U.S. patent application number 16/319099 was filed with the patent office on 2019-08-01 for methods for fluorescence imaging microscopy.
The applicant listed for this patent is ALTIUS INSTITUTE FOR BIOMEDICAL SCIENCES, UNIVERSITY OF WASHINGTON. Invention is credited to Shreeram AKILESH, John Stamatoyannopoulos.
Application Number | 20190234874 16/319099 |
Document ID | / |
Family ID | 60992821 |
Filed Date | 2019-08-01 |
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United States Patent
Application |
20190234874 |
Kind Code |
A1 |
Stamatoyannopoulos; John ;
et al. |
August 1, 2019 |
METHODS FOR FLUORESCENCE IMAGING MICROSCOPY
Abstract
Disclosed herein are methods of detecting a regulatory element,
determining the localization of a regulatory element, and/or
measuring the activity of a regulatory element.
Inventors: |
Stamatoyannopoulos; John;
(Seattle, WA) ; AKILESH; Shreeram; (Seattle,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALTIUS INSTITUTE FOR BIOMEDICAL SCIENCES
UNIVERSITY OF WASHINGTON |
Seattle
Seattle |
WA
WA |
US
US |
|
|
Family ID: |
60992821 |
Appl. No.: |
16/319099 |
Filed: |
July 19, 2017 |
PCT Filed: |
July 19, 2017 |
PCT NO: |
PCT/US17/42896 |
371 Date: |
January 18, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62364245 |
Jul 19, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 19/34 20130101;
G01N 33/56966 20130101; G01N 21/6458 20130101; A01N 1/0221
20130101; C12Q 1/6837 20130101; C40B 40/06 20130101; C12N 5/0634
20130101; C40B 30/04 20130101; C12Q 1/6841 20130101; G01N 33/582
20130101; G01N 2015/0038 20130101; G01N 21/64 20130101; C12Q 1/6841
20130101; G01N 33/574 20130101; G01N 1/30 20130101; G01N 21/78
20130101; C12N 5/0694 20130101; C12Q 2521/301 20130101; C12Q
2563/107 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; G01N 21/78 20060101 G01N021/78; C12P 19/34 20060101
C12P019/34; A01N 1/02 20060101 A01N001/02; C12N 5/09 20060101
C12N005/09; C12N 5/078 20060101 C12N005/078; G01N 1/30 20060101
G01N001/30 |
Goverment Interests
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with the support of the United
States government under Grant number RM1-HG007743-02 by the Center
for Photogenomics.
Claims
1-102. (canceled)
103: An imaging-based method for detecting a regulatory element,
the method comprising: contacting a cell sample with a detection
agent; binding the detection agent to the regulatory element; and
analysing a detection profile from the detection agent to determine
the presence or absence of the regulatory element.
104: The method of claim 103, wherein the detection agent comprises
a set of fluorescently labeled probes between 20 nucleotides to 60
nucleotides in length.
105: The method of claim 103, wherein the regulatory element is an
activated DNaseI hypersensitive site (DHS).
106: The method of claim 103, further comprising: incubating the
cell sample with a set of fluorescently labeled probes, wherein
each probe hybridizes to a DNaseI hypersensitive site (DHS);
measuring a fluorescent signature of the set of fluorescently
labeled probes; based on the fluorescent signature, determining a
DHS profile; and comparing the DHS profile to a control, wherein a
correlation with the control indicates the activity level of the
regulatory element in the cell sample.
107: The method of claim 103, further comprising: incubating the
cell sample with a set of non-labeled probes; and incubating the
cell sample with a set of fluorescently labeled probes, wherein
each of the fluorescently labeled probes in the set of
fluorescently labeled probes interacts with a non-labeled probe
within the set of non-labeled probes, thereby generating a set of
fluorescently labeled probes.
108: The method of claim 104 comprising an additional set of
fluorescently-labeled probes.
109: The method of claim 108, wherein the combination of
fluorescent moieties in each of set of fluorescently labeled probes
are different, and wherein each set of fluorescently labeled probes
comprises a spectrally distinct bar code.
110: A method for generating a chromatin map, comprising:
contacting a cell sample with a set of detection agents; binding
the set of detection agents to one or more regulatory elements; and
analysing a detection profile from the set of detection agents to
generate a chromatin map.
111: The method of claim 110, further comprising generating a
3-dimensional map from the detection profile.
112: The method of claim 110, further comprising generating a
chromatin map for a cell type, wherein each cell type comprises a
different chromatin pattern.
113: The method of claim 112, further comprising determining at
least one unique marker within the chromatin map that is associated
with a specific cell type.
114: The method of claim 110, wherein the chromatin map allows for
determination of genomic activity or chromatin compaction.
115: The method of claim 110, wherein the set of detection agents
is a set of fluorescently labeled probes.
116: A method of measuring the activity of a target regulatory
element, the method comprising: contacting a cell sample with a
first set and a second set of detection agents, wherein the first
set of detection agents interact with a target regulatory element
within the cell, and the second set of detection agents interact
with at least one product of the target regulatory element; and
analysing a fluorescent profile from the first set and the second
set of detection agents, wherein the presence or the absence of the
at least one product indicates the activity of the target
regulatory element.
117: The method of claim 116, wherein the first set and the second
set of detection agents are a first set and a second set of
fluorescently labeled probes.
118: The method of claim 116, wherein the target regulatory element
is a DNaseI hypersensitive site (DHS).
119: The method of claim 117, wherein a fluorescent moiety of the
first set of fluorescently labeled probes and a fluorescent moiety
of the second set of fluorescently labeled probes are
different.
120: The method of claim 116, further comprising incubating the
cell sample with a set of non-labeled nucleic acid probes and a set
of non-labeled antibody-oligonucleotide probes.
121: The method of claim 120, further comprising incubating the
cell sample with a first set of fluorescently labeled
oligonucleotides, wherein each of the fluorescently labeled
oligonucleotides in the first set of fluorescently labeled
oligonucleotides hybridizes to a non-labeled nucleic acid probe
within the set of non-labeled nucleic acid probes, thereby
generating the first set of fluorescently labeled probes.
122: The method of claim 121, further comprising incubating the
cell sample with a second set of fluorescently labeled
oligonucleotides, wherein each of the fluorescently labeled
oligonucleotides in the second set of fluorescently labeled
oligonucleotides hybridizes to a non-labeled
antibody-oligonucleotide probe within the set of non-labeled
antibody-oligonucleotide probes, thereby generating the second set
of fluorescently labeled probes.
Description
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/364,245, filed Jul. 19, 2016, which application
is incorporated herein by reference in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0003] Imaging techniques such as fluorescence in situ
hybridization (FISH) allows for visualization of DNA or RNA
regions, and/or assessment of gene expression, chromosome position,
and/or protein localization. In some instances, these imaging
methods are limited by small field of view and/or limited
resolution. As such, data acquisition from large number of cells
requires multiple fields of view and thereby presents challenges in
obtaining high throughput and high resolution imaging data.
SUMMARY OF THE INVENTION
[0004] In some aspects, a method of detecting a regulatory element
in situ is provided to determine the presence, absence or activity
of the regulatory element. In other aspects, a method of detecting
different types of regulatory elements simultaneously is provided
utilizing a heterogeneous set of detection agents, and translating
the molecular information from the different types of regulatory
elements to determine the activity state of a cell. In additional
aspects, methods of determining the localization of a regulatory
element and methods of measuring the activity of a regulatory
element are provided.
[0005] In certain aspects, provided herein is an imaging-based
method of detecting a regulatory element, the method comprising (a)
contacting a cell sample with a detection agent; (b) binding the
detection agent to the regulatory element; and (c) analyzing a
detection profile from the detection agent to determine the
presence or absence of the regulatory element. The analyzing the
detection profile from the detection agent can further determine an
activity of the regulatory element. The detection agent can
comprise a set of fluorescently labeled probes between about 20
nucleotides to about 60 nucleotides in length. The method can
further comprise hybridizing the set of fluorescently labeled
probes to the regulatory element. The regulatory element can
comprise DNA, RNA, polypeptides, or a combination thereof. The
regulatory element can be DNA. The regulatory element can be RNA.
The RNA can be an enhancer RNA (eRNA). The eRNA can be between
about 50 base pairs to about 3 kilobase pairs. The eRNA can be at
least 200 base pairs in length. The presence of an eRNA can
correlate to an activated regulatory element. The regulatory
element can be an activated DNaseI hypersensitive site (DHS). The
method can further comprise (a) incubating a cell sample with a set
of fluorescently labeled probes, wherein each probe hybridizes to a
DNaseI hypersensitive site (DHS); (b) measuring a fluorescent
signature of the set of fluorescently labeled probes; (c) based on
the fluorescent signature, determining a DHS profile; and (d)
comparing the DHS profile to a control, wherein a correlation with
the control indicates the activity level of the regulatory element
in the cell sample. The regulatory element can be a polypeptide.
The polypeptide can comprise a transcription factor protein, a
DNA-binding protein, a RNA-binding protein, or a gene product. The
regulatory element can comprise chromatin. The method can further
comprise generating a chromatin profile. The chromatin profile can
comprise DNA density pattern and activated DHS. The method can
further comprise (a) incubating a cell sample with a set of
fluorescently labeled probes specific to target sites on a
chromatin in the presence of an exogenous agent or condition; (b)
measuring a fluorescent signature of the set of fluorescently
labeled probes; (c) based on the fluorescent signature, generating
a fluorescent profile of the chromatin; and (d) comparing the
fluorescent profile of step c) with a second fluorescent profile of
a chromatin obtained from an equivalent sample incubated with an
equivalent set of fluorescently labeled probes in the absence of
the exogenous agent or condition, wherein a difference between the
two sets of fluorescent profiles indicates a change in the
chromatin density induced by the exogenous agent or condition. The
exogenous agent or condition can comprise a drug or a small
molecule. The exogenous agent or condition can comprise an
environmental factor. The environmental factor can comprise a
change in temperature, pH, nutrient, or a combination thereof. The
detection profile can comprise signal intensity of the detection
agent, the location of the detection agent, and/or signal size of
the detection agent. The method can further comprise (a) incubating
the cell sample with a set of non-labeled probes; and (b)
incubating the cell sample with a set of fluorescently-labeled
probes prior to the hybridizing step, wherein each of the
fluorescently-labeled probe in the set of fluorescently-labeled
probe interact with a non-labeled probe within the set of
non-labeled probes, thereby generating the set of
fluorescently-labeled probes. The fluorescently-labeled probe can
comprise a fluorescently-labeled oligonucleotide or a
fluorescently-labeled protein. Each fluorescently-labeled
oligonucleotide within the set of fluorescently-labeled
oligonucleotides can further comprise a spectrally distinct bar
code. The combination of fluorophores within the set of
fluorescently-labeled oligonucleotides can further comprise a
spectrally distinct bar code. The set of fluorescently labeled
probes can comprise at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 18, 20, 30, 40, 50, 60, or more probes. Each of the
fluorescently labeled probes can be independently labeled with a
fluorescent moiety. Each fluorescent moiety within the set of
fluorescently labeled probes can be the same. The method can
further comprise an additional set of fluorescently labeled probes.
The combination of fluorescent moieties in each set of
fluorescently labeled probes can be different, wherein each set of
the fluorescently labeled probe comprise a spectrally distinct bar
code. The detection profile can be obtain from a synthetic aperture
optics (SAO) instrumentation. The analyzing further can comprise
utilizing a SAO method. The fluorescent moiety can comprise Cy3,
Cy5, Cy5.5, Cy7, Q570, Alexa488, Alexa555, Alexa594, Alexa647,
Alexa680, Alexa 750, Alexa 790, Atto488, Atto532, Atto647N,
TexasRed, CF610, Propidium iodide, Q670, IRDye700, IRDye800,
Indocyanine green, Pacific Blue dye, Pacific Green dye, or Pacific
Orange dye. The fluorescent moiety can comprise a quantum dot. The
quantum dot can comprise QDot525, QDot 545, QDot 565, QDot 585,
QDot 605, or QDot 655. The fluorescently labeled probe can comprise
at least one unnatural base. The unnatural base can comprise
2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl,
2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP),
2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP), T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O-NMA) modified, locked nucleic acid
(LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',
5'-anhydrohexitol nucleic acids (HNA), morpholino,
methylphosphonate nucleotides, thiolphosphonate nucleotides, or
2'-fluoro N3-P5'-phosphoramidites. The fluorescently labeled probe
can comprise DNA probe, RNA probe, protein probe, or a combination
thereof. The fluorescently labeled probe can comprise a locked
nucleic acid probe, a peptide nucleic acid (PNA) probe, an
oligonucleotide, an oligopaint, an ECHO probe, a molecular beacon
probe, a toe-hold probe, a TALE probe, a ZFN probe, or a CRISPR
probe. The fluorescently labeled probe can further be crosslinked
to the regulatory element. The fluorescently labeled probe can
further comprise at least one conjugating moiety. The conjugating
moiety can be attached at the 5' terminus, 3' terminus, or at an
internal site. The conjugating moiety can comprise a hapten group.
The hapten group can be a biotin. The conjugating moiety can be a
conjugating functional group. The conjugating functional group can
be an azido group or an alkyne group. The fluorescently labeled
probe can be designed based on Primer3 algorithm. The fluorescently
labeled probe can be further optimized to reduce off-target binding
activity. The cell sample can be further treated after incubation
with the fluorescently labeled probe. The cell sample can be fixed.
The fixation condition can comprise alcohol-based or
formaldehyde-based fixatives. The cell sample can be denatured. The
denaturing agent can comprise formamide or ethylene carbonate. The
cell sample can be cryopreserved. The cell sample can be a fresh
cell sample. The cell sample can comprise cells obtained from
blood, urine, stool, saliva, lymph fluid, cerebrospinal fluid,
synovial fluid, cystic fluid, ascites, pleural effusion, amniotic
fluid, chorionic villus sample, vaginal fluid, interstitial fluid,
buccal swab sample, sputum, bronchial lavage, Pap smear sample, or
ocular fluid. The cell sample can comprise cells obtained from a
blood sample, an aspirate sample, or a smear sample. The cell
sample can be a circulating tumor cell sample. The circulating
tumor cell sample can comprise lymphoma cells, fetal cells,
apoptotic cells, epithelia cells, endothelial cells, stem cells,
progenitor cells, mesenchymal cells, osteoblast cells, osteocytes,
hematopoietic stem cells, foam cells, adipose cells, transcervical
cells, circulating cardiocytes, circulating fibrocytes, circulating
cancer stem cells, circulating myocytes, circulating cells from
kidney, circulating cells from gastrointestinal tract, circulating
cells from lung, circulating cells from reproductive organs,
circulating cells from central nervous system, circulating hepatic
cells, circulating cells from spleen, circulating cells from
thymus, circulating cells from thyroid, circulating cells from an
endocrine gland, circulating cells from parathyroid, circulating
cells from pituitary, circulating cells from adrenal gland,
circulating cells from islets of Langerhans, circulating cells from
pancreas, circulating cells from hypothalamus, circulating cells
from prostate tissues, circulating cells from breast tissues,
circulating cells from circulating retinal cells, circulating
ophthalmic cells, circulating auditory cells, circulating epidermal
cells, circulating cells from the urinary tract, or combinations
thereof. The cell sample can be a peripheral blood mononuclear cell
sample. The cell sample can comprise cells obtained from a biopsy
sample. The cell sample can be a cancerous cell sample.
[0006] In certain aspects, provided herein is a method of
generating a chromatin map, comprising (a) contacting a cell sample
with a set of detection agents; (b) binding the set of detection
agents to one or more regulatory elements; and (c) analyzing a
detection profile from the set of detection agents to generate a
chromatin map. The method can further comprise generating a
3-dimensional map from the detection profile. The method can
further comprise generating a chromatin map for a cell type,
wherein each cell type comprises a different chromatin pattern. The
method can further comprise determining at least one unique marker
within the chromatin map that is associated with a specific cell
type. The at least one unique marker can comprise a genomic
sequence, a DHS, or a combination thereof. The chromatin map can
allow for determination of genomic activity or chromatin
compaction. The cell can comprise an epithelia cell, a connective
tissue cell, a muscle cell, a nerve cell, a hormone-secreting cell,
a blood cell, an immune system cell, or a stem cell. The cell can
comprise a cancerous cell. The set of detection agent can be a set
of fluorescently labeled probes. The fluorescent moiety can
comprise Cy3, Cy5, Cy5.5, Cy7, Q570, Alexa488, Alexa555, Alexa594,
Alexa647, Alexa680, Alexa 750, Alexa 790, Atto488, Atto532,
Atto647N, TexasRed, CF610, Propidium iodide, Q670, IRDye700,
IRDye800, Indocyanine green, Pacific Blue dye, Pacific Green dye,
or Pacific Orange dye. The fluorescent moiety can comprise a
quantum dot. The quantum dot can comprise QDot525, QDot 545, QDot
565, QDot 585, QDot 605, or QDot 655. The fluorescently labeled
probe can comprise at least one unnatural base. The unnatural base
can comprise 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE),
2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl
(2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O-NMA) modified, locked nucleic acid
(LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',
5'-anhydrohexitol nucleic acids (HNA), morpholino,
methylphosphonate nucleotides, thiolphosphonate nucleotides, or
2'-fluoro N3-P5'-phosphoramidites. The fluorescently labeled probe
can comprise DNA probe, RNA probe, protein probe, or a combination
thereof. The fluorescently labeled probe can comprise a locked
nucleic acid probe, a peptide nucleic acid (PNA) probe, an
oligonucleotide, an oligopaint, an ECHO probe, a molecular beacon
probe, a toe-hold probe, a TALE probe, a ZFN probe, or a CRISPR
probe. The fluorescently labeled probe can further be crosslinked
to the regulatory element. The fluorescently labeled probe can
further comprise at least one conjugating moiety. The conjugating
moiety can be attached at the 5' terminus, 3' terminus, or at an
internal site. The conjugating moiety can comprise a hapten group.
The hapten group can be a biotin. The conjugating moiety can be a
conjugating functional group. The conjugating functional group can
be an azido group or an alkyne group. The fluorescently labeled
probe can be designed based on Primer3 algorithm. The fluorescently
labeled probe can be further optimized to reduce off-target binding
activity. The cell sample can be further treated after incubation
with the fluorescently labeled probe. The cell sample can be fixed.
The fixation condition can comprise alcohol-based or
formaldehyde-based fixatives. The cell sample can be denatured. The
denaturing agent can comprise formamide or ethylene carbonate. The
cell sample can be cryopreserved. The cell sample can be a fresh
cell sample. The cell sample can comprise cells obtained from
blood, urine, stool, saliva, lymph fluid, cerebrospinal fluid,
synovial fluid, cystic fluid, ascites, pleural effusion, amniotic
fluid, chorionic villus sample, vaginal fluid, interstitial fluid,
buccal swab sample, sputum, bronchial lavage, Pap smear sample, or
ocular fluid. The cell sample can comprise cells obtained from a
blood sample, an aspirate sample, or a smear sample. The cell
sample can be a circulating tumor cell sample. The circulating
tumor cell sample can comprise lymphoma cells, fetal cells,
apoptotic cells, epithelia cells, endothelial cells, stem cells,
progenitor cells, mesenchymal cells, osteoblast cells, osteocytes,
hematopoietic stem cells, foam cells, adipose cells, transcervical
cells, circulating cardiocytes, circulating fibrocytes, circulating
cancer stem cells, circulating myocytes, circulating cells from
kidney, circulating cells from gastrointestinal tract, circulating
cells from lung, circulating cells from reproductive organs,
circulating cells from central nervous system, circulating hepatic
cells, circulating cells from spleen, circulating cells from
thymus, circulating cells from thyroid, circulating cells from an
endocrine gland, circulating cells from parathyroid, circulating
cells from pituitary, circulating cells from adrenal gland,
circulating cells from islets of Langerhans, circulating cells from
pancreas, circulating cells from hypothalamus, circulating cells
from prostate tissues, circulating cells from breast tissues,
circulating cells from circulating retinal cells, circulating
ophthalmic cells, circulating auditory cells, circulating epidermal
cells, circulating cells from the urinary tract, or combinations
thereof. The cell sample can be a peripheral blood mononuclear cell
sample. The cell sample can comprise cells obtained from a biopsy
sample. The cell sample can be a cancerous cell sample.
[0007] In certain aspects, provided herein is a method of measuring
the activity of a target regulatory element, the method comprising
(a) contacting a cell sample with a first set and a second set of
detection agents, wherein the first set of detection agents
interact with a target regulatory element within the cell, and the
second set of detection agents interact with at least one product
of the target regulatory element; and (b) analyzing a fluorescent
profile from the first set and the second set of detection agents,
wherein the presence or the absence of the at least one product
indicates the activity of the target regulatory element. The first
set and second set of detection agents can be a first set and a
second set of fluorescently labeled probes. The method can further
comprise hybridizing the first set of fluorescently labeled probes
to the target regulatory element. The method can further comprises
hybridizing the second set of fluorescently labeled probes to the
at least one product of the target regulatory element. The target
regulatory element can be DNA, RNA, a polypeptide, or a combination
thereof. The target regulatory element can be DNA. The target
regulatory element can be a DNaseI hypersensitive site (DHS). The
at least one product can be RNA, a polypeptide, or a combination
thereof. The at least one product can be RNA. The RNA can be an
enhancer RNA (eRNA). The presence of an eRNA can correlate with
target gene transcription. The at least one product can be a
polypeptide. The fluorescent moiety of the first set of
fluorescently labeled probes and the fluorescent moiety of the
second set of fluorescently labeled probes can be different. The
method can further comprise incubating the cell sample with a set
of non-labeled nucleic acid probes and a set of non-labeled
antibody-oligonucleotide probes. The method can further comprise
incubating the cell sample with a first set of
fluorescently-labeled oligonucleotides, wherein each of the
fluorescently-labeled oligonucleotide in the first set of
fluorescently-labeled oligonucleotides hybridizes to a non-labeled
nucleic acid probe within the set of non-labeled nucleic acid
probes, thereby generating the first set of fluorescently labeled
probes. The method can further comprise incubating the cell sample
with a second set of fluorescently-labeled oligonucleotides,
wherein each of the fluorescently-labeled oligonucleotide in the
second set of fluorescently-labeled oligonucleotides hybridizes to
a non-labeled antibody-oligonucleotide probe within the set of
non-labeled antibody-oligonucleotide probes, thereby generating the
second set of fluorescently labeled probes. The fluorescent moiety
can comprise Cy3, Cy5, Cy5.5, Cy7, Q570, Alexa488, Alexa555,
Alexa594, Alexa647, Alexa680, Alexa 750, Alexa 790, Atto488,
Atto532, Atto647N, TexasRed, CF610, Propidium iodide, Q670,
IRDye700, IRDye800, Indocyanine green, Pacific Blue dye, Pacific
Green dye, or Pacific Orange dye. The fluorescent moiety can
comprise a quantum dot. The quantum dot can comprise QDot525, QDot
545, QDot 565, QDot 585, QDot 605, or QDot 655. The fluorescently
labeled probe can comprise at least one unnatural base. The
unnatural base can comprise 2'-O-methyl, 2'-O-methoxyethyl
(2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro,
2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O-NMA) modified, locked nucleic acid
(LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA), 1',
5'-anhydrohexitol nucleic acids (HNA), morpholino,
methylphosphonate nucleotides, thiolphosphonate nucleotides, or
2'-fluoro N3-P5'-phosphoramidites. The fluorescently labeled probe
can comprise DNA probe, RNA probe, protein probe, or a combination
thereof. The fluorescently labeled probe can comprise a locked
nucleic acid probe, a peptide nucleic acid (PNA) probe, an
oligonucleotide, an oligopaint, an ECHO probe, a molecular beacon
probe, a toe-hold probe, a TALE probe, a ZFN probe, or a CRISPR
probe. The fluorescently labeled probe can further be crosslinked
to the regulatory element. The fluorescently labeled probe can
further comprise at least one conjugating moiety. The conjugating
moiety can be attached at the 5' terminus, 3' terminus, or at an
internal site. The conjugating moiety can comprise a hapten group.
