U.S. patent number 8,703,734 [Application Number 13/732,029] was granted by the patent office on 2014-04-22 for nanoprobes for detection or modification of molecules.
This patent grant is currently assigned to N/A, The United States of America, as represented by the Secretary, Department of Health and Human Services. The grantee listed for this patent is N/A, The United States of America, as represented by the Secretary, Department of Health and Human Services, The United States of America, as represented by the Secretary, Department of Health and Human Services. Invention is credited to Ilya G. Lyakhov, Danielle Needle, Thomas D. Schneider.
United States Patent |
8,703,734 |
Lyakhov , et al. |
April 22, 2014 |
Nanoprobes for detection or modification of molecules
Abstract
The disclosure provides probes for one or more target molecules.
In particular examples, the probes include a molecular linker and
first and second functional groups linked and spaced by the
molecular linker, wherein the functional groups are capable of
interacting with one another or with the target biomolecule in a
predetermined reaction, and wherein the molecular linker maintains
the first and second functional groups sufficiently spaced from one
another such that the functional groups do not substantially
interact in an absence of the target biomolecule. In the presence
of the target biomolecule the functional groups interact (with each
other, with the target biomolecule, or both), and in some examples
a detectable signal is produced. In some examples, the functional
groups can detect or modify a target molecule. Also provided are
methods of using the probes, for example to detect or modify a
target molecule.
Inventors: |
Lyakhov; Ilya G. (Frederick,
MD), Schneider; Thomas D. (Frederick, MD), Needle;
Danielle (Frederick, MD) |
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Services
N/A |
Washington |
DC |
US |
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Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Services (Washington, DC)
N/A (N/A)
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Family
ID: |
48281008 |
Appl.
No.: |
13/732,029 |
Filed: |
December 31, 2012 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20130122502 A1 |
May 16, 2013 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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11638160 |
Dec 12, 2006 |
8344121 |
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60749729 |
Dec 12, 2005 |
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60749858 |
Dec 12, 2005 |
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Current U.S.
Class: |
514/44R; 436/94;
435/6.1 |
Current CPC
Class: |
G01N
33/542 (20130101); G01N 21/6486 (20130101); C12Q
1/6818 (20130101); C12Q 1/6869 (20130101); C12Q
1/6818 (20130101); C12Q 2563/107 (20130101); C12Q
2525/197 (20130101); C12Q 2521/30 (20130101); C12Q
1/6869 (20130101); C12Q 2565/101 (20130101); C12Q
2535/107 (20130101); C12Q 2525/197 (20130101); Y10T
436/143333 (20150115) |
Current International
Class: |
A61K
48/00 (20060101); G01N 33/48 (20060101); C07H
21/02 (20060101); C07H 21/04 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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1 226 436 |
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Apr 2005 |
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EP |
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WO 97/30366 |
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Aug 1997 |
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WO |
|
WO 97/40191 |
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Oct 1997 |
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WO |
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WO 98/33939 |
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Aug 1998 |
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WO |
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WO 98/40477 |
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Sep 1998 |
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WO |
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WO 99/05315 |
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Feb 1999 |
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WO |
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WO 00/53805 |
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Sep 2000 |
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WO |
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WO 00/70073 |
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Nov 2000 |
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WO |
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WO 01/16375 |
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Mar 2001 |
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WO |
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WO 02/04680 |
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Jan 2002 |
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WO |
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WO 02/090987 |
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Nov 2002 |
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WO |
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WO 2004/074503 |
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Sep 2004 |
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WO |
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WO 2005/077065 |
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Aug 2005 |
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WO |
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Other References
Adair, "Colloidal Lessons Learned for Dispersion of Nanosize
Particulate Suspensions," Lessons in Nanotechnology from
Traditional and Advanced Ceramics, pp. 93-94, 2005. cited by
applicant .
Allen et al., "Resonance Energy Transfer Measurements Between
Substrate Binding Sites Within the Large (Klenow) Fragment of
Escherichia coli DNA Polymerase I," Biochem. 28:9586-9593 (1989).
cited by applicant .
Ason et al., "A Model for Escherichia coli DNA Polymerase III
Holoenzyme Assembly at Primer/Template Ends," J. Biol. Chem.
275:3006-3015 (2000). cited by applicant .
Baubet et al., "Chimeric Green Fluorescent Protein-Aequorin as
Bioluminiscent Ca.sup.2+ Reporters at the Single-Cell Level," Proc.
Natl. Acad. Sci. USA 97:7260-7265 (2000). cited by applicant .
Beaucage et al., "Persistence Length of Isotactic Poly(hydroxy
butyrate)," Macromolecules 30:4158-4162 (1997). cited by applicant
.
Bentley, "Whole-Genome Re-Sequencing," Curr. Opin. Genetics. Dev.
16:545-552, 2006. cited by applicant .
Blessing et al., "Different Strategies for Formation of Pegylated
EGF-Conjugated PEI/DNA Complexes for Targeted Gene Delivery,"
Bioconjugate Chem. 12:529-537 (2001). cited by applicant .
Blossey and Carlon, "Reparametrizing the Loop Entropy Weights:
Effect on DNA Melting Curves," Phys. Rev. E68:061911 (2003). cited
by applicant .
Braslaysky et al., "Sequence Information can be Obtained from
Single DNA Molecules," Proc. Natl. Acad. Sci. 100:3960-3964 (2003).
cited by applicant .
Burns et al., "Studies in Fluorescence Histochemistry. X. Optimum
Conditions of the Acetic Anhydride-Salicylhydrazide-Zinc (or
Fluorescent Ketone) Technique for Demonstrating C-Terminal Carboxyl
Groups of Proteins," Histochemie 26:279-288 (1971). cited by
applicant .
Clegg, "Fluorescence Resonance Energy Transfer and Nucleic Acids,"
Methods in Enzymol. 211:353-388 (1992). cited by applicant .
Delagrave et al., "Red-Shifted Excitation Mutants of the Green
Fluorescent Protein," Bio/Tech. 13:151-154 (1995). cited by
applicant .
Ehrig et al., "Green-Fluorescent Protein Mutants with Altered
Fluorescence Excitation Spectra," FEBS Lett. 367:163-166 (1995).
cited by applicant .
Fang and Tan, maging Single Fluorescent Molecules at the Interface
of an Optical Fiber Probe by Evanescent Wave Excitation, Anal.
Chem. 71:3101-3105 (1999). cited by applicant .
Funatsu et al., "Imaging of Single Fluorescent Molecules and
Individual ATP Turnovers by Single Myosin Molecules in Aqueous
Solution," Nature 374:555-559 (1995). cited by applicant .
Furey et al., "Use of Fluorescence Resonance Energy Transfer to
Investigate the Conformation of DNA Substrates Bound to the Klenow
Fragment," Biochemistry 37:2979-2990 (1998). cited by applicant
.
Gordon et al., "Quantitative Fluorescence Resonance Energy Transfer
Measurements Using Fluorescence Microscopy," Biophys J.
74:2702-2313 (1998). cited by applicant .
Grant et al., "Development of Dual Receptor Biosensors: An Analysis
of FRET Pairs," Biosens. Bioelectron. 16:231-237 (2001). cited by
applicant .
Grant et al., "Viability of a FRET Dual Binding Technique to Detect
Calpastatin," Biosens. Bioelectron. 21:438-444 (2005). cited by
applicant .
Ha et al., Probing the Interaction Between Two Single Molecules:
Fluorescence Resonance Energy Transfer Between a Single Donor and a
Single Acceptor, Proc. Natl. Acad. Sci. USA 93:6264-6268 (1996).
cited by applicant .
Halpin and Harbury, "DNA Display II. Genetic Manipulation of
Combinatorial Chemistry Libraries for Small-Molecule Evolution,"
PLOS Biol. 2:1022-1030 (2004). cited by applicant .
Harada et al., "Mechanochemical Coupling in Actomyosin Energy
Transduction Studied by in Vitro Movement Assay," J. Mol. Biol.
216:49-68 (1990). cited by applicant .
Harms et al., "Single-Molecule Anisotrophy Imaging," Biophys. J.
77:2864-2870 (1999). cited by applicant .
Hayashi et al., "A Single Expression System for the Display,
Purification and Conjugation of Single-Chain Antibodies," Gene
160:129-130 (1995). cited by applicant .
Hengen et al., "Molecular Flip-Flops Formed by Overlapping Fis
Sites," Nucl. Acid. Res. 31:6663-6673 (2003). cited by applicant
.
Heyduk and Heyduk, "Architecture of a Complex Between the
.sigma..sup.70 Subunit of Escherichia coli RNA Polymerase and the
Nontemplate Strand Oligonucleotide," J. Biol. Chem. 274:3315-3322
(1999). cited by applicant .
Heyduk and Heyduk, "Thiol-Reactive, Luminescent Europium Chelates:
Luminescence Probes for Resonance Energy Transfer Distance
Measurements in Biomolecules," Anal. Biochem. 248:216-227 (1997).
cited by applicant .
Hung et al., "Cyanine Dyes with High Absorption Cross Section as
Donor Chromophores in Energy Transfer Primers," Anal. Biochem.
243:15-27 (1996). cited by applicant .
Inouye and Tsuji, "Aequorea Green Fluorescent Protein. Expression
of the Gene and Fluorescence Characteristics of the Recombinant
Protein," FEBS Lett. 341:277-280 (1994). cited by applicant .
Itakura et al., "Force-Generating Domain of Myosin Motor," Biochem.
Biophys. Res.Comm. 196:1504-1510 (1993). cited by applicant .
Johnson et al., "Amino-Terminal Dimerization of an Erythropoietin
Mimetic Peptide Results in Increased Erythropoietic Activity,"
Chem. Biol. 4:939-950, 1997. cited by applicant .
Kaku et al., "Binding to the Naturally Occurring Double p53 Binding
Site of the Mdm2 Promoter Alleviates the Requirement for p53
C-Terminal Activation," Nucleic Acids Res. 29:1989-1993 (2001).
cited by applicant .
Karger et al., "Multiwavelength Fluorescence Detection for DNA
Sequencing Using Capillary Electrophoresis," Nucleic Acids Res.
19:4955-4962 (1991). cited by applicant .
Kheterpal and Mathies, "Capillary Array Electophoresis DNA
Sequencing," Analy. Chem. News & Features, pp. 31A-37A (1999).
cited by applicant .
Khidekel et al., "A Chemoenzymatic Approach Toward the Rapid and
Sensitive Detection of O-GlcNAc Posttranslational Modifications,"
J. Am. Chem. Soc. 125:16162-16163 (2003). cited by applicant .
Kitamura et al., "A Single Myosin Head Moves Along an Actin
Filament with Regular Steps of 5.3 Nanometres," Nature 397:129-134
(1999). cited by applicant .
Knoll and Heyduk, "Unimolecular Beacons for Detection of DNA
Binding Proteins," Anal. Chem. 76:1156-1164 (2004). cited by
applicant .
Ko and Grant, "A Novel FRET-Based Optical Fiber Biosensor for Rapid
Detection of Salmonella typhimurium," Biosens. Bioelectron.
21:1283-1290 (2006). cited by applicant .
Korlach et al., "Spontaneous Nucleotide Exchange in Low Molecular
Weight GTPases by Fluorescently Labeled .gamma.-Phosphate-Linked
GTP Analogs," Proc Natl. Acad. Sci. USA 101:2800-2805 (2004). cited
by applicant .
Kozlov et al., "Efficient Strategies for the Conjugation of
Oligonucleotides to Antibodies Enabling Highly Sensitive Protein
Detection," Biopolymers 73:621-630 (2004). cited by applicant .
Kumar et al., "Silanized Nucleic Acids: A General Platform for DNA
Immobilization," Nucleic Acids Res. 28:e71 (2000). cited by
applicant .
Lemon and Grossman, "Localization of Bacterial DNA Polymerase:
Evidence for a Factory Model of Replication," Science 282:1516-1519
(1998). cited by applicant .
Marko and Siggia, "Stretching DNA," Macromolecules 28:8759-8770
(1995). cited by applicant .
Marras et al., "Multiplex Detection of Single-Nucleotide Variations
Using Molecular Beacons," Genetic Analysis: Biomolecular
Engineering 14:151-156 (1999). cited by applicant .
Mazzola and Fodor, "Imaging Biomolecule Arrays by Atomic Force
Microscopy," Biophys. J. 68:1653-1660 (1995). cited by applicant
.
Minor and Kulesz-Martin, "DNA Binding Specificity of Proteins
Derived from Alternatively Spliced Mouse p53 mRNAs," Nucleic Acids
Res. 25:1319-1326 (1997). cited by applicant .
Mitra et al., "Fluorescence Resonance Energy Transfer Between
Blue-Emitting and Red-Shifted Excitation Derivatives of the Green
Fluorescent Protein," Gene 173:13-17 (1996). cited by applicant
.
Muller, et al., "A Strategy for the Chemical Synthesis of
Nanostructures," Science 268:272-273 (1995). cited by applicant
.
Nakatani et al., "Highly Sensitive Detection of GG Mismatchd DNA by
Surfaces Immobilized Naphthyridine Dimer Through Poly(ethylene
oxide) Linkers," Bioorganic Med. Chem. Lett. 14:1105-1108 (2004).
cited by applicant .
Ng and Bergstrom, "Protein-DNA Footprinting by Endcapped Duplex
Oligodexyribonucleotides," Nucleic Acids Res. 32:e107 (2004). cited
by applicant .
Niemeyer et al., "Oligonucleotide-Directed Self-Assembly of
Proteins: Semisynthetic DNA--Streptavidin Hybrid Molecules as
Connectors for the Generation of Macroscopic Arrays and the
Construction of Supramolecular Bioconjugates," Nucleic Acids Res.
22:5530-5539 (1994). cited by applicant .
Park et al., "Block Copolymer Lithography: Periodic Arrays of
.about.10.sup.11 Holes in 1 Square Centimeter," Science
276:1401-1404 (1997). cited by applicant .
Park and Raines, "Green Fluorescent Protein as a Signal for
Protein-Protein Interactions," Protein Science 6:2344-2349 (1997).
cited by applicant .
Perkins et al., "Relaxation of a Single DNA Molecule Observed by
Optical Microscopy," Science 264:822-826 (1994). cited by applicant
.
Pierce et al., "Imaging Individual Green Fluorescent Proteins,"
Nature 388:338 (1997). cited by applicant .
Sinclair, "Sequence or Die--Automated Instrumentation for the
Genome Era," The Scientist, pp. 18-20, Apr. 12, 1999. cited by
applicant .
Slate et al., "Engineering of Five 88-Residue Receptor-Adhesive
Modular Proteins Containing a Parallel .alpha.-Helical Coiled Coil
and Two RGD Ligand Sites," Int. J. Peptide Protein Res. 45:290-298,
1995. cited by applicant .
Szollosi et al., "Application of Fluorescence Resonance Energy
Transfer in the Clinical Laboratory: Routine and Research,"
Cytometry 34:159-179 (1998). cited by applicant .
Travis, "Physics Festival Brightens Rainy San Jose," Science
268:30-31 (1995). cited by applicant .
Unger et al., "Single-Molecule Fluorescence Observed with Mercury
Lamp Illumination," BioTechniques 27:1008-1014 (1999). cited by
applicant .
Wang et al., "Size, Shape, and Stability of InAs Quantum Dots on
the GaAs(001) Substrate," Phys. Rev. B 62:1897-1904 (2000). cited
by applicant .
Weiss, "Fluorescence Spectroscopy of Single Biomolecules," Science
283:1676-1683 (1999). cited by applicant .
Wenner et al., "Salt Dependence of the Elasticity and
Overstretching Transition of Single Dna Molecules," Biophys. J.
82:3160-3169 (2002). cited by applicant .
Zahavy et al., "Detection of Frequency Resonance Energy Transfer
Pair on Double-Labeled Microsphere and Bacillus anthracis Spores by
Flow Cytometry," Appl. Environ. Microbiol. 69:2330-2339 (2003).
cited by applicant.
|
Primary Examiner: Bowman; Amy
Attorney, Agent or Firm: Klarquist Sparkman, LLP
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a continuation-in-part application of U.S. Ser. No.
11/638,160, filed Dec. 12, 2006, now U.S. Pat. No. 8,344,121, which
claims priority to U.S. Provisional Application Nos. 60/749,729 and
60/749,858 both filed Dec. 12, 2005 and herein incorporated by
reference.
Claims
We claim:
1. A method of detecting a target biomolecule, comprising:
contacting a sample with a probe for a target biomolecule, wherein
the probe is attached to a solid substrate, and wherein the probe
comprises: a molecular linker comprising a tether and a molecular
rod, wherein the length of the rod is shorter than the persistence
length of the rod material and the length of the tether is greater
than the persistence length of the tether material; a first
functional group comprising an acceptor fluorophore at one end of
the molecular linker; and a second functional group comprising a
donor fluorophore at the other end of the molecular linker, wherein
the first and second functional groups are linked and spaced by the
molecular linker, wherein the functional groups are capable of
interacting with one another or with the target biomolecule in a
predetermined reaction, wherein the molecular linker links the
first and second functional groups sufficiently spaced from one
another such that the functional groups do not substantially
interact in an absence of the target biomolecule but interact in a
presence of the target biomolecule to provide a signal of the
presence of the biomolecule, allowing the functional groups to
specifically interact with the target biomolecule; applying laser
light that can excite the donor fluorophore to enter the solid
substrate; trapping the laser light in the solid substrate by total
internal reflection; exciting the donor fluorophore, wherein when
interaction of the functional groups in the presence of the target
brings the acceptor fluorophore into a proximity with a donor
fluorophore to permit excitation of the acceptor fluorophore by the
donor fluorophore and results in the production of a signal by the
acceptor fluorophore; and detecting the signal from the acceptor
fluorophore when the probe interacts with target biomolecule.
2. The method of claim 1, wherein the sample comprises a blood
sample.
3. The method of claim 1, wherein the method permits detection of a
plurality of targets, and wherein: the solid substrate comprises a
plurality of probes at unique spacial locations, wherein each
location comprises a probe specific for a particular target; or the
solid substrate comprises a plurality of probes, wherein the probe
for a particular target comprises a unique combination of donor and
acceptor fluorophores, wherein detection of a signal from a
particular acceptor fluorophore indicates the presence of a
particular target.
4. The method of claim 1, wherein the target biomolecule comprises
a protein or a nucleic acid molecule.
5. The method of claim 1, wherein the solid substrate comprises a
glass slide or an optic fiber without its cladding.
6. The method of claim 1, wherein the solid substrate comprises a
well in which the sample is introduced.
7. The method of claim 1, wherein the target biomolecule is a
target nucleic acid molecule, detecting the target nucleic acid
molecule comprises sequencing the target target nucleic acid
molecule: contacting the sample comprising the target nucleic acid
molecule comprising contacting the sample with an oligonucleotide
primer and the probe in the presence of a mixture of non-labeled
hydrolyzable nucleotides, the first functional group comprises a
polymerase, the second functional group comprises a plurality of
second functional groups comprising non-hydrolyzable dNTPs at the
other end of the molecular linker, wherein each non-hydrolyzable
dNTP includes a label that emits a signal corresponding to the
particular nucleotide complementary to a nucleotide on the sample
nucleic acid sequence; detecting the signal from the acceptor
fluorophore comprises detecting the signal as each non-hydrolyzable
dNTP is exposed to the target nucleic acid molecule; and the method
further comprises allowing one of the non-labeled hydrolyzable
nucleotides to be incorporated into a synthesized nucleic acid
molecule complementary to the target nucleic acid molecule.
8. The method of claim 7, wherein at least a portion of the
molecular linker comprises a molecular rod having a persistence
length at least as great as a persistence length of a
double-stranded DNA (dsDNA) of at least 10-150 nucleotides.
9. The method of claim 7, wherein the tether comprises polyethylene
glycol (PEG).
10. The method of claim 7, wherein the molecular rod comprises a
double-stranded DNA (dsDNA) molecule of at least 10
nucleotides.
11. The method of claim 1, wherein the polymerase comprises a donor
fluorophore and each non-hydrolyzable dNTP comprises a different
acceptor fluorophore.
12. The method of claim 11, wherein detecting the signal comprises
detecting a fluorescent signal emitted from the acceptor
fluorophore.
13. The method of claim 12, wherein the sample is obtained from a
subject, and the method is performed ex vivo.
Description
FIELD
This application relates to probes that can be used to detect or
modify molecules, such as proteins and nucleic acid molecules, as
well as methods of their use.
BACKGROUND
Several methods are currently available for detecting proteins and
other molecules (such as nucleic acid molecules). For example,
proteins can be detected using western blotting, flow cytometry,
and ELISA methods. In addition, nucleic acids can be detected using
Southern or northern blotting, microarrays, quantitative or
non-quantitative PCR, chemical footprinting, and other methods
known in the art. However, these methods require multiple steps and
long detection times. Therefore, agents that permit detection with
fewer steps and less time are needed. In addition, agents that
permit detection in vivo are needed.
Methods are also currently available for modifying proteins and
nucleic acid molecules, such as the use of antisense or siRNA
molecules. However, agents having broader applications are
needed.
SUMMARY
The disclosure is directed to molecular agents (referred to herein
as nanoprobes) that can be used for detecting (such as
quantitating) or modifying (such as destroying) one or more target
molecules, such as biomolecules, organic molecules, and other
molecules such as nylon. For example, the probes can be used to
detect or modify a protein or a nucleic acid molecule. Although the
application provides probes to use for particular biomolecules, one
skilled in the art will recognize that the disclosed probes can be
adapted to detect or modify any molecule of interest, for example
by using particular functional groups.
In one example, a probe for a target biomolecule includes a
molecular linker and first and second functional groups linked and
spaced by the molecular linker. The functional groups are capable
of interacting with one another or with the target biomolecule in a
predetermined reaction, wherein the molecular linker links the
first and second functional groups sufficiently spaced from one
another such that the functional groups do not substantially
interact in an absence of the target biomolecule. In particular
examples, the molecular linker links the first and second
functional groups sufficiently spaced a distance from one another
to avoid substantial entanglement of the first and second
functional groups in an absence of the target biomolecule. In some
examples, the molecular linker (or at least a portion thereof) is
of sufficient rigidity to reduce interaction of the first and
second functional groups in the absence of the target biomolecule.
In particular examples, the molecular linker is of a sufficient
length to substantially avoid interaction of the first and second
functional groups in the absence of the target biomolecule, and
allow interaction of the first and second functional groups in the
presence of the target biomolecule.
The molecular linker (or a portion thereof, such as a molecular rod
that is part of the molecular linker) has a sufficient length in
view of its flexibility to space the functional groups sufficiently
apart to avoid the undesired interaction in the absence of the
target biomolecule, but retain sufficient flexibility to allow the
functional groups to interact with each other or the target when
one or more functional groups bind to the target. For example, at
least part of the linker can have a persistence length that permits
at least a portion of the molecular linker to be of sufficient
rigidity and length to reduce interaction of the first and second
functional groups in the absence of the target biomolecule, and
allow interaction of the first and second functional groups in the
presence of the target biomolecule. In particular examples, the
total length of the molecular linker is different than (such as
greater or less than) the persistence length of one or more
components that make up the linker, such as a double- or
single-stranded nucleic acid molecule. However, in particular
examples, the total length of the molecular linker does not exceed
a length beyond which significant interaction occurs between the
first and second functional groups in the absence of the target
biomolecule, while allowing significant interaction of the first
and second functional groups in the presence of the target
biomolecule. Such interactions can be measured using methods known
in the art, for example by measuring acceptor emission fluorescence
when one functional group includes a donor fluorophore and one or
more other functional groups include a corresponding acceptor
fluorophore of a FRET pair. In other examples, a functional group
is substantially maintained at a distance of at least twice the
Forster radius (such as a Forster radius of 22 to 90 .ANG.) from
the other functional group in the absence of the target.
Persistence length (lp) is the average local conformation for a
linear chain, which reflects the sum of the average projections of
all chain segments on a direction described by a given segment.
Therefore, persistence length is a measure of the rigidity or
stiffness of a polymer chain. In particular examples, persistence
length is the degree of bending (and hence the effective stiffness
of the chain) which, in effect, measures the contour distance over
which there occurs, on the average, a 68.40.degree. bend.
Therefore, the persistence length will vary depending on the
composition of the molecular linker. For example, the persistence
length for a double-stranded DNA (dsDNA) molecule will differ from
that of a single-stranded DNA (ssDNA) molecule and from
polyethylene glycol (PEG). In particular examples, dsDNA has a
persistence length of about 400-500 .ANG., and dsRNA has a
persistence length of 700-750 .ANG., for example at an ionic
strength of about 0.2 M and at a temperature of 20.degree. C. In
particular examples, ssDNA has a persistence length of about 40
.ANG. (for example at 20.degree. C.) (Clossey and Carlon, Phys.
Rev. E. Stat. Nonlin. Soft. Matter. Phys. 68(6 Pt 1):061911, 2003).
In particular examples, PEG has a persistence length of about 3.8
.ANG..
Particular examples of molecular linkers include, but are not
limited to, tethers, molecular rods, or combinations thereof. For
example, the molecular linker of sufficient rigidity can include a
molecular rod, for example a molecular rod composed of a
double-stranded DNA molecule (dsDNA). In some examples, the
molecular linker of sufficient rigidity includes multiple molecular
rods linked by tethers, or multiple tethers linked by molecular
rods. One particular example of a tether is a molecule composed of
(or in some examples consisting of) polyethylene glycol (PEG).
The functional groups include molecules that can interact with one
another or with the target biomolecule (or both) to provide a
predetermined reaction, such as a detectable signal or a
modification of a target biomolecule. The functional groups can be
linked in a spatially separated orientation by a molecular linker
so that the functional groups do not interact to provide the
reaction in the absence of the target molecule. However, the
molecular linker permits the functional groups, under predetermined
conditions, to be brought into sufficient proximity with one
another to interact and produce a predetermined reaction, such as a
detectable signal or modification of a target biomolecule to which
the probe binds or hybridizes. For example, the functional groups
can include a targeting moiety (such as an antibody, protein, or
nucleic acid probe) that binds to one or more sites on the target
biomolecule to bring the functional groups in sufficient proximity
to one another (or in sufficient proximity to the target
biomolecule) for the interaction to occur. In another example, the
functional groups can include an activatable moiety, such as a
labeling moiety or a biomolecule modifying moiety (such as a
proteinase or RNase). For example, at least one of the labeling
moieties can be activated when brought into sufficient proximity to
another labeling moiety, such as the excitation of an acceptor
fluorophore labeling moiety by a donor fluorophore labeling moiety
when the donor and acceptor are in sufficient proximity with one
another. However, the activatable moiety can have biological
activity in the absence of the interaction of the functional group
with the target biomolecule, but have increased biological activity
towards the target biomolecule when the functional groups are in
sufficient proximity with the target biomolecule. In particular
examples, a functional group includes both a targeting moiety and
an activatable moiety.
Particular examples of functional groups include, but are not
limited to, targeting agents (such as nucleic acid molecules,
protein detection agents (for example antibodies), proteins, and
nucleotides), as well as activation agents (such as a label or
biomolecule modifying agent, for example a proteinase or nuclease),
or combinations thereof. For example, a functional group can
include a labeled antibody. One specific example of a label
includes a FRET donor or a FRET acceptor fluorophore. Nanoprobes
that include donor and acceptor fluorophore labeling moieties, for
example in combination with a targeting moiety, will ideally
produce little or no detectable background fluorescence emission
from the acceptor fluorophore in the absence of the target
biomolecule. The targeting moieties are selected to recognize and
bind selectively or substantially only to targets of the
biomolecule that bring the labeling (or other activatable) moieties
into sufficient proximity for the targeting reaction to occur and
signal the presence of the biomolecule. In the presence of the
biomolecule, one or more of the other functional groups (such as
antibodies or nucleic acid molecules) interact with the target
biomolecule, which brings the donor and acceptor fluorophore into
sufficient proximity such that the resonance with the donor
fluorophore can excite the acceptor fluorophore, thereby resulting
in an acceptor fluorophore emission spectrum or decrease in donor
emission that can be detected.
Also provided are methods of using the nanoprobes, for example to
detect or modify a biomolecule, or both. For example, methods are
provided for using a nanoprobe to treat a subject having a disease
that could be treated by decreasing the activity or expression of
the target biomolecule. In a particular example, methods are
providing for using a nanoprobe to sequence a sample nucleic acid
molecule.
Methods of using the nanoprobes in combination with total internal
reflection (TIR) to detect one or more target biomolecules are also
provided. For example, one or nanoprobes specific for one or more
targets can be attached to a solid substrate, such as a glass slide
or optic fiber (e.g., an optic fiber with its cladding removed). In
some examples, the nanoprobes include an appropriate FRET donor and
acceptor pair. For example, if multiple targets are to be detected,
the nanoprobes for each target can have a unique donor/acceptor
pair, which permits determination of which target is detected. The
sample known or suspected of containing the one or more target
biomolecules is applied to the substrate. A laser that produces
light with excitation frequency for donor fluorophores present on
the nanoprobes can be used. The laser light enters the substrate
(e.g., an optic fiber or glass slide). The light is trapped in the
substrate by TIR. In the presence of the target(s), nanoprobes on
the surface of the substrate will bind to the target molecule and
provide a FRET signal using energy from the TIR evanescent wave.
Some of this light propagates downwards through the substrate, for
example through a filter that removes stray light from the laser,
and passes light from FRET to a detector. The resulting signal from
the acceptor fluorophore is detected if the target is present, but
no significant acceptor signal is detected if the target is absent
from the sample.
The foregoing and other features of the disclosure will become more
apparent from the following detailed description of a several
examples which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a schematic drawing showing an exemplary nanoprobe that
includes functional groups linked by a tether.
FIG. 1B is a schematic drawing showing an exemplary nanoprobe that
includes functional groups linked by a molecular rod and
tethers.
FIG. 1C is a schematic drawing showing an exemplary nanoprobe that
includes functional groups linked by multiple molecular rods and
tethers.
FIG. 2A is a schematic drawing showing an exemplary nanoprobe that
can be used to detect a DNA binding protein.
FIG. 2B is a schematic drawing showing binding of the exemplary
nanoprobe of FIG. 1A to a target DNA binding protein, thereby
generating a detectable signal.
FIG. 3A is a schematic drawing showing an exemplary nanoprobe that
can be used to detect an antigenic compound, such as a protein.
FIG. 3B is a schematic drawing showing binding of the exemplary
nanoprobe of FIG. 2A to a target antigenic compound, thereby
generating a detectable signal.
FIG. 4A is a schematic drawing showing an exemplary nanoprobe,
which can be used to detect an antigenic compound, that further
includes a molecular rod.
FIG. 4B is a schematic drawing showing an exemplary nanoprobe that
includes anti-IgG molecules, which can be used to detect an
antigenic compound. In particular examples, this nanoprobe does not
include the anti-target moiety, and the anti-target moiety is
selected by a user.
FIG. 5A is a schematic drawing showing an exemplary nanoprobe that
includes multiple tethers and a molecular rod, which can be used to
detect an antigenic compound.
FIG. 5B is a schematic drawing showing binding of the exemplary
nanoprobe of FIG. 5A to a target antigenic compound, thereby
generating a detectable signal.
FIG. 5C is a schematic drawing showing binding of the exemplary
nanoprobe of FIG. 5A to a target antigenic compound that is a
complex of at least two biomolecules, thereby generating a
detectable signal.
FIG. 6 is a schematic drawing showing an exemplary nanoprobe that
includes antisense functional groups, which can be used to detect a
target mRNA molecule.
FIG. 7 is a schematic drawing showing an exemplary mRNA nanoprobe,
which can be used to detect a target mRNA molecule, that includes
antisense functional groups and a ligase. The ligase can "seal" the
probe so that even if the target RNA has been destroyed or
dissociates from the probe, the signal from the labeling moiety
will continue.
FIGS. 8A-C are schematic drawings showing an exemplary nanoprobes
that can be used to sequence a nucleic acid molecule.
FIG. 9 is a schematic drawing showing an exemplary nanoprobe that
includes antisense and RNase H functional groups, which can be used
to destroy a target mRNA molecule.
FIG. 10A is a schematic drawing showing an exemplary nanoprobe that
includes a DNA binding region and proteinase K functional groups,
which can be used to cleave a DNA binding protein.
FIG. 10B is a schematic drawing showing an exemplary nanoprobe that
includes a protein binding agent and proteinase K functional
groups, which can be used to cleave a target protein.
FIG. 11 is a schematic drawing showing an exemplary nanoprobe that
includes antisense DNA and RNase H functional groups, which can be
used to quantitate a target mRNA molecule.
FIG. 12A is a schematic drawing showing an exemplary nanoprobe that
includes antisense and fluorophore functional groups, which can be
used to detect a target mRNA molecule.
FIG. 12B is a schematic drawing showing how an exemplary nanoprobe
1000 can be used to detect a target mRNA molecule 1020, thereby
forming a bound nanoprobe 1050.
FIG. 13A is a plot showing the relative intensity of emission at
different wavelengths for the two states of the nanoprobe of FIG.
12A (see FIG. 12B) in the presence or absence of the target
sequence. The peak at 520 nm is 6-FAM emission and the peak at 620
nm is Texas Red emission.
FIG. 13B is a plot showing the relative intensity of emission at
different wavelengths for the two states of the nanoprobe of FIG.
12A (see FIG. 12B) in the presence or absence of the target
sequence, and in the presence or absence of DNaseI. The peak at 520
nm is 6-FAM emission and the peak at 620 nm is Texas Red
emission.
