttgactagt acatgaccac
tt 8212482DNAArtificial SequenceSynthetic aptamer sequence
124gggaagagaa ggacatatga ttattctgtt cttcaaaaat cttttagcgt
atacgctatt 60tttgactagt acatgaccac tt 8212582DNAArtificial
SequenceSynthetic aptamer sequence 125gggaagagaa ggacatatga
tttccttatg ttcggtcaac agggactgct gcagcaccgg 60cttgactagt acatgaccac
tt 8212682DNAArtificial SequenceSynthetic aptamer sequence
126gggaagagaa ggacatatga ttaagcgcac tcaacagggt ctatgatccg
cgccgatcat 60gttgactagt acatgaccac tt 8212782DNAArtificial
SequenceSynthetic aptamer sequence 127gggaagagaa ggacatatga
tccgctttcc attgagatta taagctgtta gagacttatt 60tttgactagt acatgaccac
tt 8212882DNAArtificial SequenceSynthetic aptamer sequence
128gggaagagaa ggacatatga ttttcgaaac gtttctttca agttcttaat
cattcccatt 60tttgactagt acatgaccac tt 8212982DNAArtificial
SequenceSynthetic aptamer sequence 129gggaagagaa ggacatatga
tcattagatg cgcagttcga agccggtaca gctggcgcgc 60gttgactagt acatgaccac
tt 8213082DNAArtificial SequenceSynthetic aptamer sequence
130gggaagagaa ggacatatga taaagaataa ccttaaaata acaccaccgc
ctcacagcat 60attgactagt acatgaccac tt 8213182DNAArtificial
SequenceSynthetic aptamer sequence 131gggaagagaa ggacatatga
taaattgatc tattcttttc ggtgctattt atcttccatt 60tttgactagt acatgaccac
tt 8213282DNAArtificial SequenceSynthetic aptamer sequence
132gggaagagaa ggacatatga tctactcgcg cggcggacaa aagcgcaacc
cagcacccat 60gttgactagt acatgaccac tt 8213382DNAArtificial
SequenceSynthetic aptamer sequence 133gggaagagaa ggacatatga
ttcttagttt gtaattactt ttccttcctt ttattctatt 60tttgactagt acatgaccac
tt 8213482DNAArtificial SequenceSynthetic aptamer sequence
134gggaagagaa ggacatatga taacccgcgc agacttacaa gcgcgcaaaa
aaagggtacg 60tttgactagt acatgaccac tt 8213582DNAArtificial
SequenceSynthetic aptamer sequence 135gggaagagaa ggacatatga
tattccttta tgccgcatca ttttattgtt tatgacaatt 60tttgactagt acatgaccac
tt 8213682DNAArtificial SequenceSynthetic aptamer sequence
136gggaagagaa ggacatatga tatttcgtac tacttttctt ccaagcttca
atcgcccatt 60tttgactagt acatgaccac tt 8213782DNAArtificial
SequenceSynthetic aptamer sequence 137gggaagagaa ggacatatga
ttcactcatt cgcaacacaa ttgtattcgc atctgcgatt 60tttgactagt acatgaccac
tt 8213882DNAArtificial SequenceSynthetic aptamer sequence
138gggaagagaa ggacatatga tattatttcc acagttcctt tatccacaca
tcttctcatt 60tttgactagt acatgaccac tt 8213982DNAArtificial
SequenceSynthetic aptamer sequence 139gggaagagaa ggacatatga
taaactcgtt atctattcgt ttatttgcat ctctttcatt 60tttgactagt acatgaccac
tt 8214082DNAArtificial SequenceSynthetic aptamer sequence
140gggaagagaa ggacatatga tccaacctct aaagtactgg tcgcctaggg
agactgttcg 60gttgactagt acatgaccac tt 8214182DNAArtificial
SequenceSynthetic aptamer sequence 141gggaagagaa ggacatatga
tttccttttt gctatttccg ttaatgtaaa ctctcctatt 60tttgactagt acatgaccac
tt 8214282DNAArtificial SequenceSynthetic aptamer sequence
142gggaagagaa ggacatatga tccttatggc ctagtaggga tccgggcgcc