The hapten group can be a biotin. The conjugating moiety can be a
conjugating functional group. The conjugating functional group can
be an azido group or an alkyne group. The fluorescently labeled
probe can be designed based on Primer3 algorithm. The fluorescently
labeled probe can be further optimized to reduce off-target binding
activity. The cell sample can be further treated after incubation
with the fluorescently labeled probe. The cell sample can be fixed.
The fixation condition can comprise alcohol-based or
formaldehyde-based fixatives. The cell sample can be denatured. The
denaturing agent can comprise formamide or ethylene carbonate. The
cell sample can be cryopreserved. The cell sample can be a fresh
cell sample. The cell sample can comprise cells obtained from
blood, urine, stool, saliva, lymph fluid, cerebrospinal fluid,
synovial fluid, cystic fluid, ascites, pleural effusion, amniotic
fluid, chorionic villus sample, vaginal fluid, interstitial fluid,
buccal swab sample, sputum, bronchial lavage, Pap smear sample, or
ocular fluid. The cell sample can comprise cells obtained from a
blood sample, an aspirate sample, or a smear sample. The cell
sample can be a circulating tumor cell sample. The circulating
tumor cell sample can comprise lymphoma cells, fetal cells,
apoptotic cells, epithelia cells, endothelial cells, stem cells,
progenitor cells, mesenchymal cells, osteoblast cells, osteocytes,
hematopoietic stem cells, foam cells, adipose cells, transcervical
cells, circulating cardiocytes, circulating fibrocytes, circulating
cancer stem cells, circulating myocytes, circulating cells from
kidney, circulating cells from gastrointestinal tract, circulating
cells from lung, circulating cells from reproductive organs,
circulating cells from central nervous system, circulating hepatic
cells, circulating cells from spleen, circulating cells from
thymus, circulating cells from thyroid, circulating cells from an
endocrine gland, circulating cells from parathyroid, circulating
cells from pituitary, circulating cells from adrenal gland,
circulating cells from islets of Langerhans, circulating cells from
pancreas, circulating cells from hypothalamus, circulating cells
from prostate tissues, circulating cells from breast tissues,
circulating cells from circulating retinal cells, circulating
ophthalmic cells, circulating auditory cells, circulating epidermal
cells, circulating cells from the urinary tract, or combinations
thereof. The cell sample can be a peripheral blood mononuclear cell
sample. The cell sample can comprise cells obtained from a biopsy
sample. The cell sample can be a cancerous cell sample.
[0008] In certain aspects, provided herein is a method of site
specific labeling of a cell sample for visualization, the method
comprising: a) incubating a plurality of cells on a coverslip with
a crosslinking agent to fix the plurality of cells on the
coverslip; b) contacting the fixed cells with a permeabilizing
agent to enable permealization of the fixed cells; c) contacting
the treated cells of step b) with an endonuclease for a first time
sufficient to generate site specific DNA cut sites; d) incubating
the plurality of cells of step c) with a solution comprising
terminal deoxynucleotide transferase (TdT) and
5-Ethynyl-2'-deoxyuridine 5'-triphosphate (5-EdUTP) for a second
time sufficient to generate a plurality of 5-EdUTP labeled DNA; and
e) derivatizing the plurality of 5-EdUTP labeled DNA with an azide
tagged fluorophore for visualization. The endonuclease can be DNase
I. The first time sufficient to generate the site specific DNA cut
sites can be at least 1 minute, 2 minutes, 3 minutes, 4 minutes, 5
minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, or more.
The second time sufficient to generate a plurality of 5-EdUTP
labeled DNA can be at least 5 minutes, 10 minutes, 15 minutes, 20
minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 1.5 hour, 2
hours, or more. The crosslinking agent can be formaldehyde. The
permeabilizing agent can be NP-40. The method can further comprise
a washing step after each of step a), step b), step c), step d) and
step e). The method can further comprise coating the coverslip with
cells prior to step a). The cells can be obtained from a cancer
sample. The cells can be obtained from a tissue sample, a blood
sample, an aspirate sample, or a smear sample. The cells can be
A549. The fluorophore can comprise Cy3, Cy5, Cy5.5, Cy7, Q570,
Alexa488, Alexa555, Alexa594, Alexa647, Alexa680, Alexa 750, Alexa
790, Atto488, Atto532, Atto647N, TexasRed, CF610, Propidium iodide,
Q670, IRDye700, IRDye800, Indocyanine green, Pacific Blue dye,
Pacific Green dye, or Pacific Orange dye. The coverslip can be
incubated at 37.degree. C.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] Various aspects of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0010] FIG. 1 represents a conceptual illustration of methods
described herein.
[0011] FIG. 2 illustrates a conceptual schematic of an exemplary
computer server to be used for processing a method described
herein.
[0012] FIG. 3A shows a two color SPDM image (experimental) of
chromatin (blue) with DNA sensitive element sites (red), showing
anti-colocalization of the DNA sensitive element sites with
chromatin. Scale bars: 1000 nm, inserts: 100 nm. The bottom right
panel shows chromatin (blue), the middle right panel shows DNA
sensitive element sites (red), and the top right panel shows the
overlay and the anti-colocalization of the DNA sensitive element
with chromatin. FIG. 3B is the inset of FIG. 3A.
[0013] FIG. 4A and FIG. 4B illustrate the localization precision
and nearest neighbor distances for DNA and DNase sensitive
elements.
[0014] FIG. 5A and FIG. 5B illustrate multi-omics imaging via
encoding of molecular information with ssDNA tags. FIG. 5A shows a
schematic of simultaneous labeling and multiplexed imaging of mRNA
and protein targets with multicolor QDots via DNA encoding. In
general, each molecular target is encoded by target-specific
ssDNA-tagged affinity molecule (e.g., an antibody, aptamer,
oligonucleotide, etc.). The resulting array of target-bound ssDNA
tags can be sequentially or simultaneously labeled by complementary
imaging probes, enabling multiplexed imaging of all targets of
interest (e.g., via fluorescence microscopy with hyperspectral
imaging, HSI). FIG. 5B shows an exemplary multiplexed labeling of
GAPDH and HSP90-alpha mRNA and corresponding proteins with QDots.
DNA encoding methodology enables ssDNA tagging of mRNA targets via
in situ hybridization and protein targets via immunorecognition by
antibody-ssDNA bioconjugates. All ssDNA tags were simultaneously
converted into distinctive optical signals by hybridization with
complementary QDot-ssDNA' probes. Fluorescence microscopy with
hyperspectral imaging (HIS) was employed for cell imaging and 4
individual QDot channels were unmixed. Individual grayscale
channels were false-colored and merged into a composite 4-color
image. Scale bar, 50 .mu.m.
[0015] FIG. 6 shows a workflow for target encoding and labeling via
in situ hybridization, immunorecognition, and multi-omics
procedures. DNA encoding methodology allows for labeling of
different types of targets (mRNA and proteins in this
proof-of-concept study) under conditions optimized for selective
target binding in separate steps. As a result, all targets are
converted into a uniform array of intermediate ssDNA tags, which
are then simultaneously labeled by complementary QDot-ssDNA' probes
for multiplexed imaging.
[0016] FIG. 7A and FIG. 7B illustrate a schematic and
characterization of QDot-ssDNA probe preparation. FIG. 7A shows
amine crosslinking by a homobifunctional reagent BS3 used for
covalent conjugation of 5' amine-terminated ssDNA oligonucleotides
and PEG-coated amine-functionalized QDots. ssDNA is activated by an
excess BS3, purified by desalting, and reacted with QDots
overnight. QDot-ssDNA probes are purified from excess unbound ssDNA
by ultrafiltration. Agarose gel electrophoresis in FIG. 7B shows an
increase in QDot gel motility upon conjugation of
negatively-charged ssDNA oligonucleotides, confirming successful
preparation of QDot-ssDNA probes.
[0017] FIG. 8A and FIG. 8B show a schematic and characterization of
antibody-ssDNA bioconjugate preparation via maleimide-mediated
crosslinking. FIG. 8A shows rabbit anti-mouse IgG is partially
reduced by treatment with TCEP to expose sulfhydryl groups for
ssDNA conjugation. At the same time, 5' amine-terminated ssDNA
oligonucleotides are activated by sulfo-SMCC. Mixing and a 4-hour
incubation of activated ssDNA with reduced IgG yields 1/2IgG-ssDNA
bioconjugates. PAGE analysis of bioconjugation products in FIG. 8B
confirmed formation of primarily 1/2IgG with one ssDNA along with
smaller fractions of 1/2IgG conjugated to two and three ssDNA
tags.
[0018] FIG. 9 illustrates evaluation of a 6-color QDot panel for
protein labeling via DNA encoding. FIG. 9A shows specific staining
of .beta.-tubulin via incubation with mouse anti-.beta.-tubulin
primary antibody and ssDNA-conjugated rabbit anti-mouse secondary
antibody followed by immuno-labeling with anti-rabbit QDot655-2'Ab
probes preserved functionality of 2'Ab-ssDNA bioconjugates.
Consistent .beta.-tubulin staining achieved via hybridization with
complementary QDot-ssDNA probes in FIG. 9B confirmed successful
preparation of a functional 6-color QDot-ssDNA panel. A lack of
non-specific binding in FIG. 9C by QDot-ssDNA probes in control
experiments that skipped incubation with primary and secondary
antibodies corroborates the utility of such probes for highly
specific target labeling via DNA encoding. True-color images for
target staining (FIG. 9B) vs. control (FIG. 9C) were obtained at
consistent exposure time for each QDot color. Scale bar, 50
.mu.m.
[0019] FIG. 10A, FIG. 10B, and FIG. 10C show a schematic and
characterization of antibody-ssDNA bioconjugate preparation using
the Thunder-Link oligo conjugation system. A 2-step amine
crosslinking strategy as illustrated in FIG. 10A was employed for
preparation of covalent antibody-ssDNA bioconjugates with intact
IgG. Antibody and 5' amine-terminated ssDNA were simultaneously
activated by respective activation reagents, purified via
desalting, and reacted overnight, producing IgG with varying number
of attached ssDNA tags. The reducing PAGE analysis of FIG. 10B
highlights the presence of multiple higher-MW bands corresponding
to heavy and light chains conjugated to varying number of ssDNA
tags. In the four reaction conditions performed with goat
anti-rabbit secondary antibodies, the relative volume ratios of
activated IgG to ssDNA were 1) 50+50, 2) 50+30, 3) 50+20, and 4)
50+10. As expected, increasing amount of ssDNA in the reaction
leads to more ssDNA tags being conjugated to each IgG molecule. In
FIG. 10C, the staining of Lamin A via incubation with rabbit
anti-Lamin A primary antibody and goat anti-rabbit 2'Ab-ssDNA
bioconjugates followed by labeling with QDot605-ssDNA' probes
confirmed the preserved specificity of ssDNA-tagged antibodies and
successful antibody-ssDNA bioconjugation. At the same time,
increasing non-specific binding by 2'Ab-ssDNA bioconjugates was
observed with increasing number of ssDNA tags per IgG in a control
experiment in which incubation with primary antibody was skipped.
Thus, a volume ratio of Ab:ssDNA=2:1 in Thunder-Link reaction is
considered optimal. All true-color images were obtained at
consistent exposure for direct comparison of staining intensity.
Scale bar, 250 .mu.m.
[0020] FIG. 11A, FIG. 11B, FIG. 11C, FIG. 11D, and FIG. 11E show
multiplexed protein labeling via DNA encoding with a panel of 1'
antibody-ssDNA bioconjugates. Primary antibodies against
HSP90-alpha, GAPDH, Lamin A, and .beta.-tubulin were conjugated to
ssDNA tags using Thunder-Link oligo conjugation system. Reducing
PAGE shows consistent formation of IgG-ssDNA bioconjugates for all
antibodies (FIG. 11A). Conventional 2-step immunofluorescence with
unmodified antibodies and QDot565-2'Ab probes shows characteristic
staining pattern for the 4 proteins of interest (FIG. 11B). Protein
labeling in FIG. 11C with 1'Ab-ssDNA bioconjugates and QDot565-2'Ab
probes yielded staining patterns consistent with the unmodified
antibodies of FIG. 11B, confirming the preservation of
antigen-binding functionality of 1'Ab-ssDNA. Single-color staining
with 1'Ab-ssDNA bioconjugates and complementary QDot-ssDNA' probes
further corroborates successful ssDNA conjugation and preparation
of an antibody-ssDNA panel suitable for protein labeling via DNA
encoding (FIG. 11D). Multiplexed staining via DNA encoding yielded
consistent staining patterns for all four proteins in respective
spectral channels of the same hyperspectral image (HSI) (FIG. 11E).
Individual grayscale channels were false-colored for clarity. Scale
bar, 50 .mu.m.
[0021] FIG. 12 shows characterization of mRNA labeling intensity
and specificity via DNA encoding. GAPDH mRNA was labeled via
indirect FISH procedure with 41nt FISH probe set (see Table 2)
followed by staining with QDot605-ssDNA probes (left panels) or
AlexaFluor555-labeled streptavidin-ssDNA probes (right panels).
Consistent characteristic punctuate staining pattern was observed
with both complementary imaging probes (top row). At the same time,
non-complementary probes (bottom row) failed to hybridize to mRNA
in situ hybridization (ISH) probes, confirming staining specificity
of the DNA encoding methodology. "Match" and "mismatch" true-color
images were obtained at consistent exposure for direct comparison
of staining intensity. Scale bar, 50 .mu.m.
[0022] FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D illustrates the
effect of a dsDNA spacer in an in situ hybridization (ISH) probe on
mRNA labeling intensity. Physical separation of mRNA-recognition
and QDot-binding portions of 41nt ssDNA ISH probes with a 16 bp
dsDNA spacer prevents formation of secondary structures, promotes
hybridization to target mRNA, and reduces steric hindrance to QDot
binding. As a result, a substantial increase in mRNA staining
intensity was realized with such probes (FIG. 13A) in comparison to
41nt ssDNA FISH probes (FIG. 13B). At the same time, longer 60nt
ssDNA probes without pre-hybridized dsDNA spacers experienced
greater degree of secondary structure formation, which interfered
with mRNA and QDot binding and failed to produce robust mRNA
staining (FIG. 13C) above non-specific QDot binding levels (FIG.
13D). All images were obtained with HSI and normalized for direct
comparison of signal intensity. Scale bar, 50 .mu.m.
[0023] FIG. 14 shows multi-omics QDot staining via DNA encoding.
Protein and mRNA targets were encoded with ssDNA tags in separate
steps, each using conditions optimal for binding of a specific
target type. Consequently, DNA sequence code was converted into an
optical signal by hybridization with complementary QDot-ssDNA
probes. Specifically, GAPDH mRNA was labeled with a 41nt in situ
hybridization (ISH) probe set followed by labeling of
.beta.-tubulin with Ab-ssDNA bioconjugates. Finally, both ssDNA
tags were simultaneously hybridized with respective QDot-ssDNA'
probes. Clear microtubule staining pattern of .beta.-tubulin
(false-colored green) and punctuate pattern of GAPDH mRNA
(false-colored red) were observed in dual-labeled specimen (top
row), whereas only .beta.-tubulin staining was present in a control
specimen that was not hybridized with GAPDH FISH probe set (bottom
row). Nuclei were counter-stained with DAPI (false-colored blue).
Scale bar, 100 .mu.m.
[0024] FIG. 15 illustrates the heterogeneity in GAPDH RNAi
following forward transfection with siRNA. Cells were seeded into a
24-well plate, allowed to attach, grown overnight, and then
transfected with GAPDH siRNA (or non-targeting control siRNA) for
24 hrs. GAPDH mRNA was encoded via in situ hybridization (ISH) with
mRNA ISH probes and then labeled with QDot605-ssDNA' probes.
Imaging of different areas within the well highlights heterogeneity
in GAPDH knock-down, likely resulting from heterogeneity in cell
transfection with siRNA. Specifically, complete GAPDH mRNA
degradation was observed throughout cells in the well center (top
right panel), whereas cells at the crowded well edge still
expressed regular levels of GAPDH mRNA (bottom right panel)
consistent with GAPDH expression in cells transfected with control
siRNA (left panels). Substantial number of non-transfected cells
might explain an average silencing efficiency of 78% as determined
by RT-PCR. Insets: control experiments showed lack of QDot
non-specific binding in the absence of complementary ssDNA probes.
All images were obtained with true-color camera at the same
exposure time for direct comparison of signal intensity. Scale bar,
250 .mu.m.
[0025] FIG. 16 illustrates the heterogeneity in GAPDH RNAi
following reverse transfection with siRNA. Cells were mixed with
GAPDH siRNA (or non-targeting control siRNA) in suspension and then
seeded to 24-well plate for transfection and growth for 24 hrs.
GAPDH mRNA was encoded via in situ hybridization (ISH) with mRNA
ISH probes and then labeled with QDot605-ssDNA' probes. As evident
from imaging of different areas within the well, reverse
transfection achieved a more uniform transfection and GAPDH
knock-down compared to forward transfection (see FIG. 12). Complete
GAPDH mRNA degradation was observed throughout majority of cells,
with only occasional colonies with full GAPDH expression forming
from non-transfected cells, which is consistent with an improved
average silencing efficiency of 95% as determined by RT-PCR.
Insets: control experiments showed lack of QDot non-specific
binding in the absence of complementary ssDNA probes. All images
were obtained with true-color camera at the same exposure time for
direct comparison of signal intensity. Scale bar, 250 .mu.m.
[0026] FIG. 17 shows the comparison of RNAi effect on GAPDH mRNA
expression following forward vs. reverse transfection with siRNA.
Both transfection methods had no effect on GAPDH expression when
non-targeting control siRNA was used (left panels) and yielded
efficient GAPDH knock-down with GAPDH-targeting siRNA (middle
panels), as evident from the lack of mRNA staining above
non-specific QDot background (right panels). At the same time,
small fraction of cells failed to get transfected and, as a result,
expressed normal levels of GAPDH mRNA consistent with control
experiments. This observation corroborates an all-on/all-off effect
of RNAi regardless of the transfection method used. All images were
obtained with hyperspectral imaging (HIS) and were normalized for
direct comparison of signal intensity. Scale bar, 50 .mu.m.
[0027] FIG. 18 shows assessment of heterogeneity in cell
transfection with siRNA. Dual-labeling of GAPDH and HSP90-alpha
mRNA with QDots enables direct visualization of siRNA transfection
effect at a single-cell level. Cells were either grown under
regular culture conditions (FIG. 18A, FIG. 18B, and FIG. 18C),
transfected with control non-targeting siRNA (FIG. 18D, FIG. 18E,
and FIG. 18F), or transfected with GAPDH-targeting siRNA (FIG. 18G,
FIG. 18H, and FIG. 18I). After a 24-hour treatment with GAPDH
siRNA, the majority of cells had completely degraded GAPDH mRNA, as
evident from the lack of GAPDH mRNA staining (FIG. 18G). At the
same time, HSP90-alpha mRNA not targeted by RNAi machinery remained
unperturbed (FIG. 18H). Interestingly, a single cell in the field
of view failed to transfect with GAPDH siRNA (FIG. 18G, FIG. 18H,
and FIG. 18I), expressing regular levels of GAPDH mRNA consistent
with cells treated with control siRNA (FIG. 18D, FIG. 18E, and FIG.
18F) and reference cells not transfected with siRNA (FIG. 18A, FIG.
18B, and FIG. 18C), suggesting an all-on/all-off effect of RNAi.
Dual-color images were obtained with hyperspectral imaging (HIS)
and were unmixed in QDot channels. Panels for individual channels
(FIG. 18A, FIG. 18B, FIG. 18D, FIG. 18E, FIG. 18G, and FIG. 18H)
were normalized for direct comparison of signal intensity. In
merged 2-color images (FIG. 18C, FIG. 18F, and FIG. 18I) The GAPDH
channel was false-colored green and the HSP90-alpha channel was
false-colored red. Scale bar, 50 .mu.m.
[0028] FIG. 19 shows assessment of GAPDH RNAi heterogeneity at mRNA
and protein levels with multi-omics imaging. Dual labeling of GAPDH
mRNA and protein 24 hrs post-transfection with GAPDH-targeting
siRNA highlights heterogeneity in mRNA expression levels (bottom
left panel) along with the lack of RNAi effect on the protein level
(bottom middle panel) at this time point. Transfection with
non-targeting control siRNA (top row) failed to affect GAPDH
expression, yielding uniform mRNA and protein staining throughout
all cells. Dual-color images were obtained with hyperspectral
imaging (HSI), and individual channels were normalized for direct
comparison of signal intensity. The GAPDH mRNA channel was
false-colored red and the GAPDH protein channel was false-colored
green in a composite 2-color image. Scale bar, 50 .mu.m.
[0029] FIG. 20A and FIG. 20B show assessment of disparity in RNAi
kinetics at mRNA and protein levels. HeLa cells were transfected
with GAPDH siRNA for 24 hours (FIG. 20A) and 48 hours (FIG. 20B).
GAPDH and HSP90-alpha mRNA, along with corresponding proteins, were
simultaneously assessed with QDot-based multi-omics imaging
methodology. Consistent with mRNA-only analysis, multi-omics
imaging highlights complete and selective degradation of GAPDH mRNA
24 hours post-transfection, whereas GAPDH protein level remained
nearly unperturbed (FIG. 20A). Lagging mRNA knock-down 48 hours
post-transfection selective degradation of GAPDH protein was
observed (FIG. 20B). All grayscale images were normalized to HSP90
protein channel for direct comparison of staining intensities. In a
merged 4-color image the GAPDH protein channel was false-colored
yellow, the HSP90-alpha protein channel was false-colored blue, the
GAPDH mRNA channel was false-colored green, and the HSP90-alpha
mRNA channel was false-colored red. Scale bar, 50 .mu.m.
[0030] FIG. 21A and FIG. 21B show multi-omics evaluation of GAPDH
and HSP90-alpha expression at mRNA and protein levels under regular
cell culture conditions. To provide a reference of normal GAPDH and
HSP90 expression levels to RNAi experiments, cells were grown under
regular cell culture conditions for 24 hrs (FIG. 21A) and 48 hrs
(FIG. 21B). All targets of interest were labeled via a 2+2 encoding
procedure to produce a 4-plex staining. Consistent with expected
fast growth of HeLa cells, cell density increased with time.
However, GAPDH and HSP90 expression remained constant through 48
hrs of incubation, as evident from consistent intensity of mRNA and
protein labeling. Multiplex images were obtained with hyperspectral
imaging (HIS), and individual channelsw were normalized for direct
comparison of signal intensity. The GAPDH mRNA channel was
false-colored green, the HSP90 mRNA channel was false-colored red,
the GAPDH protein channel was false-colored yellow, and the HSP90
protein channel was false-colored blue in a composite 4-color
image. Scale bar, 50 .mu.m.