FIG. 14A is a schematic drawing showing an exemplary universal
nanoprobe that includes antisense and fluorophore functional
groups, which can be used to detect a target nucleic acid
molecule.
FIG. 14B is a graph showing the relative intensity of emission at
different wavelengths for the two states of the universal nanoprobe
of FIG. 14B in the presence or absence of the target sequence. The
peak at 615 nm is Texas Red emission.
FIG. 15A is a schematic drawing showing an exemplary tetherless
nanoprobe that includes antisense and fluorophore functional
groups, but not tethers, which can be used to detect a target mRNA
molecule.
FIG. 15B is a graph showing the ratio of emission fluorescence
between nanoprobes that include and omit a PEG tether and the
thermodynamics of nanoprobe-target sequence complex formation.
FIG. 15C is a graph comparing the kinetics of nanoprobe-target
sequence complex formation between nanoprobes that include and omit
a PEG tether.
FIG. 16A is a bar graph showing the effect of NaCl and MgCl.sub.2
concentration on nanoprobe-target sequence complex formation.
FIG. 16B is a graph showing the kinetics of nanoprobe-target
sequence complex formation in the presence of 250 mM NaCl and 100
mM MgCl.sub.2.
FIGS. 17A-F are graphs showing the distance between fluorophores
and FRET at several molecular rod lengths. A rod has two tethers
and FRET is measured between the tether tips. This is a computer
simulation. The tethers are 120 .ANG. long and consist of segments
that have the persistence length of PEG (3.8 .ANG.). (E and F) rod
length 0 .ANG.. (C and D) rod length 60 .ANG.. (A and B) rod length
120 .ANG.. The data were generated using the bite program and
graphed using the genhis and genpic programs.
FIGS. 18A-D are graphs showing the effect of tether length on FRET
at various rod lengths. The FRET distance, R.sub.0, is 60 .ANG..
Each graph shows the FRET efficiency versus the rod length. The
color corresponds to the frequency that the nanoprobe gives a
particular FRET signal. (A) tether length 2 .ANG.; (B) tether
length 60 .ANG.; (C) tether length 120 .ANG.; (D) tether length 240
.ANG.. The data of the graphs of FIG. 17 can be obtained from the
lower left part of FIG. 18 (such as FIG. 18C, with a tether length
of 120 .ANG.) by taking vertical slices at rod lengths of 0 .ANG.,
60 .ANG. and 120 .ANG.. The data were generated using the bite
program and graphed using programs genhis and denplo.
FIGS. 19A and 19B show a schematic drawing showing a nanoprobe
attached to a surface.
FIG. 20 is a schematic drawing showing a nanoprobe that includes a
non-specific targeting moiety (such as dodecyl sulfate) and a
specific targeting moiety (such as an antibody) that can be used to
detect a target protein.
FIGS. 21A-C are a schematic drawings showing nanoprobes that
include a non-specific targeting moiety (such as a intercalating
fluorophore) and a specific targeting moiety (such as a
complementary nucleic acid molecule) that can be used to detect a
mRNA molecule.
FIGS. 22A and B show an exemplary nanoprobe that uses coomassie as
an alternative to a fluorophore in the (A) absence or (B) presence
of the target molecule.
FIG. 23 is a schematic drawing showing how quencher
containing-oligonucleotides can be used to decrease background
fluorescence from a nanoprobe.
FIG. 24 is a schematic drawing showing how the nanoprobes provided
herein can be used in combination with TIR to detect a target
biomolecule.
SEQUENCE LISTING
The nucleic acid sequences listed in the accompanying sequence
listing are shown using standard letter abbreviations for
nucleotide bases. In particular examples, only one strand of a
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand
(for example in the case of a dsDNA molecular rod).
SEQ ID NOS: 1-4 are exemplary nucleic acid sequences that include a
p53 binding site.
SEQ ID NO: 5 is an exemplary molecular rod sequence.
SEQ ID NOS: 6 and 7 are exemplary sequences that can be used to
generate the nanoprobe shown in FIG. 4A.
SEQ ID NOS: 8-11 are exemplary sequences that can be used to
generate the nanoprobe shown in FIG. 5A.
SEQ ID NO: 12 is an exemplary target nucleic acid sequence.
SEQ ID NOS: 13-18 are exemplary sequences that can be used to
generate the nanoprobe shown in FIG. 12A.
SEQ ID NOS: 16-18 are nucleic acid sequences that will specifically
hybridize to a portion of SEQ ID NO: 12 to form different sized
molecular rods (FIG. 12A, 1002).
SEQ ID NOS: 13-18 and 19-24 are exemplary sequences that can be
used to generate the nanoprobe shown in FIG. 14A.
SEQ ID NO: 25 is an exemplary target nucleic acid sequence.
SEQ ID NOS: 26-28 are exemplary sequences that can be used to
generate the nanoprobe shown in FIG. 7.
SEQ ID NOS: 29-38 are exemplary target sequences that can be
detected with the antisense sequence shown in SEQ ID NOS: 39-48,
respectively.
SEQ ID NOS: 26-27 and 49 are sequences that can be used to generate
the probe shown in FIG. 11.
SEQ ID NOS: 50-57 are exemplary DNA binding target sequences that
can be detected with the sequence shown in SEQ ID NOS: 58-61,
respectively.
SEQ ID NO: 62 is a sequence that can be used to generate the
nanoprobe shown in FIG. 10B.
SEQ ID NOS: 63-90 are sequences that can be used to generate the
nanoprobe shown in FIG. 8C.
SEQ ID NOS: 91-95 are exemplary quencher-containing
oligonucleotides.
SEQ ID NO: 96 is an exemplary target p53 nucleic acid sequence.
DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
Abbreviations and Terms
The following explanations of terms and methods are provided to
better describe the present disclosure and to guide those of
ordinary skill in the art in the practice of the present
disclosure. As used herein, "comprising" means "including" and the
singular forms "a" or "an" or "the" include plural references
unless the context clearly dictates otherwise. For example,
reference to "a tether" includes one or a plurality of such
tethers, and reference to "an antibody" includes reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth. The term "or" refers to a single element
of stated alternative elements or a combination of two or more
elements, unless the context clearly indicates otherwise. For
example, the phrase "detecting or modifying" refers to detecting,
modifying, or a combination of both detecting and modifying.
Unless explained otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present disclosure, suitable methods and materials are described
below. The materials, methods, and examples are illustrative only
and not intended to be limiting. Other features and advantages of
the disclosure are apparent from the following detailed description
and the claims.
.ANG. angstrom
dsDNA double-stranded DNA
FRET Forster resonance energy transfer
GFP green fluorescent protein
LNA locked nucleic acid
PEG polyethylene glycol
PNA peptide nucleic acid
RT reverse transcriptase
ssDNA single-stranded DNA
Acceptor fluorophore: Compounds which absorb energy from a donor
fluorophore, for example in the range of about 400 to 900 nm (such
as in the range of about 500 to 800 nm). Acceptor fluorophores
generally absorb light at a wavelength which is usually at least 10
nm higher (such as at least 20 nm higher), than the maximum
absorbance wavelength of the donor fluorophore, and have a
fluorescence emission maximum at a wavelength ranging from about
400 to 900 nm. Acceptor fluorophores have an excitation spectrum
which overlaps with the emission of the donor fluorophore, such
that energy emitted by the donor can excite the acceptor. Ideally,
an acceptor fluorophore is capable of being attached to the
disclosed nanoprobes.
Exemplary acceptor fluorophores include, but are not limited to,
rhodamine and its derivatives (such as
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA),
6-carboxy-X-rhodamine (ROX)), fluorescein derivatives (such as
5-carboxyfluorescein (FAM) and
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE)), green
fluorescent protein (GFP), BODIPY
(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) and cyanine dyes.
In a particular example, an acceptor fluorophore is a dark
quencher, such as, Dabcyl, Black Hole Quenchers.TM. from Glen
Research, Eclipse.TM. Dark Quencher from Epoch Biosciences, Iowa
Black.TM. from Integrated DNA Technologies. In such an example,
instead of detecting an increase in emission signal from the
acceptor fluorophore when in sufficient proximity to the donor
fluorophore, a decrease in the emission signal from the donor
fluorophore can be detected when in sufficient proximity to the
quencher.
Activating or activatable moiety: A component of the functional
group that acts on the target or with another functional group. For
example, if the target is a protein, the activating moiety may be a
protease. In another example, the activating moiety permits
detection of the target biomolecule, for example a label
moiety.
Administration: To provide or give a subject an agent, such as
composition which includes the agent, by any effective route. In
particular examples the agent includes one or more of the disclosed
nanoprobes, alone or in the presence of a pharmaceutically
acceptable carrier or other therapeutic agents. Exemplary routes of
administration include, but are not limited to, oral, injection
(such as subcutaneous, intramuscular, intradermal, intraperitoneal,
and intravenous), sublingual, rectal, transdermal, intranasal, and
inhalation routes.
Antibody: Immunoglobulin (Ig) molecules and immunologically active
portions of 1 g molecules, such as molecules that contain an
antigen binding site which specifically binds (immunoreacts with)
an antigen. Monoclonal and polyclonal immunoglobulin, as well as
immunologically effective portions ("fragments") thereof, are
encompassed by the disclosure. Antibodies can be used as functional
groups in the disclosed nanoprobes.
An exemplary immunoglobulin is IgG. Naturally occurring IgG
includes four polypeptide chains, two heavy chains and two light
chains inter-connected by disulfide bonds. However, the
antigen-binding function of an antibody can be performed by
fragments of a naturally occurring antibody. Thus, these
antigen-binding fragments are also intended to be designated by the
term "antibody". Examples of binding fragments encompassed within
the term antibody include (i) an Fab fragment that includes the
variable light (VL), variable heavy (VH), constant light (CL) and
constant heavy (CH1) domains; (ii) an Fd fragment that includes the
VH and CH1 domains; (iii) an Fv fragment that includes the VL and
VH domains of a single arm of an antibody, (iv) a dAb fragment that
consists of a VH domain; and (v) an F(ab').sub.2 fragment, a
bivalent fragment that includes two Fab fragments linked by a
disulfide bridge at the hinge region.
In a particular example, an antibody is a humanized antibody (or
immunologically effective portion thereof) or a chimeric antibody.
Another particular example of an antibody is a nanobody (antibodies
from camelids). Nanobodies have a heavy chain equivalent from which
single-domain antibody fragments can be obtained, but not a light
chain.
Antisense: Molecules that are specifically hybridizable (for
example under highly stringent hybridization conditions) or
specifically complementary to either RNA or the plus strand of DNA,
for example a target mRNA or DNA sequence. In a particular example,
one or more antisense molecules include one or more of the
functional groups of the disclosed nanoprobes. Antisense molecules
ideally contain a sufficient number of nucleotides to permit a
specific interaction with a target nucleic acid sequence.
In particular examples, an antisense molecule is of a length that
the melting temperature of the antisense:nucleic acid molecule
hybrid formed is greater than about 45.degree. C. For example, 20
base pairs provides a melting temperature of about 60.degree. C.
Therefore, at reaction temperatures of 25-45.degree. C., the
antisense:nucleic acid molecule hybrid formed would not
sufficiently separate. Therefore, in particular examples, an
antisense molecule includes at least 10 nucleotides, such as at
least 12, at least 15, at least 20, at least 30, at least 40, or at
least 50 nucleotides, for example 10-100 nucleotides (such as
10-50, 20-40, or 20-30 nucleotides).
Binding: An association between two or more molecules, such as the
formation of a complex. Generally, the stronger the binding of the
molecules in a complex, the slower their rate of dissociation.
Specific binding refers to a preferential binding between an agent
and a target.
Particular examples of specific binding include, but are not
limited to, hybridization of one nucleic acid molecule to a
complementary nucleic acid molecule, the association of an antibody
with a peptide or other antigen, or the association of a protein
with a target protein or target nucleic acid molecule.
In a particular example, a protein is known to bind to another
protein or another biomolecule if a sufficient amount of the
protein forms chemical bonds to the protein or other biomolecule,
for example a sufficient amount to permit detection of that
binding, such as detection using the disclosed nanoprobes.
In one example, an oligonucleotide molecule (such as an antisense
molecule) is observed to bind to a target nucleic acid molecule if
a sufficient amount of the oligonucleotide molecule forms base
pairs or is hybridized to its target nucleic acid molecule to
permit detection of that binding, for example detection using the
disclosed nanoprobes. The binding between an oligonucleotide and
its target nucleic acid molecule is frequently characterized by the
temperature (T.sub.m) at which 50% of the oligonucleotide is melted
from its target. A higher (T.sub.m) means a stronger or more stable
complex relative to a complex with a lower (T.sub.m).
In a particular example, binding is assessed by detecting labels
present on the nanoprobe. For example, the fluorescent signal
generated following the interaction of donor and acceptor
fluorophores can be measured as an indication of binding between
one or more functional groups on a nanoprobe and one or more target
biomolecules.
In a particular example, an antibody specifically binds to a target
biomolecule if the antibody specifically immunoreacts with the
target biomolecule. Specific binding is typically determined from
the reference point of the ability of the antibody to
differentially bind the target biomolecule and an unrelated
biomolecule, and therefore distinguish between two different
biomolecules.
Biomolecule: An organic molecule, such as a macromolecule, present
in living organisms, such as a mammal. Particular examples of
biomolecules include, but are not limited to, proteins, nucleic
acid molecules (such as DNA and RNA molecules), saccharides,
vitamins, carbohydrates and lipids.
Detect: To determine if an agent is present or absent. In some
examples this can further include quantification. For example, use
of the disclosed nanoprobes in particular examples permits
detection of one or more biomolecules in a sample. In particular
examples, an emission signal from an acceptor fluorophore (such as
the increase in the signal) is detected. In other particular
examples, the emission signal from the donor fluorophore (such as
the decrease in the signal) is detected.
Detection can be in bulk, so that a macroscopic number of molecules
(such as at least 10.sup.23 molecules) can be observed
simultaneously. Detection can also include identification of
signals from single molecules using microscopy and such techniques
as total internal reflection to reduce background noise. The
spectra of individual molecules can be obtained by these techniques
(Ha et al., Proc. Natl. Acad. Sci. USA. 93:6264-8, 1996).
Donor Fluorophore: Fluorophores or luminescent molecules capable of
transferring energy to an acceptor fluorophore, thereby generating
a detectable fluorescent signal. Donor fluorophores are generally
compounds that absorb in the range of about 300 to 900 nm, for
example about 350 to 800 nm. Donor fluorophores have a strong molar
absorbance coefficient at the desired excitation wavelength, for
example greater than about 10.sup.3 M.sup.-1 cm.sup.-1. A variety
of compounds can be employed as donor fluorescent components,
including fluorescein (and derivatives thereof), rhodamine (and
derivatives thereof), GFP, phycoerythrin, BODIPY, DAPI
(4',6-diamidino-2-phenylindole), Indo-1, coumarin, dansyl, and
cyanine dyes. In particular examples, a donor fluorophore is a
chemiluminescent molecule, such as aequorin.
DNA-binding protein: Any protein that can specifically bind to
double- or single-stranded DNA. Examples include many proteins
involved in the regulation of gene expression (including
transcription factors), proteins involved in the packaging of DNA
within the nucleus (such as histones), nucleic acid-dependent
polymerases involved in DNA replication and transcription, or any
of many accessory proteins which are involved in these processes.
Other particular examples of DNA binding proteins include, but are
not limited to p53, Tus (terminal utilization substance which binds
to Ter, the terminus region in E. coli), F is (factor for inversion
stimulation which controls many genetic systems in E. coli), Lambda
repressor, and Lac repressor.
Emission signal: The light of a particular wavelength generated
from a fluorophore after the fluorophore absorbs light at its
excitation wavelengths.
Emission spectrum: The energy spectrum which results after a
fluorophore is excited by a specific wavelength of light. Each
fluorophore has a characteristic emission spectrum. In one example,
individual fluorophores (or unique combinations of fluorophores)
are attached to nucleotides and the emission spectra from the
fluorophores provide a means for distinguishing between the
different nucleotides.
Entangled: To be twisted together, for example in a tangled mass.
In particular examples, entanglement of a nanoprobe would reduce or
prevent the functional groups from interacting with one another or
from interacting with a target biomolecule, in the presence of the
target biomolecule. In other particular examples, entanglement of a
nanoprobe results in an undesirable interaction between the
functional groups, for example an interaction that prevents
interaction with, modification of, or detection of the target
biomolecule.
Excitation or excitation signal: The light of a particular
wavelength necessary to excite a fluorophore to a state such that
the fluorophore will emit a different, such as longer, wavelength
of light.
Fluorophore: A chemical compound, which when excited by exposure to
a particular stimulus such as a defined wavelength of light, emits
light (fluoresces), for example at a different wavelength.
Fluorophores are part of the larger class of luminescent compounds.
Luminescent compounds include chemiluminescent molecules, which do
not require a particular wavelength of light to luminesce, but
rather use a chemical source of energy. Therefore, the use of
chemiluminescent molecules eliminates the need for an external
source of electromagnetic radiation, such as a laser. Examples of
chemiluminescent molecules include, but are not limited to,
aequorin (Tsien, 1998, Ann. Rev. Biochem. 67:509).
Examples of particular fluorophores that can be used in the
nanoprobes disclosed herein are provided in U.S. Pat. No. 5,866,366
to Nazarenko et al., such as
4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid,
acridine and derivatives such as acridine and acridine
isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid
(EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5
disulfonate (Lucifer Yellow VS), N-(4-anilino-1-naphthyl)maleimide,
anthranilamide, Brilliant Yellow, coumarin and derivatives such as
coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120),
7-amino-4-trifluoromethylcouluarin (Coumaran 151); cyanosine;
4',6-diaminidino-2-phenylindole (DAPI);
5',5''-dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin;
diethylenetriamine pentaacetate;
4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid;
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid;
5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansyl
chloride); 4-dimethylaminophenylazophenyl-4'-isothiocyanate
(DABITC); eosin and derivatives such as eosin and eosin
isothiocyanate; erythrosin and derivatives such as erythrosin B and
erythrosin isothiocyanate; ethidium; fluorescein and derivatives
such as 5-carboxyfluorescein (FAM),
5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),
2'7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE),
fluorescein, fluorescein isothiocyanate (FITC), and QFITC (XRITC);
fluorescamine; IR144; IR1446; Malachite Green isothiocyanate;
4-methylumbelliferone; ortho cresolphthalein; nitrotyrosine;
pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde;
pyrene and derivatives such as pyrene, pyrene butyrate and
succinimidyl 1-pyrene butyrate; Reactive Red 4 (Cibacron.RTM.
Brilliant Red 3B-A); rhodamine and derivatives such as
6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine
rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B,
rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B,
sulforhodamine 101 and sulfonyl chloride derivative of
sulforhodamine 101 (Texas Red);
N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl
rhodamine; tetramethyl rhodamine isothiocyanate (TRITC);
riboflavin; rosolic acid and terbium chelate derivatives.
Other suitable fluorophores include thiol-reactive europium
chelates which emit at approximately 617 nm (Heyduk and Heyduk,
Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22,
1999), as well as GFP, Lissamine.TM., diethylaminocoumarin,
fluorescein chlorotriazinyl, naphthofluorescein,
4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No.
5,800,996 to Lee et al.) and derivatives thereof. In one example,
the fluorophore is a large Stokes shift protein (see Kogure et al.,
Nat. Biotech. 24:577-81, 2006). Other fluorophores known to those
skilled in the art can also be used, for example those available
from Molecular Probes (Eugene, Oreg.).
In particular examples, a fluorophore is used as a donor
fluorophore or as an acceptor fluorophore. Ideally, fluorophores
have the ability to be attached to a nanoprobe component without
sufficiently interfering with the ability of the nanoprobe to
interact with the target biomolecule, are stable against
photobleaching, and have high quantum efficiency. In examples where
multiple acceptor fluorophores are used, for example on a single
nanoprobe or for example on different nanoprobes that are used
together, the fluorophores are advantageously selected to have
distinguishable emission spectra, such that emission from one
fluorophore (or combination of two or more fluorophores) is
distinguishable from another fluorophore (or combination of two or
more fluorophores).
Forster (or fluorescence) resonance energy transfer (FRET): A
process in which an excited fluorophore (the donor) transfers its
excited state energy to a lower-energy light absorbing molecule
(the acceptor). This energy transfer is non-radiative, and due
primarily to a dipole-dipole interaction between the donor and
acceptor fluorophores. This energy can be passed over a distance,
for example a limited distance such as 10-100 .ANG.. FRET
efficiency drops off according to 1/(1+(R/R0)^6) where R0 is the
distance at which the FRET efficiency is 50%.
FRET pairs: Sets (such as pairs) of fluorophores that can engage in
fluorescence resonance energy transfer (FRET). Examples of FRET
pairs that can be used are listed below. However, one skilled in
the art will recognize that numerous other combinations of
fluorophores can be used.
FAM is most efficiently excited by light with a wavelength of 488
nm, emits light with a spectrum of 500 to 650 nm, and has an
emission maximum of 525 nm. FAM is a suitable donor fluorophore for
use with JOE, TAMRA, and ROX (all of which have their excitation
maxima at 514 nm, and will not be significantly stimulated by the
light that stimulates FAM).
The GFP mutant H9-40 (Tsien, 1998, Ann. Rev. Biochem. 67:509),
which is excited at 399 nm and emits at 511 nm, can serve as a
suitable donor fluorophore for use with BODIPY, fluorescein,
rhodamine green and Oregon green. In addition, the fluorophores
tetramethylrhodamine, Lissamine.TM., Texas Red and
naphthofluorescein can be used as acceptor fluorophores with this
GFP mutant.
The fluorophore
3-(.epsilon.-carboxy-pentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine
(CYA) is maximally excited at 488 nm and can therefore serve as a
donor fluorophore for rhodamine derivatives (such as R6G, TAMRA,
and ROX) which can be used as acceptor fluorophores (see Hung et
al., Analytical Biochemistry, 243:15-27, 1996). However, CYA and
FAM are not examples of a good FRET pair, because both are excited
maximally at the same wavelength (488 nm).
One of ordinary skill in the art can easily determine, using
art-known techniques of spectrophotometry, which fluorophores will
make suitable donor-acceptor FRET pairs. In addition, Grant et al.
(Biosens Bioelectron. 16:231-7, 2001) provide particular examples
of FRET pairs that can be used in the nanoprobes disclosed
herein.
Functional group: Any agent that can be used to detect (such as
quantitate) or modify (or both) a biomolecule. Particular examples
include, but are not limited to, targeting moieties and activating
moieties that are brought into activating proximity when the
targeting moiety binds to the target biomolecule. The targeting
moiety, for example, brings the activatable moiety into sufficient
proximity to the target for the activatable moiety to act on the
target (for example when the activatable moiety is a non-specific
proteinase that is brought sufficiently close to a protein target
biomolecule to cleave or degrade the target protein). In other
examples, each of the functional groups includes a targeting moiety
and an activating moiety, and the targeting moiety binds to the
target at a distance that selectively activates the activatable
moiety. For example, each functional group can include an antibody
or probe that binds to the target, and each functional group can
include a labeling moiety, such a member of a FRET donor and
acceptor fluorophore pair, wherein the acceptor is activated by the
donor to emit a fluorescent signal when the target protein binds
and brings the FRET pair into sufficient proximity to emit a
detectable characteristic signal from the acceptor.
Particular examples of targeting moieties include, but are not
limited to, antibodies, proteins, nucleotides and nucleic acid
molecules (such as DNA binding molecules, PNAs or LNAs). Particular
examples of activating moieties include, but are not limited to,
labels (such as fluorophores) and modifying agents (such as
enzymes, for example nucleases, ligases, or proteases, and a
crosslinkable group such as psoralen).
Green fluorescent protein (GFP): The source of fluorescent light
emission in Aequorea victoria. As used herein, GFP refers to both
the wild-type protein, and spectrally shifted mutants thereof, for
example as described in Tsien, 1998, Ann. Rev. Biochem. 67:509 and
in U.S. Pat. Nos. 5,777,079 and 5,625,048 to Tsien and Heim, herein
incorporated by reference. In particular examples, GFP is excited
using a laser. In other examples, GFP is excited using aequorin,
for example using a GFP-aequorin fusion protein.
Hybridization: To form base pairs between complementary regions of
two strands of DNA, RNA, or between DNA and RNA, thereby forming a
duplex molecule. Hybridization conditions resulting in particular
degrees of stringency will vary depending upon the nature of the
hybridization method and the composition and length of the
hybridizing nucleic acid sequences. Generally, the temperature of
hybridization and the ionic strength (such as the Na.sup.+
concentration) of the hybridization buffer will determine the
stringency of hybridization. Calculations regarding hybridization
conditions for attaining particular degrees of stringency are
discussed in Sambrook et al., (1989) Molecular Cloning, second
edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9
and 11). The following is an exemplary set of hybridization
conditions and is not limiting:
Very High Stringency (Detects Sequences that Share at Least 90%
Identity)
Hybridization: 5.times.SSC at 65.degree. C. for 16 hours
Wash twice: 2.times.SSC at room temperature (RT) for 15 minutes
each
Wash twice: 0.5.times.SSC at 65.degree. C. for 20 minutes each
High Stringency (Detects Sequences that Share at Least 80% Identity
r)
Hybridization: 5.times.-6.times.SSC at 65.degree. C.-70.degree. C.
for 16-20 hours
Wash twice: 2.times.SSC at RT for 5-20 minutes each
Wash twice: 1.times.SSC at 55.degree. C.-70.degree. C. for 30
minutes each
Low Stringency (Detects Sequences that Share at Least 50%
Identity)
Hybridization: 6.times.SSC at RT to 55.degree. C. for 16-20
hours
Wash at least twice: 2.times.-3.times.SSC at RT to 55.degree. C.
for 20-30 minutes each.
20.times.SSC is 3.0 M NaCl/0.3 M trisodium citrate.
Label: An agent capable of detection, for example by
spectrophotometry, flow cytometry, or microscopy. For example, one
or more labels can be attached to a nanoprobe, thereby permitting
detection of the target biomolecule. Exemplary labels include
radioactive isotopes, fluorophores, ligands, chemiluminescent
agents, enzymes, and combinations thereof.
Ligase: An enzyme that can catalyse the joining of two molecules
("ligation") by forming a new chemical bond. An exemplary ligase is
DNA ligase, which can link two nucleic acid molecules by forming a
phosphodiester bond between the two molecules.
Linker or molecular linker: A structure that joins one molecule to
other, such as one functional group to another functional group,
wherein one portion of the linker is operably linked to a first
functional group, and wherein another portion of the linker is
operably linked to two or more other functional groups. Particular
examples of linkers that can be used in a nanoprobe include, but
are not limited to, tethers, molecular rods, or combinations
thereof.
Locked Nucleic Acid (LNA.TM.): A bicyclic nucleic acid where a
ribonucleoside is linked between the 2'-oxygen and the 4'-carbon
atoms with a methylene unit. This link restricts the flexibility of
the ribofuranose ring of the nucleotide analog and locks it into
the rigid bicyclic N-type conformation. The LNA also induces
adjacent bases to adopt a conformation of the more
thermodynamically stable form of the A duplex.
LNA oligonucleotides can be synthesized by standard phosphoramidite
chemistry using DNA-synthesizers. In addition, LNA can be mixed
with DNA, RNA as well as other nucleic acid analogs. In particular
examples, LNA includes a functional group, such as an acceptor
fluorophore.
Luminescence Resonance Energy Transfer (LRET): A process similar to
FRET, in which the donor molecule is itself a luminescent molecule,
or is excited by a luminescent molecule, instead of for example by
a laser. Using LRET can decrease the background fluorescence. In
particular examples, a chemiluminescent molecule can be used to
excite a donor fluorophore (such as GFP), without the need for an
external source of electromagnetic radiation. In other examples,
the luminescent molecule is the donor, wherein the excited
resonance of the luminescent molecule excites one or more acceptor
fluorophores.
An example of luminescent molecule that can be used includes, but
is not limited to, aequorin. The bioluminescence from aequorin,
which peaks at 470 nm, can be used to excite a donor GFP
fluorophore (Tsien, 1998, Ann. Rev. Biochem. 67:509; Baubet et al.,
2000, Proc. Natl. Acad. Sci. U.S.A., 97:7260-5). GFP then excites
an acceptor fluorophore disclosed herein. In this example, both
aequorin and GFP can be attached to a nanoprobe.
Modify: To change an agent, for example to decrease the biological
activity of a biomolecule. For example, use of the disclosed
nanoprobes in particular examples permits modification (such as
cleavage or ligation) of one or more biomolecules in a sample.
Nanoprobe or probe: A molecular device that can be used to detect
or modify (for example cleave or ligate) a target biomolecule, such
as a protein or nucleic acid molecule. In particular examples, a
nanoprobe or probe includes one or more labels that permit
detection of the probe, such as an acceptor and donor fluorophore
pair.
Nucleic acid molecule (or sequence): A deoxyribonucleotide or
ribonucleotide polymer including without limitation, cDNA, mRNA,
genomic DNA, and synthetic (such as chemically synthesized) DNA or
RNA. The nucleic acid molecule can be double stranded (ds) or
single stranded (ss). Where single stranded, the nucleic acid
molecule can be the sense strand or the antisense strand. Nucleic
acid molecules can include natural nucleotides (such as A, T/U, C,
and G), and can also include analogs of natural nucleotides.
Nucleotide: Includes, but is not limited to, a monomer that
includes a base, such as a pyrimidine, purine, or synthetic analogs
thereof, linked to a sugar and one or more phosphate groups. A set
of bases linked to a peptide backbone, as in a peptide nucleic acid
(PNA), can be used as a substitute for a nucleic acid molecule. A
nucleotide is one monomer in a polynucleotide. A nucleotide
sequence refers to the sequence of bases in an oligonucleotide.
Nucleotide analog: A nucleotide containing one or more
modifications of the naturally occurring base, sugar, phosphate
backbone, or combinations thereof. Such modifications can result in
the inability of the nucleotide to be incorporated into a growing
nucleic acid chain. A particular example includes a
non-hydrolyzable nucleotide. Non-hydrolyzable nucleotides include
mononucleotides and trinucleotides in which the oxygen between the
alpha and beta phosphates has been replaced with nitrogen or carbon
(Jena Bioscience). HIV-1 reverse transcriptase cannot hydrolyze
dTTP with the oxygen between the alpha and beta phosphates replaced
by nitrogen (Ma et al., J. Med. Chem., 35: 1938-41, 1992).
A "type" of nucleotide analog refers to one of a set of nucleotide
analogs that share a common characteristic that is to be detected.
For example, the sets of nucleotide analogs can be divided into
four types: A, T, C and G analogs (for DNA) or A, U, C and G
analogs (for RNA). In this example, each type of nucleotide analog
can be associated with a unique tag, such as one or more acceptor
fluorophores, so as to be distinguishable from the other nucleotide
analogs in the set (for example by fluorescent spectroscopy or by
other optical means).
An exemplary nucleotide analog that can be used in place of "C" is
a G-clamp (Glen Research). G-clamp is a tricyclic
Aminoethyl-Phenoxazine 2'-deoxyCytidine analogue (AP-dC). The
G-clamp is available as a phosphoramidite and so can be synthesized
into DNA structures. Such an analog can be used in the nanoprobes
provided herein (for example the dCTP 556 shown in FIG. 8A can be
substituted with a G-clamp).
Oligonucleotide: A linear polynucleotide (such as DNA or RNA)
sequence, for example of at least 6 nucleotides, for example at
least 9, at least 15, at least 18, at least 24, at least 30, at
least 50, at least 100, at least 200 or even at least 500
nucleotides long. In particular examples, an oligonucleotide is
about 6-50 bases, for example about 10-25 bases, such as 12-20
bases.
An oligonucleotide can contain non-naturally occurring portions,
such as altered sugar moieties or inter-sugar linkages, such as a
phosphorothioate oligodeoxynucleotide. In particular examples, an
oligonucleotide containing non-naturally occurring portions can
bind to RNA or DNA, and include peptide nucleic acid (PNA)
molecules.
Peptide Nucleic Acid (PNA): A class of informational molecules
containing a neutral peptide-like backbone with nucleobases
allowing it to hybridize to complementary RNA or DNA with higher
affinity and specificity than conventional oligonucleotides. The
structure of a PNA molecule is analogous with DNA, wherein the
deoxyribose phosphate backbone has been replaced by a backbone
similar to that found in peptides. In particular examples, PNA is
resistant to nucleases and proteases. PNAs can include a functional
group at the N(5)-terminus, such as a fluorophore (for example an
acceptor fluorophore).
Persistence length (lp): The average local conformation for a
linear chain, reflecting the sum of the average projections of all
chain segments on a direction described by a given segment. In
particular examples, persistence length is the degree of bending
(and hence the effective stiffness of the chain) which, in effect,
measures the contour distance over which there occurs, on the
average, a 68.40.degree. bend.
Polyethylene glycol (PEG): A polymer of ethylene compounds,
H(OCH.sub.2CH.sub.2).sub.nOH. Pegylation is the act of adding a PEG
structure to another molecule, for example, a functional molecule
such as a targeting or activatable moiety. PEG is soluble in water,
methanol, benzene, dichloromethane and is insoluble in diethyl
ether and hexane.
Particular examples of PEG that can be used in the disclosed
nanoprobes include, but are not limited to: 1-7 units of Spacer 18
(Integrated DNA Technologies, Coralville, Iowa), such as 3-5 units
of Spacer 18, C3 Spacer phosphoramidite (such as 1-10 units),
Spacer 9 (such as 1-10 units), PC (Photo-Cleavable) Spacer (such as
1-10 units), (all available from Integrated DNA Technologies). In
other examples, lengths of PEG that can be used in the disclosed
nanoprobes include, but are not limited to, 1 to 40 monomers of
PEG.