gaccagcgcg 60attgactagt acatgaccac tt 8214382DNAArtificial
SequenceSynthetic aptamer sequence 143gggaagagaa ggacatatga
tcgtctgtct tcttcgaata cgttttgggc taagcccatt 60tttgactagt acatgaccac
tt 8214482DNAArtificial SequenceSynthetic aptamer sequence
144gggaagagaa ggacatatga ttcaaccaaa ctgccgacga ccgaggtatg
tccttatgta 60cttgactagt acatgaccac tt 8214582DNAArtificial
SequenceSynthetic aptamer sequence 145gggaagagaa ggacatatga
ttacgggtct gagcaaaagc gaaggaagca ggcgcaggga 60tttgactagt acatgaccac
tt 8214682DNAArtificial SequenceSynthetic aptamer sequence
146gggaagagaa ggacatatga ttctctcatt cgcaacacaa ttgtattcgc
atctgcgatt 60tttgactagt acatgaccac tt 8214782DNAArtificial
SequenceSynthetic aptamer sequence 147gggaagagaa ggacatatga
tgctctaaag tactaagcgt ttgcgccgat gcccggaccg 60cttgactagt acatgaccac
tt 8214882DNAArtificial SequenceSynthetic aptamer sequence
148gggaagagaa ggacatatga tacttcatta atgtgaggcc gtcagggggc
aaccttcgag 60cttgactagt acatgaccac tt 8214982DNAArtificial
SequenceSynthetic aptamer sequence 149gggaagagaa ggacatatga
ttccttattc ttgttactac tttcttttcc tatttttttc 60tttgactagt acatgaccac
tt 8215082DNAArtificial SequenceSynthetic aptamer sequence
150gggaagagaa ggacatatga tcgttatttt cattttcttg ttccccatat
gcccaggcgc 60attgactagt acatgaccac tt 8215182DNAArtificial
SequenceSynthetic aptamer sequence 151gggaagagaa ggacatatga
taccagcggc gtagaaacgt acagctcgcc tgtaacgcct 60gttgactagt acatgaccac
tt 8215282DNAArtificial SequenceSynthetic aptamer sequence
152gggaagagaa ggacatatga tcgatatggg tgcgggaatg tacgttcacc
gaatatgctc 60cttgactagt acatgaccac tt 8215382DNAArtificial
SequenceSynthetic aptamer sequence 153gggaagagaa ggacatatga
ttaacagtgc gtagtcatat cgaatgttta tcttcctatt 60tttgactagt acatgaccac
tt 8215482DNAArtificial SequenceSynthetic aptamer sequence
154gggaagagaa ggacatatga tcagactctc gcccaattcg caaggcgttg
cattgcgatt 60tttgactagt acatgaccac tt 8215582DNAArtificial
SequenceSynthetic aptamer sequence 155gggaagagaa ggacatatga
tttccaactc tccacgagag catgggtcga atgactcatt 60tttgactagt acatgaccac
tt 8215682DNAArtificial SequenceSynthetic aptamer sequence
156gggaagagaa ggacatatga tgcatcgcgc gtcactcaac tcgtgattac
cgagggcgcc 60gttgactagt acatgaccac tt 8215782DNAArtificial
SequenceSynthetic aptamer sequence 157gggaagagaa ggacatatga
tctgaatctt tccgcagccc tgtcctttta aagacaggtt 60tttgactagt acatgaccac
tt 8215882DNAArtificial SequenceSynthetic aptamer sequence
158gggaagagaa ggacatatga ttttgttact tacttcgtct atcttctgtt
gcacacagtt 60tttgactagt acatgaccac tt 8215982DNAArtificial
SequenceSynthetic aptamer sequence 159gggaagagaa ggacatatga
ttcaaatctt cagcgataat ggcacaattt ccgcgccatt 60tttgactagt acatgaccac
tt 8216082DNAArtificial SequenceSynthetic aptamer sequence
160gggaagagaa ggacatatga tttatgtgag atgatgtgtg ttcctagttt
tatcttgctc 60tttgactagt acatgaccac tt 8216182DNAArtificial
SequenceSynthetic aptamer sequence 161gggaagagaa ggacatatga
tccacttttc cattaactgt tgcgggcaag tagcaccgtt 60tttgactagt acatgaccac