[0031] FIG. 22A and FIG. 22B show multi-omics evaluation of GAPDH
and HSP90-alpha expression at mRNA and protein levels following
transfection with a control (non-targeting) siRNA. To assess an
effect of transfection on molecular expression profiles in
reference to GAPDH RNAi experiments, cells were reverse transfected
with non-targeting control siRNA for (FIG. 22A) 24 hrs and (FIG.
22B) 48 hrs. All targets of interest were labeled via a 2+2
encoding procedure to produce a 4-plex staining. Consistent with
expected lack of RNAi with control siRNA, GAPDH and HSP90
expression remained constant through 48 hrs of incubation, as
evident from consistent intensity of mRNA and protein labeling.
Multiplex images were obtained with hyperspectral imaging (HSI),
and individual channels were normalized for direct comparison of
signal intensity. The GAPDH mRNA channel was false-colored green,
the HSP90 mRNA channel was false-colored, the GAPDH protein channel
was false-colored yellow, and the HSP90 protein channel was
false-colored blue in a composite 4-color image. Scale bar, 50
.mu.m.
[0032] FIG. 23A and FIG. 23B show direct visualization of the
effect and kinetics of GAPDH RNAi via single-plex labeling of
individual protein and mRNA targets. To eliminate any potential
effect of multi-omics labeling methodology and artifacts of
hyperspectral (HSI) analysis, the GAPDH RNAi sample along with a
reference sample and a control sample were performed on separate
specimens in parallel (different wells of the same 24-well plate),
followed by a single-plex labeling of individual targets and direct
true-color imaging under consistent imaging conditions. Cells were
reverse transfected for 24 hrs (FIG. 23A) and 48 hrs (FIG. 23B)
prior to fixation and staining. Consistent with multi-omics
analysis, single-plex imaging confirmed efficient and specific
degradation of GAPDH mRNA within 24 hrs post-transfection, whereas
the RNAi effect on GAPDH protein level can be observed only 48 hrs
post-transfection. Scale bar, 50 .mu.m.
[0033] FIG. 24 shows the labeling of DNaseI cut sites in a cell's
nucleus using a terminal deoxynucleotidyl transferase dUTP nick end
labeling (TUNEL) assay.
DETAILED DESCRIPTION OF THE INVENTION
[0034] Cellular activation and extinction patterns can encode
information on cell identity, maturation state, cellular memory,
and disease state. Tissues are composites of cells which can have
one or more morphologically distinct cell types. In some instances,
all of the cells in a tissue are processed simultaneously, yielding
compounded information with limited sensitivity for cellular
activities and/or rare cell types. Alternative approaches employ
disaggregation and sorting of tissue components but in the process
can destroy cellular architecture and potentially introduce
artifacts such as biological stressors and perturbations.
[0035] Described herein are methods of detecting a cellular
regulatory element in situ utilizing a super-resolution microscopy
technique to determine the presence, absence, and/or activity of a
regulatory element. Also described herein are methods of detecting
different types of regulatory elements simultaneously utilizing a
heterogeneous set of detection agents, and translating the
molecular information from the different types of regulatory
elements to determine the activity state of a cell. The activity
state of a cell may correlate to a localization, expression level,
and/or interaction state of a regulatory element. One or more of
the methods described herein may further interpolate 2-dimensional
images to generate 3-dimensional maps which enable detection of
localization, interaction states, and activity of one or more
regulatory elements. Intrinsic properties such as size, intensity,
and location of a detection agent further may enable detection of a
regulatory element. Described herein are methods of determining the
localization of a regulatory element and measuring the activity of
a regulatory element. The methods provided herein may avoid the
introduction of artifacts such as biological stressors and
perturbations or destroys cellular architecture. Exemplary
properties associated with the methods described herein are
illustrated in FIG. 1.
[0036] One or more methods described herein may detect different
types of regulatory elements, distinguish between different types
of regulatory elements, and/or generate a map of a regulatory
element (e.g., chromatin). For example, a regulatory element may be
labeled by one or more different types of detection agents. The one
or more different types of detection agents may include DNA
detection agents, RNA detection agents, protein detection agents,
or combinations thereof. The detection agent may comprise a probe
portion, which may interact (e.g., hybridize) to a target site
within the regulatory element, and optionally comprise a detectable
moiety. The detectable moiety may include a fluorophore, such as a
fluorescent dye or a quantum dot. The detection agent may be an
unlabeled probe which can be further conjugated to an additional
labeled probe. Upon labeling, the regulatory element may be
detected by stochastic or deterministic super-resolution microscopy
method. The stochastic super-resolution microscopy method may be a
synthetic aperture optics (SAO) method. The SAO method may generate
a detection profile, which can encompass fluorescent signal
intensity, size, shape, or localization of the detection agent.
Based on the detection profile, the activity state, the
localization, expression level, and/or interaction state of the
regulatory element may be determined. A map based on the detection
profile of the regulatory element may also be generated, and may be
correlated to cell type identification (e.g., cancerous cell
identification). The regulatory element may be further analyzed in
the presence of an exogenous agent or condition, such as a small
molecule fragment or a drug, or under an environment such as a
change in temperature, pH, nutrient, or a combination thereof. The
perturbation of the activity state of the regulatory element in the
presence of the exogenous agent or condition may be measured. A
report may further be generated and provided to a user, such as a
laboratory clinician or health care provider.
Types of Regulatory Elements
[0037] A regulatory element may be DNA, RNA, a polypeptide, or a
combination thereof. A regulatory element may be DNA. A regulatory
element may be RNA. A regulatory element may be a polypeptide. A
regulatory element may be any combination of DNA, RNA, and/or
polypeptide (e.g., protein-protein complexes, protein-DNA/RNA
complexes, and the like).
[0038] A regulatory element may be DNA. A regulatory element may be
a single-stranded DNA regulatory element, a double-stranded DNA
regulatory element, or a combination thereof. The DNA regulatory
element may be single-stranded. The DNA regulatory element may be
double-stranded. The DNA regulatory element may encompass a DNA
fragment. The DNA regulatory element may encompass a gene. The DNA
regulatory element may encompass a chromosome. The DNA regulatory
element may include endogenous DNA regulatory elements (e.g.,
endogenous genes). The DNA regulatory element may include
artificial DNA regulatory elements (e.g., foreign genes introduced
into a cell).
[0039] A regulatory element may be RNA. A regulatory element may be
a single-stranded RNA regulatory element, a double-stranded RNA
regulatory element, or a combination thereof. The RNA regulatory
element may be single-stranded. The RNA regulatory element may be
double-stranded. The RNA regulatory element may include endogenous
RNA regulatory elements. The RNA regulatory element may include
artificial RNA regulatory elements. The RNA regulatory element may
include microRNA (miRNA), transfer RNA (tRNA), ribosomal RNA
(rRNA), messenger RNA (mRNA), pre-mRNA, transfer-messenger RNA
(tmRNA), heterogeneous nuclear RNA (hnRNA), short interfering RNA
(siRNA), or short hairpin RNA (shRNA). The RNA regulatory element
may be a RNA fragment. The RNA regulatory element may be an
anti-sense RNA.
[0040] An RNA regulatory element may be an enhancer RNA (eRNA). An
enhancer RNA may be a non-coding RNA molecule transcribed from an
enhancer region of a DNA molecule, and may be from about 50
base-pairs (bp) in length to about 3 kilo base pairs in length
(e.g., about 100 bp in length, about 200 bp in length, about 500 bp
in length, about 1 kb in length, about 1.5 kb in length, about 2 kb
in length, or about 2.5 kb in length). An enhancer RNA may be a 1D
eRNA or an eRNA that may be unidirectionally transcribed. An
enhancer RNA may also be a 2D eRNA or an eRNA that may be
bidirectionally transcribed. An eRNA may be polyadenylated.
Alternatively, an eRNA may be non-polyadenylated.
[0041] A regulatory element may be a DNaseI hypersensitive site
(DHS). DHS may be a region of chromatin unoccupied by transcription
factors and which is sensitive to cleavage by the DNase I enzyme.
The presence of DHS regions within a chromatin may demarcate
transcription factory occupancy at a nucleotide resolution. The
presence of DHS regions may further correlate with activation of
cis-regulatory elements, such as an enhancer, promoter, silencer,
insulator, or locus control region. DHS variation may be correlated
to variation in gene expression in healthy or diseased cells (e.g.,
cancerous cells) and/or correlated to phenotypic traits.
[0042] A DHS pattern may encode memory of prior cell fate decisions
and exposures. For example, upon differentiation, a DHS pattern of
a progeny may encode transcription factor occupancy of its parent.
Further, a DHS pattern of a cell may encode an
environmentally-induced transcription factor occupancy from an
earlier time point.
[0043] A DHS pattern may encode cellular maturity. An embryonic
stem cell may encode a set of DHSs that may be transmitted
combinatorially to a differentiated progeny, and this set of DHSs
may be decreased with each cycle of differentiation. As such, the
set of DHSs may be correlated with time, thereby allowing a DHS
pattern to be correlated with cellular maturity.
[0044] A DHS pattern may also encode splicing patterns. Protein
coding exons may be occupied by transcription factors, which may
further be correlated with codon usage patterns and amino acid
choice on evolutionary time scales and human fitness. A
transcription factory occupancy may further modulate alternative
splicing patterns, for example, by imposing sequence constraints at
a splice junction. As such, a DHS pattern may encode transcription
factor occupancy of one or more exons of interest and may provide
additional information on alternative splicing patterns.
[0045] A DHS pattern may encode a cell type. For example, within
each cell type, about 100,000 to about 250,000 DHSs may be
detected. About 5% of the detected DHSs may be located within a
transcription start site and the remaining DHSs may be detected at
a distal site from the transcription start site. Each cell type may
contain a distinct DHS pattern at the distal site and mapping the
DHS pattern at the distal site may allow identification of a cell
type. An overlap may further be present within two DHS patterns
from two different cell types, for example, an overlap of a set of
detected DHSs within the two DHS patterns. An overlap may be less
than about 70%, less than about 65%, less than about 60%, less than
about 55%, less than about 50%, less than about 45%, less than
about 40%, less than about 35%, less than about 30%, less than
about 25%, less than about 20%, less than about 15%, less than
about 10%, less than about 9%, less than about 8%, less than about
7%, less than about 6%, less than about 5%, less than about 4%,
less than about 3%, less than about 2%, or less than about 1% of
the detected DHSs. The presence of an overlap may not affect the
identification of a cell type.
[0046] A regulatory element may be a polypeptide. The polypeptide
may be a protein or a polypeptide fragment. For example, a
regulatory element may be a transcription factor, DNA-binding
protein or functional fragment, RNA-binding protein or functional
fragment, protein involved in chemical modification (e.g., involved
in histone modification), or gene product. A regulatory element may
be a transcription factor. A regulatory element may be a DNA or
RNA-binding protein or functional fragment. A regulatory element
may be a product of a gene transcript. A regulatory element may be
a chromatin.
Methods of Detecting a Regulatory Element
[0047] Described herein is a method of detecting a regulatory
element. The detection may encompass identification of the
regulatory element, determining the presence or absence of the
regulatory element, and/or determining the activity of the
regulatory element. A method of detecting a regulatory element may
include contacting a cell sample with a detection agent, binding
the detection agent to the regulatory element, and analyzing a
detection profile from the detection agent to determine the
presence, absence, or activity of the regulatory element.
[0048] The method may involve utilizing one or more intrinsic
properties associated with a detection agent to aid in detection of
the regulatory element. The intrinsic properties may encompass the
size of the detection agent, the intensity of the signal, and the
location of the detection agent. The size of the detection agent
may include the length of the probe and/or the size of the
detectable moiety (e.g., the size of a fluorescent dye molecule)
may modulate the specificity of interaction with a regulatory
element. The intensity of the signal from the detection agent may
correlate to the sensitivity of detection. For example, a detection
agent with a molar extinction coefficient of about
0.5-5.times.10.sup.6 M.sup.-1cm.sup.-1 may have a higher intensity
signal relative to a detection agent with a molar extinction
coefficient outside of the 0.5-5.times.10.sup.6 M.sup.-1cm.sup.-1
range and may have lower attenuation due to scattering and
absorption. Further, a detection agent with a longer excited state
lifetime and a large Stoke shift (measured by the distance between
the excitation and emission peaks) may further improve the
sensitivity of detection. The location of the detection agent may,
for example, provide the activity state of a regulatory element. A
combination of intrinsic properties of the detection agent may be
used to detect a regulatory element of interest.
[0049] A detection agent may comprise a detectable moiety that is
capable of generating a light, and a probe portion that is capable
of hybridizing to a target site on a regulatory element. As
described herein, a detection agent may include a DNA probe
portion, an RNA probe portion, a polypeptide probe portion, or a
combination thereof. Sometimes, a DNA or RNA probe portion may be
between about 10 and about 100 nucleotides in length, between about
15 and about 100 nucleotides in length, between about 20 and about
100 nucleotides in length, between about 20 and about 80
nucleotides in length, between about 20 and about 60 nucleotides in
length, between about 25 and about 55 nucleotides in length,
between about 30 and about 50 nucleotides in length, between about
15 and about 80 nucleotides in length, between about 15 and about
60 nucleotides in length, between about 20 and about 40 nucleotides
in length, or between about 20 and about 30 nucleotides in length.
Sometimes, a DNA or RNA probe portion can be about 10, about 15,
about 20, about 21, about 22, about 23, about 24, about 25, about
26, about 27, about 28, about 29, about 30, about 31, about 32,
about 33, about 34, about 35, about 36, about 37, about 38, about
39, about 40, about 45, about 50, about 55, about 60, about 65,
about 70, about 80, about 90, about 100, or more nucleotides in
length. A DNA or RNA probe portion may be a TALEN probe, ZFN probe,
or a CRISPR probe. A DNA or RNA probe portion may be a padlock
probe. A polypeptide probe may comprise a DNA-binding protein, a
RNA-binding protein, a protein involved in the
transcription/translation process, a protein that detects the
transcription/translation process, a protein that can detect an
open or relaxed portion of a chromatin, or a protein interacting
partner of a product of a regulatory element (e.g., an antibody or
binding fragment thereof).
[0050] A detection agent may comprise a DNA or RNA probe portion
which can be between about 10 and about 100 nucleotides in length,
between about 15 and about 100 nucleotides in length, between about
20 and about 100 nucleotides in length, between about 20 and about
80 nucleotides in length, between about 20 and about 60 nucleotides
in length, between about 25 and about 55 nucleotides in length,
between about 30 and about 50 nucleotides in length, between about
15 and about 80 nucleotides in length, between about 15 and about
60 nucleotides in length, between about 20 and about 40 nucleotides
in length, or between about 20 and about 30 nucleotides in length.
A detection agent may comprise a DNA or RNA probe portion which may
be about 10, about 15, about 20, about 21, about 22, about 23,
about 24, about 25, about 26, about 27, about 28, about 29, about
30, about 31, about 32, about 33, about 34, about 35, about 36,
about 37, about 38, about 39, about 40, about 45, about 50, about
55, about 60, about 65, about 70, about 80, about 90, about 100, or
more nucleotides in length.
[0051] A detection agent may comprise a DNA or RNA probe selected
from a TALEN probe, a ZFN probe, or a CRISPR probe.
[0052] A set of detection agents may be used to detect a regulatory
element. The set of detection agents may comprise about 2, about 3,
about 4, about 5, about 6, about 7, about 8, about 9, about 10,
about 11, about 12, about 13, about 14, about 15, about 16, about
17, about 18, about 19, about 20, about 25, about 30, about 35,
about 40, about 45, about 50, or more detection agents. Each of the
detection agents within the set of detection agents may recognize
and interact with a distinct region of a regulatory element.
Sometimes, about 1, about 2, about 3, about 4, about 5, about 6,
about 7, about 8, about 9, about 10, about 11, about 12, about 13,
about 14, about 15, about 16, about 17, about 18, about 19, about
20, or more detection agents may be used for detection of a
regulatory element. About 1 or more detection agents may be used
for detection of a regulatory element. About 2 or more detection
agents may be used for detection of a regulatory element. About 3
or more detection agents may be used for detection of a regulatory
element. About 4 or more detection agents may be used for detection
of a regulatory element. About 5 or more detection agents as used
for detection of a regulatory element. About 6 or more detection
agents may be used for detection of a regulatory element. About 7
or more detection agents may be used for detection of a regulatory
element. About 8 or more detection agents may be used for detection
of a regulatory element. About 9 or more detection agents may be
used for detection of a regulatory element. About 10 or more
detection agents may be used for detection of a regulatory element.
About 11 or more detection agents may be used for detection of a
regulatory element. About 12 or more detection agents may be used
for detection of a regulatory element. About 13 or more detection
agents may be used for detection of a regulatory element. About 14
or more detection agents may be used for detection of a regulatory
element. About 15 or more detection agents may be used for
detection of a regulatory element. About 20 or more detection
agents may be used for detection of a regulatory element.
[0053] A detection agent may comprise a polypeptide probe selected
from a DNA-binding protein, a RNA-binding protein, a protein
involved in the transcription/translation process, a protein that
detects the transcription/translation process, a protein that can
detect an open or relaxed portion of a chromatin, or a protein
interacting partner of a product of a regulatory element (e.g., an
antibody or binding fragment thereof).
[0054] A detectable moiety that is capable of generating a light
may be directly conjugated or bound to a probe portion. A
detectable moiety may be indirectly conjugated or bound to a probe
portion by a conjugating moiety. As described herein, a detectable
moiety may be a small molecule (e.g., a dye) which may be directly
conjugated or bound to a probe portion. A detectable moiety may be
a fluorescently labeled protein or molecule which may be attached
to a conjugating moiety (e.g., a hapten group, an azido group, an
alkyne group) of a probe.
[0055] A profile or a detection profile or signature may include
the signal intensity, signal location, or size of the signal of the
detection agent. The profile or the detection profile may comprise
about 100 image frames, about 500 frames, about 1000 frames, about
2000 frames, about 5000 frames, about 10,000 frames, about 20,000
frames, about 30,000 frames, about 40,000 frames, about 50,000
frames, or more frames. Analysis of the profile or the detection
profile may determine the activity of the regulatory element. The
degree of activation may also be determined from the analysis of
the profile or detection profile. Analysis of the profile or the
detection profile may further determine the optical isolation and
localization of the detection agents, which may correlate to the
localization of the regulatory element.
[0056] In additional cases, a detection agent can comprise a
polypeptide probe selected from a DNA-binding protein, a
RNA-binding protein, a protein involved in the
transcription/translation process or detects the
transcription/translation process, a protein that can detect an
open or relaxed portion of a chromatin, or a protein interacting
partner of a product of a regulatory element (e.g., an antibody or
binding fragment thereof).
[0057] Sometimes, a detectable moiety that is capable of generating
a light is directly conjugated or bound to a probe portion. Other
times, a detectable moiety is indirectly conjugated or bound to a
probe portion by a conjugating moiety. As described elsewhere
herein, a detectable moiety can be a small molecule (e.g., a dye)
which can be directly conjugated or bound to a probe portion.
Alternatively, a detectable moiety can be a fluorescently labeled
protein or molecule which can be attached to a conjugating moiety
(e.g., a hapten group, an azido group, an alkyne group) of a
probe.
[0058] In some instances, a profile or a detection profile or
signature can include the signal intensity, signal location, or
size of the signal of the detection agent. Sometimes, the profile
or the detection profile can comprise about 100 frames, 500 frames,
1000 frames, 2000 frames, 5000 frames, 10,000 frames, 20,000
frames, 30,000 frames, 40,000 frames, 50,000 frames or more images.
Analysis of the profile or the detection profile can determine the
activity of the regulatory element. In some cases, the degree of
activation can also be determined from the analysis of the profile
or detection profile. In additional cases, analysis of the profile
or the detection profile can further determine the optical
isolation and localization of the detection agents, which can
correlate to the localization of the regulatory element.
Detection of DNA and/or RNA Regulatory Elements
[0059] A regulatory element may be DNA. Described herein is a
method of detecting a DNA regulatory element, which may include
contacting a cell sample with a detection agent, binding the
detection agent to the DNA regulatory element, and analyzing a
profile from the detection agent to determine the presence,
absence, or activity of the DNA regulatory element.
[0060] A regulatory element may be RNA. Described herein is a
method of detecting a RNA regulatory element, which may include
contacting a cell sample with a detection agent, binding the
detection agent to the RNA regulatory element, and analyzing a
profile from the detection agent to determine the presence,
absence, or activity of the RNA regulatory element.
[0061] A regulatory element may be an enhancer RNA (eRNA). The
presence of an eRNA may correlate to an activated regulatory
element. For example, the production of an eRNA may correlate to
the transcription of a target gene. As such, the detection of an
eRNA element may indicate that a target gene downstream of the eRNA
element may be activated.
[0062] Provided herein is a method of detecting an eRNA regulatory
element, which may include contacting a cell sample with a
detection agent, binding the detection agent to the eRNA regulatory
element, and analyzing a profile from the detection agent to
determine the presence, absence, or activity of the eRNA regulatory
element. Described herein is an in situ method of detecting an
activated regulatory DNA site, which may include incubating a
sample with a set of detection agents (e.g., fluorescently-labeled
probes), hybridizing the set of detection agents to at least one
enhancer RNA (eRNA), and analyzing a profile (e.g., a fluorescent
profile) from the set of detection agents to determine the presence
of an eRNA, in which the presence of eRNA correlates to an
activated regulatory DNA site.
Detection of a DNaseI Hypersensitive Site, Generation of a DNaseI
Hypersensitive Site Map, and Determination of a Cell Type Based on
a DNaseI Hypersensitive Site Profile
[0063] A regulatory element may be a DNaseI hypersensitive site
(DHS). A DNaseI hypersensitive site may be an inactivated DNaseI
hypersensitive site. A DNaseI hypersensitive site may be an
activated DNaseI hypersensitive site. Described herein is a method
of detecting a DHS, which may include contacting a cell sample with
a detection agent, binding the detection agent to the DHS, and
analyzing a profile from the detection agent to determine the
presence, absence, or activity of the DHS.
[0064] The DHS may be an active DHS and may further contain a
single stranded DNA region. The single stranded DNA region may be
detected by S1 nuclease. A method of detecting a DHS may further be
extended to detect the presence of a single stranded DNA region
within a DHS. Such a method, for example, may comprise contacting a
cell sample with a detection agent, binding the detection agent to
a single stranded region of a DHS, and analyzing a profile from the
detection agent to determine the presence or absence of the single
stranded region within a DHS.
[0065] Also described herein is a method of determining the
activity level of a regulatory element, which may include
incubating a cell sample with a set of detection agents (e.g.,
fluorescently labeled probes), in which each detection agent
hybridizes to a DHS, measuring a signature (e.g., a fluorescent
signature) from the set of detection agents, and based on the
signature, determining a DHS profile, and comparing the DHS profile
with a control, in which a correlation with the control indicates
the activity level of the regulatory element in the cell sample.
The signature (e.g., the fluorescent signature) may further
correlate to a signal intensity (or a peak height). A set of signal
intensities may be compiled into a DHS profile and compared with a
control to generate a second DHS profile which comprises a set of
relative signal intensities (or relative peak heights). The set of
relative signal intensities may correlate to the activity level of
a regulatory element.