Polymerase: An enzyme which synthesizes a nucleic acid strand
complementary to a nucleic acid template. Examples of polymerases
that can be used to sequence a nucleic acid molecule include, but
are not limited to the E. coli DNA polymerase I, specifically the
Klenow fragment which has 3' to 5' exonuclease activity, Taq
polymerase, reverse transcriptase (such as HIV-1 RT), E. coli RNA
polymerase, and wheat germ RNA polymerase II.
The choice of polymerase is dependent on the nucleic acid to be
sequenced. If the template is a single-stranded DNA molecule, a
DNA-directed DNA or RNA polymerase can be used; if the template is
a single-stranded RNA molecule, then a reverse transcriptase (such
as an RNA-directed DNA polymerase) can be used.
Proteinase K: An endolytic protease that cleaves peptide bonds at
the carboxylic sides of aliphatic, aromatic or hydrophobic amino
acids. Proteinase K is classified as a serine protease. The
smallest peptide to be hydrolyzed is a tetrapeptide. Proteinase K
is commercially available, for example from Fermentas (Hanover,
Md.; #EO0491).
Quantum dots: Engineered, inorganic semiconductor crystalline
nanoparticles that fluoresce stably and possess a uniform surface
area that can be chemically modified to attach biomolecules (such
as one or more nanoprobes) to them. Although generally spherical,
quantum dots attached to nanoprobes of the present disclosure can
be of any shape (such a spherical, tubular, pyramidal, conical or
cubical), but particularly suitable nanoparticles are spherical.
The spherical surface provides a substantially smooth and
predictably oriented surface for the attachment of specific binding
agents such as antibodies, with the binders extending substantially
radially outwardly from the surface of the sphere.
Generally, quantum dots can be prepared with relative
monodispersity (for example, with the diameter of the core varying
approximately less than 10% between quantum dots in the
preparation), as has been described previously (Bawendi et al., J.
Am. Chem. Soc. 115:8706, 1993). Quantum dots known in the art have,
for example, a core selected from the group consisting of CdSe,
CdS, and CdTe (collectively referred to as "CdX").
Recombinant: A recombinant nucleic acid or protein sequence is one
that has a sequence that is not naturally occurring or has a
sequence that is made by an artificial combination of two otherwise
separated segments of sequence. This artificial combination can be
accomplished by chemical synthesis or by the artificial
manipulation of isolated segments of nucleic acid or protein
sequences, for example by genetic engineering techniques. In
particular examples, a molecular rod composed of a dsDNA is a
recombinant molecule.
RNase H (Ribonuclease H): A ribonuclease (EC 3.1.26.4) that cleaves
the 3'-O--P-bond of RNA in a DNA/RNA duplex to produce 3'-hydroxyl
and 5'-phosphate terminated products. RNAse H does not degrade DNA
or unhybridized RNA. To terminate the reaction, a chelator, such as
EDTA, can be added to sequester the required metal ions in the
reaction mixture.
Members of the RNase H family can be found in nearly all organisms.
RNase H proteins can be produced using commercially available
clones (for example from the E. coli Genome Project, University of
Wisconsin, Madison, Wis., such as clone pEKGb0214; from NEB,
Ambion, and Roche). In one example, MuLV RNAse H is used for the
nanoprobe (for example see Zhan and Crouch, J. Biol. Chem.,
272:22023-9, 1997).
Rod or molecular rod: A structure that can be included in a
nanoprobe's molecular linker to increase the rigidity of a portion
of the nanoprobe, thereby reducing the interaction of functional
groups, labels (such as donor and acceptor fluorophores), or
combinations thereof, in the absence of the target biomolecule. In
addition, molecular rods are of a length that permits the
functional groups, labels, or combinations thereof to interact in
the presence of the target biomolecule.
In a particular example, a molecular rod present in a molecular
linker has a length shorter than its persistence length. In the
absence of the target biomolecule, the rod significantly reduces
the interaction of the first and second functional groups joined by
the molecular linker that contains the rod. In one example, a
molecular rod consisting of dsDNA has a length of 10-140
nucleotides, which is shorter than the persistence length of dsDNA,
about 150 nucleotides.
Exemplary molecular rods include, but are not limited to, dsDNA
molecules, peptide nucleic acids (PNAs), carbon nanotubes, locked
nucleic acid molecules (LNAs), a microtubule, a bacterium, a linear
virus particle, virus tail fibers or other protein structures (such
as protein components containing alpha helices or beta barrels or
other protein structures, such as a leucine zipper structure). A
molecular rod can be a portion of a three-dimensional molecular
construct, such as a cube or octahedron built from DNA (for example
see Seeman, Sci. Am. 290:64-9 and 72-5, 2004). In a particular
example, a molecular rod is a dsDNA molecule of at least 10
nucleotides, at least 35 nucleotides, or 150 nucleotides or less,
such as 10-150 nucleotides, 10-140 nucleotides, 20-100 nucleotides,
20-50 nucleotides, 20-40 nucleotides, 30-50 nucleotides, or about
20, 30, or 40 nucleotides.
Sample: Specimens such as samples containing biomolecules, such as
nucleic acid molecules (for example genomic DNA, cDNA, RNA, or
mRNA) or proteins. Exemplary samples are those containing cells or
cell lysates from a subject, such as those present in peripheral
blood (or a fraction thereof such as serum), urine, saliva, tissue
biopsy, surgical specimen, fine needle aspirates, amniocentesis
samples and autopsy material. Also encompassed by this disclosure
are environmental samples, such as air, water, soil, as well as
samples obtained from swabbing a surface.
Signal: A detectable change or impulse in a physical property that
provides information. In the context of the disclosed methods,
examples include electromagnetic signals such as light, for example
light of a particular quantity or wavelength. In certain examples
the signal is the disappearance of a physical event, such as
quenching of light.
Subject: Living multi-cellular vertebrate organisms, including
human and veterinary subjects, such as cows, pigs, horses, dogs,
cats, birds, reptiles, and fish.
Target biomolecule: A biomolecule (such as a nucleic acid molecule,
protein, or antigenic compound) whose detection, modification (such
as inactivation or ligation), quantitation, qualitative detection,
or a combination thereof, is intended. The biomolecule need not be
in a purified form. Various other biomolecules can also be present
with the target biomolecule. For example, the target biomolecule
can be a specific nucleic acid molecule or a protein in a cell
(which can include host RNAs (such as mRNA), DNAs (such as genomic
or cDNA), and proteins), the detection or modification of which is
intended.
Targeting moiety: A functional group component that binds a
functional group to the target biomolecule. Examples include
antibodies that recognize protein targets, and nucleic acid
molecules that bind to nucleic acid target sequences.
Tether: A structure that can be included in a nanoprobe to link one
functional group to another, directly or indirectly. For example a
tether can be used to directly link one functional group to
another, such as a tether of 120 to 240 .ANG.. In another example,
one or more tethers, in combination with one or more molecular
rods, are used to link one functional group to another. Ideally,
the tether is a length that reduces the likelihood that the tether
will tangle with itself or with other components of the nanoprobe,
while still allowing the functional groups, labels, or combinations
thereof to interact in the presence of the target biomolecule.
Exemplary tethers disclosed herein include water soluble long chain
molecules, such as PEG, peptides (such as a peptide of at least 30
amino acids, for example at least 30 contiguous amino acids of the
RecB protein 70-amino acid-long flexible tether connecting the
helicase to the nuclease (Singleton et al., Nature 432:187-93,
2004)), sugar chains (such as 2000-14000 residues), abasic
phosphodiester spacers (such as the IDT 5' dSpacer), carbohydrate
chains (such as at least 10 sugar molecules), and polycaprolactone
chains (such as at least 10 monomers). In a particular example, a
tether is composed of PEG, for example a PEG length of about 23-600
.ANG., such as 23-400 .ANG., or 23-164 .ANG..
Therapeutically effective amount: An amount of an agent (alone or
in combination with other therapeutically effective agents)
sufficient to achieve a desired biological effect, for example an
amount that is effective to decrease the activity of a target
biomolecule by at least a desired amount. In a particular example,
it is an amount of a nanoprobe disclosed herein that is effective
to decrease the activity of a target biomolecule by at least 25%,
at least 50%, at least 75%, or at least 90%, for example as
compared to an amount of activity prior to treatment.
In some examples, it is an amount of a therapeutic nanoprobe (alone
or in combination with other therapeutically effective agents) that
can decrease the activity of a target biomolecule to improve signs
or symptoms of a disease caused by activity or expression of a
target biomolecule. An effective amount of a nanoprobe that
decreases the activity of a target biomolecule can be administered
in a single dose, or in several doses (for example daily, weekly,
or monthly) during a course of treatment. However, the effective
amount of agent may be dependent on the source of agent
administered, the subject being treated, the severity and type of
disease being treated, and the manner of administration.
Treating a disease: "Treatment" refers to a therapeutic
intervention that ameliorates a sign or symptom of a disease or
pathological condition, such as a sign or symptom of cancer.
Treatment can also induce remission or cure of a condition, such as
cancer. In particular examples, treatment includes preventing a
disease, for example by inhibiting the full development of a
disease, such as preventing development of cancer (for example
preventing metastasis of the cancer). Prevention of a disease does
not require a total absence of disease. For example, a decrease of
at least 10%, at least 25% or at least 50% can be sufficient.
Under conditions sufficient for: A phrase that is used to describe
any environment that permits the desired activity.
An example includes contacting a nanoprobe with a sample sufficient
to allow the desired activity. In particular examples, the desired
activity is the detection or modification of one or more target
biomolecules in the sample. In other particular examples, the
desired activity further includes quantitation of one or more
target biomolecules in the sample.
Unique Emission Signal: An emission signal that conveys information
about a specific event, such as the emission spectrum for a
particular fluorophore, which can be distinguished from other
signals (such as other emission spectrum signals). Examples in
association with the disclosed methods include attaching one or
more individual fluorophores or other labels to different types of
nanoprobes, where each different type of nanoprobe (for example
nanoprobes that are specific for different mutations of the same
protein) has its own individual or own combination of signals (such
as fluorophores that emit at different unique wavelengths). Each
nanoprobe class will have a unique emission signal that in the
examples is based on the fluorophore(s) present on that class of
nanoprobe. This signal can be used to determine which nanoprobe is
interacting with a target biomolecule. Similarly, by attaching one
or more individual fluorophores or other labels to each type of
nucleotide, each different type of nucleotide (A, T/U, C or G) has
its own individual or own combination of signals (such as
fluorophores that emit at different unique wavelengths). Each
nucleotide class will have a unique emission signal that in the
examples is based on the fluorophore(s) present on that class of
nucleotide. This signal can be used to determine which type of
nucleotide (A, T/U, C or G) will be added to a growing
complementary strand of nucleic acid, and these signals in
combination indicate the nucleic acid sequence.
A signal can be characterized not only by different wavelengths but
also by different intensities at various wavelengths, to form a
unique spectrum. In particular, two signals having the same set of
wavelengths can be distinguished if they have some different
intensities at particular wavelengths.
General Strategy
The disclosed nanoprobes include a linker that spaces functional
groups. The linker has a combination of length and flexibility that
substantially maintains the functional groups spaced a desired
distance in the absence of the target biomolecule, but permits them
to substantially interact in the presence of the target
biomolecule. Each functional group can include a targeting moiety
or an activating moiety (such as a labeling moiety), or
combinations thereof. The following table illustrates some such
combinations.
TABLE-US-00001 TABLE 1 Exemplary functional group combinations.
Target Functional group 1 Functional Group 2 Protein
Antibody/Donor* Antibody/Acceptor Protein Antibody/Acceptor
Antibody/Donor Protein Antibody/Donor Nucleic acid
sequence/Acceptor Protein Antibody/Acceptor Nucleic acid
sequence/Donor Protein Antibody Proteinase Protein
Antibody/Acceptor Proteinase/Donor Protein Antibody/Donor
Proteinase/Acceptor Protein Nucleic acid sequence Proteinase
Protein Nucleic acid sequence/Donor Proteinase/Acceptor/Antibody
DNA Nucleic acid probe nuclease DNA Nucleic acid probe/donor
Nucleic acid probe/acceptor DNA Nucleic acid probe/acceptor Nucleic
acid probe/donor DNA Polymerase/donor Non-hydrolyzable dNTPs/
acceptors RNA Nucleic acid probe RNase H RNA Nucleic acid
probe/acceptor RNase H Nucleic acid probe/donor RNA Nucleic acid
probe/donor Nucleic acid probe/acceptor RNA Nucleic acid
probe/acceptor Nucleic acid probe/donor *donor is a donor
fluorophore, acceptor is an acceptor fluorophore
The multi-component nanoprobes need only to maintain potentially
interacting components of the functional group outside of a minimum
distance. Also, since the location of the molecular components can
only be expressed in terms of statistical probabilities, it is
understood that absences of interaction are not absolute but
instead refer to restriction of dynamic molecular movements in a
manner that reduces undesired interactions between functional
groups to a desired level. Once the probe binds to a target
molecule (for example by protein/antibody interaction or nucleic
acid/nucleic acid hybridization) the flexibility of the linker is
sufficient to permit the functional groups to interact (for example
in a donor/acceptor fluorophore interaction) or the target and
unbounded functional group to interact (for example in a
protein/protein interaction). When each functional group includes a
labeling moiety (such as a donor/acceptor) and targeting moiety
(such as an antibody) the first targeting moiety binds to the
target. With the first targeting moiety bound to the target at the
first site, the second targeting moiety then has an increased
statistical probability of interacting with the target at a
preselected second site. With both targeting moieties bound to the
target, the donor/acceptor moieties are maintained in sufficient
proximity for a period of time that permits the donor/acceptor
interaction to occur and emit a detectable signal (or result in
quenching of a detectable signal).
Nanoprobes for Detection or Modification of a Biomolecule
The present disclosure provides nanoprobes for one or more target
biomolecules. In particular examples, the disclosed nanoprobes are
used in vitro, ex vivo, or even in vivo to detect or modify the
target biomolecule. For example, one or more nanoprobes can be
attached to a surface (such as a glass or plastic slide or a
microarray surface), such as via a linker, a biological sample
incubated with the surface, wherein detection of a signal from the
nanoprobe indicates the presence or absence of a target molecule.
The nanoprobes include two or more functional groups that are
selected based on the target biomolecule. Particular examples of
biomolecules that can be targeted include, but are not limited to,
nucleic acid molecules (such as RNA or DNA molecules), antigenic
compounds (such as an antigenic protein), as well as proteins.
In particular examples, nanoprobes provide benefits over currently
available technologies, such as ELISA. For example, the nanoprobes
can provide a rapid method of detecting or modifying a target
biomolecule, are relatively inexpensive to use and manufacture, can
be built using modular designs with interchangeable parts, and can
permit measurement of several parameters simultaneously.
One particular example of a probe for a target biomolecule includes
a molecular linker and first and second functional groups linked
and spaced by the molecular linker. For example, FIG. 1A shows a
nanoprobe 10 that includes first and second functional groups 12,
14 that are linked and spaced by the molecular linker 16. The
functional groups are capable of interacting with one another or
with the target biomolecule in a predetermined reaction, wherein
the molecular linker maintains the first and second functional
groups sufficiently spaced from one another such that the
functional groups do not substantially interact in an absence of
the target biomolecule. In particular examples, they are spaced a
distance from one another to avoid substantial entanglement of the
first and second functional groups in an absence of the target
biomolecule. However, in the presence of the target biomolecule,
the molecular linker permits the first and second functional groups
to sufficiently interact with one another or with the target
biomolecule in the predetermined reaction. For example, in the
presence of the target biomolecule the first and second functional
groups attach to the target in sufficient proximity to permit the
functional groups to interact and yield a signal (such as light) or
a target modifying effect (such as target lysis or
degradation).
As noted above, in particular examples the length of the molecular
rod is one that maintains the first and second functional groups
sufficiently spaced from one another such that the functional
groups do not substantially interact in an absence of the target
biomolecule. Methods are known in the art for determining whether
one functional group interacts with one or more other functional
groups, for example in the presence or absence of a target
molecule. In one example, to determine if a particular length
molecular linker is appropriate, a probe of the present disclosure
having a particular molecular linker length is generated using the
methods disclosed herein. In particular examples, multiple probes
are generated, each having a different molecular linker length. To
identify lengths of molecular linkers that are suitable for use, a
donor fluorophore is attached to one end of the molecular linker
and an appropriate acceptor fluorophore is attached to the other
end of the molecular linker. In particular examples, the probe
includes a FRET pair. To determine if the ends of the molecular
linkers are capable of interacting with one another, the molecular
linker can be placed in a solution in the presence and absence of
the target biomolecule, and acceptor emission fluorescence
detected, for example by spectrophotometry or fluorescence
microscopy. In particular examples, lengths of molecular linkers
that only produce significant acceptor emission fluorescence (for
example above a predetermined threshold) when the target
biomolecule is present, and produce no more than background levels
of acceptor emission fluorescence in the absence of the target
biomolecule, can be used in the probes of the present disclosure.
In contrast, in particular examples, lengths of molecular linkers
that do not produce significant acceptor emission fluorescence when
the target biomolecule is present, or produce levels of acceptor
emission fluorescence that are significantly above background in
the absence of the target biomolecule, are not used in the probes
of the present disclosure. In some examples, the length of the
molecular linker that produces a desirable result can vary
depending on the particular FRET pair used. For example, the length
of the molecular linker used if a GFP/fluorescein FRET pair is part
of the probe, may be different than the length of the molecular
linker used if an Alexa Fluor 430/BODIPY 630 FRET pair is part of
the probe.
In particular examples, the molecular linker (or at least a portion
thereof, such as a portion that includes a molecular rod) is of a
sufficient rigidity to reduce interaction of the first and second
functional groups in the absence of the target biomolecule. For
example, a portion of the molecular linker can have a persistence
length that permits that part of the molecular linker to be of
sufficient rigidity to reduce the interaction of the first and
second functional groups in the absence of the target biomolecule,
while other portions of the molecular linker (such as a tether)
allow interaction of the first and second functional groups (or
interaction of one or more functional groups with the target
biomolecule) in the presence of the target biomolecule.
The total length of the molecular linker can be the same or a
different length than the persistence length for a particular
component of the molecular linker, as long as the length
differential is insufficient to yield undesired interaction of the
functional groups. For example, if the molecular linker includes a
molecular rod that has a particular persistence length, the
molecular linker can be shorter or longer than that persistence
length. For example, if the persistence length of dsDNA is greater
than 150 nt, the total length of the linker can be greater than 150
nt, for example by having tethers or additional rods. Similarly,
the linker can be shorter than 150 nt, for example by having a rod
of 40 nt, and a tether of 2-4 or 3-4 PEG spacer 18 moieties. In
addition, the molecular rod length itself can be shorter or greater
than the persistence length of the polymer used to generate the
molecular rod. In particular examples, a molecular linker includes
a molecular rod, and the total length of the rod is shorter than
the persistence length of the molecule composing the molecular rod
(such as 0.1-times, 0.5-times, or 1-times the persistence length of
the molecule composing the molecular rod). In yet other particular
examples, the length of the linker can be greater or less than the
persistence length of any one of its components. For example, for a
molecular linker that includes a molecular rod, the total length of
the molecular linker is not more than 5-times shorter or longer
than the persistence length of the molecule composing the molecular
rod (such as 1-5 times, 1-4 times, or 1-3 times the persistence
length of the molecular linker that contains the molecular rod). In
one example, the molecular rod is composed of dsDNA, which has a
persistence length of 400-500 .ANG., and the length of the
molecular rod is greater than 400-500 .ANG. (such as 550-700 .ANG.
or 550-1000 .ANG.) or shorter than the persistence length (such as
100-350 .ANG., for example 200-350 .ANG.).
Those skilled in the art will recognize that at one persistence
length the far end of a rod is often still substantially pointing
in the same direction (68.40.degree.) as the original direction and
that a rod of this length, and hence flexibility, can still provide
a useful functional rigidity. Rods of lengths greater than the
persistence length provide a further degree of flexibility that can
be acceptable in some applications. In other applications a single
linker can consist of a single molecule of a uniform kind (as 1000
bp of dsDNA, which is substantially longer than the persistence
length) wherein certain portions of that linker are sufficiently
close (for example 40 bp) that they may act as molecular rods
locally and provide nanoprobe functions locally, while longer
portions of the linker are sufficiently far apart as to act as
molecular tethers that allow the parts to come together or not
depending on Brownian motion and the presence of target molecules
that can be bound. Such a situation occurs when local transcription
factors bind to DNA in essentially rigid positions relative to each
other, while further pieces of DNA can `loop` around to supply, for
example, an enhancer, activator or repressor (as for example in the
GalR binding sites of E. coli, Semsey et al., Genes Dev.
18:1898-907, 2004). Although such nanoprobe constructions are
possible, in general the constructions described herein distinguish
clearly between molecular rods as being not substantially larger
than the persistence length and molecular tethers as being
substantially longer than their corresponding persistence length.
Unlike the dsDNA transcriptional control systems found in nature,
generally in the nanoprobes described herein the molecular rods and
tethers are constructed by connecting different kinds of molecules
that have substantially different persistence lengths as for
example dsDNA with PEG.
The persistence length will vary depending on the composition. For
example, the persistence length for a double-stranded DNA (dsDNA)
molecule differs from that of a single-stranded DNA (ssDNA)
molecule and from polyethylene glycol (PEG). For example, dsDNA has
a persistence length of 400-500 .ANG.. In particular examples,
ssDNA has a persistence length of about 40 .ANG.. In particular
examples, PEG has a persistence length of about 3.8.+-.0.02 .ANG.
(Kienberger et al., Single Molecules 1:123-8, 2000).
To substantially avoid interaction of the first and second
functional groups in the absence of the target biomolecule, and
allow interaction of the first and second functional groups in the
presence of the target biomolecule, the length of the linker is at
least sufficient to maintain the functional groups spaced at least
the Forster radius for the particular donor and acceptor
fluorophores used, such as a distance of 22 to 90 .ANG.. In some
examples, the length of the linker is sufficient to separate
charges on the functional groups, such as a distance of 10 to 1000
.ANG.. In particular examples, the total length of the molecular
linker is about 10 to 500 .ANG., such as 10 to 300 .ANG., 10 to 200
.ANG., 20 to 200 .ANG., 20 to 187 .ANG., 20 to 150 .ANG., 60 to 120
.ANG., or 60 to 200 .ANG..
Examples of molecular linkers include, but are not limited to,
tethers, molecular rods, or combinations thereof. For example, the
molecular linker can include multiple molecular rods linked by
tethers or multiple tethers linked by molecular rods. One
particular example is shown in FIG. 1B, where the nanoprobe 20
includes first and second functional groups 12, 14 that are linked
and spaced by the molecular linker 16, wherein the molecular linker
is composed of a molecular rod 22 linked by tethers 24, 26. Another
particular example is shown in FIG. 1C, where the nanoprobe 30
includes first and second functional groups 12, 14 that are linked
and spaced by the molecular linker 16, wherein the molecular linker
is composed of multiple tethers 32, 34 linked by multiple molecular
rods 36, 38, 40. Although the first and second functional groups
shown in FIGS. 1A-C are shown as a single entity 12, 14, multiple
functional groups can be attached to a single molecular rod or a
single tether. In addition, each of the first and second functional
groups can include multiple functional groups. In a specific
example, each molecular rod is about 100 to 200 .ANG. (such as
about 120-140 .ANG.), and each tether is about 23 to 187 .ANG.
(such as about 60 .ANG.).
The functional groups include molecules that can interact with one
another or with the target biomolecule (or both) to provide a
predetermined reaction, such as a detectable signal or a
modification of a target biomolecule. The functional groups can be
maintained in a spatially separated orientation by a molecular
linker so that the functional groups do not interact to provide the
reaction in the absence of the target molecule. However, the
molecular linker permits the functional groups, under predetermined
conditions, to be brought into sufficient proximity with one
another to interact and produce a predetermined reaction, such as a
detectable signal or modification of a target biomolecule to which
the probe binds or hybridizes, or both.
Examples of functional groups include targeting moieties and
activating moieties, which are brought into activating proximity
when the targeting moiety binds to the target biomolecule. In
particular examples, a targeting moiety brings an activatable
moiety into sufficient proximity to the target for the activatable
moiety to act on the target. For example, if the activatable moiety
is a non-specific proteinase, the target moiety upon interacting
with the target protein brings the proteinase sufficiently close to
the protein target biomolecule to cleave or degrade the target
protein. Therefore, activatable moieties can be used to modify a
target biomolecule, for example to decrease the activity of the
target biomolecule. In another example, if the activatable moiety
is an acceptor fluorophore, the target moiety upon interacting with
the target biomolecule brings a donor fluorophore sufficiently
close to the acceptor fluorophore to excite the acceptor
fluorophore, thereby resulting in the production of a detectable
acceptor emission signal. Therefore, activatable moieties can be
used to permit detection of the interaction of a nanoprobe with the
target biomolecule.
Particular examples of targeting moieties include, but are not
limited to, antibodies, proteins, nucleotides, and nucleic acid
molecules. Particular examples of activating moieties include, but
are not limited to, labels (such as a fluorophore) and modifying
agents (such as such as enzymes, for example nucleases, ligases, or
proteases, and a crosslinkable group such as psoralen). In one
example, the activating moiety is an agent that can cleave the
target biomolecule (for example to decrease the biological activity
of the biomolecule), such as a proteinase or a nuclease. In another
example, the activating moiety is an agent that can stabilize the
target biomolecule, such as a ligase.
One skilled in the art will appreciate that combinations of
functional groups can be used. For example, the functional group
can include both a targeting moiety and an activating moiety, such
as in the example of a labeled antibody. Other particular
combinations of functional groups that can be included on a
nanoprobe include, but are not limited to: an antibody that can
specifically bind to the target protein (such as a DNA binding
protein), and one or more DNA binding sites that can specifically
bind to the target protein; first and second antisense
oligonucleotides that can hybridize to a target nucleic acid
sequence under high stringency conditions; a nucleic acid sequence
capable of specifically hybridizing to a target nucleic acid,
thereby forming a nucleic acid complex (such as a DNA/RNA complex),
and a protein capable of cleaving the nucleic acid complex; one or
more DNA binding sites that can specifically bind to the target
protein and a protein capable of cleaving the protein; and a
polymerase and one or more non-hydrolyzable dNTPs (or other
nucleosides or nucleoside analogues).
In particular examples, one of the functional groups is an
activating moiety that permits detection of the nanoprobe
interacting with the target biomolecule. One particular example is
a label. In particular examples the label is a functional group, or
part of a functional group (such as a labeled antibody). However,
one skilled in the art will recognize that one or more labels can
be attached to any part of the nanoprobe that results in a
significantly decreased signal in the absence of the target
biomolecule, and a detectable signal in the presence of the target
biomolecule. In a specific example, the label includes an acceptor
fluorophore or a donor fluorophore. For example, the acceptor
fluorophore and the donor fluorophores can be part of the first and
second functional groups, or can be linked or otherwise attached to
the probe (for example to the molecular linker, such as to a tether
or to a molecular rod). For example, the acceptor fluorophore can
be linked to the first functional group and the donor fluorophore
linked to the second functional group, or the reverse: the acceptor
fluorophore linked to the second functional group and the donor
fluorophore linked to the first functional group.
Functional Groups
As described above, functional groups may include targeting
moieties and activating moieties, such as agents that interact with
another functional group, with the target biomolecule, or both, to
provide a predetermined reaction, such as a detectable signal or a
modification of a target biomolecule. The disclosed nanoprobes can
include two or more functional groups, such as three or more
functional groups, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 different
functional groups.
In some examples, a functional group is a targeting moiety, such as
one that can specifically bind to a target biomolecule, such as a
protein detection agent (for example a protein, nucleic acid
molecule, or an antibody) or a nucleic acid molecule detection
agent (for example a protein, nucleic acid molecule, or a
nucleotide, for example a non-hydrolyzable dNTP). For example a
functional group can include an antibody that specifically binds to
a target protein. In one example, a functional group includes a
protein (such as a polymerase, for example reverse transcriptase)
that binds to a target nucleic acid molecule. In other examples, a
functional group is one that can specifically hybridize to the
target biomolecule, such as a nucleic acid molecule, for example an
antisense sequence. For example a functional group can include an
antisense molecule that specifically hybridizes to a target nucleic
acid sequence (for example under high stringency conditions).
In yet other examples, a functional group is an activating moiety,
such as one whose biological activity towards the target
biomolecule increases or one that is activated in the presence of
the target biomolecule. For example, the biological activity of the
activating moiety towards the target biomolecule, on a nanoprobe
that includes both a targeting moiety and an activation moiety, can
increase upon the interaction of a targeting moiety with the target
biomolecule, by bringing the activating moiety in sufficient
proximity to the target biomolecule to have the desired effect on
the biomolecule. Particular examples of activating moieties whose
biological activity towards the target biomolecule increases in the
presence of the target biomolecule, include but are not limited to,
agents that can modify the target biomolecule, for example agents
(such as enzymes) that cleave the target biomolecule (such as
nucleases and proteinases, for example RNase H, proteinase K, and
trypsin, as well as restriction enzymes), agents that alter the
interaction between the target biomolecule and the functional group
(such as ligase). For example, agents that cleave the target
biomolecule can be used to decrease the biological activity of the
target biomolecule.
In particular examples, a nanoprobe that includes both a targeting
moiety and an activation moiety decreases non-specific damage (for
example to other components in the cell or in the sample). For
example, a nanoprobe used to degrade a target biomolecule can
include a targeting moiety that will direct the probe to the
biomolecule of interest, and an activation moiety (such as an
enzyme) that can cleave the target biomolecule. Although some
non-specific degradation may occur, this non-specific degradation
is significantly reduced by the presence of the targeting
moiety.
In other examples the activating moiety specifically recognizes and
interacts with, modifies, and/or degrades a particular target such
as a pathogen or pathogenic molecule, such as amyloid protein
plaques of the type found in Alzheimer's disease or some prion
disorders (such as spongiform encephalopathies). Hence the
targeting moiety may be specific for the amyloid protein and the
activating moiety may be a ubiquitinase that specifically interacts
with the amyloid protein to target it to the proteasome without
substantially nonspecifically degrading other intracellular
proteins.
In one example, a functional group is an activating moiety that is
activated in the presence of the target biomolecule. Particular
examples of such activating moieties include labels, such as a
fluorophore. For example if one of the activating moieties is a
donor fluorophore, and another activating moiety is an acceptor
fluorophore, binding of the nanoprobe to the target biomolecule
brings the donor and acceptor fluorophores in sufficient proximity
such that the donor fluorophore can activate the acceptor
fluorophore, thereby producing a detectable acceptor fluorophore
emission signal.
The functional groups selected will depend on the target
biomolecule of interest, and whether the target biomolecule is to
be detected, modified, or both. For example, if the target
biomolecule is a protein, particular examples of targeting moieties
include antibodies and proteins that specifically bind to the
target protein. If the protein is a DNA binding protein, the
targeting moieties can include one or more DNA sequences specific
for the target protein. If the target biomolecule is a nucleic acid
sequence, particular examples of targeting moieties include nucleic
acid molecules that can specifically hybridize to the target
nucleic acid sequence, such as an antisense molecule. In particular
examples an antisense functional group can hybridize to a target
nucleic acid sequence under high stringency conditions.
If detection of the biomolecule is desired, targeting agents that
specifically bind to the target biomolecule can be used, such as a
protein binding agent, for example a protein or an antibody or a
nucleic acid sequence. For example, if detection of the target
biomolecule is desired, even if the target molecule is destroyed or
disassociates from the nanoprobe, a ligase can be used as an
activating moiety to seal the probe after the probe binds to the
target biomolecule. If modification of the biomolecule is desired,
such as reducing the biological activity of the target biomolecule
(such as a reduction of at least 50%, at least 80%, or even at
least 95%), agents that specifically cleave the target biomolecule
can be used. Examples of such agents include, but are not limited
to: proteinases (for example if the target biomolecule is a
protein), RNases (for example if the target biomolecule is an RNA
or a DNA/RNA hybrid), and DNases (for example if the target
biomolecule is a DNA sequence). In particular examples, the
specific cleaving agents can, in addition to recognizing a
particular class of target biomolecules (such as proteins or
nucleic acid molecules), specifically recognize and act upon or
cleave particular targets, such as an enzyme that acts upon a
specific protein substrate.
Other targeting agents include aptamers, which are DNA, RNA, or
protein domains that target proteins, small organic molecules, and
even entire organisms. Such aptamers can serve as targeting moiety
functional groups joined by the linker. For example, aptamers are
known for many agents including IgE (Invitrogen, Catalog Number
02-9788) and hemagglutinin of influenza (Jeon et al., J. Biol.
Chem. 279:48410-9, 2004). In some examples, the aptamer is divided
in half, and each half placed on the two sides of the rod-tether
nanoprobe (for example FIGS. 1B 12, 14). In the absence of the
target, the nanoprobe would not hold together, but in the presence
of the target the parts would come together and produce a
signal.
Tethers
Molecular linkers can include one or more tethers, which can
provide flexibility to the probe. Ideally, tethers are flexible
enough to allow movement of the functional groups, for example to
permit the functional groups to interact with one another, or to
interact with a target biomolecule. The length of the tether should
be sufficient to substantially avoid interaction of the functional
groups in the absence of the target biomolecule, and allow
interaction of the functional groups in the presence of the target
biomolecule. However, the tether is ideally not so long as to
result in entanglement of the tether or the functional groups. In
particular examples, tethers are water soluble and non-toxic.