tt 8216282DNAArtificial SequenceSynthetic aptamer sequence
162gggaagagaa ggacatatga tagagaagac cattcggaaa gagctgcgtg
tccttatgta 60cttgactagt acatgaccac tt 8216382DNAArtificial
SequenceSynthetic aptamer sequence 163gggaagagaa ggacatatga
ttcttatgta gcaagcaaaa tgtgccgccg agccgacgcc 60attgactagt acatgaccac
tt 8216482DNAArtificial SequenceSynthetic aptamer sequence
164gggaagagaa ggacatatga taagcgcata ataagccagc cagttcttgg
cgcgcggggt 60attgactagt acatgaccac tt 8216582DNAArtificial
SequenceSynthetic aptamer sequence 165gggaagagaa ggacatatga
ttagtccgca tttctatttt ctatatggct tactgccatt 60tttgactagt acatgaccac
tt 8216682DNAArtificial SequenceSynthetic aptamer sequence
166gggaagagaa ggacatatga tataaagaac acgcaaaacc acccggacac
ccggtgccgt 60gttgactagt acatgaccac tt 8216782DNAArtificial
SequenceSynthetic aptamer sequence 167gggaagagaa ggacatatga
tacacaggcg gtggagccga agggcaccgg gacaaaccga 60cttgactagt acatgaccac
tt 8216882DNAArtificial SequenceSynthetic aptamer sequence
168gggaagagaa
ggacatatga tagttccggc gcagcagcgt cctcacgttt tacgtgcccc 60attgactagt
acatgaccac tt 8216982DNAArtificial SequenceSynthetic aptamer
sequence 169gggaagagaa ggacatatga tgaccgtcgc gatcgtttat aatgttctgg
atctttcatt 60tttgactagt acatgaccac tt 8217082DNAArtificial
SequenceSynthetic aptamer sequence 170gggaagagaa ggacatatga
taagtggggc cccgacgact tttccttcct ctcttccggc 60attgactagt acatgaccac
tt 8217182DNAArtificial SequenceSynthetic aptamer sequence
171gggaagagaa ggacatatga tatcaacata ccaaaatgtc atttccaatc
ttttcccatt 60tttgactagt acatgaccac tt 8217282DNAArtificial
SequenceSynthetic aptamer sequence 172gggaagagaa ggacatatga
tagcgaacaa acaagggtgc ccaggccccc ttcgcacatc 60gttgactagt acatgaccac
tt 8217382DNAArtificial SequenceSynthetic aptamer sequence
173gggaagagaa ggacatatga tcctctgtaa cgcaaagtca agtcgcgcaa
ggccgcccgc 60gttgactagt acatgaccac tt 8217482DNAArtificial
SequenceSynthetic aptamer sequence 174gggaagagaa ggacatatga
tcttcatctg cgattacggt acactttagt gtatcgtttt 60tttgactagt acatgaccac
tt 8217582DNAArtificial SequenceSynthetic aptamer sequence
175gggaagagaa ggacatatga tgcctatgtg ctagatgcag cagcaaccgc
cggcgactgg 60attgactagt acatgaccac tt 8217682DNAArtificial
SequenceSynthetic aptamer sequence 176gggaagagaa ggacatatga
tccgcgccct aaccttctga ccaagcttcc ctggcacttg 60gttgactagt acatgaccac
tt 8217782DNAArtificial SequenceSynthetic aptamer sequence
177gggaagagaa ggacatatga tccttatgta ttatcagtca taccggacgc
agcccgctgg 60attgactagt acatgaccac tt 8217882DNAArtificial
SequenceSynthetic aptamer sequence 178gggaagagaa ggacatatga
tctaatctat actggctgct aacgcttttt cttttccatt 60tttgactagt acatgaccac
tt 8217982DNAArtificial SequenceSynthetic aptamer sequence
179gggaagagaa ggacatatga tcagtttacg cggagtcgtt tgtgtccatt
tcttctcatt 60tttgactagt acatgaccac tt 8218082DNAArtificial
SequenceSynthetic aptamer sequence 180gggaagagaa ggacatatga
ttcacgtgag atgatgtgtg ttcctagttt tatcttgctc 60tttgactagt acatgaccac