[0066] Also described herein is a method of generating a DHS map,
which may provide information on cell-to-cell variation in gene
expression, memory of early developmental fate decisions which
establish lineage hierarchies, quantitation of embryonic stem cell
DHS sites which decreases with cell passage, and presence of
oncogenic elements.
[0067] The location of a set of DHS sites may be correlated to a
cell type. For example, the location of about 1, about 5, about 10,
about 15, about 20, about 25, about 30, about 35, about 40, about
45, about 50, about 55, about 60, or more DHS sites may be
correlated to a cell type. The location of about 1, about 5, about
10, about 15, about 20, about 25, about 30, about 35, about 40,
about 45, about 50, about 55, about 60, or more DHS may be used to
determine a cell type. The cell may be a normal cell or a cancerous
cell. DHS variation may be used to determine the presence of
cancerous cells in a sample. A method of determining a cell type
(e.g., a cancerous cell) may include incubating a cell sample with
a set of detection agents (e.g., fluorescently labeled probes), in
which each detection agent hybridizes to a DHS, measuring a
signature (e.g., a fluorescent signature) from the set of detection
agents, and based on the signature, determining a DHS profile, and
comparing the DHS profile with a control, in which a correlation
with the control indicates the cell type of the sample.
[0068] A DHS site may be visualized through a terminal
deoxynucleotidyl transferase (TdT) dUTP Nick-End labeling (TUNEL)
assay. A TUNEL assay may utilize a terminal deoxynucleotidyl
transferase (TdT) which may catalyze the addition of a dUTP at the
site of a nick or strand break. A fluorescent moiety may further be
conjugated to dUTP. A TUNEL assay may be utilized for visualization
of a plurality of DHSs present in a cell. A TUNEL assay may be an
assay as described in EXAMPLE 2.
[0069] The sequence of a DHS site may be detected in situ, by
utilizing an in situ sequencing methodology. For example, the two
ends of a padlock probe may be hybridized to a target regulatory
element sequence and the two ends may be further ligated together
by a ligase (e.g., T4 ligase) when bound to the target sequence. An
amplification (e.g., a rolling circle amplification or RCA) may be
performed utilizing a polymerase (e.g., .phi.29 polymerase), which
may result in a single stranded DNA comprising at least about 1, at
least about 2, at least about 3, at least about 4, at least about
5, at least about 6, at least about 7, at least about 8, at least
about 9, at least about 10, or more tandem copies of the target
sequence. The amplified product at least about be sequenced by
ligation in situ using partition sequencing compatible primers and
labeled probes (e.g., fluorescently labeled probes). For example,
each target sequence within the amplified product may bind to a
primer and probe set resulting in a bright spot detectable by,
e.g., an immunofluorescence microscopy. The labeled probe (e.g.,
the fluorescent label on the probe) may identify the nucleotide at
the ligation site, thereby allowing the color detected to define
the nucleotide at the respective ligation position. Sometimes, at
least 1, at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 15, at
least 20, or more rounds of ligation and detection may occur for
detection of a DHS site.
[0070] A control as used herein may refer to a DHS profile
generated from a regulatory element those activity level is known.
A control may also refer to a DHS profile generated from an
inactivated regulatory element. A control may further refer to a
DHS profile generated from an activated or inactivated regulatory
element from a specific cell type. For example, the cell type may
be an epithelial cell, connective tissue cell, muscle cell, or
nerve cell type. The cell may be a cell derived from heart, lung,
kidney, stomach, intestines, liver, pancreas, brain, esophagus, and
the like. The cell type may be a hormone-secreting cell, such as a
pituitary cell, a gut and respiratory tract cell, thyroid gland
cell, adrenal gland cell, Leydig cell of testes, Theca interna cell
of ovarian follicle, Juxtaglomerular cell, Macula densa cell,
Peripolar cell, or Mesangial cell type. The cell may be a blood
cell or a blood progenitor cell. The cell may be an immune system
cell, e.g., monocytes, dendritic cell, neutrophile granulocyte,
eosinophil granulocyte, basophil granulocyte, hybridoma cell, mast
cell, helper T cell, suppressor T cell, cytotoxic T cell, Natural
Killer T cell, B cell, or natural killer cell.
Detection and Mapping of a Chromatin
[0071] A regulatory element may also be a chromatin. Provided
herein is a method of detecting a chromatin, which may include
contacting a cell sample with a detection agent, binding the
detection agent to the chromatin, and analyzing a profile from the
detection agent to determine the activity state of the chromatin.
The activity level of a chromatin may be determined based on the
presence or activity level of a nucleic acid of interest or the
presence or absence of a chromatin associated protein. The activity
level of a chromatin may be determined based on DHS locations. The
one or more DHS locations on a chromatin may be used to map
chromatin activity state. For example, one or more DHSs may be
localized in a region and the surrounding chromatin may be
decompacted and readily visualized relative to an inactive
chromatin state when a DHS is not present. The one or more DHSs
within a localized region may further form a localized DHS set and
a plurality of localized DHS sets may further provide a global map
or pattern of chromatin activity (e.g., an activity pattern).
[0072] Also included herein is a method of generating a chromatin
map based on the pattern of DNaseI hypersensitive sites, RNA
regulatory elements (e.g., eRNA), chromatin associated proteins or
gene products, or a combination thereof. The method of generating a
chromatin map may be based on the pattern of DNaseI hypersensitive
sites. The method may comprise generating a 3-dimensional map from
a detection profile (or a 2-dimensional detection profile). A
chromatin map may provide information on the compaction of
chromatin, the spatial structure, spacing of regulatory elements,
and localization of the regulatory elements to globally map
chromatin structure and accessibility.
[0073] A chromatin map for a cell type may also be generated, in
which each cell type comprises a different chromatin pattern. Each
cell type may be associated with at least one unique marker. The at
least one unique marker (or fiduciary marker) may be a genomic
sequence. The at least one unique marker (or fiduciary marker) may
be DHS. A cell type may comprise about 5, about 10, about 15, about
20, about 25, about 30, about 35, about 40, about 45, about 50,
about 60, or more unique markers (or fiduciary markers). The cell
type may be an epithelia cell, a connective tissue cell, a muscle
cell, a nerve cell, a hormone-secreting cell, a blood cell, an
immune system cell, or a stem cell type. The cell type may be a
cancerous cell type.
[0074] A chromatin profile (e.g., based on DHSs) in the presence of
an exogenous agent or condition may also be generated. The method
may comprise incubating a cell sample with a set of fluorescently
labeled probes specific to target sites (e.g., target DHSs) on a
chromatin in the presence of an exogenous agent or condition;
measuring a fluorescent signature of the set of fluorescently
labeled probes; based on the fluorescent signature, generating a
fluorescent profile of the chromatin; and comparing the fluorescent
profile with a second fluorescent profile of a chromatin obtained
from an equivalent sample incubated with an equivalent set of
fluorescently labeled probes in the absence of the exogenous agent
or condition, wherein a difference between the two sets of
fluorescent profiles indicates a change in the chromatin density
(e.g., changes in the presences or activation of DHSs) induced by
the exogenous agent or condition. The exogenous agent or condition
may comprise a small molecule or a drug. The exogenous agent may be
a small molecule, such as a steroid. The exogenous agent or
condition may comprise an environmental factor, such as a change in
pH, temperature, nutrient, or a combination thereof.
Methods of Determining the Localization of a Regulatory Element
[0075] Also described herein is a method for determining the
localization of a regulatory element. The localization of a
regulatory element may provide an activity state of the regulatory
element. The localization of a regulatory element may also provide
an interaction state with at least one additional regulatory
element. For example, the localization of a first regulatory
element with respect to a second regulatory element may provide
spatial coordinate and distance information between the two
regulatory elements, and v further provide information regarding
whether the two regulatory elements can interact with each other.
The activity state of a regulatory element may include, for
example, a transcription or translation initiation event, a
translocation event, or an interaction event with one or more
additional regulatory elements. The regulatory element may comprise
DNA, RNA, polypeptides, or a combination thereof. The regulatory
element may be DNA. The regulatory element may be RNA. The
regulatory element may be an enhancer RNA (eRNA). The regulatory
element may be a DNaseI hypersensitive site (DHS). The DHS may be
an inactive DHS or an active DHS. The regulatory element may be a
polypeptide. The regulatory element may be chromatin.
[0076] A detection agent may comprise a detectable moiety that is
capable of generating a light, and a probe portion that is capable
of hybridizing to a target site on a regulatory element. Each
detection agent within the first set of detection agents may have
the same or a different detectable moiety. Each detection agent
within the first set of detection agents may have the same
detectable moiety. A detectable moiety may comprise a small
molecule (e.g., a fluorescent dye). A detectable moiety may
comprise a fluorescently labeled polypeptide, a fluorescently
labeled nucleic acid probe, and/or a fluorescently labeled
polypeptide complex.
[0077] The concentration of the detection agents may be from about
5 nM to about 1 .mu.M. The concentration of the detection agent can
be from about 5 nM to about 900 nM, from about 10 nM to about 800
nM, from about 15 nM to about 700 nM, from about 20 nM to about 500
nM, from about 10 nM to about 500 nM, from about 10 nM to about 400
nM, from about 10 nM to about 300 nM, from about 10 nM to about 200
nM, from about 10 nM to about 100 nM, from about 50 nM to about 500
nM, from about 50 nM to about 400 nM, from about 50 nM to about 300
nM, from about 50 nM to about 200 nM, from about 100 nM to about
500 nM, from about 100 nM to about 300 nM, or from about 100 nM to
about 200 nM. The concentration of the detection agents can be
about 10 nM, about 15 nM, about 20 nM, about 30 nM, about 40 nM,
about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM,
about 100 nM, about 150 nM, about 200 nM, about 250 nM, about 300
nM, about 400 nM, about 500 nM, about 600 nM, about 700 nM, about
800 nM, about 900 nM, or more.
[0078] The burst of lights from the set of detection agents may
generate a detection profile. The detection profile may comprise
about 100 image frames, about 500 frames, about 1000 frames, about
2000 frames, about 5000 frames, about 10,000 frames, about 20,000
frames, about 30,000 frames, about 40,000 frames, about 50,000
frames, or more. The detection profile may also include the signal
intensity, signal location, or size of the signal. Analysis of the
detection profile may determine the optical isolation and
localization of the detection agents, which can correlate to the
localization of the regulatory element.
[0079] The detection profile may comprise a chromatic aberration
correction. The detection profile can comprise less than 5%, less
than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%,
less than 0.1%, or 0% chromatic aberration. The detection profile
may comprise less than 5% chromatic aberration. The detection
profile may comprise less than 4% chromatic aberration. The
detection profile may comprise less than 3% chromatic aberration.
The detection profile may comprise less than 2% chromatic
aberration. The detection profile may comprise less than 1%
chromatic aberration. The detection profile may comprise less than
0.5% chromatic aberration. The detection profile may comprise less
than 0.1% chromatic aberration. The detection profile may comprise
0% chromatic aberration.
[0080] More than one regulatory element can be detected at the same
time. At least 2, at least 3, at least 4, at least 5, at least 6,
at least 7, at least 8, at least 9, at least 10, at least 15, at
least 20, or more regulatory elements may be detected at the same
time. Each of the regulatory elements may be detected by a set of
detection agents. The detectable moiety between the different set
of detection agents may be the same. For example, two different
sets of detection agents may be used to detect two different
regulatory elements and the detectable moieties from the two sets
of detection agents may be the same. As such, at least 2, at least
3, at least 4, at least 5, at least 6, at least 7, at least 8, at
least 9, at least 10, at least 15, at least 20, or more regulatory
elements may be detected at the same time at the same wavelength.
Sometimes, the detectable moiety between the different set of
detection agents may also be different. For example, two different
sets of detection agents may be used to detect two different
regulatory elements and the detectable moiety from one set of
detection agents may be detected at a different wavelength from the
detectable moiety of the second set of detection agents. As such,
at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9, at least 10, at least 15, at least
20, or more regulatory elements may be detected at the same time in
which each of the regulatory elements can be detected at a
different wavelength. The regulatory element may comprise DNA, RNA,
polypeptides, or a combination thereof.
Methods of Measuring the Activity of a Regulatory Element
[0081] Also described herein is a method of measuring the activity
of a target regulatory element. The method may include detection of
a regulatory element and one or more products of the regulatory
element. One or more products of the regulatory element may also
include intermediate products or elements. The method may comprise
contacting a cell sample with a first set and a second set of
detection agents, in which the first set of detection agents
interact with a target regulatory element within the cell and the
second set of detection agents interact with at least one product
of the target regulatory element, and analyzing a detection profile
from the first set and the second set of detection agents, in which
the presence or the absence of the at least one product indicates
the activity of the target regulatory element.
[0082] As discussed herein, a detection agent may comprise a
detectable moiety that is capable of generating a light, and a
probe portion that is capable of hybridizing to a target site on a
regulatory element. Each detection agent within the first set of
detection agents may have the same or a different detectable
moiety. Each detection agent within the first set of detection
agents can have the same detectable moiety. A detectable moiety may
comprise a small molecule (e.g., a fluorescent dye). A detectable
moiety may comprise a fluorescently labeled polypeptide, a
fluorescently labeled nucleic acid probe, and/or a fluorescently
labeled polypeptide complex.
[0083] The regulatory element may comprise DNA, RNA, polypeptides,
or a combination thereof. The regulatory element may be DNA. The
regulatory element may be RNA. The regulatory element may be an
enhancer RNA (eRNA). The presence of an eRNA may correlate with
target gene transcription that is downstream of eRNA. The
regulatory element may be a DNaseI hypersensitive site (DHS). The
DHS may be an activated DHS. The pattern of the DHS on a chromatin
may correlate to the activity of the chromatin. The regulatory
element may be a polypeptide, e.g., a transcription factor, a DNA
or RNA-binding protein or binding fragment thereof, or a
polypeptide that is involved in chemical modification. The
regulatory element may be chromatin.
Detection Agents
[0084] Detection agents described herein may include a probe
portion. The probe portion may include a probe, or a combination of
probes. The probe portion may comprise a nucleic acid molecule, a
polypeptide, or a combination thereof. The detection agents may
further include a detectable moiety. The detectable moiety may
comprise a fluorophore. A fluorophore may be a molecule that can
absorb light at a first wavelength and transmit or emit light at a
second wavelength. A fluorophore may be a small molecule (e.g., a
dye) or a fluorescent polypeptide. A detectable moiety may be a
fluorescent small molecule (e.g., a dye). A detectable moiety may
not contain a fluorescent polypeptide.
Probes
[0085] As described herein, a probe portion may comprise a probe or
a combination of probes. A probe may be a nucleic acid probe, a
polypeptide probe, or a combination thereof A probe portion may be
an unconjugated probe that does not contain a detectable moiety. A
probe portion may be a conjugated probe which comprises a single
probe with a detectable moiety, or two or more probes in which at
least one probe may be an unconjugated probe bound to at least a
second probe which comprises a detectable moiety.
[0086] A probe may be a nucleic acid probe. The nucleic acid probe
may be a DNA probe, a RNA probe, or a combination thereof. The
nucleic acid probe may be a DNA probe. The nucleic acid probe may
be a RNA probe. The nucleic acid probe may be a double stranded
nucleic acid probe, a single stranded nucleic acid probe, or may
contain single-stranded and/or double stranded portions. The
nucleic acid probe may further comprise overhangs on one or both
termini, may further comprises blunt ends on one or both termini,
or may further form a hairpin.
[0087] The nucleic acid probe may be at least 10, at least 15, at
least 20, at least 21, at least 22, at least 23, at least 24, at
least 25, at least 26, at least 27, at least 28, at least 29, at
least 30, at least 31, at least 32, at least 33, at least 34, at
least 35, at least 36, at least 37, at least 38, at least 39, at
least 40, at least 45, at least 50, at least 55, at least 60, at
least 65, at least 70, at least 80, at least 90, at least 100, or
more nucleotides in length. The nucleic acid probe may be about 10,
about 15, about 20, about 21, about 22, about 23, about 24, about
25, about 26, about 27, about 28, about 29, about 30, about 31,
about 32, about 33, about 34, about 35, about 36, about 37, about
38, about 39, about 40, about 45, about 50, about 55, about 60,
about 65, about 70, about 80, about 90, or about about 100
nucleotides in length. The nucleic acid probe may be about 20
nucleotides in length. The nucleic acid probe may be about 21
nucleotides in length. The nucleic acid probe may be about 22
nucleotides in length. The nucleic acid probe may be about 23
nucleotides in length. The nucleic acid probe may be about 24
nucleotides in length. The nucleic acid probe may be about 25
nucleotides in length. The nucleic acid probe may be about 26
nucleotides in length. The nucleic acid probe may be about 27
nucleotides in length. The nucleic acid probe may be about 28
nucleotides in length. The nucleic acid probe may be about 29
nucleotides in length. The nucleic acid probe may be about 30
nucleotides in length. The nucleic acid probe may be about 31
nucleotides in length. The nucleic acid probe may be about 32
nucleotides in length. The nucleic acid probe may be about 33
nucleotides in length. The nucleic acid probe may be about 34
nucleotides in length. The nucleic acid probe may be about 35
nucleotides in length. The nucleic acid probe may be about 36
nucleotides in length. The nucleic acid probe may be about 37
nucleotides in length. The nucleic acid probe may be about 38
nucleotides in length. The nucleic acid probe may be about 39
nucleotides in length. The nucleic acid probe may be about 40
nucleotides in length. The nucleic acid probe may be about 45
nucleotides in length. The nucleic acid probe may be about 50
nucleotides in length. The nucleic acid probe may be about 55
nucleotides in length. The nucleic acid probe may be about 60
nucleotides in length.
[0088] A nucleic acid probe may be a non-labeled probe, or a probe
that does not contain a detectable moiety. A non-labeled probe may
further interact with a labeled probe (e.g., a labeled nucleic acid
probe). A non-labeled probe may hybridize with a labeled nucleic
acid probe. A non-labeled probe may also interact with a labeled
polypeptide probe. The labeled polypeptide probe may be a protein
that recognizes a sequence within the non-labeled probe. A labeled
probe may include a nucleic acid portion and a polypeptide tag
portion and the polypeptide tag portion can further interact with a
molecule comprising a detectable moiety. For example, a non-labeled
probe may be a nucleic acid probe comprising a streptavidin which
may interact with a biotinylated molecule comprising a detectable
moiety.
[0089] A nucleic acid probe may have about 95%, about 96%, about
97%, about 98%, about 99%, or about 100% sequence specificity or
sequence complementarity to a target site of a regulatory element.
The hybridization may be a high stringent hybridization
condition.
[0090] A nucleic acid probe may hybridize with a genomic sequence
that is present in low or single copy numbers (e.g., genomic
sequences that are not repetitive elements). As used herein,
repetitive element refers to a DNA sequence that is present in many
identical or similar copies in the genome. Repetitive elements are
not intended to refer to a DNA sequence that is present on each
copy of the same chromosome (e.g., a DNA sequence that is present
only once, but is found on both copies of chromosome 11, would not
be considered a repetitive element, and would be considered a
sequence that is present in the genuine as one copy). The genome
may consist of three broad sequence components: single copy or at
least very low copy number DNA (approximately 60% of the human
genome); moderately repetitive elements (approximately 30% of the
human genome); and highly repetitive elements (approximately 10% of
the human genome). For a review, see Human Molecular Genetics,
Chapter 7 (1999), John Wiley & Sons, Inc.
[0091] A nucleic acid probe may have reduced off-target
interaction. For example, "off-target" or "off-target interaction"
may refer to an instance in which a nucleic acid probe against a
given target hybridizes or interact with another target site (e.g.,
a different DNA sequence, RNA sequence, or a cellular protein or
other moiety).
[0092] A nucleic acid probe may further be cross-linked to a target
site of a regulatory element. For example, the nucleic acid probe
may be cross-linked by a photo-crosslinking means such as UV or by
a chemical cross-linking means such as by formaldehyde, or through
a reactive group within the nucleic acid probe. Reactive group can
include sulfhydryl-reactive linkers such as bismaleimidohexane
(BMH), and the like.
[0093] A nucleic acid probe may include natural or unnatural
nucleotide analogues or bases or a combination thereof. The
unnatural nucleotide analogues or bases may comprise modifications
at one or more of ribose moiety, phosphate moiety, nucleoside
moiety, or a combination thereof. The unnatural nucleotide
analogues or bases may comprise 2'-O-methyl, 2'-O-methoxyethyl
(2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro,
2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE),
2'-O-dimethylaminopropyl (2'-O-DMAP),
T-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or
2'-O--N-methylacetamido (2'-O-NMA) modified, locked nucleic acid
(LNA), ethylene nucleic acid (ENA), peptide nucleic acid (PNA),
5'-anhydrohexitol nucleic acids (HNA), morpholino,
methylphosphonate nucleotides, thiolphosphonate nucleotides, or
2'-fluoro N3-P5'-phosphoramidites. The nucleic acid probes may
further comprise one or more abasic sites. The abasic site may
further be functionalized with a detectable moiety.
[0094] A nucleic acid probe may be a locked nucleic acid probe
(e.g., a labeled locked nucleic acid probe), a labeled or unlabeled
peptide nucleic acid (PNA) probe, a labeled or unlabeled
oligonucleotide, an oligopaint, an ECHO probe, a molecular beacon
probe, a padlock (or molecular inversion probe), a labeled or
unlabeled toe-hold probe, a labeled TALE probe, a labeled ZFN
probe, or a labeled CRISPR probe.
[0095] A nucleic acid probe may be a labeled or unlabeled locked
nucleic acid probe or a labeled or unlabeled peptide nucleic acid
probe. Locked nucleic acid probes and peptide nucleic acid probes
are known to those of skill in the art and are described in Briones
et al., Anal Bioanal Chem (2012) 402:3071-3089.
[0096] A nucleic acid probe may be a padlock (or molecular
inversion probe). A padlock probe may be hybridized to a target
regulatory element sequence in which the two ends may correspond to
the target sequence. A padlock probe may be as described herein,
which may be ligated together by a ligase (e.g., T4 ligase) when
bound to the target sequence. An amplification (e.g., a rolling
circle amplification or RCA) may be performed utilizing for example
.PHI.29 polymerase, which may result in a single stranded DNA
comprising multiple tandem copies of the target sequence.
[0097] A nucleic acid probe may be an oligopaint as described in
U.S. Publication No. 2010/0304994; and in Beliveau, et al.,
"Versatile design and synthesis platform for visualizing genomes
with oligopaint FISH probes," PNAS 109(52): 21301-21306 (2012).
Oligopaint may refer to detectably labeled polynucleotides that
have sequences complementary to an oligonucleotide sequence, e.g.,
a portion of a DNA sequence e.g., a particular chromosome or
sub-chromosomal region of a particular chromosome. Oligopaints may
be generated from synthetic probes and arrays that are, optionally,
computationally patterned (rather than using natural DNA sequences
and/or chromosomes as a template).
[0098] A nucleic acid probe may be a labeled or unlabeled toe-hold
probe. Toe-hold probes are known to those of skill in the art as
described in Zhang et al., Optimizing the Specificity of Nucleic
Acid Hybridization, Nature Chemistry 4: 208-214 (2012).
[0099] A nucleic acid probe may be a molecular beacon. Molecular
beacons may be hairpin shaped molecules with an internally quenched
fluorophore whose fluorescence is restored when they bind to a
target nucleic acid sequence. Molecular beacons are known to those
of skill in the art as described in Guo et al., Anal. Bioanal.
Chem. (2012) 402:3115-3125.