In particular examples, the length of the tether is long enough to
separate the functional groups in the absence of the target
biomolecule, but not so long as to result in tangling of the
nanoprobe or the functional groups, and short enough to allow the
functional groups to interact with the target biomolecule when
present or with one another.
Examples of particular materials that can be used as tethers
include, but are not limited to, single-stranded DNA molecules,
sugar chains, peptides (such as the connector between two parts of
the RecB protein), and polyethylene glycol (PEG) or any other
flexible polymer having the properties disclosed herein. In a
particular example, a tether is composed of two or more of these
agents. In a specific example a tether includes, or in some
examples consists of, PEG.
In particular examples, the tether is about 10-500 .ANG., such as
20-200 .ANG., 23-187 .ANG., 100-140 .ANG., or 70-94 .ANG., for
example 120 .ANG..
In particular examples, the tether is composed of PEG, such as 3 to
7 units of 18-atom PEG spacers that are 23.4 .ANG. long, such as
2-4 or 3-4 of such spacers. PEG is non-toxic, flexible, hydrophilic
and can be inserted as spacers during DNA synthesis (SyntheGen,
Glen Research).
In one example, the tether is a single-stranded DNA (ssDNA)
molecule, for example having a length of 10-40 nucleotides, such as
10-30 nucleotides, 10-20 nucleotides, for example 10 nucleotides,
20 nucleotides, or 40 nucleotides. In particular examples, a ssDNA
tether can anneal to another nucleic acid strand, thereby
converting a flexible tether into a rigid molecular rod. Ideally,
the sequence is one that does not specifically hybridize to itself,
the functional groups, or to a nucleic acid sequence in the sample
to be analyzed.
In one example, the tether is a sugar chain (for example having a
length of 10-100 sugar moieties, such as 10-75, 10-50, or 20-40
sugar moieties).
Molecular Rods
Molecular linkers can include one or more molecular rods, which can
provide sufficient rigidity to the probe to reduce interaction of
the first and second functional groups in the absence of the target
biomolecule. However, the length of the rod is sufficient to permit
interaction of the functional groups in the presence of the target
biomolecule. In some examples, the presence of a molecular rod in
the nanoprobe reduces the likelihood of entanglement and can
increase the speed of the binding of the nanoprobe to the target
biomolecule.
The disclosed nanoprobes can include one or more molecular rods,
such as at least two molecular rods, for example 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 molecular rods. In one example, use of one or more
molecular rods reduces the required tether length, thereby reducing
the cost and size of the device.
In a particular example, the molecular rod is a double-stranded DNA
(dsDNA) sequence. The length of the dsDNA is one that allows
interaction of the functional groups in the presence of the target
biomolecule, but reduces their interaction in the absence of the
target biomolecule. If the nanoprobe includes donor and acceptor
fluorophores, the length of the dsDNA is one that allows
interaction of the fluorophores in the presence of the target
biomolecule, but reduces their interaction in the absence of the
target biomolecule. In specific examples, the dsDNA molecular rod
is a length that is about equal to the persistence length of
400-500 .ANG.. However, one skilled in the art will recognize that
lengths shorter or greater can be used, as long as the rod reduces
the interaction of functional groups in the absence of the target
biomolecule, and does not result in significant entanglement of a
molecular linker. In specific examples, the dsDNA molecular rod is
150 to 200 nucleotides, such as 10-150 nucleotides, such as 10-140
nucleotides, 20-140 nucleotides, 20-100 nucleotides, 20-50
nucleotides, 30-50 nucleotides, or 30-40 nucleotides, for example
35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides. In
specific examples, the dsDNA molecular rod is at least 10
nucleotides, such as at least 20 nucleotides. In a particular
example, the molecular rod is a dsDNA of 40 bases. Bases are 3.38
.ANG. thick so 40 base pairs is 135 .ANG. long, which is greater
than the typical FRET distance. In a particular example, the
sequence of the dsDNA is chosen using the NANEV program (Goodman et
al., BioTechniques, 38:548-50, 2005).
In other particular examples, the molecular rod is composed of DNA
molecules containing modifications or variants of the DNA, such as
peptide backbone DNA (Peptide Nucleic Acid, PNA) or locked nucleic
acids (LNAs). In particular examples, such DNA variants are used to
alter the helix thermal stability and resistance to nucleases. In
yet another example the molecular rod is composed of carbon
nanotubes (for example nanotubes that are 100-200 .ANG. in length).
In yet other examples, the molecular rod includes bacteria, virus
particles, or viral tail fibers.
Labels
In particular examples, nanoprobes disclosed herein include one or
more detectable labels, for example to permit detection of the
nanoprobe interacting with a target biomolecule. Exemplary labels
that can be used include fluorophores, chemiluminescent agents, and
charge. For example, a change in charge can be detected as the
target biomolecule approaches a capacitor.
In a particular example, a nanoprobe includes an acceptor
fluorophore and a donor fluorophore. Although the figures herein
only show a single donor and acceptor fluorophore on the nanoprobe,
multiple fluorophores can be included on the nanoprobe to increase
the signal or to provide combinations of spectra. Ideally, the
acceptor and donor fluorophores are attached to the nanoprobe in a
position that decreases their interaction in the absence of the
target biomolecule (thereby reducing detectable signal). However,
in the presence of the target biomolecule, the interaction of the
functional groups with the target biomolecule allows the acceptor
and donor fluorophores to interact, such that the donor fluorophore
excites the acceptor fluorophore and the acceptor emits at its
characteristic wavelength, thereby generating a detectable
signal.
In a particular example, the donor fluorophore has a large Stokes
shift. This decreases the excitation of the acceptor fluorophore by
the donor excitation light frequency. Appropriate filtration can
also reduce or remove the excitation wavelength, leaving only the
emission spectrum from the acceptor to be detected.
In a particular example, the donor fluorophore is Green Fluorescent
Protein (GFP). In another particular example, the donor fluorophore
is a chemiluminescent molecule, such as aequorin. Chelated
lanthanides provide bright, large stokes shift, non-bleaching
luminophores with sharp emission spectra, and can therefore be used
as donors. The use of a chemiluminescent molecule as the donor
fluorophore eliminates the need for an external light source.
Particular examples of acceptor and donor fluorophore pairs that
can be used include, but are not limited to: GFP mutant H9-40
(Tsien, 1998, Ann. Rev. Biochem. 67:509) as a suitable donor
fluorophore for use with BODIPY, fluorescein, rhodamine green,
Oregon green, tetramethylrhodamine, Lissamine.TM., Texas Red and
naphthofluorescein as acceptor fluorophores, and fluorophore
3-(.epsilon.-carboxy-pentyl)-3'-ethyl-5,5'-dimethyloxacarbocyanine
(CYA) as a donor fluorophore for fluorescein or rhodamine
derivatives (such as R6G, TAMRA, and ROX) as acceptor fluorophores.
Other particular examples of acceptor and donor fluorophore pairs
include, but are not limited to: 7-dimethylaminocoumarin-4-acetic
acid (DMACA) and fluorescein-5-isothiocyanate (FITC);
7-amino-4-methyl-3-coumarinylacetic acid (AMCA) and
fluorescein-5-isothiocyanate (FITC); and
fluorescein-5-isothiocyanate (FITC) and tetramethylrhodamine
isothiocyanate (TRITC).
Examples of fluorescent dyes that can be particularly used for
attaching a fluorophore to a molecular linker include the Alexa
Fluor series (Molecular Probes, Eugene, Oreg.). In one example,
Alexa Fluor 430 absorbs at 430 nm and, because of its high Stokes
shift, emits far away at 540 nm, and can therefore be used as a
donor fluorophore. Alexa Fluor 430 can be used in particular
examples with Alexa Fluors 546, 555, 568, 594, 647, and BODIPY 630
as acceptor fluorophores since their excitation spectra overlap the
540 nm emission peak of Alexa Fluor 430.
Donor and acceptor molecules can also be designed using bimolecular
fluorescence complementation (BiFC), a technique developed by Hu
and Kerppola (Hu et al., Nat. Biotechnol. 21:539-45, 2003; Hu et
al., Mol. Cell. 9(4):789-98, 2002). Two partial GFP fragments join
to give a complementation and hence fluorescence. The
complementation takes only a few moments but formation of the
chromophore takes a long time, t.sub.1/2=300 seconds. So the method
can be slower than FRET. Because the chromophore forms permanently,
it can be used in a nanoprobe to provide a long-lasting result.
Quantum Dots
In one example, one or more nanoprobes disclosed herein are
attached to fluorescent nanoparticles referred to as quantum dots.
The quantum dot or Cornell dot (silica coated fluorophores) can be
the donor fluorophore, while the nanoprobes attached to the quantum
dot can include one or more corresponding acceptor fluorophores. In
particular examples, the nanoprobes are attached to the quantum dot
directly, or via a linker, such as with antibodies coating the
quantum dot.
The quantum dots can be tethered together, for example with a
molecular linker of a sufficient length to prevent significant
FRET. In another example, quantum dots are tethered together using
a molecular linker. In another example, three dimensional molecular
linkers (such as tetrahedron constructions) keep some nanoprobe
functional groups a significant distance from the surface of a
quantum dot such that when a functional group on the nanoprobe
binds to the target biomolecule, the target biomolecule is brought
to the quantum dot surface and is detected by FRET.
The quantum dot can part of the nanoprobe, thereby replacing a
fluorophore, such as replace a donor, acceptor, or both.
Reducing Photobleaching
Methods of reducing photobleaching are known in the art, and the
disclosed methods are not limited so particular reduction methods.
In one example, confocal microscopy can be used to reduce
photobleaching of fluorophores (described above). Another means
that can be used to reduce photobleaching is to incubate the sample
in a solution containing an oxygen scavenger system, for example as
described by Kitamura et al. (Nature, 397:129, 1999); Okada and
Hirokawa (Science, 283:1152, 1999); Harada et al. (J. Mol. Biol.
216:49, 1990). Examples of solutions include: 1% glucose, 0.05
mg/ml glucose oxidase and 0.1 mg/ml catalase; and 0.5%
2-mercaptoethanol, 4.5 mg/ml glucose, 216 .mu.g/ml glucose oxidase,
36 .mu.g/ml catalase, 2 mM ATP in buffer.
One method that can be used to reduce photobleaching is to coat
fluorophores with calcium phosphate (also known as molecular dots,
see Adair et al., Colloidal Lessons Learned for Dispersion of
Nanosize Particular Suspensions, in Lessons in Nanotechnology from
Traditional and advanced Genetics, 2005). For example, when trapped
inside 60 nm nanoparticles, fluorophores remain extremely stable
and do not significantly decay. For the present probes, small
nanoparticles of about 0.5-2 nm (such as 1 nm to 2 nm) having one
amino group (or other unique attachment point) on the surface can
be used. For example, a tethered fluorophore having an amino group
(to permit attachment of the fluorophore to the desired location on
the nanoprobe) can be coated and attached to a nanoprobe. The
layering can be accomplished by incubating the fluorophore with
carboxyl (--COOH) groups and then adding calcium or aluminum. On
adding phosphate H.sub.2PO.sub.4.sup.-, another layer is formed. In
some examples, gold is used to coat the fluorophores. The resulting
particles have plasmon resonance, possibly enhancing the
fluorescence in addition to the protective coating (see Lakowicz,
Anal. Biochem. 298: 1-24, 2001).
Yet other methods of reducing photobleaching include placing a
nanoprobe proximal to metallic islands (Lakowicz, Anal. Biochem.
298: 1-24, 2001) and incubation in Trolox (Rasnik et al., Nat.
Methods 3:891-3, 2006).
Exemplary Nanoprobes for Detection of Biomolecules
The present disclosure provides multiple examples of nanoprobes
that can be used to detect one or more biomolecules. For example,
such nanoprobes can be used to determine whether a target
biomolecule is present or absent in a sample. In some examples, the
target biomolecule is quantitated.
One particular example of a nanoprobe 50 includes two functional
groups linked by a tether as shown in FIG. 2A. One functional group
includes a protein binding agent 52 (such as an antibody or
protein) and an acceptor fluorophore 54. The other functional group
contains a DNA molecule having one or more protein binding sites
56, and a donor fluorophore 58. The functional groups are joined
via a molecular linker 60 (such as a tether). Such a nanoprobe can
be used to detect binding of a protein to DNA (FIG. 2B). The
protein binding agent 52 specifically binds to the protein 62 that
binds to the protein binding sites 56. One skilled in the art will
appreciate that the positions of the donor fluorophore 58 and the
acceptor fluorophore 54 can be switched. As shown in FIG. 2B, in
the presence of the target biomolecule 62, the targeting moieties
(the protein binding agent 52 and the protein binding sites 56)
bind to the target biomolecule 62 in sufficient proximity to bring
the activating moieties (donor fluorophore 58 and acceptor
fluorophore 54) into sufficient proximity to allow the donor
fluorophore 58 to transfer energy 64 to the acceptor fluorophore
54. This energy transfer activates the acceptor fluorophore 54 so
that it emits at its characteristic wavelength, thereby generating
a detectable signal 66.
The protein binding agent 52 is a targeting moiety that includes
any agent that can specifically bind to a protein of interest, and
ideally is capable of attaching to a tether and a label, such as a
fluorophore. Examples of protein binding agents 52 include, but are
not limited to, antibodies and proteins. Particular examples of
protein binding agents include agents that can bind to a DNA
binding protein. DNA binding proteins include any protein that
binds to double- or single-stranded DNA. Examples include proteins
involved in the regulation of gene expression (including
transcription factors), proteins involved in the packaging of DNA
within the nucleus (such as histones), and nucleic acid-dependent
polymerases involved in DNA replication and transcription, as well
as accessory proteins involved in these processes. One particular
example of a DNA binding protein is p53.
In particular examples, the DNA binding protein is p53, and the
protein binding agent 52 is an agent that can detect p53. Examples
include, but are not limited to, an anti-p53 antibody (such as
pAb421) or a p53-binding protein (such as P53BP1, MDM2, Rad51, TBP,
P300, SRC1, and ACTr). In a particular example, a p53 activating or
non-activating antibody is used. A nanoprobe using an activating
antibody will activate p53 to bind to DNA and so will detect all
p53 molecules while a non-activating antibody will only detect p53
that has been activated by another mechanism. By using these two
nanoprobes with a common donor and different acceptor fluorophores,
the ratio of non-activated to activated p53 can be monitored. Other
p53 antibodies can be used to detect the various different
phosphorylation and acetylation modifications of p53. In addition,
antibodies can be used to detect particular p53 mutations.
The one or more protein binding sites 56 can include at least two
protein binding sites, such as at least three DNA binding sites,
for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 binding site sequences.
The sequence of the protein binding site 56 will depend on the
target protein to be detected. For example, if detection of p53
binding to DNA is desired, the protein binding site 56 can include
or consist of the sequence GAACATGCCCGGGCATGTCC (SEQ ID NO: 1),
GAACATGTCCCAAACATGTTG (SEQ ID NO: 2), or RRRCWWGYYYRRRCWWGYY (SEQ
ID NO: 3) (wherein R is any purine, Y is any pyrimidine, and W is A
or T). Other particular p53 binding sequences are known (for
example see Miner and Kulesz-Martin Nucleic Acids Res. 25:1319-26,
1997; Kaku et al., Nucleic Acids Res. 29:1989-93, 2001, herein
incorporated by reference as to the p53 binding sequences). As
shown in FIG. 2A, the one or more protein binding sites 56 are
represented by three rectangles with crosses. In one example, each
rectangle represents half of a p53 site. p53 could bind to the left
and middle as shown in FIG. 2B, or to the middle and right
rectangles. Since there are two sites for p53 to bind on the
nanoprobe 50 but only one can be bound at a time, the sensitivity
of the p53 assay can be doubled (for example see U.S. Pat. No.
6,774,222).
Another particular example of a nanoprobe that includes two
functional groups linked by a tether is shown in FIG. 3A. Each
functional group includes both a targeting moiety 102, 104 and an
activating moiety 106, 108 where the activating moiety serves as a
label. Such a nanoprobe can be used to detect any antigenic
compound of interest, such as a protein. The nanoprobe 100 includes
at least two protein binding agents 102, 104 (such as an antibody
or protein) linked via a molecular linker 110 (such as a tether).
One antibody 102 carries a donor fluorophore 106 and the other
antibody 104 carries an acceptor fluorophore 108 of a FRET pair.
One skilled in the art will appreciate that the positions of the
donor fluorophore 106 and the acceptor fluorophore 108 can be
switched. As shown in FIG. 3B, in the presence of the target
protein 112, the targeting moieties (protein binding agents 102,
104) specifically bind to the target protein 112, thereby bringing
the two activating moieties (fluorophores 106, 108) into sufficient
proximity to allow the donor fluorophore 106 to transfer energy 114
to the acceptor fluorophore 108. This energy transfer activates the
acceptor fluorophore 108 so that it emits at its characteristic
wavelength, thereby generating a detectable signal 116.
In particular examples, the protein binding agents 102, 104, are
antibodies or proteins that can specifically bind to an antigenic
compound of interest, such as a protein. For example, if the
protein is p53, p53 antibodies or p53 binding proteins can be used
in the nanoprobe shown in FIG. 3A, for example to measure total p53
concentration independently of p53 DNA binding ability. In
contrast, the nanoprobe 50 shown in FIG. 2A would only detect p53
if it were bound to the DNA binding sequence. For example,
nanoprobe 100 in FIG. 3A can be used even if p53 is mutated in a
way that interferes with p53's ability to bind to DNA, and thus
would not be detected by the nanoprobe 50 shown in FIG. 2A. In one
example, the nanoprobe 100 is used for detecting modified proteins.
For example, antibodies specific to a modified protein can
distinguish between the wild type and a modified protein (such as a
mutant protein). For example, anti-phospho-p53 and
anti-acetylated-p53 antibodies are available from commercial
sources (such as Cell Signaling Technology, Inc.). Using these
antibodies, whether or not p53 has been activated and the specific
way in which p53 has been activated can be determined.
In another specific example, the antibodies 102, 104 are matched
antibody pairs. Matched antibody pairs are antibodies that
recognize different domains (such as epitopes) of the same protein,
and therefore can be used to detect an antigenic protein of
interest (such as epidermal growth factor, human growth factor,
Il-8, IL-16, and prostate specific antigen). Such antibodies are
available from commercial sources such as Anogen (Mississauga,
Ontario, Canada). In yet another example the antibodies 102, 104
are specific for biowarfare agents (such as Bacillus anthracis) and
p450 variants. In one example, the antibodies 102, 104 detect a
cancer-specific antigen, such as BRCA1 (for example to determine if
a cancer is BRCA1 positive, or to determine if a particular
treatment modality is appropriate for a subject having cancer), p53
(for example to determine if a cancer is p53 positive, to determine
if a cancer expresses a particular p53 mutation, or to determine if
a particular treatment modality is appropriate for a subject having
cancer). In another example, the antibodies 102, 104 detect human
chorionic gonadotropin (HCG) (for example as a pregnancy test).
In particular examples, a molecular linker includes a molecular
rod, for example as shown in FIG. 4A. The inclusion of a molecular
rod, such as a dsDNA molecule, can increase the rigidity of the
molecular linker and further separate the functional groups in the
absence of the target biomolecule. For example, a nanoprobe with a
molecular rod can have at least two points about which the
antibodies or other functional groups (such as a binding protein)
will move by Brownian motion. That is, there will be at least two
"cloud spheres" each of which represents all the possible locations
of a functional group with respect to the end of a rod as allowed
by tethers. By design, these spheres will intersect to some degree.
In the absence of a target molecule, the nanoprobe can maintain
substantial sphere separation, but ideally the distance between the
two ends of the molecular rod is less than the sum of the two
tether lengths and the target molecule size when held between the
two `hands`. In the presence of the target biomolecule, both
"hands" hold onto the target, bringing the activatable moieties
(such as the donor and acceptor fluorophores) together, thereby
creating a detectable signal. In particular examples, the molecular
rod is used to decrease FRET between the donor and acceptor
fluorophores on the nanoprobe in the absence of the target
biomolecule.
FIG. 4A shows the nanoprobe of FIG. 3A with the inclusion of a
double-stranded DNA sequence as the molecular rod. The nanoprobe
200 includes functional groups 202, 204, 206, 208, linked by a
molecular linker composed of tethers 210, 212 linked by a molecular
rod 214. Each functional group includes both a targeting moiety
202, 204 and an activating moiety 206, 208 where the activating
moiety serves as a label. Such a nanoprobe can be used to detect
any antigenic compound of interest, such as a protein. One skilled
in the art will appreciate that the positions of the donor
fluorophore 206 and the acceptor fluorophore 208 can be switched.
Similar to what is shown in FIG. 3B, in the presence of the target
protein, the protein binding agents 202 204 will specifically bind
to the target protein, thereby bringing the two fluorophores 206
208 into sufficient proximity to generate a detectable signal.
The molecular rod 214 can include a dsDNA sequence that increases
the rigidity of the molecular linker, for example to reduce the
interaction of the protein binding agents in the absence of the
target antigenic compound. In particular examples the rod reduces
the FRET signal in the absence of the target antigenic compound,
while still allowing interaction of the donor and acceptor
fluorophores in the presence of the antigenic compound. In a
particular example, the molecular rod is a dsDNA sequence of 10 to
140 nucleotides, such as 20-100 nucleotides, for example 40
nucleotides. In a specific example the molecular rod is 120
angstroms (.ANG.).
FIG. 4B shows a variation of the nanoprobe 200 shown in FIG. 4A.
The nanoprobe 250 further includes anti-IgG antibodies 252 254. In
a particular example, the anti-IgG antibodies 252 254 are
anti-mouse IgG antibodies, such as a fluorescently labeled
anti-mouse IgG antibody (for example available from Jackson
ImmunoResearch Laboratories, Inc., West Grove, Pa.), which are
bound to mouse anti-target biomolecule antibodies. This approach
allows the anti-target biomolecule antibodies to be exchanged
easily. Therefore, the nanoprobe 250 can be generated without the
anti-target antibodies 202 204, and the end user can attach the
desired mouse anti-target biomolecule antibodies of interest.
By making molecular rod 214 from dsDNA, the two halves of the
nanoprobe can be constructed independently and then annealed to
create rod 214. That is, anti-IgG 252 is attached to fluorophore
206, tether 210 and the upper DNA strand of rod 214 in one
reaction. In a separate reaction anti-IgG 254 is joined to
fluorophore 208, tether 212 and the lower DNA strand of rod 214.
These two components provide a "kit" to an end user. The end user
attaches anti-target antibody 202 to the left component in one
reaction. In a separate reaction, the end user attaches anti-target
antibody 204 to the right component. The end user then joins the
left and the right components to create the final nanoprobe
containing dsDNA rod 214. By this means the anti-IgGs 252 and 254
can be identical, but every nanoprobe contains one anti-target of
type 202 and another anti-target of type 204.
Another particular example of a nanoprobe that includes a molecular
rod is shown in FIG. 5A. In contrast to the nanoprobe 200 shown in
FIG. 4, which showed the functional groups 202 and 204 attached
directly to the tether 210, 212, the nanoprobe 300 shown in FIG. 5A
shows functional groups 302, 304 attached directly to a dsDNA
oligonucleotide 306 and functional groups 308, 310 attached
directly to a dsDNA oligonucleotide 312. Each functional group of
nanoprobe 300 includes both a targeting moiety 302, 308 and an
activating moiety 304, 310 where the activating moiety serves as a
label. By replacing part of the tether 314, 316 with a molecular
rod 306, 312, as well as introducing a molecular rod 318 within the
tether, the region of space explored by the functional group 302,
304, 308 or 310 is reduced to the surfaces of two spheres rather
than the entire volume of two spheres. This can reduce or eliminate
tangling of tethers 314, 316. In one example, oligonucleotide 306,
312 (which in some examples are molecular rods) are included in a
nanoprobe, to allow for generation of a particular molecular
linker. For example, DNA and PEG constructs can be synthesized
commercially. Attachment of fluorophores and other functional
groups to such a molecular linker can be accomplished by use of an
amino group (for example an amino group at the end of the linker,
or within the linker). In particular examples, the oligonucleotides
306, 312 are dsDNA with one amino group per DNA strand. In example,
each amino group is used to attach a functional group. As shown in
FIG. 5A, oligonucleotides 306, 312 connect the functional groups
302, 308 and fluorophores 304, 310, respectively, to the nanoprobe.
Nanoprobe 300 therefore provides a means of construction in which
only one amino group is needed for each strand of DNA/PEG
synthesized.
The nanoprobe 300 can also be used to detect any antigenic compound
of interest, such as a protein. In a particular example, the
nanoprobe 300 includes at least two functional groups 302 and 308
(such as protein binding agents, for example antibodies or
proteins), that are linked together via a molecular linker composed
of two or more molecular rods 306, 312, 318 linked by two or more
tethers 314, 316. The nanoprobe 300 includes a donor fluorophore
304 and an acceptor fluorophore 310. One skilled in the art will
appreciate that the positions of the donor fluorophore 304 and the
acceptor fluorophore 310 can be switched. In a particular example,
at least two functional groups 302 and 308 are anti-mouse IgG
antibodies, which allows anti-target biomolecule antibodies to be
exchanged easily (as described above for FIG. 4B).
As shown in FIG. 5B, in the presence of the target biomolecule 320
(such as an antigenic compound), the protein binding agents 302,
308 will specifically bind to the target biomolecule 320, thereby
bringing the two fluorophores 304, 310 into sufficient proximity to
allow the donor fluorophore 304 to transfer energy 322 to the
acceptor fluorophore 310, upon excitation 324 of the donor
fluorophore 304. This energy transfer activates the acceptor
fluorophore 310 so that it emits at its characteristic wavelength,
thereby generating a detectable signal 326.
In a particular example, the nanoprobe 300 is used to detect a
protein complex. For example, as shown in FIG. 5C, if the target
biomolecule 320 is a protein complex that includes at least two
different biomolecules 328, 330 that can interact and form a
detectable complex, nanoprobes that include protein binding agents
302, 308 that can each specifically bind to one member of a protein
complex can be used. For example, protein binding agent 302 can
detect biomolecule 328 and protein binding agent 308 can detect
biomolecule 330. If the biomolecules 328, 330 interact, then the
distance between the fluorophores 304, 310 decreases, and a
detectable signal 326 will be observed.
Nanoprobes can also be used to detect target nucleic acid
molecules, for example to determine whether a particular nucleic
acid sequence is present or absent in a sample. FIG. 6 shows a
nanoprobe 400 that includes targeting moieties as functional groups
(antisense DNA oligonucleotides 402, 404 that can specifically
hybridize to a target RNA sequence 406), as well as activating
moieties (a donor fluorophore 408 is attached to the 5' end of one
antisense oligonucleotide 402, and an acceptor fluorophore 410 is
attached to the 3' end of the other antisense oligonucleotide 404).
The antisense oligonucleotides 402, 404 are linked via a molecular
linker that includes tethers 412 414 separated by a molecular rod
416. One skilled in the art will appreciate that the positions of
donor 408 and acceptor 410 fluorophores can be reversed. In the
absence of the mRNA target 406 (not part of the nanoprobe) there is
little detectable signal. However, as shown in FIG. 6, when the
antisense oligonucleotides 402, 404 specifically hybridize to the
mRNA target 406, the donor 408 and an acceptor fluorophore 410 are
in sufficient proximity to allow the acceptor fluorophore 410 to
produce a characteristic wavelength of light, resulting in a
detectable signal. In particular examples, the nanoprobe 400 shown
does not require the use of high temperatures to melt a DNA helix,
thereby permitting its use in vivo. For example, the nanoprobe 400
can be introduced into a living cell to detect mRNA inside the
cell. The detectable signal can be measured using known methods in
the art, such as fluorescence microscopy.
A variation of nanoprobe 400 is shown in FIG. 7. The nanoprobe 500
in FIG. 7 includes antisense oligonucleotides 502, 504 that can
specifically hybridize to a target RNA sequence 506, which are
linked via tethers 508 510 that are separated by a molecular rod
512.
A dsDNA molecule 514 is used to attach the ligase 516 via a tether
518. The nanoprobe 400 includes three molecules, which are
hybridized to form the nanoprobe. The first molecule includes
fluorophore 520, antisense oligonucleotide 502, tether 508, the
bottom strand of molecular rod 512, and the top strand of dsDNA
molecule 514. The second molecule includes fluorophore 522,
antisense oligonucleotide 504, tether 510, and the top strand of
molecular rod 512. When the first and second molecules are
hybridized, this results in the formation of molecular rod 512.
Therefore, the sequence of the top and bottom strand of molecular
rod 512 are complementary. The third molecule includes the bottom
strand of dsDNA molecule 514, tether 518, and ligase 516. When the
second and third molecules are hybridized, this results in the
formation of dsDNA molecule 514. Therefore, the sequences of the
top and bottom strand of dsDNA molecule 514 are complementary.
The nanoprobe 500 also includes activating moieties: donor
fluorophore 520 attached to the 5' end of antisense oligonucleotide
502, and acceptor fluorophore 522 attached to the 3' end of
antisense oligonucleotide 504. One skilled in the art will
appreciate that the position donor and acceptor fluorophores can be
reversed. In the absence of the mRNA target 506 (not part of the
nanoprobe) there is little detectable emission signal from the
acceptor fluorophore 522. However, as shown in FIG. 7, when the
antisense oligonucleotides 502, 504 specifically hybridize to the
mRNA target 506, the donor 520 and an acceptor fluorophore 522 are
in sufficient proximity to allow the donor fluorophore 520 to
transfer energy to the acceptor fluorophore 522. This energy
transfer activates the acceptor fluorophore 522 so that it emits at
its characteristic wavelength, thereby generating a detectable
signal. A probe without ligase 516 will detect the target RNA 506
but eventually they may separate again by thermal motions and the
detectable signal will be reduced or eventually disappear
completely. By adding a ligase 516, when the target mRNA 506 is
bound, the nanoprobe 500 is modified so that DNA 502 is joined to
DNA 504 so that there is a detectable signal from the acceptor 522,
even if the target RNA 506 is no longer bound to the nanoprobe. A
particular example of a ligase that can be used is T4 DNA ligase,
which can join DNA in DNA/RNA hybrids (Engler and Richardson,
(1982) P. D. Boyer (Eds.), The Enzymes, 5, pp. 3. San Diego:
Academic Press.).
Nanoprobes can also be used to determine the sequence of a target
nucleic acid molecule. FIGS. 8A-C shows nanoprobes (herein referred
to as medusa nanoprobes) that can be used to sequence a nucleic
acid molecule. As shown in FIG. 8A, the nanoprobe 550 includes a
targeting moiety (polymerase 552) linked to multiple nucleotides
554, 556, 558, 560 that cannot be added to a growing nucleic acid
chain (such as a base that contains a non-hydrolyzable
triphosphate) via molecular linkers 562. The nucleotides 554, 556,
558, 560 can be attached at the base, at the 3' hydroxyl of the
sugar, at the 5' .gamma. phosphate or to any point on a nucleotide
that does not interfere with specific nucleotide binding to the
polymerase or complementary base pairing. The nucleotides 554, 556,
558, 560 can be different nucleotides (as shown in FIG. 8A), or can
be the same nucleotides (in which case nanoprobes with each
nucleotide would be included in the sequencing reaction). The
polymerase 552 includes a donor fluorophore 564, and each
nucleotide 554, 556, 558, 560 includes an acceptor fluorophore 566,
568, 570, 572. For example, if multiple types of nucleotides are on
the same nanoprobe, each nucleotide can include a unique acceptor
fluorophore.
A variation of the nanoprobe shown in FIG. 8A is shown in FIG. 8B.
The nanoprobe 580 includes a polymerase 552 linked to multiple
nucleotides 554, 556, 558, 560 via a molecular linker 562 composed
of multiple molecular rods (for example 582) and tethers (for
example 584). The nanoprobe 580 includes a donor fluorophore 564,
and each nucleotide 554, 556, 558, 560 is associated with a
different acceptor fluorophore 566, 572, 570, 568, respectively. In
this example, primer 586 and target nucleic acid to be sequenced
588 are not part of the nanoprobe 580. However, if desired, primer
586 can be attached to the nanoprobe 580 via a molecular linker
Another variation is shown in FIG. 8C. The nanoprobe 590 includes a
polymerase 552 linked to multiple nucleotides 554, 556, 558, 560
via a molecular linker 562 composed of molecular rods (for example
561, 563, 565, 569, 571, 575, 577, 581, 582) and tethers (for
example 584, 567, 573, 579, 583, 585, 587, 589). The nanoprobe 590
includes a donor fluorophore 564, and each nucleotide 554, 556,
558, 560 is associated with a different acceptor fluorophore 566,
572, 570, 568, respectively. The primer 586 and target nucleic acid
to be sequenced 588 are not part of the nanoprobe 590. However, if
desired, primer 586 can be attached to the nanoprobe 580 via a
molecular linker
Nanoprobes can also be used to detect alternative splicing, for
example as an alternative to molecular beacons. For example, a pair
of nanoprobes with different acceptors can distinguish two
alternative splice junctions (for example, this can be done with a
nanoprobe containing three different molecular linkers). The
nanoprobe can have long homology regions (high Tm) yet if it binds
to a place which is not a target it gives no signal.
A nanoprobe that detected an alternative splice junction can be
joined with a second nanoprobe on in the same molecule. For
example, the left can include an upstream binding arm with a donor
and the right can include two or more downstream binding arms with
different acceptors. Such a probe can be generated using a single
DNA to which and multiple "arms" are annealed by hybridization. For
example, a molecular rod can include multiple tethers. The
left-most tether is for the upstream part of a splice junction. The
down stream two tethers are for the alternative splice
junctions.