tt 8218182DNAArtificial SequenceSynthetic aptamer sequence
181gggaagagaa ggacatatga ttccttgtgt accgctccga atgtgctcca
gcgcgcctcg 60gttgactagt acatgaccac tt 8218282DNAArtificial
SequenceSynthetic aptamer sequence 182gggaagagaa ggacatatga
taagccggcc cgggaacatg tcacgcgcgc gcgcaaagta 60gttgactagt acatgaccac
tt 8218382DNAArtificial SequenceSynthetic aptamer sequence
183gggaagagaa ggacatatga tcctggattt ccgaaattag agtgccgttt
cgttacggtt 60tttgactagt acatgaccac tt 8218482DNAArtificial
SequenceSynthetic aptamer sequence 184gggaagagaa ggacatatga
tcgtgtcatc cgcacaagga ggcctgcatg gcagggacac 60gttgactagt acatgaccac
tt 8218582DNAArtificial SequenceSynthetic aptamer sequence
185gggaagagaa ggacatatga tgagtagact ttttgtatca tttttttatc
gtaagatatt 60tttgactagt acatgaccac tt 8218682DNAArtificial
SequenceSynthetic aptamer sequence 186gggaagagaa ggacatatga
tccatgtgag atgatgtgtg ttcctagttt tatcttgctc 60tttgactagt acatgaccac
tt 8218782DNAArtificial SequenceSynthetic aptamer sequence
187gggaagagaa ggacatatga tctttgctct agagtgtagt ctatgaggga
caaggtagcc 60attgactagt acatgaccac tt 8218882DNAArtificial
SequenceSynthetic aptamer sequence 188gggaagagaa ggacatatga
tgttggtttt ctttctcttt cttttctttc tctttctatt 60tttgactagt acatgaccac
tt 8218982DNAArtificial SequenceSynthetic aptamer sequence
189gggaagagaa ggacatatga tcaatcgggc gggggtaaga ggcgtgcgca
gcgtggaggt 60gttgactagt acatgaccac tt 8219082DNAArtificial
SequenceSynthetic aptamer sequence 190gggaagagaa ggacatatga
tcaccgtggt gcgcaaagcc gcaacgagaa ctgcggaatc 60gttgactagt acatgaccac
tt 8219182DNAArtificial SequenceSynthetic aptamer sequence
191gggaagagaa ggacatatga ttgctttaag tctttttatc attttgtttc
cttcattttt 60tttgactagt acatgaccac tt 8219282DNAArtificial
SequenceSynthetic aptamer sequence 192gggaagagaa ggacatatga
tcgactagtt atactgcaaa ggctataagc gcgagcgcgc 60gttgactagt acatgaccac
tt 8219382DNAArtificial SequenceSynthetic aptamer sequence
193gggaagagaa ggacatatga tgagtaatag atggcgtaca caaatcggat
acgacgagcg 60cttgactagt acatgaccac tt 8219482DNAArtificial
SequenceSynthetic aptamer sequence 194gggaagagaa ggacatatga
ttttcgcttc aagattccca acgccttgta agtcaaggtt 60tttgactagt acatgaccac
tt 8219582DNAArtificial SequenceSynthetic aptamer sequence
195gggaagagaa ggacatatga tgtgtgagat gagcccctgg accagacgca
cgctcgcact 60gttgactagt acatgaccac tt 8219682DNAArtificial
SequenceSynthetic aptamer sequence 196gggaagagaa ggacatatga
tcaggatgcg gcgccggtaa ttgacttccc cctacgtagg 60attgactagt acatgaccac
tt 8219782DNAArtificial SequenceSynthetic aptamer sequence
197gggaagagaa ggacatatga tcagggaccc ggccggtgca tctccttctt
tagcgtacgc 60cttgactagt acatgaccac tt 8219882DNAArtificial
SequenceSynthetic aptamer sequence 198gggaagagaa ggacatatga
tctgctctaa agtaccaacc gcgggagcta aatgcaagcc 60gttgactagt acatgaccac
tt 8219982DNAArtificial SequenceSynthetic aptamer sequence
199gggaagagaa ggacatatga tgattgccat gcattagggg gggacgcgcg
cgaaagggag 60attgactagt acatgaccac tt 8220082DNAArtificial