[0100] A nucleic acid probe may be an ECHO probe. ECHO probes may
be sequence-specific, hybridization-sensitive. quencher-free
fluorescent probes for RNA detection, which may be designed using
the concept of fluorescence quenching caused by intramolecular
excitonic interaction of fluorescent dyes. ECHO probes are known to
those of skill in the art as described in Kubota et al., PLoS ONE.
Vol. 5, Issue 9. e13003 (2010); or Okamoto, Chem. Soc. Rev., 2011,
40, 5815-5828. Wang et al., RNA (2012). 18:166-175.
[0101] A nucleic acid probe may be a CRISPR probe. The CRISPR
system may use a Cas9 protein to recognize DNA sequences, in which
the target specificity can be solely determined by a small guide
(sg) RNA and a protospacer adjacent motif (PAM). Upon binding to
target DNA, the Cas9-sgRNA complex may generate a DNA
double-stranded break. In instances such as for imaging, a Cas9
protein may be replaced with an endonuclease-deactivated Cas9
(dCas9) protein. For example, for imaging a cell, e.g., by
fluorescence in situ hybridization, may be achieved by synthesizing
a dCas9 within the cell, synthesizing RNA within the cell to bind
genomic DNA and to complex with the dCas9 forming a dCas9/RNA
complex, labeling the dCas9/RNA complex, and imaging the labeled
dCas9/RNA complex within the live cell bound to genomic DNA. The
endonuclease-deactivated Cas9 may be synthesized in vivo by using
an integrated construct, a transiently transfected construct, by
injection into the cell of a syncitia of nuclei or via
electroporation into cells and/or nuclei.
[0102] A nucleic acid probe may comprise an
endonuclease-deactivated Cas9 (dCas9) protein as described in Chen
et al., "Dynamic imaging of genomic loci in living human cells by
an optimized CRISPR/Cas system." Cell 155(7): 1479-1491 (2013); or
Ma et al., "Multicolor CRISPR labeling of chromosomal loci in human
cells." PNAS 112(10): 3002-3007 (2015). The dCas9 protein may be
further labeled with a detectable moiety.
[0103] The RNA of the Cas9/RNA complex may be synthesized in vivo
by using an integrated construct, a transiently transfected
construct, by injection into the cell of a syncitia of nuclei or
via electroporation into cells and/or nuclei. The Cas9/RNA complex
may be labeled by making a fusion protein that includes Cas9 and a
reporter, by injection of RNA that has been attached to a reporter
into the cell or by a syncitia of nuclei including RNA that has
been attached to a reporter, by electroporation into cells or
nuclei or by indirect labeling of the RNA by hybridization with a
labeled secondary oligonucleotide. The label may be a conditional
reporter, based on the binding of Cas9/RNA to the target nucleic
acid. The label may be quenched and may then be activated upon the
Cas9/RNA complex binding to the target nucleic acid.
[0104] A nucleic acid probe may be a TALEN probe. Transcription
Activator-Like Effector Nucleases (TALEN) are engineered
restriction enzymes generated by fusing the TAL effector DNA
binding domain to a FokI DNA cleavage domain. A FokI DNA cleavage
domain may comprise an endonuclease-deactivated FokI domain. A
nucleic acid probe may be a TALEN probe comprising an
endonuclease-deactivated FokI domain.
[0105] A nucleic acid probe may be a zinc-finger nuclease (ZFN)
probe. Similar to TALEN, a zinc-finger nuclease may be an
engineered restriction enzyme generated by fusing a zinc finger
DNA-binding domain to a zinc finger nuclease. A zinc finger
nuclease may comprise an endonuclease-deactivated zinc finger
nuclease. A nucleic acid probe may be a ZFN probe comprising an
endonuclease-deactivated zinc finger nuclease.
[0106] A probe disclosed herein may be a polypeptide probe. A
polypeptide probe may include a protein or a binding fragment
thereof that interacts with a target site (e.g., a nucleic acid
target site or a protein target) of interest. A polypeptide probe
may comprise a DNA-binding protein, a RNA-binding protein, a
protein involved in the transcription/translation process or
detects the transcription/translation process, a protein that can
detect an open or relaxed portion of a chromatin, or a protein
interacting partner of a product of a regulatory element.
[0107] A polypeptide probe may be a DNA-binding protein. The
DNA-binding protein may be a transcription factor that modulates
the transcription process, polymerases, or histones. A DNA-binding
protein may comprise a zinc finger domain, a helix-turn-helix
domain, a leucine zipper domain (e.g., a basic leucine zipper
domain), a high mobility group box (HMG-box) domain, and the like.
In some instances, the DNA-binding protein interacts with a nucleic
acid region in a sequence specific manner. The DNA-binding protein
may interact with a nucleic acid region in a sequence non-specific
manner. The DNA-binding protein may interact with single-stranded
DNA. The DNA-binding protein may interact with double-stranded DNA.
The DNA-binding protein probe may further comprise a detectable
moiety.
[0108] A polypeptide probe may be a RNA-binding protein. The
RNA-binding protein may participate in forming ribunucleoprotein
complexes. The RNA-binding protein may modulate post-transcription
such as in splicing, polyadenylation, mRNA stabilization, mRNA
localization, or in translation. A RNA-binding protein may comprise
a RNA recognition motif (RRM), dsRNA binding domain, zinc finger
domain, K-Homology domain (KH domain), and the like. The
RNA-binding protein may interact with single-stranded RNA. The
RNA-binding protein may interact with double-stranded RNA. The
RNA-binding protein probe may further comprise a detectable
moiety.
[0109] A polypeptide probe may be a protein that can detect an open
or relaxed portion of a chromatin. The polypeptide probe may be a
modified enzyme that lacks cleavage activity. The modified enzyme
may be an enzyme that recognizes double-stranded or single-stranded
DNA or RNA. Examples of modified enzymes may include
oxidoreductases, transferases, hydrolases, lyases, isomerases, or
ligases. A modified enzyme may be an endonuclease (e.g., a
deactivated restriction endonuclease such as the TALEN or CRISPR
probes described herein).
[0110] A polypeptide probe may be an antibody or binding fragment
thereof. The antibody or binding fragment thereof may be a protein
interacting partner of a product of a regulatory element. The
antibody or binding fragment thereof may comprise a humanized
antibody or binding fragment thereof, murine antibody or binding
fragment thereof, chimeric antibody or binding fragment thereof,
monoclonal antibody or binding fragment thereof, monovalent Fab',
divalent Fab2, F(ab)'3 fragments, single-chain variable fragment
(scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody,
tetrabody, disulfide stabilized Fv protein (dsFv), single-domain
antibody (sdAb), Ig NAR, camelid antibody or binding fragment
thereof, or a chemically modified derivative thereof. The antibody
or binding fragment thereof may further comprise a detectable
moiety.
Detectable Moiety
[0111] A detectable moiety may be a small molecule (e.g., a dye) or
a macromolecule. A macromolecule may include polypeptides (e.g.,
proteins and/or protein fragments), nucleic acids, carbohydrates,
lipids, macrocyles, polyphenols, and/or endogenous macromolecule
complexes. A detectable moiety may be a small molecule. A
detectable moiety may be a macromolecule.
[0112] A detectable moiety may include a moiety that is detectable
by a colorimetric method or a fluorescent method. For example, a
colorimetric method may be an assay which utilizes reagents that
undergo a measurable color change in the presence of an analyte
(e.g., an enzyme, an antibody, a compound, a hormone). Exemplary
colorimetric methods may include enzyme-mediated detection method
such as tyramide signal amplification (TSA) which utilizes
horseradish peroxidase (HRP) to generate a signal when digested by
tyramide substrate and 3,3',5,5'-Tetramethylbenzidine (TMB) which
generates a blue color upon oxidation to
3,3'5,5'-tetramethylbenzidine diamine in the presence of a
peroxidase enzyme such as HRP. A detectable moiety described herein
may include a moiety that is detectable by a colorimetric
method.
[0113] A detectable moiety may also include a moiety that is
detectable by a fluorescent method. The detectable moiety may be a
fluorescent moiety. A fluorescent moiety may be a small molecule
(e.g., a dye) or a fluorescently labeled macromolecule. A
fluorescently labeled macromolecule may include a fluorescently
labeled polypeptide (e.g., a labeled protein and/or a protein
fragment), a fluorescently labeled nucleic acid molecule, a
fluorescently labeled carbohydrate, a fluorescently labeled lipid,
a fluorescently labeled macrocyle, a fluorescently labeled
polyphenol, and/or a fluorescently labeled endogenous macromolecule
complex (e.g., a primary antibody-secondary antibody complex).
[0114] A fluorescent small molecule may comprise rhodamine, rhodol,
fluorescein, thiofluorescein, aminofluorescein, carboxyfluorescein,
chlorofluorescein, methylfluorescein, sulfofluorescein,
aminorhodol, carboxyrhodol, chlororhodol, methylrhodol,
sulforhodol; aminorhodamine, carboxyrhodamine, chlororhodamine,
methylrhodamine, sulforhodamine, thiorhodamine, cyanine,
indocarbocyanine, oxacarbocyanine, thiacarbocyanine, merocyanine,
cyanine 2, cyanine 3, cyanine 3.5, cyanine 5, cyanine 5.5, cyanine
7, oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole,
benzoxadiazole, pyren derivatives, cascade blue, oxazine
derivatives, Nile red, Nile blue, cresyl violet, oxazine 170,
acridine derivatives, proflavin, acridine orange, acridine yellow,
arylmethine derivatives, auramine, crystal violet, malachite green,
tetrapyrrole derivatives, porphin, phtalocyanine, bilirubin
1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene
sulfonate, 2-p-touidinyl-6-naphthalene sulfonate,
3-phenyl-7-isocyanatocoumarin,
N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM
(Fluorescein), 6-FAM (NHS Ester), 5(6)-FAM, 5-FAM, Fluorescein dT,
5-TAMRA-cadavarine, 2-aminoacridone, HEX, JOE (NHS Ester), MAX,
TET, ROX, TAMRA, TARMA.TM. (NHS Ester), TEX 615, ATTO.TM. 488,
ATTO.TM. 532, ATTO.TM. 550, ATTO.TM. 565, ATTO.TM. Rho101, ATTO.TM.
590, ATTO.TM. 633, ATTO.TM. 647N, TYE.TM. 563, TYE.TM. 665, or
TYE.TM. 705.
[0115] A fluorescent moiety may comprise Cy3, Cy5, Cy5.5, Cy7,
Q570, Alexa488, Alexa555, Alexa594, Alexa647, Alexa680, Alexa 750,
Alexa 790, Atto488, Atto532, Atto647N, TexasRed, CF610, Propidium
iodide, Q670, IRDye700, IRDye800, Indocyanine green, Pacific Blue
dye, Pacific Green dye, or Pacific Orange dye.
[0116] In some instances, a fluorescent moiety may comprise a
quantum dot (QD). Quantum dots can be a nanoscale seminconducting
photoluminescent material, for example, as described in Alivisatos
A. P., "Semiconductor clusters, nanocrystals, and quantum dots,"
Science 271(5251): 933-937 (1996).
[0117] Exemplary QDs may include, but are not limited to, CdS
quantum dots, CdSe quantum dots, CdSe.RTM.CdS core/shell quantum
dots, CdSe.RTM.ZnS core/shell quantum dots, CdTe quantum dots, PbS
quantum dots, and/or PbSe quantum dots. As used herein,
CdSe.RTM.ZnS may mean that a ZnS shell is coated on a CdSe core
surface (e.g., "core-shell" quantum dots). The shell materials of
core-shell QDs may have a higher bandgap and passivate the core QDs
surfaces, resulting in higher quantum yield and higher stability
and wider applications than core QDs.
[0118] QDs may absorb a wide spectrum of light, and may be
physically tuned with emission bandwidths in various wavelengths.
See, e.g., Badolato, et al., Science 208:1158-61 (2005). For
example, the emission bandwidth may be in the visible spectrum
(e.g., from about 350 nm to about 750 nm), the visible-infrared
spectrum (e.g., from about 0.1 .mu.m to about 0.7 .mu.m), or in the
near-infrared spectrum (e.g., from about 0.7 .mu.m to about 2.5
.mu.m). QDs that emit energy in the visible range may include, but
are not limited to, CdS, CdSe, CdTe, ZnSe, ZnTe, GaP, and GaAs. QDs
that emit energy in the blue to near-ultraviolet range may include,
but are not limited to, ZnS and GaN. QDs that emit energy in the
near-infrared range may include, but are not limited to, InP, InAs,
InSb, PbS, and PbSe.
[0119] The radius of a QD may be modulated to manipulate the
emission bandwidth. For example, a radius of between about 5 nm and
about 6 nm QD may emit wavelengths resulting in emission colors
such as orange or red. A radius of between about 2 nm and about 3
nm may emit wavelengths resulting in emission colors such as blue
or green.
[0120] The QD may further form a QD microstructure, which may
encompass one or more layers of QD. For example, each quantum dot
containing layer may comprise a single type of quantum dot of a
specific emission color. For example, each layer may be made of any
material suitable for use that (a) allows excitation light to reach
the quantum dot and allows fluorescence generated from the quantum
dot to pass through the layer(s) for detection and (b) may be
combined with a quantum dot to form a layer. Examples of materials
that may n be used to form layers containing quantum dots may
include, but are not limited to, inorganic, organic, or polymeric
material, each with or without biodegradable properties, and
combinations thereof. The layers may comprise silica-based
compounds or polymers. Exemplary silica-based layers may include,
but are not limited to, those comprising tetramethoxy silane or
tetraethylorthosilicate. Exemplary polymer layers may include, but
are not limited to, those comprising polystyrene, poly (methyl
methacrylate), polyhydroxyalkanoate, polylactide, or co-polymers
thereof.
[0121] The quantum dot may further comprise a spacer layer which
serves as a barrier to prevent interactions between different QD
layers, and may be made of any material suitable for use that (a)
allows excitation light to reach the quantum dots in the quantum
dot containing layer(s) below it and allows fluorescence generated
from those quantum dots to pass through it and (b) can segregate
the quantum dots in one layer from those in other layers. Examples
of materials that may be used to form spacer layers are the same as
for the quantum dot containing layers.
[0122] The materials used for the quantum dot containing and spacer
layers may be the same or different. The same material may be used
in the quantum dot containing layers and the spacer layers.
[0123] The quantum dot containing layers and the spacer layers
within a given QD molecule may be any thickness and can be varied.
For example, thicker QD-containing layers may allow for the loading
of increased QDs in the shell, resulting in greater fluorescence
intensity for that layer than for a thinner layer containing the
same concentration of QDs. Thus, varying layer thickness may
facilitate preparing QD-containing layer of various intensities,
thereby generating spectrally distinct QD bar codes. The
QD-containing layers may be between 5 nm and 500 nm thick; between
10 nm and 500 nm thick; between 5 nm and 100 nm thick, or between
10 nm and 100 nm thick. Those of skill in the art will understand
that other methods for varying intensity also exist, for example,
modifying concentrations of the same QD in one microstructure with
a first unique barcode compared to a second QD microstructure with
a different fluorescent barcode. The ability to vary the
intensities for the same QD color may allow for an increased number
of distinct and distinguishable microstructures (e.g., spectrally
distinct barcodes). The spacer layers may be greater than 10 nm
thick, up to approximately 5 .mu.m thick, 10 nm to 500 nm thick, or
10 nm to 100 nm thick.
[0124] The quantum dot-containing and spacer layers may be arranged
in any order. Examples may include, but are not-limited to,
alternating QD-containing layers and spacer layers, or quantum dot
containing layers separated by more than one spacer layer. Thus, a
"spacer layer" may comprise a single layer, or may comprise two or
more such spacer layers.
[0125] The QD microstructure may comprise any number of quantum dot
containing layers suitable for use with the microstructure. For
example, a microstructure described herein may comprise 2, 3, 4, 5,
6, 7, 8, 9, 10, or more quantum dot-containing layers and an
appropriate number of spacer layers based on the number of quantum
dot-containing layers. Further, the number of quantum dot
containing layers in a given microstructure may range from 1 to
"m," where "m" is the number of quantum dots that may be used.
[0126] A defined intensity level may refer to a known amount of
quantum dots in each quantum dot containing layer, resulting in a
known amount of fluorescent intensity generated from the QD
containing layer upon appropriate stimulation. Since each QD
containing layer has a defined intensity level, each microstructure
may possess a defined ratio of fluorescence intensities generated
from the various QD-containing layers upon stimulation. This
defined ratio is referred to herein as a barcode. Thus, each type
of microstructure with the same QD layers may possess a similar
barcode that may be distinguished from microstructures with
different QD layers.
[0127] Thus, each quantum dot containing layer may comprise a
single type of quantum dot of a specific emission color and the
layer is produced to possess a defined intensity level, based on
the concentration of the QD in the layer. By varying the intensity
levels of QDs ("n") in different microstructures and using a
variety of different quantum dots ("m"), the number of different
unique barcodes (and thus the number of different unique
microstructure populations that can be produced) may be
approximated by the equation, (n.sup.m-1) unique codes. This may
provide the ability to generate a large number of different
populations of microstructures each with its own unique
barcode.
[0128] A set of QD-labeled probes may further generate a spectrally
distinct barcode. For example, each probe with the set of
QD-labeled probes may comprise a QD with a distinct excitation
wavelength and the combination of the set can generate a distinct
barcode. A set of spectrally distinct QD-labeled probes may be
utilized to detect a regulatory element. As such, when detecting
two or more regulatory elements, each regulatory element may be
spectrally barcoded.
[0129] A quantum dot provided herein may include QDot525, QDot 545,
QDot 565, QDot 585, QDot 605, or QDot 655. A probe described herein
may comprise a quantum dot. A probe described herein may comprise
QDot525, QDot 545, QDot 565, QDot 585, QDot 605, or QDot 655. A
probe described herein may comprise QDot525. A probe described
herein may comprise QDot 545. A probe described herein may comprise
QDot 565. A probe described herein may comprise QDot 585. A probe
described herein may comprise QDot 605. A probe described herein
may comprise QDot 655.
[0130] A quantum dot may comprise a quantum dot as described in Han
et al., "Quantum-dot-tagged microbeads for multiplexed optical
coding of biomolecules," Nat. Biotechnol. 19:631-635 (2001); Gao
X., "QD barcodes for biosensing and detection," Conf Proc IEEE Eng
Med Biol Soc 2009: 6372-6373 (2009); and Zrazhevskiy, et al.,
"Multicolor multicycle molecular profiling with quantum dots for
single-cell analysis," Nat Protoc 8:1852-1869 (2013).
[0131] A QD may further comprise a functional group or attachment
moiety. One example of such a QD that has a functional group or
attachment moiety is a QD with a carboxylic acid terminated
surface, such as those commercially available though, for example,
Quantum Dot, Inc., Hayward, Calif.
Conjugating Moiety
[0132] The probe may include a conjugating moiety. The conjugation
moiety may be attached at the 5' terminus, the 3' terminus, or at
an internal site. The conjugating moiety may be a nucleotide
analog, e.g., bromodeoxyuridine. The conjugating moiety may be a
conjugating functional group. The conjugating functional group may
be an azido group or an alkyne group. The probe may further be
derivatived through a chemical reaction such as click chemistry.
The click chemistry may be a copper(I)-catalyzed [3+2]-Huisgen
1,3-dipolar cyclo-addition of alkynes and azides leading to
1,2,3-triazoles. The click chemistry may be a copper free variant
of the above reaction.
[0133] The conjugating moiety may comprise a hapten group. A hapten
group may include digoxigenin, 2,4-dinitrophenyl, biotin, avidin,
or are selected from azoles, nitroaryl compounds, benzofurazans,
triterpenes, ureas, thioureas, rotenones, oxazoles, thiazoles,
coumarins, cyclolignans, heterobiaryl compounds, azoaryl compounds
or benzodiazepines. A hapten group may include biotin.
[0134] The probe comprising the conjugating moiety may further be
linked to a second probe (e.g., a nucleic acid probe or a
polypeptide probe), a fluorescent moiety (e.g., a dye such as a
quantum dot), a target regulatory element, or a conjugating partner
such as a polymer (e.g., PEG), a macromolecule (e.g., a
carbohydrate, a lipid, a polypeptide), and the like.
Samples
[0135] A sample described herein may be a fresh sample or a fixed
sample. The sample can be a fresh sample. The sample can be a fixed
sample. The sample may be subjected to a denaturing condition. The
sample may be cryopreserved.
[0136] The sample may be a cell sample. The cell sample may be
obtained from the cells of an animal. The animal cell may include a
cell from a marine invertebrate, fish, insects, amphibian, reptile,
or mammal. The mammalian cell may be obtained from a primate, ape,
equine, bovine, porcine, canine, feline, or rodent. The mammal may
be a primate, ape, dog, cat, rabbit, ferret, or the like. The
rodent may be a mouse, rat, hamster, gerbil, hamster, chinchilla,
or guinea pig. The bird cell may be from a canary, parakeet, or
parrot. The reptile cell may be from a turtle, lizard, or snake.
The fish cell may be from a tropical fish. For example, the fish
cell may be from a zebrafish (e.g., Danino rerio). The worm cell
may be from a nematode (e.g., C. elegans). The amphibian cell may
be from a frog. The arthropod cell may be from a tarantula or
hermit crab.
[0137] The cell sample may be obtained from a mammalian cell. The
mammalian cell may be an epithelial cell, connective tissue cell,
hormone secreting cell, a nerve cell, a skeletal muscle cell, a
blood cell, an immune system cell, or a stem cell.
[0138] Exemplary mammalian cells may include, but are not limited
to, 293A cell line, 293FT cell line, 293F cells, 293H cells, HEK
293 cells, CHO DG44 cells, CHO-S cells, CHO-K1 cells, Expi293F.TM.
cells, Flp-In.TM. T-REx.TM. 293 cell line, Flp-In.TM.-293 cell
line, Flp-In.TM.-3T3 cell line, Flp-In.TM.-BHK cell line,
Flp-In.TM.-CHO cell line, Flp-In.TM.-CV-1 cell line,
Flp-In.TM.-Jurkat cell line, FreeStyle.TM. 293-F cells,
FreeStyle.TM. CHO-S cells, GripTite.TM. 293 MSR cell line, GS-CHO
cell line, HepaRG.TM. cells, T-REx.TM. Jurkat cell line, Per.C6
cells, T-REx.TM.-293 cell line, T-REx.TM.-CHO cell line,
T-REx.TM.-HeLa cell line, NC-HIMT cell line, and PC12 cell
line.
[0139] The cell sample may be obtained from cells of a primate. The
primate may be a human, or a non-human primate. The cell sample may
be obtained from a human. For example, the cell sample may comprise
cells obtained from blood, urine, stool, saliva, lymph fluid,
cerebrospinal fluid, synovial fluid, cystic fluid, ascites, pleural
effusion, amniotic fluid, chorionic villus sample, vaginal fluid,
interstitial fluid, buccal swab sample, sputum, bronchial lavage,
Pap smear sample, or ocular fluid. The cell sample may comprise
cells obtained from a blood sample, an aspirate sample, or a smear
sample.