Exemplary Nanoprobes for Modification of Biomolecules
The present disclosure provides multiple examples of nanoprobes
that can be used to modify one or more biomolecules. For example,
such nanoprobes can be used to cleave a target biomolecule, for
example to reduce the biological activity of the target
biomolecule.
One particular example of a nanoprobe that can be used to cleave a
target RNA molecule (for example to inactivate the RNA) is shown in
FIG. 9. The nanoprobe 600 includes two functional groups: a
targeting moiety (an antisense oligonucleotide 602) and an
activating moiety (RNase H 604). A molecular linker 606 (such as a
tether) joins the antisense oligonucleotide 602 to the RNase H 604,
wherein the antisense oligonucleotide 602 can hybridize to the RNA
target 608. In the absence of the mRNA target 608 (not part of the
nanoprobe), RNase H 604 will not cut the mRNA target, because RNase
H cuts RNA only in RNA/DNA hybrids. However, in the presence of the
mRNA target 608, the antisense oligonucleotide 602 finds the mRNA
target 608 and will hybridize to the mRNA target. The RNase H 604
can then cleave the mRNA target 608 because the linker 606 holds
them sufficiently close together for a sufficient period of time
for the RNAse H to cut the RNA/DNA hybrid target.
One particular example of a nanoprobe that can be used to cleave a
target protein (such as a DNA binding protein, for example a
transcription factor) is shown in FIG. 10A. The nanoprobe 700
includes two functional groups: a targeting moiety (a dsDNA
molecule 702) and an activating moiety (proteinase K 704). A
molecular linker 706 (such as a tether) joins the dsDNA molecule
702 to the proteinase K 704, wherein the dsDNA molecule 702
includes a sequence that will specifically bind to the target
protein 708 (not part of the nanoprobe). If desired, the nanoprobe
700 could also include a molecular rod (for example as part of the
linker 706). In the absence of the target protein 708 (not part of
the nanoprobe), the proteinase K 704 will not specifically cleave
the protein target (though it may cleave proteins free in
solution). However, in the presence of the target protein 708, the
target protein 708 will specifically bind to the dsDNA 702, and be
in close enough proximity to the proteinase K 704 such that the
proteinase K 704 can then preferentially cleave the protein target
708, thereby significantly reducing the biological activity of the
protein.
Another particular example of a nanoprobe that can be used to
cleave a target protein (such as amyloids, virus or bacterial
components) is shown in FIG. 10B. The nanoprobe 800 includes two
functional groups: a targeting moiety (a protein binding agent 802)
and an activating moiety (proteinase K or other proteinase 804). A
molecular linker 806 joins the protein binding agent 802 to the
proteinase 804, wherein the protein binding agent 802 (such as an
antibody) specifically binds to the target protein 808 (not part of
the nanoprobe). The molecular linker 806 includes tethers 810, 812
separated by a molecular rod 814.
In particular examples, the nanoprobe 800 can further include other
activating moieties, such as a donor fluorophore 816 and an
acceptor fluorophore 818, such as a donor fluorophore 816 attached
to a tether 812 and an acceptor fluorophore 818 attached to another
tether 810. In another example (not pictured), the probe 800
further includes a single-stranded nucleic acid molecule between
tether 812 and the protein binding agent 802, as well as between
tether 810 and the proteinase K 804. The donor fluorophore 816 is
attached to an amino of a single stranded nucleic acid
complementary to the single stranded nucleic acid molecule between
tether 812 and the protein binding agent 802, and the acceptor
fluorophore 818 is attached to an amino of a single-stranded
nucleic acid complementary to the single stranded nucleic acid
molecule between tether 810 and the proteinase K 804. The
single-stranded nucleic acid molecules containing each fluorophore
are hybridized to the nanoprobe, thereby attaching the fluorophores
to the nanoprobe. One skilled in the art will appreciate that the
positions of the donor and acceptor fluorophores can be
reversed.
The molecular rod 814 ideally keeps the proteinase K 804
sufficiently separated from the protein binding agent 802, thereby
reducing or preventing hydrolysis of the protein binding agent 802.
When the target protein 808 (not part of the nanoprobe) is bound by
the protein binding agent 802, it is held in place to be cleaved by
the proteinase K 804. In particular examples, this cleavage can be
observed by detecting a signal from an acceptor fluorophore. In the
absence of the target protein 808, the donor and acceptor
fluorophores 816, 818 will not be in sufficient proximity with one
another to produce a detectable signal from the acceptor 818, and
the proteinase K 804 will not be in sufficient proximity with the
target protein 808 to degrade the target protein. In the presence
of the target protein 808, the donor and acceptor fluorophores 816,
818 are in sufficient proximity with one another to produce a
detectable signal from the acceptor 818, and the proteinase K 804
is in sufficient proximity with the target protein 808 to degrade
the target protein. Therefore, detection of an emission signal from
the acceptor fluorophore (or detection of loss of an emission
signal from the donor fluorophore) indicates that the target
protein is being cleaved.
In a particular example, the nanoprobe 800 shown in FIG. 10B can
include a protein binding agent that specifically binds to
proteinase K 804 (such as an anti-protienase K antibody) on a
tether that links the protein binding agent to the proteinase K,
for example to reduce the biological activity of proteinase K (such
as to reduce degradation of non-target biomolecules). However when
the target protein 808 is bound to the protein binding agent 802,
the protein binding agent that specifically binds to proteinase K
804 will sometimes not be bound to proteinase K, thereby releasing
proteinase K, and permitting the proteinase K to cleave the target
protein 808.
In particular examples, the disclosed nanoprobes that can modify a
target biomolecule can be used as an alternative to siRNA-based
gene silencing methods.
Exemplary Nanoprobes for Detecting and Modifying Biomolecules
The present disclosure also provides multiple examples of
nanoprobes that can be used to both detect and to modify one or
more biomolecules. For example, such nanoprobes can be used to
detect a target biomolecule, for example quantitate an amount of
the target biomolecule present, and also modify the detected
biomolecules.
As described above, nanoprobe 800 shown in FIG. 10B can be used to
detect cleavage of a target protein.
A variation of nanoprobe 600 in FIG. 9 is shown in FIG. 11. Similar
to nanoprobe 600, nanoprobe 900 can be used to cleave a target mRNA
molecule. However, nanoprobe 900 can also be used to quantitate an
amount of mRNA present, for example to determine an amount of
target mRNA present in a sample. The nanoprobe 900 in FIG. 11
includes activation moieties, a donor fluorophore 902 and acceptor
fluorophore 904 that permit detection (and in some examples
quantitation) of RNA as it is cleaved by the RNase 920 (such as
RNase H). Nanoprobe 900 includes two antisense oligonucleotides
906, 908 that can specifically hybridize to a target RNA sequence
910 (not part of the nanoprobe), which are linked via a molecular
linker that includes tethers 912, 914 that are joined by a
molecular rod 916. A dsDNA molecule 918 is used to attach the RNase
920 via a tether 922. The nanoprobe 900 includes three molecules,
which are hybridized to form the nanoprobe. The first molecule
includes fluorophore 902, antisense oligonucleotide 906, tether
912, the bottom strand of molecular rod 916, and the top strand of
dsDNA molecule 918. The second molecule includes fluorophore 904,
antisense oligonucleotide 908, tether 914, and the top strand of
molecular rod 916. When the first and second molecules are
hybridized, this results in the formation of molecular rod 916.
Therefore, the sequence of the top and bottom strands of molecular
rod 916 are complementary. The third molecule includes the bottom
strand of dsDNA molecule 918, tether 922, and RNase 920. When the
first and third molecules are hybridized, this results in the
formation of dsDNA molecule 918. Therefore, the sequences of the
top and bottom strand of dsDNA molecule 918 are complementary.
The nanoprobe 900 includes a donor fluorophore 902 attached to the
5' end of antisense oligonucleotide 906 (such as the 5'
nucleotide), and an acceptor fluorophore 904 attached to the 3' end
of antisense oligonucleotide 908 (such as the 3' nucleotide). One
skilled in the art will appreciate that the position donor and
acceptor fluorophores can be reversed. In the absence of the mRNA
target 910 (not part of the nanoprobe) there is little detectable
signal from the acceptor fluorophore 904 (or alternatively, a
strong, unquenched signal from the donor fluorophore 902). In
addition, when RNase 920 is RNaseH, RNaseH will not cut any unbound
mRNA target 910, because RNase H only cleaves RNA in RNA/DNA
hybrids. However, as shown in FIG. 11, in the presence of the mRNA
target 910 the antisense oligonucleotides 906, 908 specifically
hybridize to the mRNA target 910, allowing the donor 902 and
acceptor fluorophore 904 to be sufficient proximity to allow the
donor fluorophore 902 to transfer energy to the acceptor
fluorophore 904, thereby activating the acceptor fluorophore so
that it emits at its characteristic wavelength, and generating a
detectable signal. The detectable signal will be generated each
time the nanoprobe 900 binds to the target RNA 910. In addition,
when the antisense oligonucleotides 906, 908 specifically hybridize
to the mRNA target 910, this brings the RNase 920 in sufficient
proximity to the formed DNA/RNA hybrid between the mRNA target 910
and the antisense oligonucleotides 906, 908, so that the RNAse 920
can cleave the DNA/RNA hybrid, which will reduce or eventually
eliminate the detectable signal. The nanoprobe 900 then can repeat
the process on a new target RNA 910. Therefore, the cleavage of an
mRNA is accompanied by a FRET signal burst, which can be monitored
to count specific mRNA molecules. The mRNA molecules are destroyed
after counting so there is no duplication. In particular examples,
the total detectable signal is proportional to the number of
destroyed target RNA molecules.
In addition, the nanoprobe 900 can be used to count specific kinds
of splicing in the mRNA. For example, if the left half of the
target RNA 910 is the 3' end of an exon and the right half is the
5' end of the next exon that have been joined by splicing, then the
nanoprobe 900 will detect this particular splice alternative and
generate a detectable signal from the acceptor fluorophore 904.
Other splicing alternatives will not be detected. Therefore,
nanoprobes that are specific for various alternative splice
products (for example by having the sequences of the antisense
oligonucleotides 906, 908 specific for each splice product to be
detected) can be contacted with a sample, to determine which splice
products are present in the sample. By including a different
acceptor fluorophore 904 or combinations of acceptor fluorophores
on each nanoprobe that each recognizes a particular splice product,
two or more nanoprobes added to the sample can be differentiated by
the different acceptor fluorophore signals produced. Therefore, the
generation of specific alternative splicing products can be
monitored in real time.
Purification of Bound Nanoprobes
In some examples, nanoprobes bound to their target molecule are
purified. For example, the resulting complexes can be purified by
applying the reaction mixture to a solid medium containing the
target, such as a column containing beads coated with the target.
This will remove free nanoprobe leaving in the flow-through
nanoprobes complexed with the target. Such purification can in some
examples reduce background. Alternatively, this can be done by
conjugating the target to magnetic beads and removing excess
nanoprobes by a magnet.
Generation of Nanoprobes
Many methods are available for generating the disclosed nanoprobes.
For example, methods of attaching a fluorophore to another molecule
are known. In addition, methods of generating DNA-PEG structures
are known. Although particular methods are provided herein, the
disclosure is not limited to these methods.
DNA/PEG Synthesis and Attachments
In examples where the molecular linker includes one or more DNA
molecular rods and one or more PEG tethers, the following methods
can be used. DNA of any desired sequence can be obtained from a
variety of commercial sources (such as Invitrogen, Synthegen,
Sigma). The sequence of the DNA can be determined using the NANEV
program, which employs evolutionary methods for the design of
nucleic acid nanostructure (Goodman et al., BioTechniques,
38:548-50, 2005). This program can be used to design DNA sequences
in a nanoprobe so that only the desired structure forms by
hybridization. In particular examples a PEG tether is incorporated
as a standard phosphoramidite `spacer` anywhere within the
molecular linker. It is also possible to introduce an amino group
anywhere in the DNA sequence.
By appropriate use of DNA-DNA hybridization, a nanoprobe can be
constructed using only one amino group per DNA/PEG linker. This
allows the amino group to be used to attach a fluorophore or
protein on the nanoprobe, for example as shown in FIG. 5A.
DNA-Protein Conjugation Methods
A synthetic DNA containing an amino group can be attached to a
protein via a unique cysteine (Kukolka and Niemeyer, Org. Biomol.
Chem., 2:2203-6, 2004) or a different chemically modified residue
(Khidekel et al., J. Am. Chem. Soc., 125:16162-3, 2003; Zhang et
al., Science, 303:371-3, 2004; and Klarmann et al., Protein Expr.
Purif., 38:37-44, 2004).
In one example, a nanoprobe includes one antibody attached to a
molecular linker For example, the method of Kozlov et al.
(Biopolymers, 73:621-30, 2004) can be used to make
antibody-oligonucleotide conjugates. In addition, the complexes can
be separated by the ratio of number of antibodies per
oligonucleotide, to obtain those having a 1:1 ratio. A desired
antibody that recognizes a target biomolecule can be attached to an
oligonucleotide commercially (Biosyn, Lewisville, Tex.).
In one example, a single chain antibody (scFv) that recognizes a
target biomolecule includes a Cys on the C-terminus (for example
using the method of Hayashi et al., Gene, 160:129-30, 1995),
thereby allowing the antibody to be attached to an amino-modified
oligonucleotide.
In another example, a monoclonal antibody that recognizes a target
biomolecule (such as a commercially available monoclonal antibody),
is attached to a molecular linker, for example by using the sugars
on IgG antibodies to attach a PEG tether. The sugars are alkylated,
and reduction and amination permits attachment of a PEG tether. In
a particular example, one PEG tether is attached to the
antibody.
In another example, a monoclonal antibody that recognizes a target
biomolecule (such as a commercially available monoclonal antibody),
is attached to a molecular rod, for example by using the two
asparagine-linked oligosaccharides (one per chain) that has two
mannose sugars connected to a GlcNAc and that GlcNAc is attached to
another GlcNAc which is, in turn, attached to the asparagine
residue at the Fc region of the antibody. The enzyme Endo-H (New
England Biolabs) can be used to break the linkage between the two
GlcNAc residues, leaving two GlcNAc residues (one per chain)
attached to the asparagine residue of the Fc region of antibody.
When oxidized, this reduces the number of attached PEG tethers to
four. To reduce the number of tethers further, the Y286L mutant of
the bovine Gal-T1 enzyme or Y289L mutant of the human Gal-T1 enzyme
(Ramakrishnan and Qasba, J. Biol. Chem., 277:20833-9, 2002) can be
used to attach a modified galactose (Khidekel et al., J. Am. Chem.
Soc., 125:16162-3, 2003) resulting in only two PEG chains being
attached to the antibody.
In an example where two PEG tethers are attached to an antibody,
the PEG chains terminate in DNA strands that are complementary.
When the PEG is attached, there are four possible combinations of
DNA, but only two will anneal to each other. These are separable
from the unannealed molecules. One of the two strands will continue
with further PEG leading to the rest of the nanoprobe.
Alternatively, the two tethers can have the same DNA part; these
can be annealed to a DNA containing a direct repeat of the
complementary sequence, which in turn is attached to the remainder
of the nanoprobe. This approach allows for the use of commercially
available antibodies.
In an example where an antibody that recognizes a target
biomolecule is linked by multiple molecular rods, the nanoprobe is
used to detect a DNA-binding protein. For example, a single
antibody can have multiple PEG tethers attached thereto, followed
by DNA. The DNA has a complementary strand and on the far end is
the donor or acceptor fluorophore. The binding site for the protein
is on the far end also (so that the double helix is a spacer).
A particular example of a method that can be used to attach one or
more antibodies to a nanoprobe is provided in Martin and
Papahadjopoulos (J. Biol. Chem. 257:286-8, 1982). Briefly, the
method includes removing an Fc portion from an IgG using Pepsin.
This generates two F(Ab')2 fragments. DTT is used to separate
these, leaving a unique --SH group on each. Maleimidophenyl can be
used to connect the --SH group to the nanoprobe (for example to a
PEG tether or a rod) as described in Martin et al. (Biochemistry,
20:4229-38, 1981).
Methods of Using Nanoprobes to Detect Target Biomolecules
The disclosed nanoprobes can be used to detect biomolecules in
vivo, ex vivo, in vitro or in situ. In particular examples, such
methods are used to diagnose a disease, for example a disease that
is caused by one or more known mutations in a target biomolecule.
In some examples, the nanoprobe is attached to a surface, for
example to provide a rapid-flow, reusable, parallel-detection
method.
In particular examples, the method includes contacting a sample
with one or more of the disclosed nanoprobes under conditions
sufficient for one or more (such as two or more) functional groups
to specifically interact with the target biomolecule, wherein
interaction of the functional groups results in the production of a
detectable signal by a label on the probe. The signal is detected,
wherein the presence of a detectable signal indicates that the
probe interacted with target biomolecule. This indicates that the
target biomolecule is present in the sample. In contrast, the
absence of a detectable signal (for example a signal that is at
least twice the background signal) indicates that the probe did not
interact with the target biomolecule. This indicates that the
target biomolecule is not present in the sample (or that the target
biomolecule is sequestered, protected or destroyed).
In a particular example, the target biomolecule is a DNA binding
protein. DNA binding proteins include the zinc finger proteins,
helix-turn-helix proteins, and leucine zipper proteins. Particular
examples include, but are not limited to: p53, Tus, F is, Lambda
repressor, and Lac repressor. In this example, one functional group
can include a protein binding agent (such as an antibody or protein
that specifically binds to the DNA binding protein), and another
functional group can include a nucleic acid sequence that can
specifically bind to the DNA binding protein. For example, as shown
in FIG. 2B, the nanoprobe 50 can be used to detect a DNA binding
protein 62. When the DNA binding protein 62 binds to the binding
sites 56 and the protein binding agent 52 binds to the DNA binding
protein 62, the donor 58 fluorophore and acceptor 54 fluorophore
are brought close enough to create FRET 64, which results in an
emission signal 66 from the acceptor fluorophore 54 that can be
detected. In contrast, in the absence of the DNA binding protein
62, there is no significant acceptor emission signal 66.
In a particular example, the probe includes a donor and an acceptor
fluorophore, wherein interaction of the functional groups brings
the donor and acceptor fluorophores into proximity to permit
excitation of the acceptor fluorophore by resonance with the
excited donor fluorophore. In this case, detecting the signal can
include detecting the fluorescent signal emission from the acceptor
fluorophore or detecting a decrease in the fluorescent signal
emission from the donor. In a particular example, the acceptor
fluorophore is a quencher, and interaction of the functional groups
brings the donor and acceptor fluorophores into proximity to permit
quenching of the donor fluorophore emission by the acceptor
quencher. In this case, detecting the signal can include detecting
a decrease in the fluorescent signal emission from the donor
fluorophore.
In examples where the probe includes a donor and an acceptor
fluorophore, the method can include exposing the sample to a light
source, such as a laser, at the appropriate wavelength to excite
the donor fluorophore. However, if the donor fluorophore is
replaced or excited by a chemiluminescent molecule, the laser can
be omitted.
The sample can include any biological sample that may contain the
target biomolecule. For example, the sample can include a cell
extract that contains one or more proteins, microbes, or nucleic
acid molecules. If desired, the proteins and nucleic acid molecules
can be in a purified or concentrated form. In a particular example,
the sample is a tissue section, such as a tissue slice. For
example, a tissue array that includes specimens from many different
subjects permits screening of a large number of such specimens, for
example simultaneously. In one example, if detection of a nucleic
acid molecule is desired, the sample can be exposed to one or more
proteases. If desired, agents can be subsequently added to
substantially neutralize the proteases, or the proteases can be
removed. In another example, if detection of a protein is desired,
the sample can be exposed to one or more nucleases. If desired,
agents can be subsequently added to substantially neutralize the
nucleases, or the nucleases can be removed. In some examples, the
sample includes one or more cells that may contain the target
biomolecule. In such examples, the sample is exposed to the probe
under conditions that permit the probe to enter the cell. In
particular examples, the nanoprobe is present in a liposome,
thereby permitting entry of the nanoprobe into the cell.
In particular examples, the sample is obtained from a subject. A
biological sample from a subject (such as a cheek swab) can be used
directly, or can be manipulated, such as concentrated or purified.
In one example, proteins or nucleic acid molecules are purified
from the sample, prior to contact with the probe.
In some examples, one or more of the disclosed nanoprobes is
administered to a subject, and the detection performed in vivo. For
example, the nanoprobe can be administered on or under the skin,
and a light source (such as a laser) directed to the skin, and the
resulting fluorescence detected. In some examples where the
detection is in vivo, one or more nanoprobes are introduced into a
live cell, for example using a liposome. Upon introduction of a
nanoprobe into a cell, it should take only seconds (or less) to
detect (or modify) the target biomolecule.
Use of Total Internal Reflection (TIR) to Detect Target
In a particular example, the disclosed nanoprobes are used in
combination with total internal reflection (TIR) to detect one or
more target biomolecules. In particular examples, such methods are
used to detect the presence of a particular nucleic acid, protein,
microbe (e.g., virus, bacteria, fungi), or other biomolecule, for
example to diagnose disease or to determine if an area is
contaminated (such as a water source). The nanoprobe can be
attached to a solid substrate, such as a glass slide or optic fiber
(e.g., an optic fiber with its cladding removed; see for example
Fang and Tan, Anal. Chem. 71:3101-5, 1999, herein incorporated by
reference). In one example, the nanoprobes are attached to a
substrate, such as a glass microscope slide having a
biotin/streptavidin surface (wherein the nanoprobe would include
for example a biotin, such as a biotinylated oligonucleotide, to
permit it to be attached to the substrate).
In some examples, the nanoprobes include an appropriate FRET donor
and acceptor pair. If multiple targets are to be detected, the
nanoprobes for each target can have a unique donor/acceptor pair,
which permits for the determination of which target is detected.
For example, nanoprobe 1 specific for biomolecule 1 can have donor
A and acceptor B, while nanoprobe 2 specific for biomolecule 2 can
have donor A (or another donor, such as donor D) and acceptor C,
and so forth. In this way, each target has a unique acceptor
fluorophore associated with it. Thus, if a signal from acceptor B
is detected, but no signal for acceptor C is detected, this
indicates the presence of biomolecule 1 in the sample, but not
biomolecule 2.
In another example, if multiple targets are to be detected, the
nanoprobes for each target can have the same donor/acceptor pairs,
but be located on different regions of the surface (e.g., in
different wells of the surface) to implement spacial detection in
parallel of different substances in the same sample. For example,
nanoprobe 1 specific for biomolecule 1 can have donor A and
acceptor B and be located in well or region 1 of the substrate,
while nanoprobe 2 specific for biomolecule 2 can have donor A and
acceptor B, but be located in well or region 2 of the substrate,
and so forth. In this way, each target has a unique region on the
substrate associated with it. Thus, if a signal from well or region
1 is detected, but no signal for well or region 2 is detected, this
indicates the presence of biomolecule 1 in the sample, but not
biomolecule 2.
The sample known or suspected of containing the one or more target
biomolecules is applied to (or contacted with) the substrate
containing the nanoprobes under conditions sufficient for one or
more (such as two or more) functional groups to specifically
interact with the target biomolecule, wherein interaction of the
functional groups results in the production of a detectable signal
by a label on the probe. As discussed herein, any sample can be
used. A laser that produces light with excitation frequency for
donor fluorophores present on the nanoprobes can be used. Exemplary
lasers include a 473 nm blue laser, 532 green laser, a 635 nm red
laser, and so forth. The laser light enters the substrate (e.g., an
optic fiber or glass slide). The light is trapped in the substrate
by TIR.
In the presence of the target(s), nanoprobes on the surface of the
substrate will bind to the target molecule and provide a FRET
signal using energy from the TIR evanescent wave. Some of this
light propagates downwards through the substrate, for example
through a filter that removes stray light from the laser, and
passes light from FRET to a detector. The resulting signal from the
acceptor fluorophore is detected if the target is present. In
contrast, the absence of a detectable signal from the acceptor
fluorophore (for example a signal that is at least twice the
background signal) indicates that the probe did not interact with
the target biomolecule, which indicates that the target biomolecule
is not present in the sample (or that the target biomolecule is
sequestered, protected or destroyed).
FIG. 24 shows an example of how TIR and the nanoprobes provided
herein can be used to detect desired targets. The box on the upper
left shows how the nanoprobe works. The top image is a small filled
ellipse representing the target molecule to be detected, the middle
image is a T shape with arms representing the nanoprobe with rod
and tethers (not shown are the donor and acceptor fluorophores and
the target binding moieties), and the bottom image represents the
nanoprobe bound to target. In this mode FRET can occur. The black
rounded rectangle on the left represents the laser producing light
(rightward arrow) with excitation frequency for donor fluorophores.
The laser light enters the solid substrate, such as an optic fiber
or glass slide. The light is trapped in the substrate by TIR.
Attached to and above the substrate is a well shown in cross
section (brick pattern) containing a sample (such as a drop of
blood, large hemisphere). Red blood cells (bone shape) are opaque
but cannot block the detection process since that occurs at the
surface. Nanoprobes on the surface bind to the target molecule and
provide a FRET signal using energy from the TIR evanescent wave.
Some of this light propagates downwards through the optic fiber or
glass slide, through a filter that removes stray light from the
laser and passes light from FRET to a detector (hatched object on
bottom).
Thus, provided herein are devices that can be used to detect one or
more targets. For example, the device can include a solid substrate
(such as an optic fiber or glass slide), having attached thereto
one or more nanoprobes for one or more target molecules. The
substrate can also include structures to permit formation of a
well, which can contain a solution (for example may contain saline,
which can be removed before addition of the sample to the well).
The device can include a lid or seal (which in some examples is
opaque) over the well. When ready for use, the lid or cover can be
removed, and the sample added. The lid can be reapplied to seal the
well, and the device agitated (e.g., stirred, shaken or vibrated)
to permit the target molecules in the sample to interact with the
nanoprobes on the substrate.
Use of Nanoprobes for Sequencing
One particular type of detection includes sequencing of a target
nucleic acid molecule. In particular examples, one or more
nanoprobes are used to sequence a target nucleic acid sequence. For
example, referring to FIG. 8A, the method can include contacting a
target nucleic acid sequence 574 with an oligonucleotide primer 576
and a nanoprobe 550 (or those shown in FIGS. 8B and 8C), in the
presence of a mixture of non-labeled hydrolysable nucleotides. In
the absence of a nucleic acid molecule to be sequenced 574 (not
part of the nanoprobe) there is little detectable signal. When the
polymerase 552 is bound to a target nucleic acid sequence 574 at a
primer 576, a base 578 is exposed on the template strand 574. The
nucleotides 554 556 558 560 at the ends of the molecular linkers
562 compete for binding to base 578, but only one of the four
nucleotides (in this case 554) will be complementary to the exposed
base 578. The non-complementary bases (in this case 556 558 560)
will quickly dissociate, but the complementary base 554 will dwell
for a substantial time in the binding pocket of the polymerase 552.
During this time, the corresponding acceptor fluorophore 566 on the
complementary base 554 will be in sufficient proximity to the donor
fluorophore 564 attached to the polymerase 552 for FRET to occur,
producing a characteristic acceptor emission signal. All of the
nucleotides 554 556 558 560 will diffuse in and out of the active
site, but the linker 562 with the base that is complementary to the
template will dominate the signal because it occupies the active
site the longest. Thus the nanoprobe will report the next base that
would be incorporated into the target nucleic acid sequence 574,
but since the nucleotides 554 556 558 560 cannot be added to a
growing nucleic acid chain, no reaction will take place. A small
concentration of hydrolysable dNTPs (or NTPs for an RNA polymerase)
is provided in the reaction solution. The appropriate hydrolysable
dNTP will eventually be incorporated into the nascent strand 576
and the nanoprobe 550 will step forward one position. This exposes
the next complementary base and so a (possibly) different acceptor
fluorophore signal will be emitted. The varying acceptor
fluorophore signals correspond to the nucleotide sequence.
Methods of Using Nanoprobes to Modify Target Biomolecules
The disclosed nanoprobes can be used to modify biomolecules in
vivo, ex vivo, in vitro or in situ. In particular examples, such
methods are used to treat a disease, for example a disease that is
caused by the undesired expression of a target biomolecule. For
example, the disclosed nanoprobes can be used to cleave a target
biomolecule, thereby reducing the biological activity or even
inactivating the target biomolecule.
In particular examples, the method includes contacting a sample
with one or more of the disclosed nanoprobes under conditions
sufficient for the functional groups to specifically interact with
the target biomolecule, wherein this interaction results in the
modification of the target biomolecule. In particular examples, the
method further includes detecting the modification of the target
biomolecule, for example by detecting a signal generated by a label
on the probe. For example, if the probe includes a donor and an
acceptor fluorophore, interaction of the functional groups brings
the donor and acceptor fluorophores into a proximity to permit
excitation of the acceptor fluorophore by the donor fluorophore. In
this case, detecting the signal can include detecting the
fluorescent signal emission from the acceptor fluorophore.
In examples where the probe includes a donor and an acceptor
fluorophore, the method can include exposing the sample to a light
source, such as a laser, at the appropriate wavelength to excite
the donor fluorophore. However, if the donor fluorophore is
replaced by a chemiluminescent molecule, this step can be
omitted.
In a specific example, one of the functional groups includes a
nucleic acid sequence that can specifically hybridize to a target
nucleic acid, thereby forming a complex, and one of the functional
groups includes a protein that can cleave the complex. Upon
hybridization of the nucleic acid sequence to the target
biomolecule, for example forming a DNA/RNA complex, the protein
(such as RNase), can cleave the complex.
As described above, the sample can include any biological sample
that may contain the target biomolecule.
Methods of Using Nanoprobes for Treatment
The disclosed nanoprobes can be used to treat a subject having a
disorder related to a target biomolecule. In particular examples,
the methods are used to treat a disease, for example a disease that
is caused by the undesired expression or biological activity of a
target biomolecule. For example, the disclosed nanoprobes can be
used to cleave a target biomolecule, thereby reducing the
biological activity or even inactivating the target
biomolecule.
In particular examples, the method includes administering a
therapeutic amount of one or more of the disclosed nanoprobes to a
subject, wherein the subject has a disorder that can be treated by
decreasing the activity or expression of the target biomolecule. In
some examples, the nanoprobe includes a protease or nuclease (such
as RNAase) as one of the functional groups, and another functional
group that can specifically bind to or hybridize to the target
biomolecule.
Any mode of administration can be used, and the method can be
determined by a skilled clinician.
Example 1
Nanoprobe for Detecting DNA Binding by a Protein
This example describes a nanoprobe that includes as functional
groups an agent that can specifically bind to a DNA binding protein
(such as an antibody or a protein) and a nucleic acid molecule.
Such a nanoprobe can be used to detect protein binding to a DNA
molecule. Although particular functional groups are described for
the detection of p53 binding to a target nucleic acid sequence, one
skilled in the art will recognize that other p53 detection agents
and other p53 target nucleic acid sequences can be used to detect
the DNA/p53 protein interaction. Similarly, one skilled in the art
will recognize that other binding agents and other nucleic acid
sequences can be used to detect the DNA/protein interaction of
interest.
FIG. 2A shows an exemplary nanoprobe that can be used to detect
binding of p53 to a target nucleic acid sequence. For example, the
protein binding agent 52 can be an anti-p53 antibody, and the
protein binding site(s) 56 is a nucleic acid sequence that
specifically binds to p53.
Antibodies that specifically bind to p53 are commercially
available, or can be made using routine methods in the art.
Particular examples of commercially available p53 antibodies
include, but are not limited to, those shown in Table 2 (all
available from Calbiochem):
TABLE-US-00002 TABLE 2 p53 antibodies Anti-p53 Antibodies that
detect phosphorylation at Fluorescein - anti-p53 Anti-p53 Abs
particular Serines conjugates OP03 Anti-p53 (Ab-1) PC386
PhosphoDetect .TM. OP03F Anti-p53 (Ab-1) (Pantropic) Mouse mAb
Anti-p53 (pSer15) (Ab-3) (Pantropic) Mouse mAb (PAb421) Rabbit pAb
(PAb421) Fluorescein Conjugate OP104L Anti-p53 (Ab-11) PC461
PhosphoDetect .TM. OP43F Anti-p53 (Ab-6) (Pantropic) Mouse mAb
Anti-p53 (pSer15) (Ab-6) (Pantropic) Mouse mAb (PAb1802) Rabbit pAb
(DO-1) Fluorescein Conjugate OP140 Anti-p53 (Ab-12) DR1023
PhosphoDetect .TM. (Pantropic) Mouse mAb Anti-p53 (pSer20) Rabbit
(DO-7) pAb OP09 Anti-p53 (Ab-2) PC387 PhosphoDetect .TM.