SequenceSynthetic aptamer sequence 200gggaagagaa ggacatatga
ttcgctctaa agtaccaacc gcgggagcta aatgcaagcc 60gttgactagt acatgaccac
tt 8220182DNAArtificial SequenceSynthetic aptamer sequence
201gggaagagaa ggacatatga taaaaaaccg gggttcttaa ttttcattgt
tcgtcgtact 60tttgactagt acatgaccac tt 8220282DNAArtificial
SequenceSynthetic aptamer sequence 202gggaagagaa ggacatatga
taacccattg gtgaatcgca accacagcca gcccggcgcg 60attgactagt acatgaccac
tt 8220382DNAArtificial SequenceSynthetic aptamer sequence
203gggaagagaa ggacatatga tcgaagtgag gggatcgcgc ggggtgcacc
taaatatggg 60attgactagt acatgaccac tt 8220482DNAArtificial
SequenceSynthetic aptamer sequence 204gggaagagaa ggacatatga
tagccttatg tactatagaa gtcagctatc cgccgcacaa 60tttgactagt acatgaccac
tt 8220582DNAArtificial SequenceSynthetic aptamer sequence
205gggaagagaa ggacatatga tcgttgtttt tcccaaagct cgttagcatt
cattcctatt 60tttgactagt acatgaccac tt 8220682DNAArtificial
SequenceSynthetic aptamer sequence 206gggaagagaa ggacatatga
tgatcatcag cggaaagcac gaaacgccac gggccgcggc 60attgactagt acatgaccac
tt 8220782DNAArtificial SequenceSynthetic aptamer sequence
207gggaagagaa ggacatatga ttccttccta ttgacaatgc gcccgggcct
cttcaattgt 60attgactagt acatgaccac tt 8220882DNAArtificial
SequenceSynthetic aptamer sequence 208gggaagagaa ggacatatga
tagttgccgc gcggcgcaag attggagagt cccgggctgt 60attgactagt acatgaccac
tt 8220982DNAArtificial SequenceSynthetic aptamer sequence
209gggaagagaa ggacatatga tcataagttc gttcattccg ttaacacgcg
tatggcgttt 60tttgactagt acatgaccac tt 8221082DNAArtificial
SequenceSynthetic aptamer sequence 210gggaagagaa ggacatatga
tcctttgtct ccaaatctta ggactgaatg agtgcctatt 60tttgactagt acatgaccac
tt 8221182DNAArtificial SequenceSynthetic aptamer sequence
211gggaagagaa ggacatatga tcttctttga gaattctctt tttacaattc
cggcgccgtg 60attgactagt acatgaccac tt 8221282DNAArtificial
SequenceSynthetic aptamer sequence 212gggaagagaa ggacatatga
ttaggctaac tgtttaggga tttgatatgc atgaggagca 60cttgactagt acatgaccac
tt 8221382DNAArtificial SequenceSynthetic aptamer sequence
213gggaagagaa ggacatatga tcgtctgtct tcttcgaata cgttttgggc
taagcccatt 60tttgactagt acatgaccac tt 8221482DNAArtificial
SequenceSynthetic aptamer sequence 214gggaagagaa ggacatatga
ttaggctaac tgctcaggga tttgatatgc atgaggagca 60cttgactagt acatgaccac
tt 8221582DNAArtificial SequenceSynthetic aptamer sequence
215gggaagagaa ggacatatga ttaggctaac tgttcaggga cttgatatgc
atgaggagca 60cttgactagt acatgaccac tt 8221682DNAArtificial
SequenceSynthetic aptamer sequence 216gggaagagaa ggacatatga
ttaggctcac tgttcaggga tttgatatgc atgaggagca 60cttgactagt acatgaccac
tt 8221782DNAArtificial SequenceSynthetic aptamer sequence
217gggaagagaa ggacatatga ttaggctaac tgttcaggga tttgatatgc
atggggagca 60cttgactagt acatgaccac tt 8221882DNAArtificial
SequenceSynthetic aptamer sequence 218gggaagagaa ggacatatga
tttcttccta ttgacgatgc gcccgggcct cttcaattgt 60attgactagt acatgaccac