[0140] The cell sample may be a circulating tumor cell sample. A
circulating tumor cell sample may comprise lymphoma cells, fetal
cells, apoptotic cells, epithelia cells, endothelial cells, stem
cells, progenitor cells, mesenchymal cells, osteoblast cells,
osteocytes, hematopoietic stem cells, foam cells, adipose cells,
transcervical cells, circulating cardiocytes, circulating
fibrocytes, circulating cancer stem cells, circulating myocytes,
circulating cells from kidney, circulating cells from
gastrointestinal tract, circulating cells from lung, circulating
cells from reproductive organs, circulating cells from central
nervous system, circulating hepatic cells, circulating cells from
spleen, circulating cells from thymus, circulating cells from
thyroid, circulating cells from an endocrine gland, circulating
cells from parathyroid, circulating cells from pituitary,
circulating cells from adrenal gland, circulating cells from islets
of Langerhans, circulating cells from pancreas, circulating cells
from hypothalamus, circulating cells from prostate tissues,
circulating cells from breast tissues, circulating cells from
circulating retinal cells, circulating ophthalmic cells,
circulating auditory cells, circulating epidermal cells,
circulating cells from the urinary tract, or combinations
thereof.
[0141] A cell sample may be a peripheral blood mononuclear cell
sample.
[0142] A cell sample may comprise cancerous cells. Cancer may be a
solid tumor or a hematologic malignancy. The cancerous cell sample
may comprise cells obtained from a solid tumor. The solid tumor may
include a sarcoma or a carcinoma. Exemplary sarcoma cell sample may
include, but are not limited to, cell sample obtained from alveolar
rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma,
angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma of soft
tissue, dedifferentiated liposarcoma, desmoid, desmoplastic small
round cell tumor, embryonal rhabdomyosarcoma, epithelioid
fibrosarcoma, epithelioid hemangioendothelioma, epithelioid
sarcoma, esthesioneuroblastoma, Ewing sarcoma, extrarenal rhabdoid
tumor, extraskeletal myxoid chondrosarcoma, extraskeletal
osteosarcoma, fibrosarcoma, giant cell tumor, hemangiopericytoma,
infantile fibrosarcoma, inflammatory myofibroblastic tumor, Kaposi
sarcoma, leiomyosarcoma of bone, liposarcoma, liposarcoma of bone,
malignant fibrous histiocytoma (MFH), malignant fibrous
histiocytoma (MFH) of bone, malignant mesenchymoma, malignant
peripheral nerve sheath tumor, mesenchymal chondrosarcoma,
myxofibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic
sarcoma, neoplasms with perivascular epitheioid cell
differentiation, osteosarcoma, parosteal osteosarcoma, neoplasm
with perivascular epitheioid cell differentiation, periosteal
osteosarcoma, pleomorphic liposarcoma, pleomorphic
rhabdomyosarcoma, PNET/extraskeletal Ewing tumor, rhabdomyosarcoma,
round cell liposarcoma, small cell osteosarcoma, solitary fibrous
tumor, synovial sarcoma, or telangiectatic osteosarcoma.
[0143] Exemplary carcinoma cell sample may include, but are not
limited to, cell sample obtained from anal cancer, appendix cancer,
bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, brain
tumor, breast cancer, cervical cancer, colon cancer, cancer of
Unknown Primary (CUP), esophageal cancer, eye cancer, fallopian
tube cancer, gastroenterological cancer, kidney cancer, liver
cancer, lung cancer, medulloblastoma, melanoma, oral cancer,
ovarian cancer, pancreatic cancer, parathyroid disease, penile
cancer, pituitary tumor, prostate cancer, rectal cancer, skin
cancer, stomach cancer, testicular cancer, throat cancer, thyroid
cancer, uterine cancer, vaginal cancer or vulvar cancer.
[0144] The cancerous cell sample may comprise cells obtained from a
hematologic malignancy. A hematologic malignancy may comprise a
leukemia, a lymphoma, a myeloma, a non-Hodgkin's lymphoma, or a
Hodgkin's lymphoma. The hematologic malignancy may be a T-cell
based hematologic malignancy. The hematologic malignancy may be a
B-cell based hematologic malignancy. Exemplary B-cell based
hematologic malignancies may can include, but are not limited to,
chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma
(SLL), high risk CLL, a non-CLUSLL lymphoma, prolymphocytic
leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell
lymphoma (DLBCL), mantle cell lymphoma (MCL), Waldenstrom's
macroglobulinemia, multiple myeloma, extranodal marginal zone B
cell lymphoma, nodal marginal zone B cell lymphoma, Burkitt's
lymphoma, non-Burkitt high grade B cell lymphoma, primary
mediastinal B-cell lymphoma (PMBL), immunoblastic large cell
lymphoma, precursor B-lymphoblastic lymphoma, B cell prolymphocytic
leukemia, lymphoplasmacytic lymphoma, splenic marginal zone
lymphoma, plasma cell myeloma, plasmacytoma, mediastinal (thymic)
large B cell lymphoma, intravascular large B cell lymphoma, primary
effusion lymphoma, or lymphomatoid granulomatosis. Exemplary T-cell
based hematologic malignancies may include, but are not limited to,
peripheral T-cell lymphoma not otherwise specified (PTCL-NOS),
anaplastic large cell lymphoma, angioimmunoblastic lymphoma,
cutaneous T-cell lymphoma, adult T-cell leukemia/lymphoma (ATLL),
blastic NK-cell lymphoma, enteropathy-type T-cell lymphoma,
hematosplenic gamma-delta T-cell lymphoma, lymphoblastic lymphoma,
nasal NK/T-cell lymphomas, or treatment-related T-cell
lymphomas.
[0145] A cell sample described herein may include a tumor cell line
sample. Exemplary tumor cell line sample may include, but are not
limited to, cell samples from tumor cell lines: 600MPE, AU565,
BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF-7, MDA-MB-231,
SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460,
A2780, SKOV-3/Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948,
DLD-1, SW480, Capan-1, MC/9, B72.3, B25.2, B6.2, B38.1, DMS 153,
SU.86.86, SNU-182, SNU-423, SNU-449, SNU-475, SNU-387, Hs 817.T,
LMH, LMH/2A, SNU-398, PLHC-1, HepG2/SF, OCI-Ly1, OCI-Ly2, OCI-Ly3,
OCI-Ly4, OCI-Ly6, OCI-Ly7, OCI-Ly10, OCI-Ly18, OCI-Ly19, U2932, DB,
HBL-1, RIVA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3,
TALL-104, AML-193, THP-1, BDCM, HL-60, Jurkat, RPMI 8226, MOLT-4,
RS4, K-562, KASUMI-1, Daudi, GA-10, Raji, JeKo-1, NK-92, and
Mino.
[0146] A cell sample may comprise cells obtained from a biopsy
sample.
[0147] The cell samples (e.g., a biopsy sample) may be obtained
from an individual by any suitable means of obtaining the sample
using well-known and routine clinical methods. Procedures for
obtaining tissue samples from an individual are well known. For
example, procedures for drawing and processing tissue sample such
as from a needle aspiration biopsy are well-known and is employed
to obtain a sample for use in the methods provided. Typically, for
collection of such a tissue sample, a thin hollow needle is
inserted into a mass such as a tumor mass for sampling of cells
that, after being stained, will be examined under a microscope.
Imaging Instrumentation
[0148] One or more far-field fluorescence techniques may be
utilized for the detection, localization, activity determination,
and mapping of one or more regulatory elements described herein. A
microscopy method may be a high magnification oil immersion
microscopy method. In such method, the wide-field and confocal
fluorescent microscopes may achieve sub-cellular resolution. A
microscopy method may utilize a super-resolution microscopy, which
allows images to be taken with a higher resolution than the
diffraction limit. A super-resolution microscopy method may include
deterministic super-resolution, which utilizes a fluorophore's
nonlinear response to excitation to enhance resolution. Exemplary
deterministic super-resolution may include stimulated emission
depletion (STED), ground state depletion (GSD), reversible
saturable optical linear fluorescence transitions (RESOLFT), and
saturated structured illumination microscopy (SSIM). A
super-resolution microscopy method may also include stochastic
super-resolution, which utilizes a complex temporal behavior of a
fluorophore, to enhance resolution. Exemplary stochastic
super-resolution method may include Super-resolution optical
fluctuation imaging (SOFI), all single-molecular localization
method (SMLM) such as spectral precision determination microscopy
(SPDM), SPDMphymod, photo-activated localization microscopy (PALM),
FPALM, stochastic optical reconstruction microscopy (STORM), and
dSTROM.
[0149] A microscopy method may be a single-molecular localization
method (SMLM). A microscopy method may be a spectral precision
determination microscopy (SPDM) method. A SPDM method may rely on
stochastic burst or blinking of fluorophores and subsequent
temporal integration of signals to achieve lateral resolution at,
for example, between about 10 nm and about 100 nm.
[0150] A microscopy method may be a spatially modulated
illumination (SMI) method. A SMI method may utilize phased lasers
and interference patterns to illuminate specimens and increase
resolution by measuring the signal in fringes of the resulting
Moire patterns.
[0151] A microscopy method may be a synthetic aperture optics (SAO)
method. A SAO method may utilize a low magnification, low numerical
aperture (NA) lens to achieve large field of view and depth of
field, without sacrificing spatial resolution. For example, an SAO
method may comprise illuminating the detection agent-labeled target
(e.g., a regulatory element) with a predetermined number (N) of
selective excitation patterns, where the number (N) of selective
excitation patterns is determined based upon the detection agent's
physical characteristics corresponding to spatial frequency content
(e.g., the size, shape, and/or spacing of the detection agents on
the imaging target) from the illuminated target, optically imaging
the illuminated target at a resolution insufficient to resolve the
objects on the target, and processing optical images of the
illuminated target using information on the selective excitation
patterns to obtain a final image of the illuminated target at a
resolution sufficient to resolve the objects on the target. The
number (N) of selective excitation patterns may correspond to the
number of k-space sampling points in a k-space sampling space in a
frequency domain, with the extent of the k-space sampling space
being substantially proportional to an inverse of a minimum
distance (.DELTA.x) between the objects that is to be resolved by
SAO, and with the inverse of the k-space sampling interval between
the k-space sampling points being less than a width (w) of a
detected area captured by a pixel of a system for said optical
imaging. The number (N) may include a function of various
parameters of the imaging system (e.g., magnification (Mag) of the
objective lens, numerical aperture (NA) of the objective lens,
wavelength .lamda..sub.E of the light emitted from the imaging
target, and/or effective pixel size p of the pixel sensitive area
of the CCD, etc.).
[0152] A SAO method may analyze a set of detection agent profiles
from at least 100, at least 200, at least 250, at least 500, at
least 1000, or more cells imaged simultaneously within one field of
view utilizing an imaging instrument. The one field of view can be
a single wide field of view allowing image capture of greater than
100, greater than 200, greater than 250, greater than 500, greater
than 1000, or more cells. The single wide field of view may have
about 0.70 mm by about 0.70 mm field of view. The SAO imaging
instrument may enable a resolution of about 0.25 .mu.m with a
20.times./0.45NA lens. The SAO imaging instrument may enable a
depth of field of about 2.72 .mu.m with a 20.times./0.45NA lens.
The imaging instrument may enable a working distance of about 7 mm
with a 20.times./0.45NA lens. The imaging instrument may enable a
z-stack of 1 with a 20.times./0.45NA lens. The SAO method may
further integrate and interpolate 3-dimensional images from
2-dimensional images.
[0153] The SAO imaging instrument may be an SAO instrument as
described in U.S. Publication No. 2011/0228073 (Lightspeed
Genomics, Inc).
Digital Processing Device
[0154] The systems, apparatus, and methods described herein can
include a digital processing device, or use of the same. The
digital processing device can include one or more hardware central
processing units (CPU) that carry out the device's functions. The
digital processing device can further comprise an operating system
configured to perform executable instructions. In some instances,
the digital processing device is optionally connected to a computer
network, is optionally connected to the Internet such that it
accesses the World Wide Web, or is optionally connected to a cloud
computing infrastructure. In other instances, the digital
processing device is optionally connected to an intranet. In other
instances, the digital processing device is optionally connected to
a data storage device.
[0155] In accordance with the description herein, suitable digital
processing devices can include, by way of non-limiting examples,
server computers, desktop computers, laptop computers, notebook
computers, sub-notebook computers, netbook computers, netpad
computers, set-top computers, media streaming devices, handheld
computers, Internet appliances, mobile smartphones, tablet
computers, personal digital assistants, video game consoles, and
vehicles. Those of skill in the art will recognize that many
smartphones are suitable for use in the system described herein.
Those of skill in the art will also recognize that select
televisions, video players, and digital music players with optional
computer network connectivity are suitable for use in the system
described herein. Suitable tablet computers can include those with
booklet, slate, and convertible configurations, known to those of
skill in the art.
[0156] The digital processing device can include an operating
system configured to perform executable instructions. The operating
system can be, for example, software, including programs and data,
which can manage the device's hardware and provides services for
execution of applications. Those of skill in the art will recognize
that suitable server operating systems can include, by way of
non-limiting examples, FreeBSD, OpenBSD, NetBSD.RTM., Linux,
Apple.RTM. Mac OS X Server.RTM., Oracle.RTM. Solaris.RTM., Windows
Server.RTM., and Novell.RTM. NetWare.RTM.. Those of skill in the
art will recognize that suitable personal computer operating
systems include, by way of non-limiting examples, Microsoft.RTM.
Windows.RTM., Apple.RTM. Mac OS X.RTM., UNIX.RTM., and UNIX-like
operating systems such as GNU/Linux.RTM.. In some cases, the
operating system is provided by cloud computing. Those of skill in
the art will also recognize that suitable mobile smart phone
operating systems include, by way of non-limiting examples,
Nokia.RTM. Symbian.RTM. OS, Apple.RTM. iOS.RTM., Research In
Motion.RTM. BlackBerry OS.RTM., Google.RTM. Android.RTM.,
Microsoft.RTM. Windows Phone.RTM. OS, Microsoft.RTM. Windows
Mobile.RTM. OS, Linux.RTM., and Palm.RTM. WebOS.RTM.. Those of
skill in the art will also recognize that suitable media streaming
device operating systems include, by way of non-limiting examples,
Apple TV.RTM., Roku.RTM., Boxee.RTM., Google TV.RTM., Google
Chromecast.RTM., Amazon Fire.RTM., and Samsung.RTM. HomeSync.RTM..
Those of skill in the art will also recognize that suitable video
game console operating systems include, by way of non-limiting
examples, Sony PS3.RTM., Sony PS4.RTM., Microsoft.RTM. Xbox
360.RTM., Microsoft Xbox One, Nintendo.RTM. Wii.RTM., Nintendo.RTM.
Wii U.RTM., and Ouya.RTM..
[0157] In some instances, the device can include a storage and/or
memory device. The storage and/or memory device can be one or more
physical apparatuses used to store data or programs on a temporary
or permanent basis. In some instances, the device is volatile
memory and requires power to maintain stored information. In other
instances, the device is non-volatile memory and retains stored
information when the digital processing device is not powered. In
still other instances, the non-volatile memory comprises flash
memory. The non-volatile memory can comprise dynamic random-access
memory (DRAM). The non-volatile memory can comprise ferroelectric
random access memory (FRAM). The non-volatile memory can comprise
phase-change random access memory (PRAM). The device can be a
storage device including, by way of non-limiting examples, CD-ROMs,
DVDs, flash memory devices, magnetic disk drives, magnetic tapes
drives, optical disk drives, and cloud computing based storage. The
storage and/or memory device can also be a combination of devices
such as those disclosed herein.
[0158] The digital processing device can include a display to send
visual information to a user. The display can be a cathode ray tube
(CRT). The display can be a liquid crystal display (LCD).
Alternatively, the display can be a thin film transistor liquid
crystal display (TFT-LCD). The display can further be an organic
light emitting diode (OLED) display. In various cases, on OLED
display is a passive-matrix OLED (PMOLED) or active-matrix OLED
(AMOLED) display. The display can be a plasma display. The display
can be a video projector. The display can be a combination of
devices such as those disclosed herein.
[0159] The digital processing device can also include an input
device to receive information from a user. For example, the input
device can be a keyboard. The input device can be a pointing device
including, by way of non-limiting examples, a mouse, trackball,
track pad, joystick, game controller, or stylus. The input device
can be a touch screen or a multi-touch screen. The input device can
be a microphone to capture voice or other sound input. The input
device can be a video camera or other sensor to capture motion or
visual input. Alternatively, the input device can be a Kinect.TM.,
Leap Motion.TM., or the like. In further aspects, the input device
can be a combination of devices such as those disclosed herein.
Non-Transitory Computer Readable Storage Medium
[0160] In some instances, the systems, apparatus, and methods
disclosed herein can include one or more non-transitory computer
readable storage media encoded with a program including
instructions executable by the operating system of an optionally
networked digital processing device. In further instances, a
computer readable storage medium is a tangible component of a
digital processing device. In still further instances, a computer
readable storage medium is optionally removable from a digital
processing device. A computer readable storage medium can include,
by way of non-limiting examples, CD-ROMs, DVDs, flash memory
devices, solid state memory, magnetic disk drives, magnetic tape
drives, optical disk drives, cloud computing systems and services,
and the like. In some cases, the program and instructions are
permanently, substantially permanently, semi-permanently, or
non-transitorily encoded on the media.
Computer Program
[0161] The systems, apparatus, and methods disclosed herein can
include at least one computer program, or use of the same. A
computer program includes a sequence of instructions, executable in
the digital processing device's CPU, written to perform a specified
task. In some embodiments, computer readable instructions are
implemented as program modules, such as functions, objects,
Application Programming Interfaces (APIs), data structures, and the
like, that perform particular tasks or implement particular
abstract data types. In light of the disclosure provided herein,
those of skill in the art will recognize that a computer program,
in certain embodiments, is written in various versions of various
languages.
[0162] The functionality of the computer readable instructions can
be combined or distributed as desired in various environments. A
computer program can comprise one sequence of instructions. A
computer program can comprise a plurality of sequences of
instructions. In some instances, a computer program is provided
from one location. In other instances, a computer program is
provided from a plurality of locations. In additional cases, a
computer program includes one or more software modules. Sometimes,
a computer program can include, in part or in whole, one or more
web applications, one or more mobile applications, one or more
standalone applications, one or more web browser plug-ins,
extensions, add-ins, or add-ons, or combinations thereof.
Web Application
[0163] A computer program can include a web application. In light
of the disclosure provided herein, those of skill in the art will
recognize that a web application, in various aspects, utilizes one
or more software frameworks and one or more database systems. In
some cases, a web application is created upon a software framework
such as Microsoft.RTM. .NET or Ruby on Rails (RoR). In some cases,
a web application utilizes one or more database systems including,
by way of non-limiting examples, relational, non-relational, object
oriented, associative, and XML database systems. Sometimes,
suitable relational database systems can include, by way of
non-limiting examples, Microsoft.RTM. SQL Server, mySQL.TM., and
Oracle.RTM.. Those of skill in the art will also recognize that a
web application, in various instances, is written in one or more
versions of one or more languages. A web application can be written
in one or more markup languages, presentation definition languages,
client-side scripting languages, server-side coding languages,
database query languages, or combinations thereof. A web
application can be written to some extent in a markup language such
as Hypertext Markup Language (HTML), Extensible Hypertext Markup
Language (XHTML), or eXtensible Markup Language (XML). In some
embodiments, a web application is written to some extent in a
presentation definition language such as Cascading Style Sheets
(CSS). A web application can be written to some extent in a
client-side scripting language such as Asynchronous Javascript and
XML (AJAX), Flash.RTM. Actionscript, Javascript, or
Silverlight.RTM.. A web application can be written to some extent
in a server-side coding language such as Active Server Pages (ASP),
ColdFusion.RTM., Perl Java.TM., JavaServer Pages (JSP), Hypertext
Preprocessor (PHP), Python.TM., Ruby, Tcl, Smalltalk, WebDNA.RTM.,
or Groovy. Sometimes, a web application can be written to some
extent in a database query language such as Structured Query
Language (SQL). Other times, a web application can integrate
enterprise server products such as IBM.RTM. Lotus Domino.RTM.. In
some instances, a web application includes a media player element.
In various further instances, a media player element utilizes one
or more of many suitable multimedia technologies including, by way
of non-limiting examples, Adobe.RTM. Flash.RTM., HTML 5, Apple.RTM.
QuickTime.RTM., Microsoft.RTM. Silverlight.RTM., Java.TM., and
Unity.RTM..
Mobile Application
[0164] A computer program can include a mobile application provided
to a mobile digital processing device. In some cases, the mobile
application is provided to a mobile digital processing device at
the time it is manufactured. In other cases, the mobile application
is provided to a mobile digital processing device via the computer
network described herein.
[0165] In view of the disclosure provided herein, a mobile
application is created by techniques known to those of skill in the
art using hardware, languages, and development environments known
to the art. Those of skill in the art will recognize that mobile
applications are written in several languages. Suitable programming
languages include, by way of non-limiting examples, C, C++, C#,
Objective-C, Java.TM., Javascript, Pascal, Object Pascal,
Python.TM., Ruby, VB.NET, WML, and XHTML/HTML with or without CSS,
or combinations thereof.
[0166] Suitable mobile application development environments are
available from several sources. Commercially available development
environments include, by way of non-limiting examples, AirplaySDK,
alcheMo, Appcelerator.RTM., Celsius, Bedrock, Flash Lite, .NET
Compact Framework, Rhomobile, and WorkLight Mobile Platform. Other
development environments are available without cost including, by
way of non-limiting examples, Lazarus, MobiFlex, MoSync, and
Phonegap. Also, mobile device manufacturers distribute software
developer kits including, by way of non-limiting examples, iPhone
and iPad (iOS) SDK, Android.TM. SDK, BlackBerry.RTM. SDK, BREW SDK,
Palm.RTM. OS SDK, Symbian SDK, webOS SDK, and Windows.RTM. Mobile
SDK.
[0167] Those of skill in the art will recognize that several
commercial forums are available for distribution of mobile
applications including, by way of non-limiting examples, Apple.RTM.
App Store, Android.TM. Market, BlackBerry App World, App Store for
Palm devices, App Catalog for webOS, Windows.RTM. Marketplace for
Mobile, Ovi Store for Nokia.RTM. devices, Samsung.RTM. Apps, and
Nintendo.RTM. DSi Shop.
Standalone Application
[0168] A computer program can include a standalone application,
which is a program that is run as an independent computer process,
not an add-on to an existing process, e.g., not a plug-in. Those of
skill in the art will recognize that standalone applications are
often compiled. A compiler is a computer program(s) that transforms
source code written in a programming language into binary object
code such as assembly language or machine code. Suitable compiled
programming languages include, by way of non-limiting examples, C,
C++, Objective-C, COBOL, Delphi, Eiffel, Java.TM., Lisp,
Python.TM., Visual Basic, and VB .NET, or combinations thereof.
Compilation is often performed, at least in part, to create an
executable program. A computer program can include one or more
executable complied applications.
Web Browser Plug-in
[0169] The computer program can include a web browser plug-in. In
computing, a plug-in is one or more software components that add
specific functionality to a larger software application. Makers of
software applications support plug-ins to enable third-party
developers to create abilities which extend an application, to
support easily adding new features, and to reduce the size of an
application. When supported, plug-ins enable customizing the
functionality of a software application. For example, plug-ins are
commonly used in web browsers to play video, generate
interactivity, scan for viruses, and display particular file types.
Those of skill in the art will be familiar with several web browser
plug-ins including, Adobe.RTM. Flash.RTM. Player, Microsoft.RTM.