(Pantropic) Mouse mAb Anti-p53 (pSer392) (Ab-4) (PAb1801) Rabbit
pAb OP29 Anti-p53 (Ab-3) PC387T PhosphoDetect .TM. (Mutant) Mouse
mAb Anti-p53 (pSer392) (Ab-4) (PAb240) Rabbit pAb OP32 Anti-p53
(Ab-4) 506133 PhosphoDetect .TM. (Wild type) Mouse mAb Anti-p53
(pSer392) Mouse (PAb246) mAb (9F4) OP33 Anti-p53 (Ab-5) DR1024
PhosphoDetect .TM. (Wild type) Mouse mAb Anti-p53 (pSer46) Rabbit
(PAb1620) pAb OP43 Anti-p53 (Ab-6) (Pantropic) Mouse mAb (DO-1)
PC35 Anti-p53 (Ab-7) (Pantropic) Sheep pAb OP73 Anti-p53 (Ab-8)
(Pantropic) Mouse mAb (BP53.12)
Both p53 activating and non-activating antibodies can be used. When
a nanoprobe includes a p53 activating antibody, the nanoprobe can
detect activated or non-activated p53 binding DNA. In contrast, if
the nanoprobe includes a p53 antibody that does not activate p53,
only previously activated p53 can be detected by its binding to the
nanoprobe. In one example, the nanoprobe includes p53 antibodies
that can detect different phosphorylation and acetylation
modifications of the p53. In yet another example, the nanoprobe
includes p53 antibodies that specifically bind to wild-type or
mutated p53, permitting the detection of a particular p53 mutation.
Particular examples of p53 antibodies that can be used include, but
are not limited to: pAb421, and those available from Biodesign
International (Saco, Me.), Calbiochem (San Diego, Calif.) and
Epitomics (Burlingame, Calif.).
As an alternative to using an antibody as the protein binding agent
52 (FIGS. 2A and 2B), a p53-binding protein can be used. Examples
of p53 binding proteins that can be used include, but are not
limited to: p53BP1, p53BP2, MDM2, Rad51, TBP, P300, SRC1, BRCA1,
and ACTr. Such proteins can be made recombinantly using standard
molecular biology methods. For example, Origene (Rockville, Md.)
commercially provides clones that can be used to produce the
following p53 binding proteins: TC116621 NM.sub.--005657 Homo
sapiens tumor protein p53 binding protein, 1 (TP53BP1); TC111040
NM.sub.--005426 Homo sapiens tumor protein p53 binding protein, 2
(TP53BP2), transcript variant 2; TC108990 NM.sub.--007294 Homo
sapiens breast cancer 1, early onset (BRCA1), transcript variant
BRCA1a; TC123899 NM.sub.--007296 Homo sapiens breast cancer 1,
early onset (BRCA1), transcript variant BRCA1a'; TC108993
NM.sub.--007297 Homo sapiens breast cancer 1, early onset (BRCA1),
transcript variant BRCA1-delta2-10; TC115590 NM.sub.--007306 Homo
sapiens breast cancer 1, early onset (BRCA1), transcript variant
BRCA1-exon4; and TC118660 NM.sub.--002392 Homo sapiens Mdm2,
transformed 3T3 cell double minute 2, p53 binding protein (mouse)
(MDM2), transcript variant MDM2.
The protein binding site(s) 56 that include a nucleic acid sequence
that specifically binds to p53, can include at least one binding
site, such as at least two, or at least three binding sites, for
example 1, 2, 3, 4, or 5 binding sites. One particular example of a
protein binding site sequence that can be used to detect p53
includes, but is not limited to:
TABLE-US-00003 (SEQ ID NO: 4)
GGACATGTCCGGACATGTCCGCGAAGCGGACATGTCCGGACATGTCC.
In one example, the protein binding site(s) 56 includes at least
two binding sites. For example, when a DNA binding protein 62 such
as p53 has two adjacent sites that overlap, only one can be bound
at a time, so the effective binding constant is doubled. As shown
in FIG. 2A, each half binding site 56 is represented by a rectangle
with a cross (so there are two distinct overlapping DNA binding
sites shown in FIG. 2A). Each rectangle in one example represents
half of a p53 site. p53 could bind to the left and middle as shown
in FIG. 2B, or to the middle and right rectangles.
In a particular example, a nanoprobe that can be used to detect p53
is generated as follows. The protein binding agent 52 (such as an
antibody) is attached to a PEG tether 60 of oligonucleotide
containing p53 binding sites (such as SEQ ID NO: 4) by using a
bifuncational cross-linker, such as succinimidyl
4-hydrazinonicotinateacetone hydrazone (SANH), EDC
(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), or
AMAS (N-(.alpha.-Maleimidoacetoxy)succinimide ester) (for example
for amine-sulfhydryl cross-linking). For example the protein
binding agent 52 includes a Cys, and the PEG includes an NH.sub.2
group on the end, the NH.sub.2 and Cys can be linked using AMAS as
follows. Briefly, the protein binding agent 52 is dissolved in the
manufacturer's Conjugation Buffer at 0.1 mM (such as 5 mg in 1 ml
for a 50 kDa protein). The cross-linker is added to the protein
binding agent 52 at 1 mM final (=10-fold molar excess) by
dissolving 2.52 mg AMAS in 1 ml DMSO (makes 10 mM) and then adding
100 .mu.l/ml of protein binding agent 52. The mixture is incubated
for 30 minutes at room temperature or 2 hours at 4.degree. C.
Excess cross-linker is removed using a desalting column
equilibrated with Conjugation Buffer. The commercial desalting
column product instructions allow one to determine which fractions
contain protein binding agent 52. Alternatively, the protein
binding agent 52 can be located by measuring for fractions having
peak absorbance at 280 nm; however, the NHS-ester leaving group
also absorbs strongly at 280 nm. The protein binding agent 52-SH
and desalted protein binding agent 52-NH.sub.2 are combined and
mixed in a molar ratio corresponding to that desired for the final
conjugate and consistent with the relative number of sulfhydryl and
activated amines that exist on the two proteins. Incubate the
reaction mixture at room temperature for 30 minutes or 2 hours at
4.degree. C. However, there is generally no harm in allowing the
reaction to proceed for several hours or overnight, although
usually the reaction will be complete in the specified time. To
terminate the conjugation reaction before completion, add buffer
containing reduced cysteine at a concentration several times
greater than the sulfhydryls of antibody-SH.
A FRET pair will be placed on the protein binding site and the
p53-binding agent (such as an antibody or protein) 58 such that
proximity of the fluorophores will produce a characteristic signal
after p53 binds to the DNA.
Example 2
Nanoprobe to Detect Antigenic Compounds
This example describes a nanoprobe that includes as functional
groups agents that can specifically bind to a protein (such as an
antibody or a protein). Such a nanoprobe can be used to detect one
or more target proteins. Although particular functional groups are
described for the detection of p53 (for example total p53
concentration independent of p53 binding ability), one skilled in
the art will recognize that other specific binding agents can be
used to detect p53. Similarly, one skilled in the art will
recognize that other binding agents can be used to detect the
target protein of interest.
In a particular example, the nanoprobe shown in FIG. 3A includes
anti-p53 antibodies as the protein binding agents 102, 104 that are
connected by a PEG tether 110. In a particular example, the
anti-p53 antibodies are pAb421, or those available from Biodesign
International (Saco, Me.), Calbiochem (San Diego, Calif.) (see
Table 2) and Epitomics (Burlingame, Calif.). One antibody 102
carries a donor fluorophore 106 and the other antibody 104 carries
an acceptor fluorophore 108 of a FRET pair. In a particular
example, the donor fluorophore 106 is FAM and the acceptor
fluorophore 108 is Texas Red.
In one example, the nanoprobe 100 is used for detecting modified
proteins. Antibodies specific to a modified protein can distinguish
between the wild type and a modified protein. For example,
anti-phospho-p53 and anti-acetylated-p53 antibodies are available
from commercial sources (such as Cell Signaling Technology, Inc.
and Calbiochem). Using these antibodies, whether or not p53 has
been activated and the specific way in which p53 has been activated
can be determined
In a particular example, a nanoprobe that can be used to detect an
antigenic compound is generated as follows. The protein binding
agents 102, 104 are attached to a PEG tether 110 by using a
bifuncational cross-linker, such as succinimidyl
4-hydrazinonicotinateacetone hydrazone (SANH), EDC
(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride), or
AMAS (N-(.alpha.-Maleimidoacetoxy)succinimide ester), for example
using the method described in Example 1.
A donor fluorophore is attached to one of the protein binding
agents and the acceptor fluorophore is attached to the other
protein binding agent. In one example, antibodies are labeled with
a fluorophore using a commercially available kit, using the
manufacturer's instructions (for example from Pierce, Rockford,
Ill.).
The FRET pair will produce a characteristic signal upon binding of
the protein binding agents 102, 104 to the antigenic compound, such
as p53.
Example 3
Nanoprobes with a DNA Separation Rod
This example describes a nanoprobe that includes a DNA sequence as
the molecular rod. Such a nanoprobe can be used to detect a target
antigenic compound, such as a protein. Although particular DNA
molecular rods are described, one skilled in the art will recognize
that variations can be made. For example, the length of the
molecular rod can be changed, and the exact sequence of the DNA can
be changed.
In a particular example, the nanoprobe 200 shown in FIG. 4A is used
to detect p53. For example, the nanoprobe 200 can include anti-p53
antibodies as the protein binding agents 202, 204 that are
connected by a molecular rod that includes a PEG tether 210, 212
(each tether is 2-4 units of Spacer 18) separated by a dsDNA
molecular rod 214 of 40 nucleotides (such as
GACGCTAGTATCTTATGAAGCTTTCCTGACTGCGGCATTA (SEQ ID NO: 5) hybridized
to its complementary strand). In a particular example, the donor
fluorophore 206 is 6-FAM and the acceptor fluorophore 208 is Texas
Red.
In a particular example, the nanoprobe shown in FIG. 4A is
generated by synthesizing half-nanoprobe molecules, which are then
joined together by annealing complementary single stranded DNAs to
form the molecular rod 214. Half-nanoprobe molecules are
synthesized that have (1) --NH.sub.2 or --SH groups on the terminus
for attachment to an antibody, (2) one or more fluorescently
labeled nucleotides (all donor or acceptor for a particular
half-nanoprobe), (3) PEG linkers, and (4) 40 bases (for example) of
unique DNA sequence (single copy sequence). Two such
half-nanoprobes are constructed that have complementary single copy
DNA sequences. After each half-nanoprobe is attached to a
corresponding antibody, the two half-nanoprobes are annealed to
create the full nanoprobe (FIG. 4A).
For example, the molecule
NH.sub.2-[PEG18][PEG18]-GACGCTAGTATCTTATGAAGCTTTCCTGACTGCGGCATTA
(SEQ ID NO: 6) and the molecule
NH.sub.2-[PEG18][PEG18]-TAATGCCGCAGTCAGGAAAGCTTCATAAGATACTAGCGTC
(SEQ ID NO: 7) are generated (for example by Integrated DNA
Technologies), wherein [PEG18] is 1 unit of Spacer 18 (Integrated
DNA Technologies). The antibodies are attached to the NH.sub.2
group, and the two molecules incubated under conditions that permit
the two complementary DNA strands to hybridize, thereby generating
the nanoprobe shown in FIG. 4A. Exemplary incubation conditions
include overnight incubation in binding buffer (100 mM NaCl, 10 mM
Tris HCl pH=7.5, 100 .mu.M EDTA) at room temperature.
The 40 bases of DNA separate the fluorescent donor from the
acceptor well beyond the FRET limit Bases are 3.38 .ANG. thick so
40 base pairs are 135 .ANG. long, which is greater than the typical
FRET distance. However, the exact length used can be varied.
Commercially available 18-atom PEG spacers are 23 .ANG. long, so
2-4 of them are sufficient to bring the antibodies together at the
center of the nanoprobe. This can significantly reduce the
background FRET signal. Yet, when the two antibodies bind to a
common target biomolecule, the FRET signal will be enhanced. By
using multiple fluorescent nucleotides, the signal can be enhanced
and become less sensitive to bleaching.
Example 4
Nanoprobes with Further Separated Functional Groups
This example describes a nanoprobe that includes two or more
tethers and two or more molecular rods, for example to permit
further separation of the functional groups from one another.
Although particular functional groups and molecular rods are
described, one skilled in the art will appreciate that others can
be used. For example, different antibodies can be used to detect
the target biomolecule of interest.
FIG. 5A shows a nanoprobe 300 that further separates the protein
binding agents 302, 308 in the absence of the target biomolecule.
In a particular example, the protein binding agents 302, 308 are
anti-p53 antibodies (for example see Example 1), the molecular rods
306, 312, 318 are dsDNA molecules (such as 40 nucleotides, for
example SEQ ID NO: 5 hybridized to its complementary strand), and
the tethers 314, 316 are PEG (such as 2-4 units of Spacer 18). A
single amino group on the end of the molecular rods 306, 312, 318
can be used to attach the fluorophores 304, 310. For example Texas
Red.RTM.-X NHS Ester (IDT) can be attached to an amino-modified
oligonucleotide (Integrated DNA Technologies).
In addition, a single amino group on the end of the molecular rods
306, 312 can be used to attach the antibodies 302, 308. An antibody
can be cross-linked to an amino group containing DNA by the
following method. Briefly, a 5'-aldehyde group is introduced into
molecular rods 306, 312, for example using 5'-aldehyde-modifier C2
phosphoramidite (Glen Research). The antibodies are reconstituted
to a final concentration of 0.5 mg/mL in PBS. The antibodies are
concentrated to 2 mg/mL in PBS, using a 50 000 MWCO Microcon
filtration device (Millipore). Then, 20 mole equivalents of
succinimidyl 4-hydrazinonicotinateacetone hydrazone (SANH;
Solulink) prepared in DMF is added to the 2 mg/mL antibody solution
and incubated in the dark at room temperature for 2.5 hours.
Purification is performed by size exclusion chromatography using a
NAP-5 column pre-equilibrated with 100 mM citrate buffer, pH 6.0,
150 mM NaCl. The eluent is concentrated in a 50 000 MWCO spin
filter and the filter washed once with citrate buffer. The modified
antibody is resuspended to 1 mg/mL in citrate buffer. To produce
DNA-antibody conjugates, an oligonucleotide modified with an
aldehyde moiety at the 5' end are added to the antibody solution at
a minimum ratio of 10:1, DNA/Ab. The reaction is carried out
overnight at room temperature.
Restriction sites can be placed in the dsDNA sequences 306, 312,
318 (for example to help characterize the nanoprobe to ensure it
was properly constructed).
In a particular example, the nanoprobe shown in FIG. 5A is
generated as follows. For example, the following molecules can be
produced by Integrated DNA Technologies or Midland Certified
Reagent Company, Inc., Midland, Tex.:
TABLE-US-00004 (SEQ ID NO: 8) 1. GTGCCGTCGAATTCTCGCTA-[6-FAM] (SEQ
ID NO: 9) 2. [NH.sub.2]-TAGCGAGAATTCGACGGCAC-[PEG18][PEG18]-
GACGCTAGTATCTTATGAAGCTTTCCTGACTGCGGCATTA (SEQ ID NO: 10) 3.
[NH.sub.2]-CGATAGGGATCCATTACTGC-[PEG18][PEG18]-
TAATGCCGCAGTCAGGAAAGCTTCATAAGATACTAGCGTC (SEQ ID NO: 11) 4.
GCAGTAATGGATCCCTATCG-[Texas Red]
[PEG18] is 1 unit of Spacer 18 (Integrated DNA Technologies).
Molecule 1 will hybridize to molecule 2, molecule 3 will hybridize
to molecule 4, and molecules 2 and 3 will hybridize, due to the
complementarity of the nucleic acid sequences. Antibody 302 is
attached to the NH.sub.2 group of molecule 2, and antibody 308 is
attached to the NH.sub.2 group of molecule 3. After attaching the
antibodies, the four molecules are incubated under conditions that
permit the complementary DNA strands to hybridize, thereby
generating the nanoprobe shown in FIG. 5A. Exemplary incubation
conditions include overnight incubation in binding buffer (100 mM
NaCl, 10 mM Tris HCl pH=7.5, 100 .mu.M EDTA) at room
temperature.
In one example, instead of using an NH.sub.2 group to attach an
antibody to an oligonucleotide sequence, the method of Niemeyer et
al. is used (Nucleic Acids Res. 22:5530-9, 1994) or Kozlov et al.
(Biopolymers 73:621-30, 2004) (all herein incorporated by reference
as to these methods). In yet another example, to attach an antibody
to an oligonucleotide, a single chain antibody (scFv) having a Cys
on the C-terminus is attached to an amino-modified oligonucleotide
(for example see Hayashi et al. Gene. 160(1):129-30, 1995)
Example 5
Nanoprobes for Detecting Interactions Between Two Targets
This example describes a nanoprobe that includes at least two
different protein binding agents, wherein each protein binding
agent recognizes different target biomolecules that can interact.
Although particular functional groups, molecular rods, and tethers
are described, one skilled in the art will appreciate that others
can be used. For example, different antibodies can be used to
detect the target biomolecules of interest.
FIG. 5C shows how the nanoprobe 300 of FIG. 5A can be used to
detect a protein complex that is the target biomolecule 320 (not
part of the nanoprobe). The protein complex 320 includes at least
two different biomolecules 328, 330 that can interact, thereby
forming a detectable complex. The nanoprobe 300 includes protein
binding agents 302, 308 which are antibodies that each specifically
bind to two members of a protein complex. Particular examples of
protein complexes, and the corresponding antibodies that can be
used in the nanoprobe, are listed in Table 3. The molecular rods
and tethers can be as described in Example 4.
TABLE-US-00005 TABLE 3 Exemplary protein complexes that can be
detected with the disclosed nanoprobes, and the antibodies that can
be used. Protein Complex Antibodies* p53-p53BP1 Anti-p53 Binding
Protein 1 Mouse mAb (BP13) p53-BRCA1 Anti-BRCA1 (Ab-1) Mouse mAb
(MS110) p53-Mdm2 Anti-MDM2 (Ab-1) Mouse mAb (IF2) p53-p300 Anti-APC
(Ab-1) Mouse mAb (FE9) p53-SV40 T antigen Anti-SV40 T Antigen
(Ab-1) Mouse mAb (PAb419) *All available from EMD Biosciences,
Inc., San Diego, CA. Particular examples of p53 antibodies are
provided in Table 2.
The nanoprobe shown in FIG. 5C can be generated using the methods
disclosed in Example 4, wherein the antibodies are selected for
their ability to specifically bind to a particular member of the
target biomolecule complex.
Example 6
Nanoprobes for Detecting DNA
This example describes a nanoprobe that can be used to detect DNA,
for example in vivo, in situ, or in vitro. Although particular
sequences are described, one skilled in the art will recognize that
other sequences can be used to detect any target biomolecule of
interest, and the sequence of such molecules can be determined by
those skilled in the art. In addition, although this example
describes a molecule to detect DNA, one skilled in the art will
appreciate that similar methods can be used to construct a
nanoprobe that can detect mRNA.
A variant of the nanoprobe 400 shown in FIG. 6 is shown in FIG.
12A. The nanoprobe 1000 shown in FIG. 12A includes a dsDNA rod 1002
with PEG tethers 1004, 1006 at both ends of the molecular rod 1002.
As shown in FIG. 12A, the dsDNA rod 1002 can be different lengths,
such as 20 nucleotides (m), 30 nucleotides (nml), or 40 nucleotides
(onmlk) Attached to the PEG tethers 1004, 1006 are antisense
oligonucleotides 1008, 1010. To each of the oligonucleotides 1008,
1010, an oligonucleotide 1012, 1014 complementary to at least a
portion of oligonucleotides 1008, 1010 is hybridized, respectively.
One of the complementary oligonucleotides 1012 includes a donor
fluorophore 1016 and the other complementary oligonucleotide 1014
includes an acceptor fluorophore 1018. In a particular example, the
donor fluorophore 1016 is 6-FAM and the acceptor fluorophore 1018
is Texas Red.
In one example, the nanoprobe shown in FIG. 12A is used to detect
the target sequence TCTATACGGATCCTTACGCTCACCCAGTCTCGCGAATTCCGGCCTT
(SEQ ID NO: 12) 1020 (not part of the probe). In such an example,
the nanoprobe shown in FIG. 12A can be generated as follows. For
example, the following molecules can be produced by Integrated DNA
Technologies, Invitrogen, IBA GmbH (Germany) or Midland Certified
Reagent Company, Inc. (Midland, Tex.).
TABLE-US-00006 (SEQ ID NO: 13) 1.
TAGCGAGAATTCGACGGCACAGCGTAAGGATCCGTATAGA- [PEG18][PEG18][PEG18]-
GACGCTAGTATCTTATGAAGCTTTCCTGACTGCGGCATTA- [PEG18][PEG18][PEG18]-
AAGGCCGGAATTCGCGAGACCGATAGGGATCCATTACTGC (FIG. 12A, 1010, 1006,
1002, 1004, 1008). (SEQ ID NO: 14) 2. [5'
6-FAM]-GTGCCGTCGAATTCTCGCTA (FIG. 12A, 1014) (SEQ ID NO: 15) 3.
GCAGTAATGGATCCCTATCG-[3' Texas Red] (FIG. 12A, 1012) (SEQ ID NO:
16) 4. TAATGCCGCAGTCAGGAAAGCTTCATAAGATACTAGCGTC; (SEQ ID NO: 17)
CCGCAGTCAGGAAAGCTTCATAAGATACTA; or (SEQ ID NO: 18)
GTCAGGAAAGCTTCATAAGA.
Each of the molecule 4 sequences will hybridize to molecule 1,
thereby producing a molecular rod of different lengths, depending
which molecule 4 sequence is used. [PEG18] is 1 unit of Spacer 18
(Integrated DNA Technologies), wherein three units are about 7 nm
long. Molecules 2 and 3 will hybridize to molecule 1, and molecule
4 will hybridize to molecule 1, due to the complementarity of the
nucleic acid sequences. The four molecules are incubated under
conditions that permit the complementary DNA strands to hybridize,
thereby generating the nanoprobe shown in FIG. 12A. Exemplary
incubation conditions include overnight incubation in TNE binding
buffer (50 mM NaCl, 10 mM Tris HCl pH=8.0, 1 mM EDTA) at room
temperature. Molecule 1 (SEQ ID NO: 13) can be biotinylated to
permit purification of the bound nanoprobe by streptavidin coated
magnetic beads.
This design makes constructing variations of the nanoprobe
possible, because different parts can be made separately and
exchanged with other parts having different properties. For
example, fluorophores can be easily replaced by resynthesizing the
oligonucleotides 1014, 1012 (e.g. SEQ ID NOS: 14 and 15).
The nanoprobe shown in FIG. 12A can be used to detect a target
sequence (such as SEQ ID NO: 12) as shown in FIG. 12B. The
nanoprobe 1000 can bind to the target 1020, thereby generating the
bound nanoprobe 1050.
The nanoprobe 1000 shown in FIG. 12A (1 .mu.M) was incubated with
(1) 1 .mu.M of the target oligonucleotide (SEQ ID NO: 12), (2) 1
.mu.M of a non-specific oligonucleotide
(CCCGGACGATATTGAACAATGGTTCACTGAAGACCCAGGTCCAGATGAAGCT; SEQ ID NO:
96), or (3) with no DNA, in 20 .mu.l 1.times.TNE buffer at room
temperature for 1 hour, followed by emission scans (FIG. 13A). The
spectra were measured on a SpectraMax Gemini EM microplate
spectrofluorometer (Molecular Devices). Emission scans for a
combination of 6-FAM and Texas Red fluorophores were made using the
following parameters: excitation wavelength 470 nm, excitation
cut-off filter wavelength 495 nm, emission wavelength range 505-650
nm
As shown in FIG. 13A, when excited at 470 nm, the nanoprobe alone
and nanoprobe incubated with non-specific target show both the
6-FAM donor emission at 520 nm and a low 615 nm emission background
of the acceptor (Texas Red) excitation. In contrast, the nanoprobe
incubated with the specific target shows decreased fluorescence at
520 nm and increased fluorescence at 615 nm caused by FRET between
the donor and acceptor fluorophores that are kept close to each
other by the target sequence. Therefore, the nanoprobe specifically
recognizes the target oligonucleotide, but not a non-specific
oligonucleotide.
That the appearance of the 615 nm FRET signal is mediated by DNA
was confirmed by incubating the complexes with DNaseI. The
nanoprobe (1 .mu.M) 1000 was incubated with or without 1 .mu.M of
the target oligonucleotide (SEQ ID NO: 12) at room temperature
overnight in TNE buffer. Half of each sample was treated with 0.2
u/.mu.l DNaseI (2 u/.mu.l, Ambion, Inc.) for 1 hour and emission
spectra were scanned as described above.
As shown in FIG. 13B, DNaseI added to the nanoprobe alone does not
affect the emission spectrum because in the unbound state the donor
and acceptor fluorophore are sufficiently apart and do not
significantly interact. This is unchanged when the unbound
nanoprobe is destroyed, completely separating the fluorophores. The
nanoprobes form a complex with the target sequence resulting in 615
nm FRET emission. DNaseI treatment destroys the nanoprobe-target
complex, and the 615 nm signal disappears. Therefore, the nanoprobe
complex 1050 is sensitive to DNaseI, demonstrating that the
observed FRET signal in FIGS. 13A and 13B is mediated by DNA
contacts.
Example 7
Universal Nanoprobe
This example describes methods that can be used to generate a
universal nanoprobe. In particular examples, such a universal
nanoprobe does not require rebuilding the entire "core" each time a
new sequence is targeted. For example, the nanoprobe 2000 shown in
FIG. 14A is a universal probe that can be used to detect a target
DNA or RNA molecule, such as a p53 mRNA or DNA sequence. Although
particular DNA targeting sequences are described, one skilled in
the art will recognize that the targeting sequences can be altered
to any sequence that will specifically bind to the target sequence
of interest. In addition, other changes to the probe can be made,
without significantly affecting the function of the probe. For
example, the length of the molecular rod can be changed, and the
exact sequence of the DNA rod can be changed.
The nanoprobe 2000 shown in FIG. 14A is similar to the nanoprobe
shown in FIG. 12A. The nanoprobe 2000 includes a dsDNA rod 2002
with PEG tethers 2004, 2006 at both ends of the molecular rod 2002.
As shown in FIG. 14A, the dsDNA rod 2002 can be different lengths,
such as 20 nucleotides (m), 30 nucleotides (nml), or 40 nucleotides
(onmlk) Attached to the PEG tethers 2004, 2006 are antisense
oligonucleotides 2008, 2010. To each of the oligonucleotides 2008,
2010 an oligonucleotide 2012, 2014 complementary to at least a
portion of oligonucleotides 2008, 2010 is hybridized, respectively.
One of the complementary oligonucleotides 2014 includes a donor
fluorophore 2016 and the other complementary oligonucleotide 2012
includes an acceptor fluorophore 2018. In a particular example, the
donor fluorophore 1016 is 6-FAM and the acceptor fluorophore 2018
is Texas Red.
Oligonucleotides 2020, 2022 that contain a portion that is
complementary to the core portion of the nanoprobe (for example to
a portion of 2010 and 2008) and a portion that is complementary to
the target sequence 2024 can be generated by an end user, and
hybridized to probe 2000, thereby generating a complete probe as
shown in FIG. 14A. This makes probe 2000 universal, as any target
oligonucleotide sequences can be used. For example, oligonucleotide
2020 includes portion 2026 that is complementary to a portion of
2010 and portion 2028 that is complementary to a portion of target
sequence 2024. Similarly, oligonucleotide 2022 includes portion
2030 that is complementary to a portion of 2008 and portion 2032
that is complementary to a portion of target sequence 2024.
Therefore, to generate a nanoprobe specific for a different
sequence new oligonucleotides 2020 and 2022 can be designed.
In one example, the nanoprobe shown in FIG. 14A is used to detect
the target sequence (SEQ ID NO: 17, a fragment of p53) 2024 (not
part of the probe). In such an example, the nanoprobe shown in FIG.
14A can be generated as follows. For example, the following
molecules can be produced by Integrated DNA Technologies,
Invitrogen, IBA GmbH (Germany) or Midland Certified Reagent
Company, Inc., Midland, Tex.
TABLE-US-00007 (SEQ ID NO: 13) 1.
TAGCGAGAATTCGACGGCACAGCGTAAGGATCCGTATAGA- [PEG18][PEG18][PEG18]-
GACGCTAGTATCTTATGAAGCTTTCCTGACTGCGGCATTA-[PEG18] [PEG18][PEG18]-
AAGGCCGGAATTCGCGAGACCGATAGGGATCCATTACTGC (FIG. 14A, 2010, 2006,
2002, 2004, 2008). (SEQ ID NO: 14) 2. [5'
6-FAM]-GTGCCGTCGAATTCTCGCTA (FIG. 14A, 2014) (SEQ ID NO: 15) 3.
GCAGTAATGGATCCCTATCG-[3' Texas Red] (FIG. 14A, 2012) (SEQ ID NO:
16) 4. TAATGCCGCAGTCAGGAAAGCTTCATAAGATACTAGCGTC; (SEQ ID NO: 17)
CCGCAGTCAGGAAAGCTTCATAAGATACTA; or (SEQ ID NO: 18)
GTCAGGAAAGCTTCATAAGA. (FIG. 14A, bottom strands for 2002) (SEQ ID
NO: 19) 5. TCTATACGGATCCTTACGCTCCATTGTTCAATATCGTCCG; (SEQ ID NO:
20) TCTATACGGATCCTTACGCTTCCATTGTTCAATATCGTCCG; (SEQ ID NO: 21)
TCTATACGGATCCTTACGCTTTCCATTGTTCAATATCGTCCG (FIG. 14A, 2020, first
underlined portion is 2026 and italicized portion is 2028) (SEQ ID
NO: 22) 6. TCATCTGGACCTGGGTCTTCGTCTCGCGAATTCCGGCCTT; (SEQ ID NO:
23) TCATCTGGACCTGGGTCTTCTGTCTCGCGAATTCCGGCCTT; (SEQ ID NO: 24)
TCATCTGGACCTGGGTCTTCTTGTCTCGCGAATTCCGGCCTT (FIG. 14A, 2022, first
italicized portion is 2032 and second underlined portion is
2030)
[PEG18] is 1 unit of Spacer 18 (Integrated DNA Technologies). Each
of the molecule 4 sequences will hybridize to molecule 1, thereby
producing a molecular rod of different lengths, depending which
molecule 4 sequence is used. Molecules 2 and 3 will hybridize to
molecule 1 due to the complementarity of the nucleic acid
sequences. Molecules 5 and 6 will hybridize to molecule 1 (as well
as to a target nucleic acid). The difference between the
oligonucleotide sequences within the sets (molecules 5 and 6) is
the sequence between the part recognizing the nanoprobe core and
the part responsible for target sequence recognition.
All components of the nanoprobe (1 .mu.M each) were incubated in
the presence or absence of the target sequence (SEQ ID NO: 17)
under conditions that permit the complementary DNA strands to
hybridize, thereby generating the nanoprobe shown in FIG. 14A (one
skilled in the art will appreciate that the probe can be
constructed in one or more steps). The molecules were incubated
overnight in buffer containing 10 mM Tris 8.0, 50 mM NaCl, and 1 mM
EDTA at room temperature in a reaction volume of 20 .mu.l. The
resulting nanoprobe 2000 included p53 target-specific sequences
2028, 2032 (portions of molecules 5 and 6 above) that hybridize to
the p53 target nucleic acid sequence 2024 (SEQ ID NO: 17). The
donor fluorophore 2016 was 6-FAM and the acceptor fluorophore 2018
was Texas Red. Emission spectra were scanned using .lamda.ex=484
nm, .lamda.Cut off filter=530 nm as described in Example 6. The
best ratio between the 615 nm signals of nanoprobe with and without
the target sequence was observed with the combination of
oligonucleotide 2020 (SEQ ID NOS: 19-21) and oligonucleotide 2022
(SEQ ID NOS: 22-24) (FIG. 14B). As shown in FIG. 14B, emission
signal at 615 nm was only observed in the presence of the target
sequence. It was also observed that the combination of SEQ ID NOS:
19 and 23 provided the best signal.
Example 8
Effect of PEG Tethers
This example describes a nanoprobes with and without PEG tethers
that can be used to detect a nucleic acid molecule (such as DNA or
mRNA), for example in vivo, in situ, or in vitro. Although
particular sequences are described, one skilled in the art will
recognize that other sequences can be used to detect a target
biomolecule of interest, and the sequence of such molecules can be
determined by those skilled in the art.
To demonstrate the role of PEG tethers 100 nM of the nanoprobe
described in Example 6 (FIG. 12A, 1000), or a tetherless nanoprobe
3000 (FIG. 15A) were incubated with and without 100 nM of the
target sequence (SEQ ID NO: 12) in a buffer containing 10 mM Tris
8.0, 50 mM NaCl, 1 mM EDTA at room temperature for 2 hours and
emission spectra were scanned as described in Example 6.
FIG. 15A shows a tetherless nanoprobe 3000 that includes antisense
oligonucleotides 3002, 3004. To each of the oligonucleotides 3002,
3004 an oligonucleotide 3006, 3008 complementary to at least a
portion of oligonucleotides 3002, 3004 is hybridized, respectively.
One of the complementary oligonucleotides 3006 includes a donor
fluorophore 3010 and the other complementary oligonucleotide 3008
includes an acceptor fluorophore 3012. In a particular example, the
donor fluorophore 3010 is 6-FAM and the acceptor fluorophore 3012
is Texas Red. In one example, the nanoprobe 3000 shown in FIG. 15A
is used to detect the target sequence shown in SEQ ID NO: 12 3014
(not part of the probe). In such an example, the nanoprobe shown in
FIG. 15A can be generated as follows. For example, the following
molecules can be produced by Integrated DNA Technologies,
Invitrogen, IBA GmbH (Germany) or Midland Certified Reagent
Company, Inc. (Midland, Tex.).
TABLE-US-00008 (nucleotides 1-40 of SEQ ID NO: 13) 1.
TAGCGAGAATTCGACGGCACAGCGTAAGGATCCGTATAGA (FIG. 15A, 3002).