tt 8221982DNAArtificial SequenceSynthetic aptamer sequence
219gggaagagaa ggacatatga ttaggctaac tgttcgggga tttgatatgc
atgaggagca 60cttgactagt acatgaccac tt 8222082DNAArtificial
SequenceSynthetic aptamer sequence 220gggaagagaa ggacatatga
ttaggctaac tgttcaggga tttgatatgc acgaggagca 60cttgactagt acatgaccac
tt 8222182DNAArtificial SequenceSynthetic aptamer sequence
221gggaagagaa ggacatatga ttaggttaac tgttcaggga tttgatatgc
atgaggagca 60cttgactagt acatgaccac tt 8222282DNAArtificial
SequenceSynthetic aptamer sequence 222gggaagagaa ggacatatga
ttaggctaac tgttcaggga tttgatgtgc atgaggagca 60cttgactagt acatgaccac
tt 8222350DNAArtificial SequenceSynthetic aptamer sequence
223atgtgtgttc ctagttttat cttgctcttt gactagtaca tgaccacttg
5022431DNAArtificial SequenceSynthetic aptamer sequence
224ttcctagttt tatcttgctc tttgactagt a 3122521DNAArtificial
SequenceSynthetic aptamer sequence 225ggacatatga ttcatgtgag a
2122625DNAArtificial SequenceSynthetic aptamer sequence
226ttcgcaaaag caggctgagt gcggc 2522729DNAArtificial
SequenceSynthetic aptamer sequence 227caggcgcgtg ttgactagta
catgaccac 2922844DNAArtificial SequenceSynthetic aptamer sequence
228gggaagagaa ggacatatga tatttcgtac tacttttctt ccaa
4422950RNAArtificial SequenceSynthetic aptamer sequence
229auguguguuc cuaguuuuau cuugcucuuu gacuaguaca ugaccacuug
5023031RNAArtificial SequenceSynthetic aptamer sequence
230uuccuaguuu uaucuugcuc uuugacuagu a 3123121RNAArtificial
SequenceSynthetic aptamer sequence 231ggacauauga uucaugugag a
2123225RNAArtificial SequenceSynthetic aptamer sequence
232uucgcaaaag caggcugagu gcggc 2523329RNAArtificial
SequenceSynthetic aptamer sequence 233caggcgcgug uugacuagua
caugaccac 2923444RNAArtificial SequenceSynthetic aptamer sequence
234gggaagagaa ggacauauga uauuucguac uacuuuucuu ccaa
4423516RNAArtificial SequenceSynthetic aptamer
sequencemisc_feature(7)..(8)n is a, c, g, or u 235uuccuannuu uaucuu
1623616DNAArtificial SequenceSynthetic aptamer
sequencemisc_feature(7)..(8)n is a, c, g, or t 236ttcctanntt tatctt
1623710RNAArtificial SequenceSynthetic aptamer
sequencemisc_feature(7)..(7)n is a, c, g, or u 237uucgcanaag
1023814RNAArtificial SequenceSynthetic aptamer
sequencemisc_feature(8)..(8)n is a, c, g, or u 238ugcggcangc gcgu
1423910DNAArtificial SequenceSynthetic aptamer
sequencemisc_feature(7)..(7)n is a, c, g, or t 239ttcgcanaag
1024014DNAArtificial SequenceSynthetic aptamer
sequencemisc_feature(8)..(8)n is a, c, g, or t 240tgcggcangc gcgt
1424110RNAArtificial SequenceSynthetic aptamer sequence
241cuuuucuucc 1024210DNAArtificial SequenceSynthetic aptamer
sequence 242cttttcttcc 1024310RNAArtificial SequenceSynthetic
aptamer sequencemisc_feature(1)..(1)n is a, c, g, or
umisc_feature(10)..(10)n is a, c, g, or u 243ngcgcgcgcn
1024410DNAArtificial SequenceSynthetic aptamer
sequencemisc_feature(1)..(1)n is a, c, g, or
tmisc_feature(10)..(10)n is a, c, g, or t 244ngcgcgcgcn 10
* * * * *
References