Silverlight.RTM., and Apple.RTM. QuickTime.RTM.. In some
embodiments, the toolbar comprises one or more web browser
extensions, add-ins, or add-ons. In some embodiments, the toolbar
comprises one or more explorer bars, tool bands, or desk bands.
[0170] In view of the disclosure provided herein, those of skill in
the art will recognize that several plug-in frameworks are
available that enable development of plug-ins in various
programming languages, including, by way of non-limiting examples,
C++, Delphi, Java.TM., PHP, Python.TM., and VB .NET, or
combinations thereof.
[0171] Web browsers (also called Internet browsers) can be software
applications, designed for use with network-connected digital
processing devices, for retrieving, presenting, and traversing
information resources on the World Wide Web. Suitable web browsers
include, by way of non-limiting examples, Microsoft.RTM. Internet
Explorer.RTM., Mozilla.RTM. Firefox.RTM., Google.RTM. Chrome,
Apple.RTM. Safari.RTM., Opera Software.RTM. Opera.RTM., and KDE
Konqueror. In some embodiments, the web browser is a mobile web
browser. Mobile web browsers (also called mircrobrowsers,
mini-browsers, and wireless browsers) are designed for use on
mobile digital processing devices including, by way of non-limiting
examples, handheld computers, tablet computers, netbook computers,
subnotebook computers, smartphones, music players, personal digital
assistants (PDAs), and handheld video game systems. Suitable mobile
web browsers include, by way of non-limiting examples, Google.RTM.
Android.RTM. browser, RIM BlackBerry.RTM. Browser, Apple.RTM.
Safari.RTM., Palm.RTM. Blazer, Palm.RTM. WebOS.RTM. Browser,
Mozilla.RTM. Firefox.RTM. for mobile, Microsoft.RTM. Internet
Explorer.RTM. Mobile, Amazon.RTM. Kindle.RTM. Basic Web, Nokia.RTM.
Browser, Opera Software.RTM. Opera.RTM. Mobile, and Sony.RTM.
PSP.TM. browser.
Software Modules
[0172] The systems and methods disclosed herein can include
software, server, and/or database modules, or use of the same. In
view of the disclosure provided herein, software modules can be
created by techniques known to those of skill in the art using
machines, software, and languages known to the art. The software
modules disclosed herein can be implemented in a multitude of ways.
A software module can comprise a file, a section of code, a
programming object, a programming structure, or combinations
thereof. A software module can comprise a plurality of files, a
plurality of sections of code, a plurality of programming objects,
a plurality of programming structures, or combinations thereof. In
various aspects, the one or more software modules comprise, by way
of non-limiting examples, a web application, a mobile application,
and a standalone application. In some instances, software modules
are in one computer program or application. In other instances,
software modules are in more than one computer program or
application. In some cases, software modules are hosted on one
machine. In other cases, software modules are hosted on more than
one machine. Sometimes, software modules can be hosted on cloud
computing platforms. Other times, software modules can be hosted on
one or more machines in one location. In additional cases, software
modules are hosted on one or more machines in more than one
location.
Databases
[0173] The methods, apparatus, and systems disclosed herein can
include one or more databases, or use of the same. In view of the
disclosure provided herein, those of skill in the art will
recognize that many databases are suitable for storage and
retrieval of analytical information described elsewhere herein. In
various aspects described herein, suitable databases can include,
by way of non-limiting examples, relational databases,
non-relational databases, object oriented databases, object
databases, entity-relationship model databases, associative
databases, and XML databases. A database can be internet-based. A
database can be web-based. A database can be cloud computing-based.
Alternatively, a database can be based on one or more local
computer storage devices.
Services
[0174] Methods and systems described herein can further be
performed as a service. For example, a service provider can obtain
a sample that a customer wishes to analyze. The service provider
can then encodes the sample to be analyzed by any of the methods
described herein, performs the analysis and provides a report to
the customer. The customer can also perform the analysis and
provides the results to the service provider for decoding. In some
instances, the service provider then provides the decoded results
to the customer. In other instances, the customer can received
encoded analysis of the samples from the provider and decodes the
results by interacting with softwares installed locally (at the
customer's location) or remotely (e.g., on a server reachable
through a network). Sometimes, the softwares can generate a report
and transmit the report to the costumer. Exemplary customers
include clinical laboratories, hospitals, industrial manufacturers,
and the like. Sometimes, a customer or party can be any suitable
customer or party with a need or desire to use the methods provided
herein.
Server
[0175] The methods provided herein can be processed on a server or
a computer server (FIG. 2). The server 401 can include a central
processing unit (CPU, also "processor") 405 which can be a single
core processor, a multi core processor, or plurality of processors
for parallel processing. A processor used as part of a control
assembly can be a microprocessor. The server 401 can also include
memory 410 (e.g., random access memory, read-only memory, flash
memory); electronic storage unit 415 (e.g., hard disk);
communications interface 420 (e.g., network adaptor) for
communicating with one or more other systems; and peripheral
devices 425 which includes cache, other memory, data storage,
and/or electronic display adaptors. The memory 410, storage unit
415, interface 420, and peripheral devices 425 can be in
communication with the processor 405 through a communications bus
(solid lines), such as a motherboard. The storage unit 415 can be a
data storage unit for storing data. The server 401 can be
operatively coupled to a computer network ("network") 430 with the
aid of the communications interface 420. A processor with the aid
of additional hardware can also be operatively coupled to a
network. The network 430 can be the Internet, an intranet and/or an
extranet, an intranet and/or extranet that is in communication with
the Internet, a telecommunication or data network. The network 430
with the aid of the server 401, can implement a peer-to-peer
network, which can enable devices coupled to the server 401 to
behave as a client or a server. The server can be capable of
transmitting and receiving computer-readable instructions (e.g.,
device/system operation protocols or parameters) or data (e.g.,
sensor measurements, raw data obtained from detecting metabolites,
analysis of raw data obtained from detecting metabolites,
interpretation of raw data obtained from detecting metabolites,
etc.) via electronic signals transported through the network 430.
Moreover, a network can be used, for example, to transmit or
receive data across an international border.
[0176] The server 401 can be in communication with one or more
output devices 435 such as a display or printer, and/or with one or
more input devices 440 such as, for example, a keyboard, mouse, or
joystick. The display can be a touch screen display, in which case
it functions as both a display device and an input device.
Different and/or additional input devices can be present such an
enunciator, a speaker, or a microphone. The server can use any one
of a variety of operating systems, such as for example, any one of
several versions of Windows.RTM., or of MacOS.RTM., or of
Unix.RTM., or of Linux.RTM..
[0177] The storage unit 415 can store files or data associated with
the operation of a device, systems or methods described herein.
[0178] The server can communicate with one or more remote computer
systems through the network 430. The one or more remote computer
systems can include, for example, personal computers, laptops,
tablets, telephones, Smart phones, or personal digital
assistants.
[0179] A control assembly can include a single server 401. In other
situations, the system can include multiple servers in
communication with one another through an intranet, extranet and/or
the Internet.
[0180] The server 401 can be adapted to store device operation
parameters, protocols, methods described herein, and other
information of potential relevance. Such information can be stored
on the storage unit 415 or the server 401 and such data is
transmitted through a network.
[0181] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the claimed subject matter belongs. It
is to be understood that the foregoing general description and the
following detailed description are exemplary and explanatory only
and are not restrictive of any subject matter claimed. In this
application, the use of the singular includes the plural unless
specifically stated otherwise. It must be noted that, as used in
the specification and the appended claims, the singular forms "a,"
"an" and "the" include plural referents unless the context clearly
dictates otherwise. In this application, the use of "or" means
"and/or" unless stated otherwise. Furthermore, use of the term
"including" as well as other forms, such as "include", "includes,"
and "included," is not limiting.
[0182] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 .mu.L" means "about 5 .mu.L" and also "5
.mu.L." Generally, the term "about" includes an amount that would
be expected to be within experimental error.
[0183] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
EXAMPLES
[0184] These examples are provided for illustrative purposes only
and not to limit the scope of the claims provided herein.
Example 1
DNase Treatment and TUNEL Assay
[0185] A TUNEL assay as described below may be used to label DNaseI
cut sites on a global cell. For example, all of the DNaseI cut
sites within a cell's nucleus may be labeled.
[0186] Cells were prepared for a 2-color SPDM for DNA density and
DNase I sensitivity (TUNEL) assay.
[0187] An adherent cell line, A549 (lung adenocarcinoma), was used
for these experiments. They were plated overnight on uncoated 18 mm
(#1 thickness) coverslips. Cells were deliberately plated sparsely
to be .about.20% confluent on the day of the assay.
[0188] For all coverslips, cells were fixed with 4% formaldehyde in
PBS for 10 minutes at room temperature, and then equilibrated in
buffer A at room temperature for 15 minutes. The cells were
permeabilized with 0.1% NP-40 in buffer A for 10 minutes at room
temperature.
[0189] The DNaseI assay was performed with 80 U/ml DNaseI for 3
minutes at 37.degree. C. Cells were then post fixed in 4%
formaldehyde in buffer A for 10 minutes at room temperature. The
coverslips were permeabilized for 20 minutes with buffer A with
0.25% TX-100, and washed twice with distilled water and were
equilibrated with 100 .mu.l of TdT reaction buffer for 10 minutes
at room temperature. The terminal deoxynucleotide transferase (TdT)
reaction with EdUTP-alkyne (100 .mu.l per coverslip) was performed
for 1 hour at 37.degree. C. At the end of the TdT reaction, the
coverslips were washed twice with 3% BSA/PBS. The ClickIT reaction
was then performed for 2 coverslips to add Alexafluor647 to
incorporated EdUTP-alkyne. This reaction was performed for 30
minutes at room temperature, in the dark. The other coverslips were
kept in 3% BSA/PBS at room temperature. The coverslips were washed
once with 3% BSA/PBS before being stained with Vybrant Violet
staining and imaged by a SMLM method.
[0190] FIG. 3A shows a two color SPDM image (experimental) of
chromatin (blue) with a DNA sensitive element (red), showing
anti-colocalization of the DNA sensitive element with chromatin.
Scale bars: 1000 nm, inserts: 100 nm. FIG. 3B is the inset of FIG.
3A.
[0191] FIG. 4A and FIG. 4B illustrate the localization precision
and nearest neighbor distances for DNA and DNase sensitive
elements.
Example 2
DNA Encoding of Molecular Targets on a Multi-Omics Imaging
Platform
[0192] Integration of imaging data across different molecular
target types may provide in-depth insights into cell physiology and
pathology. A multi-omics imaging platform is utilized which enables
simultaneous visualization of multiple molecular targets
irrespective of target type and imaging probes used. The
multi-omics imaging platform comprises (i) decoupling of target
binding and labeling steps, (ii) translation of heterogeneous
molecular information into an intermediate standardized molecular
code amenable to read-out via imaging probes, and (iii) employing
encoding capacity and self-assembly capabilities of DNA bonding.
Specifically, molecular targets of interest are first encoded with
unique ssDNA tags via binding by ssDNA-conjugated
target-recognition moieties under optimized conditions favoring
specific target binding. Individual ssDNA tags are then converted
into detectable signals via sequence-specific hybridization with
complementary ssDNA'-conjugated imaging probes under
probe-optimized conditions. As such, molecular target uniqueness,
localization, abundance, and specimen morphology information are
preserved through all steps of labeling procedure, producing
comprehensive molecular signatures of a physiological or
pathological process.
Methods
[0193] Oligonucleotide Probe Design.
[0194] Sequences for 6 ssDNA/ssDNA' encoding pairs were selected
from a random pool. Selection criteria were: continuous 16 bp
complementarity, balanced nucleotide composition, lack of stable
secondary structures at room temperature, lack of substantial
cross-hybridization between mismatch pairs. See TABLE 1 for a
complete list of ssDNA/ssDNA' encoding pairs.
[0195] Sequences for human GAPDH mRNA (NM_002046.5) and HSP90-alpha
mRNA (NM_001271969.1) were obtained from NCBI. Sets of mRNA in situ
hybridization (ISH) probes were designed using Stellaris RNA FISH
Probe Designer (Biosearch Technologies). Probe sets contained 36
unique probes for GAPDH mRNA and 48 probes for HSP90-alpha mRNA.
Each probe featured 5' terminal 20nt-long region complementary to
mRNA, a spacer (either AAAAA for smaller 41nt probes or
AAAA-dsSpacer-AAAA for longer 60nt probes), and a 16nt-long QDot
binding tag. The ISH probe strand of the dsSpacer was
5'-TTCCCAAGCGTCATCT-3' (SEQ ID NO: 6), pre-hybridized with a
complementary 5'-AGATGACGCTTGGGAA-3' ssDNA (SEQ ID NO: 98) at a 1:1
molar ratio to form a 16 bp dsDNA spacer prior to specimen
labeling. See TABLE 2 and TABLE 3 for a complete list of ISH
probes. All oligonucleotides were purchased from IDT DNA.
[0196] Antibody-ssDNA Conjugation.
[0197] Purified primary and secondary antibodies in PBS were
purchased from Sigma-Aldrich. Amine-terminated HPLC purified ssDNA
tags were purchased from IDT DNA (see TABLE 1, Tag IDs 1B-6B).
Covalent antibody-ssDNA bioconjugation was achieved either a) via
maleimide-mediated amine-sulfhydryl crosslinking or b) using
Thunder-Link oligo conjugation system (Innova Biosciences).
[0198] For maleimide-mediated crosslinking, IgG was partially
reduced by TCEP to expose free sulfhydryl groups, while 5'
amine-terminated ssDNA oligonucleotides were activated by
sulfo-SMCC (Thermo Scientific). IgG was diluted to 1 mg/mL in 100
.mu.L PBS with 10 mM EDTA, mixed with 0.5 mM TCEP, and incubated
for 30 min at 37.degree. C. At the same time, ssDNA was diluted to
40 .mu.M in 100 .mu.L PBS, mixed with 10 mM sulfo-SMCC, and
incubated for 30 min at RT. Reduced IgG and activated ssDNA were
then purified by 3 rounds of desalting in Zeba desalting spin
columns (Thermo Scientific) pre-washed with PBS/10 mM EDTA, mixed,
and reacted for 4 hrs at room temperature (RT). Finally, unreacted
sulfhydryl groups were capped by addition of 1 mM sulfo-SMCC
pre-quenched by excess glycine. Antibody-ssDNA bioconjugates were
purified by ultrafiltration for at least 6 times with Amicon Ultra
50 KDa MWCO centrifugal filter (Millipore) and stored in PBS
solution at 4.degree. C.
[0199] For antibody-ssDNA conjugation with Thunder-Link oligo
conjugation system, IgG was diluted to 1 mg/mL in 100 .mu.L PBS,
activated by the Antibody Activation Reagent for 1 Hr at RT, and
purified using desalting column. At the same time, 5'
amine-terminated ssDNA oligonucleotides were diluted to 80 .mu.M in
100 .mu.L PBS, activated by the Oligo Activation Reagent for 1 Hr
at RT, and desalted. Activated IgG and ssDNA were mixed at a volume
ratio of 2:1 (200 .mu.L IgG+100 .mu.L ssDNA+100 .mu.L wash buffer),
reacted overnight at RT, and stored at 4.degree. C. For
optimization studies, following IgG:ssDNA volume ratios were
tested: 50+50, 50+30, 50+20, and 50+10.
[0200] QDot-ssDNA Conjugation.
[0201] Amine-functionalized PEG-coated QDots (Qdot ITK amino (PEG)
quantum dots, Invitrogen) with emission peaks centered at 525, 545,
565, 585, 605, and 655 nm were used for the preparation of
QDot-ssDNA probes. Amine-terminated HPLC purified 16nt-long ssDNA
tags were purchased from IDT DNA (see TABLE 1, Tag IDs 1A-6A).
Oligonucleotides were activated with bifunctional cross-linker BS3
(Bis[sulfosuccinimidyl] suberate, Thermo Scientific), followed by
covalent conjugation with QDots. 100 .mu.L 40 .mu.M ssDNA solution
in PBS was mixed with 500 molar excess of BS3 and incubated for 30
minutes at room temperature. Excess crosslinker was removed by 3
rounds of desalting in Zeba desalting spin columns (Thermo
Scientific) pre-washed with PBS. Activated ssDNA was then mixed
with 25 .mu.L 8 .mu.M stock QDot solution. The reaction was
incubated overnight at room temperature and purified by
ultrafiltration for at least 6 times with Amicon Ultra 100 KDa MWCO
centrifugal filter (Millipore). Purified QDot-ssDNA probes were
stored in PBS solution at 4.degree. C.
[0202] Agarose gel electrophoresis was used for characterization of
QDot-ssDNA probes. Procedure was performed on a 2% agarose gel in
1.times.TBE at 90V for 2 hrs.
[0203] Cell Culture and Processing.
[0204] Human cervical cancer cell line HeLa (ATCC) was used as a
model specimen for evaluation of the multi-omics imaging via DNA
encoding. Cells were grown in glass-bottom 24-well plates (Greiner
Bio-One) in a humidified atmosphere at 37.degree. C. with 5%
CO.sub.2 to a density of 80-90% using MEM culture medium with
L-glutamine (Gibco) supplemented with 10% fetal bovine serum
(Gibco). Prior to labeling, cells were rinsed with PBS, fixed with
4% formaldehyde in PBS for 5 min at room temperature followed by 15
min at 4.degree. C., permeabilized with ice-cold 0.5% TritonX-100
(Thermo Scientific) in PBS for 15 min at 4.degree. C., and washed
with PBS. For mRNA imaging, cells were immediately processed for in
situ hybridization to minimize degradation of mRNA prior to
labeling. For protein imaging only, fixed cells could be stored in
PBS with 0.03% sodium azide at 4.degree. C. for several days.
[0205] Encoding Via Immunorecognition.
[0206] Encoding of protein targets in formalin-fixed cells was
performed via incubation with antibody-ssDNA bioconjugates. Prior
to labeling, cells were blocked by 2% BSA (from 10% BSA/PBS
solution, Thermo Scientific), 0.5% Western blot blocking reagent
(from 10% solution, Roche), 0.1% low MW dextran sulfate (9-20 kDa
MW, Sigma-Aldrich), 0.1 mg/mL shredded salmon sperm DNA
(Invitrogen), and 1.times.PBS for 30 min at RT. Antibodies were
used at a final concentration of 5 .mu.g/mL diluted in 2% BSA, 0.1%
dextran sulfate, 0.1 mg/mL shredded salmon sperm DNA, and
1.times.PBS and incubated with cells for 1-2 hrs at RT. Following
labeling, cells were washed with PBS.
[0207] For reference studies, cell labeling with unmodified
antibodies was performed in a similar fashion.
[0208] Encoding Via In Situ Hybridization (ISH).
[0209] Encoding of mRNA targets was performed via hybridization
with ssDNA-tagged mRNA ISH probes. Cells were equilibrated with 10%
formamide (Thermo Scientific), 2 mM RVC (New England BioLabs),
2.times.SSC (Invitrogen) buffer for 30 min at RT and then incubated
with 400 .mu.L/well 250 nM mix of mRNA ISH probes in 1% dextran
sulfate (>500 kDa MW, Sigma-Aldrich), 1 mg/mL tRNA (from E.
coli, Roche), 10% formamide, 2 mM RVC, 2.times.SSC hybridization
buffer for 4 Hrs (or overnight) at 37.degree. C. Following
hybridization, cells were washed with warm 10% formamide,
2.times.SSC buffer for 30 min at 37.degree. C., two changes of
1.times.PBS for 10 min at RT, and blocked by 2% BSA, 0.5% Western
blot blocking reagent, 0.1% low MW dextran sulfate, 0.1 mg/mL
shredded salmon sperm DNA, 1.times.PBS for 30 min at RT.
[0210] Encoding for Multi-Omics Studies.
[0211] Encoding of protein and mRNA targets on the same specimen
was performed by combining immunorecognition and in situ
hybridization procedures. First, cells were hybridized with
ssDNA-tagged mRNA ISH probes as described above. Following
hybridization and washing, cells were blocked, incubated with
antibody-ssDNA bioconjugates for 1-2 Hrs at RT, and washed with
PBS.
[0212] Specimen Labeling with QDot Probes.
[0213] Following encoding of targets with ssDNA tags, cells were
simultaneously labeled with complementary QDot-ssDNA' probes. QDots
were used at a final concentration of 5 nM in 2% BSA, 0.1% low MW
dextran sulfate, 0.1 mg/mL shredded salmon sperm DNA, 1.times.PBS
and incubated with cells for 2-4 Hrs at RT. Following staining
cells were washed with PBS. Optionally, nuclei could be
counter-stained by a 5-min incubation with DAPI.
[0214] For reference immunofluorescence studies, cell staining with
QDots functionalized with secondary Ab fragments (Qdot goat F(ab')2
anti-mouse or anti-rabbit IgG conjugates (H+L), Invitrogen) was
performed in a similar fashion.
[0215] RNAi.
[0216] Knock-down of GAPDH expression was done via cell
transfection with GAPDH siRNA (Ambion). For forward transfection,
cells were grown in a glass-bottom 24-well plate overnight and then
treated with 500 .mu.l/well culture medium containing 25 nM GAPDH
siRNA and 0.5 .mu.l/well DharmaFECT-2 transfection reagent
(Dharmacon) for 24 hrs. For reverse transfection, cells were grown
in a 10 cm TC-treated dish, trypsinized, mixed in suspension with
culture medium containing 25 nM GAPDH siRNA and 0.5 .mu.l/well
DharmaFECT-2 transfection reagent, seeded into a glass-bottom
24-well plate at 500 .mu.l/well cell suspension, and incubated for
24 hrs or 48 hrs. Following transfection, cells were processed for
staining. Triplicate samples were also prepared for RT-PCR
analysis.
[0217] Rt-PCR Analysis.
[0218] Total RNA was isolated from cell pellets using TRIzol
reagent (Invitrogen) according to the manufacturer's protocol. Two
hundred nanograms of RNA was converted to cDNA using random hexamer
primer and MultiScribe Reverse Transcriptase Reagent (Applied
Biosystems). One hundred nanograms of cDNA was amplified by the
Real-Time PCR using SensiFAST.TM. Real-Time PCR Kits (Bioline, UK)
on Chromo4 Real-Time PCR detection system (Bio-Rad). The primers
used for GAPDH amplification were 5'-TCGCTCTCTGCTCCTCCTGTTC-3'
(forward primer; SEQ ID NO: 99) and 5'-CGCCCAATACGACCAAATCC-3'
(reverse primer; SEQ ID NO: 100). Cyclophilin A (PPIA) was used as
an internal control, and the primers were
5'-GTCAACCCCACCGTGTTCTTC-3' (forward primer; SEQ ID NO: 101) and
5'-TTTCTGCTGTCTTTGGGACCTTG-3' (reverse primer SEQ ID NO: 102). To
confirm the PCR specificity, PCR products were subjected to a
melting-curve analysis. The comparative threshold (C.sub.t) method
was used to calculate the relative mRNA amount of the treated
sample in comparison to control samples. Mean value from triplicate
samples was reported.
[0219] Imaging and Signal Analysis.