(nucleotides 83-122 of SEQ ID NO: 13) 2.
AAGGCCGGAATTCGCGAGACCGATAGGGATCCATTACTGC (FIG. 15A, 3004). (SEQ ID
NO: 14) 3. [5' 6-FAM]-GTGCCGTCGAATTCTCGCTA (FIG. 15A, 3006) (SEQ ID
NO: 15) 4. GCAGTAATGGATCCCTATCG-[3' Texas Red] (FIG. 15A, 3008)
Molecules 3 and 4 will hybridize to molecules 1 and 2,
respectively, due to the complementarity of the nucleic acid
sequences. The four molecules are incubated under conditions that
permit the complementary DNA strands to hybridize, thereby
generating the tetherless nanoprobe shown in FIG. 15A. Exemplary
incubation conditions include overnight incubation in TNE buffer
(10 mM Tris 8.0, 50 mM NaCl, 1 mM EDTA) for one hour.
FIG. 15B shows the ratio of fluorescence between a nanoprobe in the
presence or absence of a target sequence (SEQ ID NO: 12). In the
absence of a nanoprobe, the ratio is flat and close to 1.0.
Tetherless nanoprobes (3000 FIG. 15A) show decreased fluorescence
at 520 nm and increased FRET signal at 615 nm resulting from
binding to the target sequence. However, this effect is much
greater for nanoprobes containing a tether (1000 FIG. 12A).
Therefore the thermodynamics of target binding by the nanoprobe is
enhanced by the presence of PEG tethers.
The presence of tethers also enhanced the kinetics of
nanoprobe-target sequence complex formation, as shown in FIG. 15C.
Tethered (1000, FIG. 12A) and tetherless nanoprobes (3000, FIG.
15A) were incubated with the target sequence (SEQ ID NO: 12) as
described above, and FRET signals at 615 nm detected. In the
absence of a nanoprobe, the signal does not change with time. As
shown in FIG. 15C, tetherless nanoprobes show slow binding
kinetics, while the kinetics of tethered nanoprobe formation is
faster.
Example 9
Effect of NaCl and MgCl.sub.2 of Nanoprobe-Target Sequence Complex
Formation
This example describes methods used to determine the effect of NaCl
and MgCl2 concentration on the formation of complexes between a
nanoprobe and its target sequence.
The nanoprobe shown in FIG. 12A (see Example 6) (100 nM) was
incubated with 100 nM of a target oligonucleotide (SEQ ID NO: 12)
at room temperature for 5 minutes in 50 mM Tris-HCl pH 8.0 with
different concentrations of NaCl and MgCl.sub.2 (see FIG. 16A).
Emission spectra were obtained as described in Example 6. As shown
in FIG. 16A, both NaCl and MgCl.sub.2 enhance nanoprobe-target
sequence complex formation.
The effect of NaCl and MgCl.sub.2 on kinetics was determined by
incubating the nanoprobe shown in FIG. 12A (see Example 6) (50 nM)
with and without 50 nM target oligonucleotide (SEQ ID NO: 12) in
buffer containing 25 mM Tris-HCl pH8.0, 250 mM NaCl, 100 mM
MgCl.sub.2 and FRET emission at 615 nm was measured as a function
of time as described in Example 6. As shown in FIG. 16B, formation
of the nanoprobe-target sequence complex was completed in five
minutes. In contrast, in the absence of MgCl.sub.2, the formation
of the nanoprobe-target sequence complex took at least 15 minutes
and was not saturated by an hour (see FIG. 15C). Therefore, the
presence of Mg.sup.2+ ions enhances the kinetics of
nanoprobe-target sequence complex formation, and thus Mg.sup.2+
ions can be included in reaction buffers (for example providing
MgCl.sub.2 at a concentration of at least 10 mM, at least 50 mM, or
at least 100 mM in the reaction buffer).
Example 10
Nanoprobe for Detecting Ligation-Mediated mRNA-Antisense
Complexes
This example describes a variant of the nanoprobe described in
Example 6, that can also be used to detect target mRNA molecules,
for example in vivo or in vitro. Although particular antisense
sequences are described, one skilled in the art will recognize that
other antisense sequences can be used to detect the RNA target
biomolecule of interest, and the sequence of such antisense
molecules determined by those skilled in the art. Similarly, one
skilled in the art will recognize that modifications can be made to
the ligase, molecular rods and tethers.
The nanoprobe 500 shown in FIG. 7 includes a DNA ligase 516. In a
particular example, the nanoprobe 500 in FIG. 7 includes antisense
oligonucleotides 502, 504 that can specifically hybridize to a
target RNA sequence 506, which are linked via PEG tethers 508, 510
separated by a molecular rod 512. In one example, the nanoprobe
shown in FIG. 7 is used to detect the target sequence 506
CGATAGGGATCCATTACTGCTAGCGAGAATTCGACGGCAC (SEQ ID NO: 25). In such
an example, the nanoprobe shown in FIG. 7 can be generated as
follows. For example, the following molecules can be produced by
Integrated DNA Technologies or Midland Certified Reagent Company,
Inc.
TABLE-US-00009 (SEQ ID NO: 26) 1. [6-FAM]-GCAGTAATGGATCCCTATCG
[PEG18][PEG18]- TAATGCCGCAGTCAGGAAAGCTTCATAAGATACTAGCGTC (SEQ ID
NO: 27) 2. GTGCCGTCGAATTCTCGCTA-[T-Texas Red]-[PEG18]
[PEG18]-GACGCTAGTATCTTATGAA (SEQ ID NO: 28) 3.
ligase-[PEG18][PEG18]-GCTTTCCTGACTGCGGCATTA
[PEG18] is 1 unit of Spacer 18 (Integrated DNA Technologies). When
the first and second molecules are hybridized, this results in the
formation of molecular rod 512, and when the first and third
molecules are hybridized, this results in the formation of dsDNA
molecule 514. This provides a detectable label on the nanoprobe.
All of the molecules are incubated under conditions that permit the
complementary DNA strands to hybridize, thereby generating the
nanoprobe shown in FIG. 7. If desired, the nanoprobe can be
generated in stages, by hybridizing different portions at different
times. Exemplary incubation conditions include overnight incubation
in TNE buffer (50 mM NaCl, 10 mM Tris HCl pH=8, 1 mM EDTA) at room
temperature.
When using this construct, in particular examples the incubation
conditions include ATP (for a T4 ligase) or NADH (for an E. coli
ligase). Manganese ions can be used to increase ligase
efficiency.
Example 11
Nanoprobe for Gene Silencing by mRNA Degradation
This example describes a nanoprobe that can be used to decrease
gene expression, for example in vivo or in vitro. Such a nanoprobe
can be used as an alternative to (or in addition to) antisense- or
siRNA-based therapies. Although particular antisense sequences are
described, one skilled in the art will recognize that other
antisense sequences can be used to hybridize to the RNA target
biomolecule of interest, and the sequence of such antisense
molecules determined by those skilled in the art.
FIG. 9 shows a nanoprobe 600 that in particular examples includes a
PEG tether 606 of 2-4 units of Spacer 18, with RNase H 604 on one
end of the tether 606, and an antisense oligonucleotide 602 that
can hybridize to an mRNA target 608 (not part of the nanoprobe) on
the other end of the tether 606. RNase H proteins can be produced
using commercially available clones (for example from the E. coli
Genome Project, University of Wisconsin, Madison, Wis., such as
clone pEKGb0214).
Particular examples of targets, and the corresponding antisense
sequence that can be used in the nanoprobe, are listed in Table 4.
For example, the antisense sequence can be attached to 2-3 units of
Spacer 18, and RNase H attached to the other end of the tether, for
example by using a bi-functional cross-linking reagent (see Example
1).
TABLE-US-00010 TABLE 4 Exemplary target sequences and the
corresponding antisense sequence. Exemplary Target Sequence
Antisense sequence on nanoprobe Protein (SEQ ID NO) (SEQ ID NO) p53
CGGACGATATTGAACAATGGTTC CCATTGTTCAATATCGTCCG (29) (39)
ACTGAAGACCCAGGTCCAGATGA TCATCTGGACCTGGGTCTTC (30) (40) Hypoxia
TCAGCTATTTGCGTGTGAGGAAA CCTCACACGCAAATAGCTGA inducible factor (31)
(41) 1 (HIF1) CTTCTGGATGCTGGTGATTTGGA TCCAAATCACCAGCATCCAG (32)
(42) hexose-6- GCCAGTACCGCCAACTGAAGACG CTTCAGTTGGCGGTACTGGC
phosphate (33) (43) dehydrogenase GCCGAGGACTATCAGGCCCTGAA
TTCAGGGCCTGATAGTCCTC (glucose 1- (34) (44) dehydrogenase) (H6PD)
leucine proline- TGGTGATGGACGGCGTAATCTCT GATTACGCCGTCCATCACCA
enriched (35) (45) proteoglycan GACCACGAGTGTCAGGAGCTGCA
TGCAGCTCCTGACACTCGTG (leprecan) 1 (36) (46) (LEPRE1) Homo sapiens
TGGGCTTTGACAAACAGCTCTCA GAGCTGTTTGTCAAAGCCCA chromosome 1 (37) (47)
open reading CAGGACCTGGCTGTCAACCTCCT AGGAGGTTGACAGCCAGGTC frame 50
(38) (48) (C1orf50),
Example 12
Nanoprobe for Quantitating mRNA
This example describes a nanoprobe that can be used to quantitate
specific mRNA molecules, for example in vivo or in vitro. Although
particular antisense sequences are described, one skilled in the
art will recognize that other antisense sequences can be used to
hybridize to the RNA target biomolecule of interest, and the
sequence of such antisense molecules determined by those skilled in
the art.
FIG. 11 shows a nanoprobe 900 that includes donor fluorophore 902
and acceptor fluorophore 904 that permit detection (and in some
examples quantitation) of RNA as it is cleaved by the RNase H 920.
In a particular example, the nanoprobe 900 in FIG. 11 includes
antisense oligonucleotides 906, 908 that can specifically hybridize
to a target RNA sequence 910 (not part of the probe), which are
linked by PEG tethers 912, 914 separated by a molecular rod 916. In
one example, the nanoprobe shown in FIG. 11 is used to cleave SEQ
ID NO: 25. In such an example, the nanoprobe shown in FIG. 11 can
be generated as follows. For example, the following molecules can
be produced by Integrated DNA Technologies or Midland Certified
Reagent Company, Inc.
TABLE-US-00011 (SEQ ID NO: 26) 1. [6-FAM]-GCAGTAATGGATCCCTATCG
[PEG18][PEG18]- TAATGCCGCAGTCAGGAAAGCTTCATAAGATACTAGCGTC (SEQ ID
NO: 27) 2. GTGCCGTCGAATTCTCGCTA-[T-Texas Red]-[PEG18]
[PEG18]-GACGCTAGTATCTTATGAA (SEQ ID NO: 49) 3.
RNaseH-[PEG18][PEG18]-GCTTTCCTGACTGCGGCATTA.
[PEG18] is 1 unit of Spacer 18 (Integrated DNA Technologies). When
the first and second molecules are hybridized, this results in the
formation of molecular rod 916, and when the first and third
molecules are hybridized, this results in the formation of dsDNA
molecule 918. This provides a detectable label on the nanoprobe.
All of the molecules are incubated under conditions that permit the
complementary DNA strands to hybridize, thereby generating the
nanoprobe shown in FIG. 11. If desired, the nanoprobe can be
generated in stages, by hybridizing different portions at different
times. Exemplary incubation conditions include overnight incubation
in TNE buffer (50 mM NaCl, 10 mM Tris HCl pH=8, 1 mM EDTA) at room
temperature.
Example 13
Nanoprobe for Cleaving DNA-Binding Proteins
This example describes a nanoprobe that can be used to reduce the
activity of a DNA-binding protein, for example in vivo or in vitro.
Such a nanoprobe is an alternative to antisense- or siRNA-based
therapies. Although particular DNA binding site sequences and PEG
tethers are described, one skilled in the art will recognize that
other sequences and tethers can be used. For example, the DNA
binding site sequence can be selected based on the target DNA
binding protein to be inactivated.
FIG. 10A shows a nanoprobe 700, which in particular examples
includes a protein binding site sequence 702 linked to proteinase K
704 via a PEG linker 706 of 2-4 units of Spacer 18. The DNA
containing a binding site 702 linked to proteinase K 704 will bind
and then destroy a DNA-binding protein 708 (not part of the
nanoprobe), such as a transcription factor. Although the proteinase
K can cleave other proteins in the sample or the subject into which
it is administered, it will attack the bound target protein at a
higher rate because of the high effective concentration.
In a specific example, the nanoprobe 700 includes a DNA binding
site sequence that can specifically hybridize to a DNA binding
protein. Particular examples of targets, and the corresponding
sequence that can be used in the nanoprobe, are listed in Table 5.
For example, the nucleotide sequence can be attached to 2-3 units
of Spacer 18, and a proteinase (such as proteinase K) attached to
the other end of the tether (for example using a bi-functional
cross-linker as described in Example 1).
TABLE-US-00012 TABLE 5 Exemplary target sequences and the
corresponding nanoprobe sequence. Exemplary Target Sequence
Sequence on nanoprobe Protein (SEQ ID NO) (SEQ ID NO) p53
GGACATGTCCGGACATGTCC (50) GGACATGTCCGGACAT
GGACATGTCCGGACATGTCCGCGAAGC (51) GTCC (58) Hypoxia
TCTCACACACGTACACACACGTGTC (52) GACACGTGTGTGTACG inducible
TCTCACACACGTACACACACGTGTCGCGAAGC TGTGTGAGA (59) factor 1 (53)
(HIF1) NF- GGGACATTCCGGGACATTCC (54) GGAATGTCCCGGAATG Kappa B
GGGACATTCCGGGACATTCCGCGAAGC (55) TCCC (60) consensus STAT1
GTCGACATTTCCCGTAAATCGTCGA (56) TCGACGATTTACGGGA
GTCGACATTTCCCGTAAATCGTCGAGCGAAGC AATGTCGAC (61) (57)
Example 14
Nanoprobe for Cleaving Target Proteins
This example describes a nanoprobe that can be used to reduce the
activity of a target protein, for example in vivo, in situ, or in
vitro. Such a nanoprobe is an alternative to antisense- or
siRNA-based therapies. Although particular protein binding agents,
PEG tethers, and molecular rods are described, one skilled in the
art will recognize that other binding agents, tethers, and
molecular rods can be used. For example, the protein binding agent
can be selected based on the target protein to be cleaved, thereby
decreasing the biological activity of the target protein.
FIG. 10B shows a nanoprobe 800, which in particular examples
includes proteinase K 804 linked to an antibody that recognizes an
amyloidogenic or prion target protein 808 (for example an
anti-Prion Protein (PrP) (Ab-3) Mouse mAb (F89/160.1.5) from EMD
Biosciences, Inc.) via a PEG tethers 810, 812 each composed of 2-4
units of Spacer 18 separated by a dsDNA molecular rod 814 having
the sequence shown in SEQ ID NO: 66 hybridized to its complementary
strand. The nanoprobe 800 also includes a donor fluorophore 816
attached to the PEG tether 812 and an acceptor fluorophore 818
attached to PEG tether 810.
In a particular example, the nanoprobe 800 shown in FIG. 10B can
include a second antibody on a tether that binds to the proteinase
K, for example to reduce the function of proteinase K. However when
the target protein is bound, the close proximity of the proteinase
K to the target protein results in an increased activity of the
proteinase K towards the target protein. For example, the following
molecule can be produced by Integrated DNA Technologies or Midland
Certified Reagent Company, Inc.:
[PEG18][PEG18]-TAATGCCGCAGTCAGGAAAGCTTCATAAGATACTAGCGTC
[PEG18][PEG18]-[Texas Red] (SEQ ID NO: 62)
[PEG18] is 1 unit of Spacer 18 (Integrated DNA Technologies). The
antibody 808 can be fluorescently labeled with 6-FAM using a
commercial kit, and attached to the PEG using the methods described
in Example 1. In addition, the proteinase K can be attached to the
other end using the methods described in Example 1.
Example 15
Nanoprobe for Sequencing Nucleic Acid Molecules
This example describes a particular probe that can be used to
sequence a target nucleic acid molecule. Although particular
fluorophores, molecular linkers, and polymerases are described, one
skilled in the art will appreciate that variations to these can be
made, based on the teachings herein.
The design is based on FIG. 8C, but the method of attachment of the
molecular linker to the polymerase is changed by removing the
tethers and dsDNA 582 that link the polymerizing agent 552 to the
molecular linker 562. dsDNA 582 is replaced by a continuous dsDNA,
without any break, that contains a binding site (ter) for the Tus
protein. The Tus protein is translationally fused to HIV-1 RT.
The DNA sequences were designed using the NANEV program and checked
to ensure that the restriction sites shown in FIG. 8C are unique.
In addition, a MaeIII restriction site naturally appears in the ter
sequence and this is unique in the probe. The Tus sequence used is
the consensus bases from nine known Tus sites. Given the
constraints that certain pairs of ssDNA sequences are complementary
to each other and that some sequences contain the restriction sites
shown in FIG. 8C, the NANEV program was used to evolve
structure.
NANEV uses single letter names for dsDNA strands. Lower case
letters (a, c, g, t, e, h, b, p, m) represent a segment of ssDNA
that is to be hybridized to the corresponding ssDNA labeled with an
upper case letter (A, C, G, T, E, H, B, P, M). Each dsDNA branch is
named by the corresponding non-hydrolyzable base (A, C, G, T) while
the `hub` parts are named by restriction enzymes that cut them (E,
H, B, P, M). For example, for the branch 561 that has a
non-hydrolyzable adenosine 554, one oligonucleotide is named
566-561-1a, and it is bound to fluorophore 566. 566-561-1a will
anneal with 563-584-561-585-554-11E-2A.
The 14 dsDNA parts designed using NANEV are shown in Table 6:
TABLE-US-00013 TABLE 6 Sequences to generate nanoprobe SEQ ID #
name Sequence NO: 1 a GGCCTCCGTCCTCGGCAGTA 63 2 A
TACTGCCGAGGACGGAGGCC 64 3 c CGATAATGCCTGTCATGCAT 65 4 C
ATGCATGACAGGCATTATCG 66 5 g GAACGTCTAGACTTATCGC 67 6 G
GCGATAAGTCTAGACGTTCC 68 7 t CTCTCGCTCCGTGCCGTAAG 69 8 T
CTTACGGCACGGAGCGAGAG 70 9 eMh CGCTCCTGAATTCGACGTACGCTATATATTTA 71
GT ATGTTGTAACTAAAGTCCAGCGCGAAGCTTA ATGACT 10 pmb
ATTCAGTCTGCAGGAAGGCCGACTTTAGTTA 72 CAA
CATACTAAATATATAGCCAGTAAGGGATCCG ATCTCG 11 E GTACGTCGAATTCAGGAGCG 73
12 B CGAGATCGGATCCCTTACTG 74 13 H AGTCATTAAGCTTCGCGCTG 75 14 P
GGCCTTCCTGCAGACTGAAT 76
Some of these 14 components are joined by PEG and linked to
appropriate fluorophores to create ten oligonucleotides. The ten
oligonucleotides can be synthesized commercially by IDT
(Coralville, Iowa) or Midland (Midland, Tex.) as follows:
TABLE-US-00014 (SEQ ID NO: 77) >566-561-1a:
[fluorophore-566]-GGCCTCCGTCCTCGGCAGTA where [fluorophore-566] is
Rhodamine Red(TM)-X (Absorbance Max: 574 nm, Emission Max: 594)
"GREEN" (SEQ ID NO: 78) >572-569-3c:
CGATAATGCCTGTCATGCAT-[fluorophore-572] where [fluorophore-572] is
Cy3 .RTM. (Absorbance Max: 550 nm, Emission Max: 564) "BLUE" (SEQ
ID NO: 79) >570-571-5g: GGAACGTCTAGACTTATCGC-[fluorophore-570]
where [fluorophore-570] is Texas Red .RTM.-X (Absorbance Max: 598
nm, Emission Max: 617) "YELLOW" (SEQ ID NO: 80) >568-581-7t:
[fluorophore-568]-CTCTCGCTCCGTGCCGTAAG where [fluorophore-568] is
Cy5(TM) (Absorbance Max: 648 nm, Emission Max: 668) "RED" (SEQ ID
NO: 81) >563-584-561-585-NH2-11E-2A:
GTACGTCGAATTCAGGAGCG-[PEG18]-[PEG18]-[PEG18]-
TACTGCCGAGGACGGAGGCC-[PEG9]-NH2 (SEQ ID NO: 82)
>NH2-583-569-567-565-4C-12B:
NH2-[PEG9]-ATGCATGACAGGCATTATCG-[PEG18]-[PEG18]-
[PEG18]-CGAGATCGGATCCCTTACTG (SEQ ID NO: 83)
>NH2-587-571-573-575-6G-13H:
NH2-[PEG9]-GCGATAAGTCTAGACGTTCC-[PEG18]-[PEG18]-
[PEG18]-AGTCATTAAGCTTCGCGCTG (SEQ ID NO: 84)
>577-579-581-589-NH2-14P-8T:
GGCCTTCCTGCAGACTGAAT-[PEG18]-[PEG18]-[PEG18]-
CTTACGGCACGGAGCGAGAG-[PEG9]-NH2 >563-582-575-9eMh: (SEQ ID NO:
85) CGCTCCTGAATTCGACGTACGCTATATATTTAGTATGTTGTAACTAAAGT
CCAGCGCGAAGCTTAATGACT >577-582-565-10pmb: (SEQ ID NO: 86)
ATTCAGTCTGCAGGAAGGCCGACTTTAGTTACAACATACTAAATATATAG
CCAGTAAGGGATCCGATCTCG
Four non-hydrolyzable dNTPs are synthesized (for example by Jena
Bioscience): dGMPCPP, dAMPCPP, dCMPCPP, and TMPCPP, where C
represents a CH.sub.2 group instead of the oxygen between the
.alpha. and .beta. phosphates. Note that the older terminology
TMPCPP means dTMPCPP that is, deoxyribo-TMPCPP. Jena Bioscience can
also provide dGMPNPP, dAMPNPP, dCMPNPP, and TMPNPP where N
represents an NH group instead of the oxygen between the .alpha.
and .beta. phosphates. Note that the older terminology TMPNPP means
dTMPNPP that is, deoxyribo-TMPNPP. In addition, Jena Bioscience
provides aminoallyl-dUpCpp (NU-826) labeled with various
fluorescent dyes.
Each non-hydrolyzable dNTP is covalently attached by its .gamma.
phosphate to an amino group on the corresponding oligonucleotide
branch using the following reaction protocol derived from Pierce
Technical Resource TR0030.1 "Modify and label oligonucleotide 5'
phosphate groups" except that the roles of label and
oligonucleotide are reversed.
1. Dissolve the non-hydrolyzable dNTP in 10 .mu.l reaction buffer.
(The reaction buffer recommended by Pierce is "Reaction Buffer,
such as phosphate buffered saline (PBS) with EDTA: 10 mM sodium
phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. Avoid using PBS with
>10 mM phosphate, which will interfere with the intended
reaction. Other amine free and carboxylate-free buffers can be
substituted, but avoid Tris, which contains a primary amine that
will quench the reaction.")
2. Dissolve the oligonucleotide to a final concentration of 1 mM in
10 .mu.l of 0.1 M Imidazole, pH 6.
3. Weigh 1.25 mg (6.52 micromol) of EDC
(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride,
Pierce Product No. 22980.) into a microcentrifuge tube.
4. Add 7.5 .mu.l of the prepared non-hydrolyzable dNTP to the tube
containing the EDC and immediately add 5 .mu.l of the
oligonucleotide/imidazole solution.
5. Vortex tube until contents are completely dissolved, and then
briefly centrifuge the tube to gather contents.
6. Add an additional 20 .mu.l of 0.1 M imidazole, pH 6.
7. Incubate the reaction overnight at Room Temperature.
8. Separate the unreacted oligonucleotide from the reaction product
on a 10% polyacrylamide gel.
It is also possible to purify the product by its ability to bind to
a DNA polymerase. The unreacted nucleotides are first removed by
using a size-exclusion column or dialysis. Then a column is created
that has HIV-1 RT or another polymerase attached (for example HIV-1
RT with a histidine-6 tag bound to a nickel column). A template DNA
and annealed primer DNA can be added. This polymerase column should
retard the oligonucleotide to which is attached a nucleotide,
compared to the unreacted oligonucleotide that does not have a
tethered nucleotide. A description of the carbodiimide
cross-linking reaction described above is given in Chatterji and
Gopal (Methods Enzymol. 274:456-78, 1996). This protocol is
performed separately for each oligonucleotide.
The oligonucleotide: is attached to:
563-584-561-585-NH2-11E-2A (SEQ ID NO: 81) non-hydrolyzable
dAMPCPP
NH2-583-569-567-330-4C-12B (SEQ ID NO: 82) non-hydrolyzable
dCMPCPP
NH2-587-571-573-575-6G-13H (SEQ ID NO: 83) non-hydrolyzable
dGMPCPP
577-579-581-589-NH2-14P-8T (SEQ ID NO: 84) non-hydrolyzable
TMPCPP
This creates the following structures:
TABLE-US-00015 (SEQ ID NO: 87) >563-584-561-585-554-11E-2A:
GTACGTCGAATTCAGGAGCG-[PEG18]-[PEG18]-[PEG18]-
TACTGCCGAGGACGGAGGCC-[PEG9]-NH-POOH-O-POOH-CH2- POOH-deoxyadenosine
(SEQ ID NO: 88) >577-579-581-589-560-14P-8T:
GGCCTTCCTGCAGACTGAAT-[PEG18]-[PEG18]-[PEG18]-
CTTACGGCACGGAGCGAGAG-[PEG9]-NH-POOH-O-POOH-CH2- POOH-deoxythymidine
(SEQ ID NO: 89) >558-587-571-573-575-6G-13H:
deoxyguanosine-POOH-CH2-POOH-O-POOH-NH-[PEG9]-
GCGATAAGTCTAGACGTTCC-[PEG18]-[PEG18]-[PEG18]- AGTCATTAAGCTTCGCGCTG
(SEQ ID NO: 90) >556-593-569-567-565-4C-12B:
deoxycytidine-POOH-CH2-POOH-O-POOH-NH-[PEG9]-
ATGCATGACAGGCATTATCG-[PEG18]-[PEG18]-[PEG18]-
CGAGATCGGATCCCTTACTG
The 10 oligonucleotides:
563-584-561-585-554-11E-2A (SEQ ID NO: 87);
577-579-581-589-560-14P-8T (SEQ ID NO: 88);
558-587-571-573-575-6G-13H (SEQ ID NO: 89);
556-583-569-567-565-4C-12B (SEQ ID NO: 90); 566-561-1a (SEQ ID NO:
77); 572-569-3c (SEQ ID NO: 78); 570-571-5g (SEQ ID NO: 79);
568-581-7t (SEQ ID NO: 80); 563-582-575-9eMh (SEQ ID NO: 85);
577-582-565-10pmb (SEQ ID NO: 86); are then hybridized together to
form the molecular linker 562 of the probe shown in FIG. 8C. The
molecular linker 562 is then purified by gel electrophoresis, an
exclusion column or sucrose gradient. The structure of the
generated molecular linker 562 can be tested by digesting with the
five restriction enzymes separately and in combinations and by
observing the products on polyacrylamide gels.
The tus (termination utilization substance) gene from E. coli has
been cloned in pBAD33tus (Henderson et al., Mol. Genet. Genomics
265:941-53, 2001, Guzman et al., J. Bacteriol. 77:4121-30, 1995).
The HIV-1 RT p66 subunit has been cloned and modified to replace
all solvent-accessible cysteine residues with serine residues (C38S
and C280S) and to substitute a unique cysteine for the lysine at
287, K287C (Kensch et al., J. Mol. Biol. 301:1029-39, 2000). The
unique cysteine at 287 is on the "thumb" of the polymerase, close
to the active site of the polymerase, but far enough away so as not
to interfere with DNA binding or the active site. There are only
two cysteines in the Tus protein at CYS99 and CYS255 and they are
both completely buried, (PDB 1ECR Kamada et al., Nature
383:598-603, 1996), so it is not necessary to engineer Tus to avoid
exposed cysteines. Tus is cloned in a translational fusion with the
mutated HIV-1 RT.
As seen in the three dimensional structures of HIV-1 RT (PDB entry
1RTD Huang et al., Science 282:1669-1675, 1998) and Tus bound to
DNA (1ECR Kamada et al., Nature 383:598-603, 1996) the N and C
termini of both proteins are on their surfaces well away from the
active sites, so fusion of the two proteins will not interfere with
their structures or functions. The hydrophylic polypeptide that
connects the two parts of the RecB protein (PDB entry 1W36,
Singleton et al., Nature 432:187-93, 2004) is used to connect Tus
to HIV-1 RT to create Tus-HIV-1 RT.
Those skilled in the art will recognize that either Tus or HIV-1 RT
protein can be placed at the N terminus of the fusion and that they
can be interchanged. Those skilled in the art will also recognize
that 6-histidine tags can be placed on either end of the
construction to help isolation. A 6-histidine tag on the N terminus
of Tus has little effect on binding, while a 6-histidine tag on the
C terminus of HIV-1 has no known effect on polymerase activity.
The donor fluorophore is attached to the unique cysteine in
Tus-HIV-1 RT by using the maleimide labeling reagent
Fluorescein-5-Maleimide (Pierce, Rockford, Ill., using the
manufacturer's instructions). This donor fluorophore forms FRET
pairs with each of the four acceptor pairs described above. Those
skilled in the art will also recognize that additional acceptor
fluorophores can be added to the corresponding oligonucleotides to
adjust for the relative signal strength, if desired. Those skilled
in the art will recognize that many other possible combinations of
fluorophores are possible.
The Tus-HIV-1 RT protein is added to the core to create the
completed probe. The probe is then purified by gel electrophoresis,
an exclusion column or sucrose gradient. The final probe structure
is checked by digesting with the five restriction enzymes
separately and in combinations and by observing the products on
polyacrylamide gels.
In a second example, the reverse transcriptase is modified by
directed mutagenesis of F227A to reduce its error frequency
(Wisniewski et al., J. Biol. Chem. 274:28175-84, 1999). In a third
example, the connection between Tus and HIV-1 RT is determined as
it is for single chaing Fv (scFv) linker sequences. The classical
sequence used is (Gly.sub.4Ser).sub.3, but phage display technology
can be used to obtain other variations (Tang et al., J. Biol. Chem.
271:15682-6, 1996; Hennecke et al., Protein Eng. 11:405-10,
1998).
Those skilled in the art will recognize that many other design
variations are possible for the nanoprobe of the present
disclosure.
Example 16
Calculation of Distance Between Fluorophores with Different Rod
Lengths
As described above, the disclosed nanoprobes can include two or
more functional groups connected by a tether, for example a tether
composed of PEG (for example see FIGS. 2A and 3A). However, with no
force to keep them separated, the free PEG chains may condense to
form cloud around the same point. Therefore, as described in some
of the examples above, the addition of one or more molecular rods
to the nanoprobe (for example as shown in FIGS. 1B, 4A, 4B, 5A-C,
6, 7, 8B, 8C, 10B, and 11) can be included to further separate the
functional groups, for example to further separate the
fluorophores.
This example describes computer simulations used to determine how a
FRET signal is affected by the length of a molecular rod, which can
be, for example, composed of dsDNA. FIG. 1B shows the situation in
the simulation. Two tethers 22, 24 are connected to a rod 26 and
the distance between fluorophores 12 and 14 is measured. For a
single tether, assuming that the tether is infinitely thin (does
not have a problem intersecting with itself) the distance from the
attachment point to the end of the tether is a random walk in three
dimensions. In each single dimension this is the sum of small
random steps, which would give a Gaussian distribution. In three
dimensions it will be the spherically symmetric Maxwell gas
distribution (Schneider, J. Theor. Biol., 148:83-123, 1991). It is
not appropriate to merely integrate over the intersection of two
such distributions with a given separation because individual
molecules will be at specific directions and distances. For this
reason, an explicit simulation was performed.
To summarize the method, for each rod length, the two tethers were
grown and the distance between the tips and then the FRET
efficiency was computed. In the simulation, encoded by the program
Bite (bi-tether), two polymers were attached to the ends of a fixed
rod. Each polymer was generated by a series of random steps,
starting from a rod end. The size of the steps is given by the
persistence length of the chain. For PEG, the persistence length is
3.8.+-.0.02 .ANG.. The direction of each step was chosen randomly.
The FRET signal was computed for each pair of randomly extended
chains. This signal is a function the final distance between the
tether chain ends R, and the FRET radius R.sub.0 according to the
FRET efficiency, E=1/(R/R.sub.0).sup.6+1) (1)
The parameters used by the program include the persistence length,
the length of the tethers L and the rod length D that separates the
tether points. This process was repeated 1000 times to obtain the
distributions shown in FIG. 17.
When the distance, R, between the fluorophores is R.sub.0, the
transfer efficiency is 50%. For example, for a FRET pair with an
R.sub.0=60 .ANG., which is a typical distance, and a tether length
of 120 .ANG., FIGS. 17A-F show the effect of varying the rod
length. With a rod length of 0 (FIGS. 17E and 17F) the tethers
gather around a common point (FIG. 17E) leading to a large FRET
signal (FIG. 17F). As the rod length is increased to 60 .ANG. (FIG.
17C), the FRET distribution signal spreads out (FIG. 17D).
Sometimes the two ends are close and some FRET will be observed.
Strikingly, when the rod length is 120 .ANG. (FIGS. 17A and 17B),
the FRET signal is almost completely eliminated (FIG. 17B). This
happens when the tether length is twice the rod length, so there is
plenty of potential overlap of the tethers, allowing the tethers to
bind to the target. Yet the FRET signal is almost undetectable.
These results demonstrate particular advantages of including a rod
in a molecular linker
Example 17
FRET with Different Tether Lengths
This example describes methods used to determine the effect of
changing the length of a tether on a FRET signal.
The Bite program was run with various tether lengths (FIG. 18). In
the upper left (FIG. 18A) a very short tether of 2 .ANG. results in
the basic FRET curve given by equation (1). As the tether length is
increased from 2 to 60 .ANG., 120 .ANG. and then 140 .ANG. (FIGS.
18 B, C and D), the basic FRET curve remains, but the distribution
becomes more spread out. With a molecular rod length of about 120
.ANG. and tethers of 120 .ANG., there is little FRET even though
the tethers could overlap significantly. Therefore, use of a
molecular rod of about 120 .ANG. and tethers of 120 .ANG., results
in almost no FRET until the two tethers are brought together by
binding to the target molecule.
Even with tethers that are 240 .ANG. long (FIG. 18D), the right
side of the distribution is almost the same as with 120 .ANG. long
tethers (FIG. 18C). That is, tether length makes little difference
to the FRET results, but molecular rod length has a significant
effect.
In summary, the length of the tether had little effect on FRET,
while the length of the molecular rod made a significant
difference. Including a rod in the molecular linker of a nanoprobe
reduces FRET to almost undetectable levels, even when tethers are
more than sufficiently long to reach the target. In contrast, in
the presence of a target the FRET signal can be large. This
provides a strong molecular switch on the output signal based on
the presence or absence of the target. An example of a useful
rod-tether combination for a nanoprobe uses a rod of 120 .ANG. with
two tethers also of 120 .ANG.. These are conveniently constructed
from 40 nucleotides of dsDNA, to create the rod, and 5 to 6 PEG 18
spacers of 23 .ANG. each, to create tethers.
Two time scales can be considered in understanding the operation of
molecular nanoprobes. On the time scale of molecular vibrations,
picoseconds, the tethers will explore a large variety of
possibilities and the joining of two tether tips that are separated
by a molecular rod appears to take a long time. For example, this
process could take several orders of magnitude longer than
molecular vibrations, for example, 100 milliseconds. Although 100
milliseconds is a long time from the viewpoint of molecular
motions, it is only 1/10th of a second on the human time scale.
Thus a detection process using a molecular probe may appear to be
quite rapid.
Example 18
Targeted Nanoprobes
Nanoprobes can include molecules that can be used to direct the
nanoprobe to a particular cell, or to a particular cellular
compartment.
For example, commercially available fluorescent proteins that
localize to actin filaments, mitochondria, endoplasmic reticulum,
nuclei, Golgi apparatus, peroxisomes, and endosomes (for example
from BD Biosciences--Clontech) can be used as a targeting moiety
attached to a nanoprobe (see Table 7). Similarly, nanoprobes that
include a targeting moiety (such as an antibody) can be used to
direct a nanoprobe to a particular type of cell. Such antibodies
are known in the art, and can be attached to a nanoprobe using the
methods described herein.
TABLE-US-00016 TABLE 7 Exemplary targeting moieties from Clontech
Product Catalog number pAcGFP1-Actin 632453 pAcGFP1-Endo 632490
pAcGFP1-Golgi 632464 pAcGFP1-Hyg-C1 632492 pAcGFP1-Hyg-N1 632489
pAcGFP1-Mem 632491 pAcGFP1-Mito 632432 pAcGFP1-Nuc 632431
pAcGFP1-Tubulin 632488 pDsRed-Monomer-Actin 632479 pDsRed-Monomer-F
632493 pDsRed-Monomer-Golgi 632480 pDsRed-Monomer-Hyg-C1 632495
pDsRed-Monomer-Hyg-N1 632494 pDsRed2-Mito 632421 pDsRed2-Nuc 632408
pDsRed2-Peroxi 632418 pHcRed1-Mito 632434 pHcRed1-Nuc 632433
Example 19
Reaction Rate Acceleration
In this nanomachine design, the ends of the molecular linker
contain two different enzymes. There are also antibodies for a
target on the ends of the molecular linker. The solution contains
an initial substrate. The molecular linker can include one or more
tethers separated by a molecular rod, so that when the target
biomolecule is absent the reaction proceeds, but slowly. When the
target biomolecule is present the reaction is accelerated. By this
means alternative metabolic pathways could be selected using
arbitrary external controlling substances.
Example 20
Methods of Detecting a Biomolecule
This example describes methods that can be used to detect a
biomolecule, for example in vitro, in situ, or in vivo. Although
particular examples are provided for the detection of particular
biomolecules (such as proteins or nucleic acid molecules), one
skilled in the art will appreciate that based on the teachings
herein, other biomolecules can be detected, for example by
modifying the particular nanoprobes disclosed herein. In contrast
to currently available assays, which can take hours to obtain a
result, it should only take a few seconds to detect a signal using
a nanoprobe following introduction into a cell or following contact
with a sample.
In one example, the nanoprobe shown in FIG. 2A that includes the
particular functional groups described in Example 1, is used to
detect p53. Generally, the method includes incubating one or more
nanoprobes for detecting p53 binding to DNA with a sample (for
example from a subject), under conditions that permit the
nanoprobe(s) to specifically bind to p53 when bound to DNA. One or
more signals generated from the nanoprobe are then detected, and in
some examples quantitated. For example, the presence or absence of
an acceptor fluorophore emission signal can be detected.
In some examples, multiple nanoprobes are incubated with a sample.
For example, if the sample is incubated with two nanoprobes having
a common donor and different acceptor fluorophores, wherein one
nanoprobe has an antibody that detects non-activated p53 and the
other has an antibody that detects activated p53, the ratio of
non-activated to activated p53 can be determined, by comparing the
two FRET signals. In another example, if the sample is incubated
with two nanoprobes having a common donor and different acceptor
fluorophores, wherein one nanoprobe has an antibody that detects
one p53 mutation and the other has an antibody that detects a
different p53 mutation, the presence of a particular p53 mutation
can be determined, by determining which of the two acceptor
fluorophore emission signals is detected.
In a particular example, p53 binding to DNA is detected in vivo. In
some examples, the nanoprobe is introduced into cells using
liposomes or targeted to certain cells with immunoliposomes (for
example see Yu et al., Nucleic Acids Res. 32:e48).
In some examples, detection of the target molecule is performed in
vitro. For example, as shown in FIGS. 19A and 19B, one or more
nanoprobes 4000 can be attached to a surface 4002 (such as the
surface of an array, glass slide, plastic slide, or membrane). Such
a surface can include controls. For example, a control nanoprobe
can include an additional molecular linker with the target molecule
attached on the end. Methods of attaching a probe to a surface are
known. For example, as shown in FIG. 19B, DNA 4000 can be
synthesized onto the surface 4002, and then two molecular linkers
4004 4006 containing fluorophores 4008 4010 are annealed. In
another example, the nanoprobe 4000 includes a biotinylated
oligonucleotide or a PEG tether tether terminated with a biotin,
which can be attached to a surface 4002 containing
streptavidin.
The biological sample can be added to the surface 4002, and an
emission signal detected (for example using a photometer), wherein
the presence of a signal indicates the presence or absence of the
target molecule (such as p53) (depending on the type of fluorophore
used). In some examples, the biological sample is applied using a
capillary tube.
If desired, the biological target can be quantitated. In some
examples, the nanoprobe-target complexes are allowed to form, and
then the complexes detected and quantitated. In another example,
the nanoprobes are monitored continuously, and the initial slope of
the exponential saturation curve observed. The initial slope will
depend on the concentration, independently of the total volume.
In some examples, nanoprobes are attached to the surface of an
optic fiber or a flat glass or plastic (such as a slide or array)
and illuminated using total internal reflection (TIR), which
excites fluorophores within about 100 nm of the surface. When the
biological sample is placed on the surface, the target molecules
bind, leaving other components in solution. For example, red blood
cells are large compared to the nanoprobes and therefore would not
significantly interfere with binding because they mostly stay away
from the surface (out of the excitation range of TIR). Some of the
output FRET light signal will pass through the surface to a
detector on the other side. In some examples, the sample is treated
with a DNase to remove DNA when detecting proteins or proteinase to
remove proteins when detecting nucleic acids, before application to
the surface.
In some examples, the sample is concentrated. For example, a thin
electrode (such as gold) can be evaporated or attached to the glass
surface. A second electrode is placed elsewhere, behind the sample
so that the sample is between the electrodes. Electrophoresis can
then be used to drive any charged molecules, including target
molecules, to the detection surface that has the immobilized
nanoprobes. This allows removal of the target molecules from the
biological sample to concentrate them near the nanoprobes. If
target molecules contact the electrodes they may be oxidized. To
reduce this possibility, the electrode surface can be coated or
covered with a membrane that will not allow the DNA to reach the
electrode. In some examples, the electrodes are used to extract
target molecules from cells in the sample by `reverse
electroporation` of the samples. A strong but short electrical
pulse will open the membranes of cell. For example, cells can be
exposed to a pulse voltage of about 1-20 kV/cm for a pulse time of
about 1-10 seconds. This can be followed by passive diffusion or by
active transport of the molecules by electrophoresis to bring the
targets to the nanoprobe detection surface.
In some examples, addition of the biological sample rehydrates a
dried nanoprobe on a surface. For example, adding saliva or blood
would supply moisture to the dried probes. A signal from the probe
will be generated if the target is present.
Example 21
Nanoprobe for Detecting Prostate Specific Antigen (PSA)
This example describes a nanoprobe that can be used to detect
PSA.
In one example, the nanoprobe shown in FIG. 4A includes antibodies
that can specifically bind to PSA as the targeting moieties 202,
204. PSA antibodies are commercially available and known in the art
(such as those available from GeneTex Inc., San Antonio, Tex. and
Abcam, Cambridge, Mass.). In another example matched antibody pairs
are used that bind to two distinct epitopes on the target (such as
those available from Anogen--YES Biotech Laboratories Ltd.,
Mississauga, Ontario). Such a nanoprobe can be generated using the
methods disclosed herein (for example using the methods described
in Example 3, except that PSA antibodies are used instead of p53
antibodies).
Generally, the method includes incubating one or more nanoprobes
for detecting PSA with a sample (for example from a subject) under
conditions that permit the nanoprobe(s) to specifically bind to
PSA. Particular examples of samples include, but are not limited
to, saliva and blood (or a fraction thereof such as serum). One or
more signals generated from the nanoprobe are then detected, and in
some examples quantitated. For example, the presence or absence of
an acceptor fluorophore emission signal can be detected, wherein
the presence of detectable signal indicates the presence of
PSA.
Example 22
Methods of Modifying a Biomolecule
This example describes methods that can be used to modify a
biomolecule, for example in vitro or in vivo. Although particular
examples are provided for the modification of particular
biomolecules (such as proteins or nucleic acid molecules), one
skilled in the art will appreciate that based on the teachings
herein, other biomolecules can be modified, for example by changing
the activating moiety used on the particular nanoprobes disclosed
herein.
In one example, the nanoprobe shown in FIG. 9 or 11 that includes
the particular functional groups described in Examples 8 and 9, is
used to degrade a p53 RNA sequence, for example to decrease
expression of a p53 protein. Generally, the method includes
incubating one or more nanoprobes for cleaving p53 with a sample
(for example from a subject), under conditions that permit the
nanoprobe(s) to specifically hybridize to p53 RNA, for example
under high stringency conditions. In some examples, one or more
signals generated from the nanoprobe are detected, for example to
quantitate an amount of p53 RNA in the sample. For example, the
presence or absence of an acceptor fluorophore emission signal can
be detected.
In a particular example, the p53 RNA is degraded in vivo. In some
examples, the nanoprobe is introduced into cells using liposomes or
targeted to certain cells with immunoliposomes (for example see Yu
et al., Nucleic Acids Res. 32:e48).
Example 23
Nanoprobes with Specific and Non-Specific Recognizers
This example describes nanoprobes that include a specific and a
non-specific targeting moiety instead of two specific targeting
moieties. Such nanoprobes can be used to detect target molecules,
such as target proteins or target nucleic acid molecules (such as a
target mRNA). Although particular examples are provided, those
skilled in the art will recognize how to make appropriate
substitutions.
Detection of a Target Protein
As shown in FIG. 20, the nanoprobe 5000 includes oligonucleotides
5002, 5004. To each of the oligonucleotides 5002, 5004 an
oligonucleotide 5006, 5008 complementary to at least a portion of
oligonucleotides 5002, 5004 is hybridized, respectively. One of the
complementary oligonucleotides 5006 includes a donor fluorophore
5010 and the other complementary oligonucleotide 5008 includes an
acceptor fluorophore 5012. In a particular example, the donor
fluorophore 5010 is 6-FAM and the acceptor fluorophore 5012 is
Texas Red. The nanoprobe 5000 also includes a molecular linker 5014
attached to the oligonucleotides 5002, 5004 that can be used to
separate the specific targeting moiety 5030 and the non-specific
targeting moiety 5026. In some examples, the molecular linker
includes PEG molecules 5016, 5018 and a molecular rod 5020. The
nanoprobe 5000 further includes an oligonucleotide 5022, 5024
complementary to at least a portion of oligonucleotides 5002, 5004,
respectively. One of the oligonucleotides 5022 includes a
non-specific targeting moiety (for example dodecyl sulfate) 5026
attached via a molecular linker 5028. Other exemplary non-specific
targeting moieties 5026 that can be used include ionic and
non-ionic detergents, SYPRO Ruby or SYPRO Rose dyes, and
poly-arginine or poly-glutamate polypeptides. The other
oligonucleotide 5024 includes a specific targeting moiety 5030 (for
example an antibody).
In one example, the nanoprobe 5000 shown in FIG. 20 is used to
detect a protein 5032 (not part of the probe). The hydrophobic part
of dodecyl sulfate 5026 penetrates the protein core and remains
inside the protein 5032. The dodecyl sulfate group 5026 and the
antibody 5030 specific for the target protein 5032 bring a FRET
acceptor 5012 and a donor 5010 together, resulting in a FRET signal
(FIG. 20). In some examples, an aptamer is used in place of the
antibody 5030.
Detection of a Target Nucleic Acid Molecule
A specific example of a nanoprobe containing specific and a
non-specific recognizers that can be used to detect a target mRNA
is shown in FIG. 21A. As shown in FIG. 21A, the nanoprobe 6000
contains a specific complementary oligonucleotide 6002 linked to a
dsDNA intercalating fluorophore 6004 by a molecular linker 6006
(such as a PEG tether, for example a PEG tether of 5 nm to 10 nm).
In some examples, the dsDNA intercalating fluorophore 6004 can act
as a FRET donor, such as a FRET donor of Texas Red. A specific
example of a dsDNA intercalating fluorophore 6004 that can be used
is SYBR Green. The oligonucleotide 6002 also includes a fluorophore
6008, such as a FRET acceptor (for example Texas Red or TAMRA).
Binding of the oligonucleotide 6002 to its complementary target
sequence 6010 (not part of the probe) results in FRET between the
donor 6004 and the acceptor 6006. The fluorescence intensity of
SYBR Green is enhanced over 100-fold on binding to double stranded
DNA, therefore there is no background fluorescence before DNA
binding.
Another specific example of a nanoprobe containing specific and
non-specific recognizers that can be used to detect a target mRNA
is shown in FIG. 21B. As shown in FIG. 21B, the nanoprobe 6050
contains a specific complementary oligonucleotide 6052 linked to
two dsDNA intercalating fluorophores 6054 6056 (the non-specific
targeting moieties) by a molecular linker 6058 6060 (such as a PEG
tether, for example a PEG tether of 5 nm to 10 nm), respectively.
In one example, the dsDNA intercalating fluorophores 6054 6056 are
a red and a green intercalating fluorophore, such as ethidium
bromide and SYBR Green. Upon binding of the nanoprobe 6050 to its
target mRNA 6062 (not part of the probe), the two intercalating
dyes form a FRET pair that can be detected. There is no significant
donor or acceptor fluorophore background fluorescence before the
specific binding.
Another specific example of a nanoprobe containing specific and a
non-specific recognizers that can be used to detect a target mRNA
is shown in FIG. 21C. As shown in FIG. 21C, the nanoprobe 6076
contains a specific complementary oligonucleotide 6078 linked to a
dsDNA intercalating fluorophore 6080 (the non-specific targeting
moiety) by a molecular linker 6082 (such as a PEG tether, for
example a PEG tether of 5 nm to 10 nm). Upon binding of the
specific complementary oligonucleotide 6078 to the mRNA target 6084
(not part of the probe), the dsDNA intercalating fluorophore 6080
(such as SYBR Green) intercalates the complex formed between the
oligonucleotide 6078 and its target 6084, thereby producing a
detectable fluorescent signal.
Example 24
Generating Nanoprobes In Vivo
This example describes methods that can be used to produce a
nanoprobe in vivo. For example, the nanoprobes disclosed herein can
be produced in a eukaryotic or prokaryotic host cells, such as E.
coli or a yeast cell. Methods of making recombinant molecules are
known in the art.
In particular examples the nanoprobes expressed in vivo include
only nucleic acid and protein elements. For example, tmRNAs which
attach an RNA to a protein, can be used to make an RNA nanoprobe.
Nanoprobes having single chain antibodies, such as
ssAB-CFP-YFP-ssAB, can be generated in vivo. In addition, such a
construct can be used to detect a target protein in vivo. In one
example the nanoprobe has the structure: ssAB-CFP-RecBCD
tether-YFP-ssAB. The ssABs can be replaced by RNA binding proteins.
The RNA can be synthesized inside the cell and would automatically
bind, so nucleic acid detecting nanoprobes could also be grown.
In one example, the nanoprobe includes proteins, such as two
protein-based fluorophores (such as CFP 477 nm/YFP 514 nm, EGFP 508
nm/YFP), two single chain antibodies and a tether or tethers with a
separating rod or other protein. The nanoprobe could include a
purification tag, such as streptavidin or a His tag, to permit
purification of the probe from the cells.
Example 25
"Self-Staining" Nanoprobes
This example describes nanoprobes that include coomassie brilliant
blue as a label. Such molecules can avoid the use of a fluorometer
for signal detection.
Molecules that change their absorbance spectrum after binding to
another molecule and a spectrophotometer can be used for signal
detection. An example of such a molecule is Coomassie protein
stain. Coomassie dye (Coomassie Brilliant Blue G-250) in acid
solution has an absorbance shift from 465 nm to 595 nm when it is
bound to protein. This dye specifically binds to proteins at
arginine, tryptophan, tyrosine, histidine and phenylalanine
residues. One target for Coomassie is Poly-Arg.
As shown in FIG. 22A, the nanoprobe 7000 contains specific
complementary oligonucleotides 7002, 7004 attached to one another
via a molecular linker 7006 that can include PEG tethers 7008, 7010
and a DNA rod 7012. To each of the oligonucleotides 7002, 7004 an
oligonucleotide 7014, 7016 complementary to at least a portion of
oligonucleotides 7002, 7004 is hybridized, respectively. One of the
complementary oligonucleotides 7014 includes Coomassie 7018 and the
other complementary oligonucleotide 7016 includes Poly-Arg 7020.
The molecular linker 7006 can be used to separate the Coomassie
7018 and the Poly-Arg 7020 in the absence of the target molecule.
In the absence of the target molecule, the Poly-Arg 7020 and
Coomassie 7018 groups are significantly separated and no
significant amount of blue color is detected (FIG. 22A).
As shown in FIG. 22B, after binding to the target molecule 7022
(not part of the probe), Poly-Arg 7020 and Coomassie 7018 interact,
and Coomassie 7018 changes color to blue (FIG. 22B).
The Coomassie moiety 7018 can be substituted with a pH-sensitive
dye, for example phenolphthalein. Phenolphthalein is white if pH is
lower than 8, and it becomes pink if pH is above 10. A Poly-Lys or
any basic group can make high "local" pH. If phenolphthalein is
near the basic group, it turns pink. Also, low pH-sensitive
molecules, and Poly-Glu or any acidic group can be used.
In some examples, a molecular linker is included between the
Coomassie moiety 7018 and oligonucleotide 7014, between the
Poly-Arg 7020 and oligonucleotide 7016, or combinations
thereof.
The approach described in this example can be extended to detecting
target biological molecules by providing suitable adapters for
connection between such biomolecules and activation compounds. For
example, the dye can be used for DNA recognition if an adapter,
that binds to the phosphate groups, also includes an arginine
residue(s).
Example 26
Reducing Background
This example describes methods used to reduce background signal
detected from a nanoprobe in the absence of a target molecule.
Although particular examples are provided for when the target is a
nucleic acid molecule, such methods can be used for any target
(such as a protein) (for example by attaching a quencher attached
to a protein instead of a quencher-containing oligonucleotide).
In FIG. 13A, the nanoprobe bound to the target shows an increased
615 nm FRET emission and a decreased 520 nm donor emission. The
fraction of the nanoprobe which is not bound to the target shows a
strong 520 nm emission signal of the donor and background
fluorescence of the acceptor due to the direct excitation at 470
nm. Therefore the unbound nanoprobe produced strong background
signals.
As shown at the top of FIG. 23, when nanoprobe 8000 is not bound it
its target molecule 8002, background fluorescence can be detected
from the donor 8004 and acceptor 8006 fluorophores. As shown at the
bottom left of FIG. 23, when nanoprobe 8000 binds to its target
8002, FRET can be detected due to the interaction between the donor
8004 and acceptor 8006 fluorophores.
Background signals can be reduced or even eliminated by treatment
of the reaction mixture after binding of the target 8002 to the
nanoprobe 8000 with quencher-containing oligonucleotides 8008 8010
that are complementary to the F and G parts of the nanoprobe core.
As shown at the bottom right of FIG. 23, when nanoprobe 8000 binds
to the oligonucleotides 8008 8010, fluorescence of the donor 8004
and acceptor 8006 are significantly quenched by the quenchers 8012
and 8014, respectively. Integrated DNA Technologies provides
quenchers that can be used on the oligonucleotides 8008 8010 (Table
8). Attaching the quencher containing oligonucleotides 8008 8010 by
a tether to the nanoprobe 8000 can be used to increase the binding
affinity and speed of binding.
TABLE-US-00017 TABLE 8 Exemplary quenchers Absor- Extinction bance
Dye to Name Coefficient Max quench Position Iowa Black FQ .TM.
13344 531 nm 6-FAM 5', 3' Iowa Black RQ .TM. 50457 656 nm Texas Red
5', 3' Black Hole Quencher .TM. 1 8000 534 nm 6-FAM 3' Black Hole
Quencher .TM. 2 8000 578 nm Texas Red 3'
Specific non-limiting examples of quencher-containing
oligonucleotides that can be used to quench when the target
sequence is SEQ ID NO: 12 are: TCTATACGGATCCTTACGCT-[Iowa Black
FQ.TM.] (SEQ ID NO: 91); [Iowa Black RQ.TM.]GTCTCGCGAATTCCGGCCTT
(SEQ ID NO: 92); TCTATACGGATCCTTACGCT[Black Hole Quencher.TM. 1]
(SEQ ID NO: 93); [Black Hole Quencher.TM. 2)]GTCTCGCGAATTCCGGCCTT
(SEQ ID NO: 94); TCTATACGGATCCTTACGCT[Tamra] (SEQ ID NO: 95).
In view of the many possible embodiments to which the principles of
our invention may be applied, it should be recognized that the
illustrated examples are only examples of the disclosure and should
not be taken as a limitation on the scope of the invention. Rather,
the scope of the invention is defined by the following claims. We
therefore claim as our invention all that comes within the scope
and spirit of these claims.
SEQUENCE LISTINGS
1
96120DNAHomo sapiens 1gaacatgccc gggcatgtcc 20221DNAHomo sapiens
2gaacatgtcc caaacatgtt g 21319DNAArtificial sequencep53 binding
sequence 3rrrcwwgyyy rrrcwwgyy 19447DNAHomo sapiens 4ggacatgtcc
ggacatgtcc gcgaagcgga catgtccgga catgtcc 47540DNAArtificial
sequenceexemplary molecular rod sequence 5gacgctagta tcttatgaag
ctttcctgac tgcggcatta 40640DNAArtificial sequenceexemplary sequence
that can be used in making a nanoprobe. 6gacgctagta tcttatgaag
ctttcctgac tgcggcatta 40740DNAArtificial sequenceexemplary sequence
that can be used in making a nanoprobe. 7taatgccgca gtcaggaaag
cttcataaga tactagcgtc 40820DNAArtificial sequenceexemplary sequence
that can be used in making a nanoprobe. 8gtgccgtcga attctcgcta
20960DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 9tagcgagaat tcgacggcac gacgctagta tcttatgaag
ctttcctgac tgcggcatta 601060DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 10cgatagggat
ccattactgc taatgccgca gtcaggaaag cttcataaga tactagcgtc
601120DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 11gcagtaatgg atccctatcg 201246DNAArtificial
sequenceexemplary target sequence 12tctatacgga tccttacgct
cacccagtct cgcgaattcc ggcctt 4613120DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 13tagcgagaat
tcgacggcac agcgtaagga tccgtataga gacgctagta tcttatgaag 60ctttcctgac
tgcggcatta aaggccggaa ttcgcgagac cgatagggat ccattactgc
1201420DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 14gtgccgtcga attctcgcta 201520DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
15gcagtaatgg atccctatcg 201640DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 16taatgccgca
gtcaggaaag cttcataaga tactagcgtc 401730DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
17ccgcagtcag gaaagcttca taagatacta 301820DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
18gtcaggaaag cttcataaga 201940DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 19tctatacgga
tccttacgct ccattgttca atatcgtccg 402041DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
20tctatacgga tccttacgct tccattgttc aatatcgtcc g 412142DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
21tctatacgga tccttacgct ttccattgtt caatatcgtc cg
422240DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 22tcatctggac ctgggtcttc gtctcgcgaa ttccggcctt
402341DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 23tcatctggac ctgggtcttc tgtctcgcga attccggcct t
412442DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 24tcatctggac ctgggtcttc ttgtctcgcg aattccggcc
tt 422540DNAArtificial sequenceexemplary target sequence
25cgatagggat ccattactgc tagcgagaat tcgacggcac 402660DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
26gcagtaatgg atccctatcg taatgccgca gtcaggaaag cttcataaga tactagcgtc
602740DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 27gtgccgtcga attctcgcta tgacgctagt atcttatgaa
402821DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 28gctttcctga ctgcggcatt a 212923DNAArtificial
sequenceexemplary target sequence 29cggacgatat tgaacaatgg ttc
233023DNAArtificial sequenceexemplary target sequence 30actgaagacc
caggtccaga tga 233123DNAArtificial sequenceexemplary target
sequence 31tcagctattt gcgtgtgagg aaa 233223DNAArtificial
sequenceexemplary target sequence 32cttctggatg ctggtgattt gga
233323DNAArtificial sequenceexemplary target sequence 33gccagtaccg
ccaactgaag acg 233423DNAArtificial sequenceexemplary target
sequence 34gccgaggact atcaggccct gaa 233523DNAArtificial
sequenceexemplary target sequence 35tggtgatgga cggcgtaatc tct
233623DNAArtificial sequenceexemplary target sequence 36gaccacgagt
gtcaggagct gca 233723DNAArtificial sequenceexemplary target
sequence 37tgggctttga caaacagctc tca 233823DNAArtificial
sequenceexemplary target sequence 38caggacctgg ctgtcaacct cct
233920DNAArtificial sequenceexemplary nanoprobe sequence
39ccattgttca atatcgtccg 204020DNAArtificial sequenceexemplary
nanoprobe sequence 40tcatctggac ctgggtcttc 204120DNAArtificial
sequenceexemplary nanoprobe sequence 41cctcacacgc aaatagctga
204220DNAArtificial sequenceexemplary nanoprobe sequence
42tccaaatcac cagcatccag 204320DNAArtificial sequenceexemplary
nanoprobe sequence 43cttcagttgg cggtactggc 204420DNAArtificial
sequenceexemplary nanoprobe sequence 44ttcagggcct gatagtcctc
204520DNAArtificial sequenceexemplary nanoprobe sequence
45gattacgccg tccatcacca 204620DNAArtificial sequenceexemplary
nanoprobe sequence 46tgcagctcct gacactcgtg 204720DNAArtificial
sequenceexemplary nanoprobe sequence 47gagctgtttg tcaaagccca
204820DNAArtificial sequenceexemplary nanoprobe sequence
48aggaggttga cagccaggtc 204921DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 49gctttcctga
ctgcggcatt a 215020DNAArtificial sequenceexemplary target sequence
50ggacatgtcc ggacatgtcc 205127DNAArtificial sequenceexemplary
target sequence 51ggacatgtcc ggacatgtcc gcgaagc 275225DNAArtificial
sequenceexemplary target sequence 52tctcacacac gtacacacac gtgtc
255332DNAArtificial sequenceexemplary target sequence 53tctcacacac
gtacacacac gtgtcgcgaa gc 325420DNAArtificial sequenceexemplary
target sequence 54gggacattcc gggacattcc 205527DNAArtificial
sequenceexemplary target sequence 55gggacattcc gggacattcc gcgaagc
275625DNAArtificial sequenceexemplary target sequence 56gtcgacattt
cccgtaaatc gtcga 255732DNAArtificial sequenceexemplary target
sequence 57gtcgacattt cccgtaaatc gtcgagcgaa gc 325820DNAArtificial
sequenceexemplary nanoprobe sequence 58ggacatgtcc ggacatgtcc
205925DNAArtificial sequenceexemplary nanoprobe sequence
59gacacgtgtg tgtacgtgtg tgaga 256020DNAArtificial sequenceexemplary
nanoprobe sequence 60ggaatgtccc ggaatgtccc 206125DNAArtificial
sequenceexemplary nanoprobe sequence 61tcgacgattt acgggaaatg tcgac
256240DNAArtificial sequenceexemplary nanoprobe sequence
62taatgccgca gtcaggaaag cttcataaga tactagcgtc 406320DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
63ggcctccgtc ctcggcagta 206420DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 64tactgccgag
gacggaggcc 206520DNAArtificial sequenceexemplary sequence that can
be used in making a nanoprobe. 65cgataatgcc tgtcatgcat
206620DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 66atgcatgaca ggcattatcg 206719DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
67gaacgtctag acttatcgc 196820DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 68gcgataagtc
tagacgttcc 206920DNAArtificial sequenceexemplary sequence that can
be used in making a nanoprobe. 69ctctcgctcc gtgccgtaag
207020DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 70cttacggcac ggagcgagag 207171DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
71cgctcctgaa ttcgacgtac gctatatatt tagtatgttg taactaaagt ccagcgcgaa
60gcttaatgac t 717271DNAArtificial sequenceexemplary sequence that
can be used in making a nanoprobe. 72attcagtctg caggaaggcc
gactttagtt acaacatact aaatatatag ccagtaaggg 60atccgatctc g
717320DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 73gtacgtcgaa ttcaggagcg 207420DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
74cgagatcgga tcccttactg 207520DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 75agtcattaag
cttcgcgctg 207620DNAArtificial sequenceexemplary sequence that can
be used in making a nanoprobe. 76ggccttcctg cagactgaat
207720DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 77ggcctccgtc ctcggcagta 207820DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
78cgataatgcc tgtcatgcat 207920DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 79ggaacgtcta
gacttatcgc 208020DNAArtificial sequenceexemplary sequence that can
be used in making a nanoprobe. 80ctctcgctcc gtgccgtaag
208140DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 81gtacgtcgaa ttcaggagcg tactgccgag gacggaggcc
408240DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 82atgcatgaca ggcattatcg cgagatcgga tcccttactg
408340DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 83gcgataagtc tagacgttcc agtcattaag cttcgcgctg
408440DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 84ggccttcctg cagactgaat cttacggcac ggagcgagag
408571DNAArtificial sequenceexemplary sequence that can be used in
making a nanoprobe. 85cgctcctgaa ttcgacgtac gctatatatt tagtatgttg
taactaaagt ccagcgcgaa 60gcttaatgac t 718671DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
86attcagtctg caggaaggcc gactttagtt acaacatact aaatatatag ccagtaaggg
60atccgatctc g 718740DNAArtificial sequenceexemplary sequence that
can be used in making a nanoprobe. 87gtacgtcgaa ttcaggagcg
tactgccgag gacggaggcc 408840DNAArtificial sequenceexemplary
sequence that can be used in making a nanoprobe. 88ggccttcctg
cagactgaat cttacggcac ggagcgagag 408940DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
89gcgataagtc tagacgttcc agtcattaag cttcgcgctg 409040DNAArtificial
sequenceexemplary sequence that can be used in making a nanoprobe.
90atgcatgaca ggcattatcg cgagatcgga tcccttactg 409120DNAArtificial
sequencequencher oligonucleotide 91tctatacgga tccttacgct
209220DNAArtificial sequencequencher oligonucleotide 92gtctcgcgaa
ttccggcctt 209320DNAArtificial sequencequencher oligonucleotide
93tctatacgga tccttacgct 209420DNAArtificial sequencequencher
oligonucleotide 94gtctcgcgaa ttccggcctt 209520DNAArtificial
sequencequencher oligonucleotide 95tctatacgga tccttacgct
209652DNAArtificial sequenceoligonucleotide 96cccggacgat attgaacaat
ggttcactga agacccaggt ccagatgaag ct 52
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