[0220] IX-71 inverted fluorescence microscope (Olympus) equipped
with a true-color CCD (QColor5, Olympus) and a hyperspectral
imaging camera (Nuance, 420-720 nm spectral range, CRI, now
PerkinElmer) was used for cell imaging. Low-magnification images
were obtained with .times.20 dry objective (NA 0.75, Olympus) and
high-magnification with .times.40 (NA 1.30, Olympus) and .times.100
(NA 1.40, Olympus) oil-immersion objectives. Wide UV filter cube
(330-385 nm band-pass excitation, 420 nm long-pass emission,
Olympus) was used for imaging of all QDot probes, while Rhodamine
LP cube (530-560 nm band-pass excitation, 572 nm long-pass
emission, Chroma) was used for Alexa Fluor 555 detection. All
images were acquired with cells attached to the coverslip bottom of
the well and immersed in PBS without use of anti-fading
reagents.
[0221] Nuance image analysis software was used to unmix the
obtained multispectral images based on the reference spectra of
each QDot component along with an extra channel for background
fluorescence. In a false-color composite image, brightness and
contrast of each channel was automatically adjusted for best visual
representation and clear depiction of relative target distribution,
unless noted otherwise. For direct comparison of QDot staining
intensity individual QDot channels were normalized.
[0222] DNA Encoding for Multi-Omic Imaging Studies.
[0223] To demonstrate the DNA encoding for multi-omics imaging
studies concurrent analysis of single-cell molecular expression
profiles at mRNA and protein levels were performed. Fluorescent
quantum dot probes (QDots) in combination with fluorescence
microscopy and hyperspectral imaging (HSI) were employed for
simultaneous visualization of all ssDNA tags following separate
encoding of mRNA and protein targets (FIG. 5A). For example, GAPDH
and HSP90-alpha mRNA molecules and their respective product
proteins can be readily labeled by 4-color QDots to highlight
relative intracellular distribution and abundance of the two target
types at a single-cell level (FIG. 5B). Unlike direct labeling
procedures performed at a single incubation condition fixed for all
targets and probes, DNA encoding enables tuning of conditions to
favor recognition of individual target types and hybridization with
detection probes in separate steps, offering great flexibility in
choice of specimens, targets, and imaging systems (FIG. 6).
[0224] QDot-Based Multi-Omics Imaging Platform.
[0225] To implement and systematically characterize the model
QDot-based multi-omics imaging platform, a set of 6 unique 16 bp
ssDNA/ssDNA' linkers was developed for encoding of up to 6
different molecular targets (TABLE 1) along with a library of
complementary 6-color QDot-ssDNA probes (FIG. 7A and FIG. 7B) and a
control set of 6 secondary antibody-ssDNA (2'Ab-ssDNA)
bioconjugates (FIG. 8A and FIG. 8B). Indirect labeling of
.beta.-tubulin in HeLa cells via a 3-step procedure involving
incubation with unmodified primary antibodies, 2'Ab-ssDNA
bioconjugates, and complementary QDot-ssDNA' probes demonstrated
preserved antigen-recognition functionality of ssDNA-modified
antibodies and high specificity of QDot staining via DNA
hybridization (FIG. 9).
[0226] Mutiplex Protein Immuno-Labeling.
[0227] Multiplexed protein immuno-labeling was realized through
preparation of a library of primary antibody-ssDNA (1'Ab-ssDNA)
bioconjugates (FIG. 10A, FIG. 10B, and FIG. 10C; and FIG. 11A, FIG.
11B, FIG. 11C, FIG. 11D, and FIG. 11E). Characterization of such
bioconjugates with PAGE and cell staining confirmed preserved
stability and antigen-binding functionality of antibodies,
specificity of target staining with QDots in a 2-step procedure,
and consistent target identification with different QDot colors in
a multiplexed imaging format (FIG. 11A, FIG. 11B, FIG. 11C, FIG.
11D, and FIG. 11E). Nuclear envelope protein Lamin A, microtubule
.beta.-tubulin, and cytoplasmic proteins HSP90-alpha and GAPDH were
labeled as model target molecules with distinct characteristic
intracellular localization.
[0228] Labeling of model GAPDH and HSP90-alpha mRNA molecules via
an indirect in situ hybridization (ISH) procedure was done with
modified mRNA ISH oligonucleotide probes featuring 5' 20nt
mRNA-recognition portion and a 3' 16nt QDot-binding tag separated
by a single-stranded AAAAA spacer (TABLE 2 and TABLE 3).
Hybridization of oligonucleotide probes under optimized ISH
conditions yielded labeling of each mRNA molecule with multiple
ssDNA tags (up to 36 for GAPDH and 48 for HSP90-alpha), producing
distinct spots upon staining with complementary QDot-ssDNA probes
consistent with results achieved with conventional mRNA ISH
protocols (FIG. 12). In some instances, non-complementary
QDot-ssDNA probes failed to hybridize to exposed ssDNA tags,
producing minimal non-specific staining background. To explore
effects of potential secondary structure formation in 41nt ssDNA
oligonucleotides as well as steric hindrance experienced by QDots
approaching tightly spaced ssDNA tags, an alternative mRNA ISH
probe set was designed with each probe containing a 16 bp dsDNA
spacer between 5' mRNA-recognition and 3' QDot-binding portions.
Indeed, physical separation of functional ssDNA portions improved
mRNA staining intensity in comparison to linear 41nt ssDNA
oligonucleotides (FIG. 13A, FIG. 13B, FIG. 13C, and FIG. 13D),
offering one strategy for enhancing per-spot signal intensity and
improving signal-to-noise ratio.
[0229] Separation of target-recognition and QDot-labeling events
via an intermediate DNA encoding enabled straightforward
implementation of a model multi-omics imaging protocol, with both
mRNA and protein targets being robustly labeled by respective QDot
probes and accurately identified through hyperspectral imaging and
analysis (FIG. 14), corroborating broad applicability of the DNA
encoding strategy for simultaneous detection and imaging of various
types of targets within the same specimen.
[0230] Multi-omics imaging platform was then applied to study gene
knock-down via RNAi at a single-cell level. HeLa cells were
transfected with GAPDH-targeting siRNA (as well as non-targeting
siRNA for control) for 24 hrs, and GAPDH mRNA abundance was
assessed with RT-PCR and QDot-based imaging. In some cases, bulk
GAPDH mRNA measurement by RT-PCR indicated silencing efficiency of
78% with forward transfection and 95% with reverse transfection. At
the same time, imaging revealed heterogeneity in RNAi, likely
resulting from heterogeneous cell transfection with siRNA
throughout different regions of cell culture. For example, forward
transfection failed to achieve efficient GAPDH mRNA degradation in
dense cell populations, yielding areas of completely silenced cells
along with patches of cells with normal GAPDH mRNA expression
levels (FIG. 15). In contrast, reverse transfection achieved a more
uniform cell transfection in suspension, producing a greater
proportion of silenced cells with only a few wild-type clones (FIG.
16). Direct comparison of mRNA imaging results obtained from
forward vs. reverse transfection further corroborated complete mRNA
degradation upon successful transfection with either method along
with unperturbed GAPDH mRNA levels in non-transfected cells (FIG.
17), suggesting an all-on/all-off mode of GAPDH RNAi and
attributing incomplete silencing observed with bulk RT-PCR analysis
to heterogeneity in siRNA transfection.
[0231] Selectivity of GAPDH RNAi was confirmed by performing
dual-target imaging of GAPDH mRNA and HSP90-alpha mRNA.
Target-selective siRNA should trigger degradation of only its
complementary target mRNA, having no immediate effect on
non-targeted mRNA molecules. This was indeed observed with GAPDH
RNAi studies (FIG. 18). Indirect dual-target ISH produced robust
staining of both mRNA species in reference HeLa cells grown in
culture medium. Similarly, cell transfection with non-targeting
control siRNA failed to produce any effect on mRNA expression.
Transfection with GAPDH-targeting siRNA, however, triggered rapid
degradation of GAPDH mRNA within 24 hrs post-transfection, while
leaving non-targeted HSP90-alpha mRNA intact. A single
non-transfected cell within the field of view features intact
expression of both GAPDH and HSP90 mRNA, consistent with discussion
above.
[0232] Imaging of mRNA unambiguously demonstrated heterogeneity in
RNAi stemming from incomplete cell transfection with siRNA.
However, such heterogeneity could not be detected at the protein
level, as GAPDH protein remained unperturbed 24 Hrs
post-transfection in both transfected and non-transfected cells, as
was evident from dual labeling of GAPDH mRNA and protein (FIG. 19).
To further investigate the disparity between RNAi effect at mRNA
and protein levels, HeLa cells were reverse transfected with
GAPDH-targeting siRNA for 24 and 48 Hrs and processed for
multiplexed imaging of GAPDH and HSP90-alpha mRNA and their
respective protein products. Consistent with studies discussed
earlier, 24 Hrs post-transfection a complete degradation of GAPDH
mRNA was observed, whereas GAPDH protein level remained unperturbed
(FIG. 20A). In contrast, 48 hrs post-transfection a substantial
reduction of GAPDH protein level could be observed, with GAPDH mRNA
remaining below the detection limit (FIG. 20B). HSP90 mRNA and
protein levels remained unperturbed through 48 hours, confirming
selectivity of GAPDH silencing. Further, all molecular targets
exhibited consistent unperturbed levels in reference
non-transfected cells (FIG. 21A and FIG. 21B) and cells transfected
with non-targeting siRNA (FIG. 22A and FIG. 22B) throughout the
study, corroborating that the observed GAPDH knock-down indeed
resulted from RNAi mechanism. Multiplexed analysis was fully
confirmed by a series of single-plex studies to mitigate any
artifacts that could potentially be introduced from the multi-omics
labeling methodology, HSI, and image analysis (FIG. 23A and FIG.
23B).
[0233] In some cases, delay in RNAi effect at the protein level is
present, as proteins are typically degraded and cleared slower in
comparison to siRNA-mediated mRNA degradation. In other cases,
heterogeneity in cell transfection can modulate assessing RNAi
efficiency with bulk RT-PCR measurement and downstream phenotypic
and molecular signaling analysis. Non-transfected cells might gain
growth advantage and achieve substantial clonal expansion during
the time it takes for higher-level manifestations of RNAi to occur,
thus distorting observed RNAi effect at a population level.
Imaging-based analysis at a single-cell level can by-pass this
ambiguity and can offer a more accurate insight into molecular
processes.
TABLE-US-00001 TABLE 1 List of ssDNA/ssDNA' tag pairs for encoding
of molecular targets Tag ID Sequence* SEQ ID NO: QDot-coupled 1A
5'-/5AmMC6/iSp18/CGTCGCACCAAGAAAT-3' 1 2A
5'-/5AmMC6/iSp18/TAGACTTGCCATACGT-3' 2 3A
5'-/5AmMC6/iSp18/AATTCTTGAGACCAGG-3' 3 4A
5'-/5AmMC6/iSp18/ATCTGCCCAAACTCCA-3' 4 5A
5'-/5AmMC6/iSp18/TTCCCAAGCGTCATCT-3' 5 6A
5'-/5AmMC6/iSp18/TCTATCGGACGCTGTA-3' 6 IgG-coupled 1B
5'-/5AmMC6/AAAAAAAAAAATTTCTTGGTGCGACG-3' 7 2B
5'-/5AmMC6/AAAAAAAAAAACGTATGGCAAGTCTA-3' 8 3B
5'-/5AmMC6/AAAAAAAAAACCTGGTCTCAAGAATT-3' 9 4B
5'-/5AmMC6/AAAAAAAAAATGGAGTTTGGGCAGAT-3' 10 5B
5'-/5AmMC6/AAAAAAAAAAAGATGACGCTTGGGAA-3' 11 6B
5'-/5AmMC6/AAAAAAAAAATACAGCGTCCGATAGA-3' 12 *all ssDNA tags have 5'
terminal amino group (/5AmMC6/) for bioconjunction separated from
the pairing sequence by either a hexa-ethyleneglycol spacer
(/iSp18/) for QDot-coupled tags or 10A oligonucleotide spacer
(AAAAAAAAAA; SEQ ID NO: 97) for IgG-coupled tags.
TABLE-US-00002 TABLE 2 Sequences of GAPDH mRNA ISH probes (with 2B
encoding tag) # mRNA-recognition region encoding tag 2B SEQ ID NO:
1 5'-ATTTATAGAAACCGGGGGCG ACGTATGGCAAGTCTA-3' 13 2
5'-CGAACAGGAGGAGCAGAGAG ACGTATGGCAAGTCTA-3' 14 3
5'-GCTGGCGACGCAAAAGAAGA ACGTATGGCAAGTCTA-3' 15 4
5'-CATGGTGTCTGAGCGATGTG ACGTATGGCAAGTCTA-3' 16 5
5'-TACGACCAAATCCGTTGACT ACGTATGGCAAGTCTA-3' 17 6
5'-CAGAGTTAAAAGCAGCCCTG ACGTATGGCAAGTCTA-3' 18 7
5'-GGGTCATTGATGGCAACAAT ACGTATGGCAAGTCTA-3' 19 8
5'-AACCATGTAGTTGAGGTCAA ACGTATGGCAAGTCTA-3' 20 9
5'-GGGTGGAATCATATTGGAAC ACGTATGGCAAGTCTA-3' 21 10
5'-TTGACGGTGCCATGGAATTT ACGTATGGCAAGTCTA-3' 22 11
5'-CATTGATGACAAGCTTCCCG ACGTATGGCAAGTCTA-3' 23 12
5'-TCCTGGAAGATGGTGATGGG ACGTATGGCAAGTCTA-3' 24 13
5'-CCACTTGATTTTGGAGGGAT ACGTATGGCAAGTCTA-3' 25 14
5'-GGACTCCACGACGTACTCAG ACGTATGGCAAGTCTA-3' 26 15
5'-TTCTCCATGGTGGTGAAGAC ACGTATGGCAAGTCTA-3' 27 16
5'-AGAGATGATGACCCTTTTGG ACGTATGGCAAGTCTA-3' 28 17
5'-GACGAACATGGGGGCATCAG ACGTATGGCAAGTCTA-3' 29 18
5'-CATACTTCTCATGGTTCACA ACGTATGGCAAGTCTA-3' 30 19
5'-ATTGCTGATGATCTTGAGGC ACGTATGGCAAGTCTA-3' 31 20
5'-CTAAGCAGTTGGTGGTGCAG ACGTATGGCAAGTCTA-3' 32 21
5'-CCACGATACCAAAGTTGTC AACGTATGGCAAGTCTA-3' 33 22
5'-TCTTCTGGGTGGCAGTGATG ACGTATGGCAAGTCTA-3' 34 23
5'-TAGAGGCAGGGATGATGTTC ACGTATGGCAAGTCTA-3' 35 24
5'-TCAGCTCAGGGATGACCTTG ACGTATGGCAAGTCTA-3' 36 25
5'-CACTGACACGTTGGCAGTGG ACGTATGGCAAGTCTA-3' 37 26
5'-CAGGTTTTTCTAGACGGCAG ACGTATGGCAAGTCTA-3' 38 27
5'-CACCTTCTTGATGTCATCAT ACGTATGGCAAGTCTA-3' 39 28
5'-GCTGTTGAAGTCAGAGGAGA ACGTATGGCAAGTCTA-3' 40 29
5'-CGTCAAAGGTGGAGGAGTGG ACGTATGGCAAGTCTA-3' 41 30
5'-AGTGGTCGTTGAGGGCAATG ACGTATGGCAAGTCTA-3' 42 31
5'-TCATACCAGGAAATGAGCTT ACGTATGGCAAGTCTA-3' 43 32
5'-CCTGTTGCTGTAGCCAAATT ACGTATGGCAAGTCTA-3' 44 33
5'-TGAGGAGGGGAGATTCAGTG ACGTATGGCAAGTCTA-3' 45 34
5'-CTCTTCAAGGGGTCTACATG ACGTATGGCAAGTCTA-3' 46 35
5'-TACATGACAAGGTGCGGCTC ACGTATGGCAAGTCTA-3' 47 36
5'-TGAGCACAGGGTACTTTATT ACGTATGGCAAGTCTA-3' 48 Note:
mRNA-recognition region and encoding tag are separated by a spacer
(bolded and italicized). Shorter 41nt mRNA ISH probe contain
-AAAAA- single strand spacer. Longer 60nt mRNA ISH probes contain
pre-hybridized 16 bp double-stranded spacer flanked by -AAAA-
single-stranded linkers.
TABLE-US-00003 TABLE 3 Sequences of HSP90-alpha mRNA ISH probes
(with 4B encoding tag) # mRNA-recognition region encoding tag 4B
SEQ ID NO: 1 5'-AGGAGTATGATTGTCAACCC TGGAGTTTGGGCAGAT-3' 49 2
5'-CCTATATAAGGCGAAGCAC ATGGAGTTTGGGCAGAT-3' 50 3
5'-GAGTGACTCGAGAGAGCTAC TGGAGTTTGGGCAGAT-3' 51 4
5'-ATAGTGAGCAACGTAGGCTT TGGAGTTTGGGCAGAT-3' 52 5
5'-GGACATGAGTTGGGCAATTT TGGAGTTTGGGCAGAT-3' 53 6
5'-GAGATCAACTCCCGAAGGAA TGGAGTTTGGGCAGAT-3' 54 7
5'-AATCTTGTCCAAGGCATCAG TGGAGTTTGGGCAGAT-3' 55 8
5'-AACTTCGAAGGGTCTGTCAG TGGAGTTTGGGCAGAT-3' 56 9
5'-GGTTGGGGATGATGTCAATT TGGAGTTTGGGCAGAT-3' 57 10
5'-TACCAAAGTCAGGGTACGTT TGGAGTTTGGGCAGAT-3' 58 11
5'-TGAGATCAGCTTTGGTCATG TGGAGTTTGGGCAGAT-3' 59 12
5'-TTGGCAATGGTTCCCAAATT TGGAGTTTGGGCAGAT-3' 60 13
5'-CTGAAGAGCCTCCATGAATG TGGAGTTTGGGCAGAT-3' 61 14
5'-CCACCAAGTAGGCAGAATAA TGGAGTTTGGGCAGAT-3' 62 15
5'-TGCTTTGTGATCACAACCAC TGGAGTTTGGGCAGAT-3' 63 16
5'-CAGAAGACTCCCAAGCATAC TGGAGTTTGGGCAGAT-3' 64 17
5'-AGCACGCACAGTGAAGGAAC TGGAGTTTGGGCAGAT-3' 65 18
5'-TCTAGGTACTCTGTCTGATC TGGAGTTTGGGCAGAT-3' 66 19
5'-TAAAGGGTGATGGGATAGCC TGGAGTTTGGGCAGAT-3' 67 20
5'-TGTTTAGTTCTTCCTGATCA TGGAGTTTGGGCAGAT-3' 68 21
5'-AGGGTTTCTGGTCCAAATAG TGGAGTTTGGGCAGAT-3' 69 22
5'-TCATTAGTGAGGCTCTTGTA TGGAGTTTGGGCAGAT-3' 70 23
5'-AAAGTGCTTGACTGCCAAGT TGGAGTTTGGGCAGAT-3' 71 24
5'-TGAATTCCAACTGACCTTCT TGGAGTTTGGGCAGAT-3' 72 25
5'-GAGCCCGACGAGGAATAAAT TGGAGTTTGGGCAGAT-3' 73 26
5'-TGAACACACGGCGGACATAG TGGAGTTTGGGCAGAT-3' 74 27
5'-ATCAACTCATCACAGCTGTC TGGAGTTTGGGCAGAT-3' 75 28
5'-AAGATTTTGCTCTGCTGGAG TGGAGTTTGGGCAGAT-3' 76 29
5'-AGAGAAGAGCTCAAGGCACT TGGAGTTTGGGCAGAT-3' 77 30
5'-GTGGATTCCAAGCTTGAGAT TGGAGTTTGGGCAGAT-3' 78 31
5'-AGACTGGGAGGTATGATAGC TGGAGTTTGGGCAGAT-3' 79 32
5'-CTCTGACAGAGATGTCATCT TGGAGTTTGGGCAGAT-3' 80 33
5'-TAGATGGACTTCTGTGTCTC TGGAGTTTGGGCAGAT-3' 81 34
5'-GCTCCACAAAAGCTGAGTTG TGGAGTTTGGGCAGAT-3' 82 35
5'-CATATATACCACCTCGAAGC TGGAGTTTGGGCAGAT-3' 83 36
5'-ACACAGTACTCGTCAATGGG TGGAGTTTGGGCAGAT-3' 84 37
5'-TTCCCATCAAATTCCTTGAG TGGAGTTTGGGCAGAT-3' 85 38
5'-GAGATTGTCACCTTCTCAAC TGGAGTTTGGGCAGAT-3' 86 39
5'-TGCAGCAAGGTGAAGACACA TGGAGTTTGGGCAGAT-3' 87 40
5'-GCTTTTTGGCCATCATATAG TGGAGTTTGGGCAGAT-3' 88 41
5'-AACTGCCTTATCATTCTTGT TGGAGTTTGGGCAGAT-3' 89 42
5'-ATCCTCAAGGGAAAAGCCAG TGGAGTTTGGGCAGAT-3' 90 43
5'-TGATCATGCGATAGATGCGG TGGAGTTTGGGCAGAT-3' 91 44
5'-CATCAGGAACTGCAGCATTG TGGAGTTTGGGCAGAT-3' 92 45
5'-CAAGGGCACAAGTTTTCCAA TGGAGTTTGGGCAGAT-3' 93 46
5'-TACTGCCTTCAACACAAGGA TGGAGTTTGGGCAGAT-3' 94 47
5'-AGAGTAGAGAGGGAATGGGG TGGAGTTTGGGCAGAT-3' 95 48
5'-TACACAACATCCAATCCTGC TGGAGTTTGGGCAGAT-3' 96 Note:
mRNA-recognition portion and encoding tag are separated by a spacer
(bolded and italicized). Shorter 41nt mRNA ISH probes contain
-AAAAA- single stranded spacer. Longer 60nt mRNA ISH probes contain
pre-hybridized 16 bp double-stranded spacer flanked by -AAAA-
single-stranded linkers.
Example 3
Global In Situ Visualization of the DNaseI Hypersensitivity Site
(DHS) Compartment of a Cell
[0234] This example shows the global in situ visualization of the
DNaseI Hypersensitivity Site (DHS) compartment of a cell, which
allows for identification of nuclear compartments where regulatory
DNA activation occurs. As shown in the graphic on the left side of
FIG. 24, K562 cells were fixed with Paxgene reagent, treated with
DNaseI, DNaseI-induced nicks were labeled using terminal
transferase (TdT) and ethynyl-dUTP (EdUTP) (TUNEL assay),
Alexafluor-488 (AF488) was conjugated to the EdUTP via copper click
chemistry, and then SPDM imaging was performed. FIG. 24 shows
multiple images of this. The top left image is of the raw signal
data. The local density map image (top middle) shows a ring of
condensation at the nuclear lamina, which is simlar to findings
reported by the Weintraub lab 30 years ago (Weintraub, Cell (1985)
43:471-482); see FIG. 24 top right reproduced image). Approximately
18.4% of the localized points are within the ring density at the
nuclear lamina, as shown the calculations in the lower right box,
in which the image data calculation was based off the the image on
the lower left of FIG. 24. The image data calculation is similar to
the proportion of K562 DHS within lamina-associated domains (LADS).
These findings indicate labeling of DNaseI cut sites in a cell's
nucleus using a TUNEL assay may be used for better understanding of
the nuclear localization of regulatory DNA activation.
[0235] The examples and embodiments described herein are for
illustrative purposes only and various modifications or changes
suggested to persons skilled in the art are to be included within
the spirit and purview of this application and scope of the
appended claims.
* * * * *