U.S. patent application number 10/830544 was filed with the patent office on 2004-10-07 for methods for detecting aggregations of proteins.
Invention is credited to Matray, Tracy, Salimi-Moosavi, Hossein, Singh, Sharat.
Application Number | 20040197815 10/830544 |
Document ID | / |
Family ID | 32034246 |
Filed Date | 2004-10-07 |
United States Patent
Application |
20040197815 |
Kind Code |
A1 |
Singh, Sharat ; et
al. |
October 7, 2004 |
Methods for detecting aggregations of proteins
Abstract
Families of compositions are provided as labels, referred to as
eTag reporters for attaching to polymeric compounds and assaying
based on release of the eTag reporters from the polymeric compound
and separation and detection. For oligonucleotides, the eTag
reporters are synthesized at the end of the oligonucleotide by
using phosphiste or phosphate chemistry, whereby mass-modifying
regions, charge-modifying regions and detectable regions are added
sequentially to produce the eTag labeled reporters. By using small
building blocks and varying their combination large numbers of
different eTag reporters can be readily produced attached to the
oligonucleotide of interest for identification. Protocols are used
that release the eTag reporter when the target sequence is present
in the sample.
Inventors: |
Singh, Sharat; (San Jose,
CA) ; Matray, Tracy; (San Lorenzo, CA) ;
Salimi-Moosavi, Hossein; (Sunnyvale, CA) |
Correspondence
Address: |
ACLARA BIOSCIENCES, INC.
1288 PEAR AVENUE
MOUNTAIN VIEW
CA
94043
US
|
Family ID: |
32034246 |
Appl. No.: |
10/830544 |
Filed: |
April 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10830544 |
Apr 22, 2004 |
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10420549 |
Apr 18, 2003 |
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10420549 |
Apr 18, 2003 |
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09698846 |
Oct 27, 2000 |
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6627400 |
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09698846 |
Oct 27, 2000 |
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09602586 |
Jun 21, 2000 |
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6514700 |
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09602586 |
Jun 21, 2000 |
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09561579 |
Apr 28, 2000 |
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6682887 |
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Current U.S.
Class: |
435/6.12 ;
435/6.1; 536/25.32 |
Current CPC
Class: |
C40B 50/16 20130101;
C07H 21/00 20130101; C40B 70/00 20130101 |
Class at
Publication: |
435/006 ;
536/025.32 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1-48. (canceled)
49. A method of performing a multiplexed assay for the
determination of a plurality of target proteins in an aggregation
of proteins, the method comprising the steps of: providing a
binding compound for each of the plurality of target proteins, each
binding compound having one or more eTag reporters attached thereto
by a cleavable linkage, the one or more eTag reporters of each
different binding compound having a different electrophoretic
mobility so that eTag reporters of each different binding compound
form distinct peaks upon electrophoretic separation; combining with
the aggregation a binding compound for each of the plurality of
target proteins such that in the presence of a target protein a
complex is formed between each target protein and the binding
compound specific therefor; cleaving the cleavable linkage of each
binding compound forming such complex so that eTag reporters are
released; and electrophoretically separating and identifying the
released eTag reporters to determine the plurality of target
proteins in the aggregation.
50. The method of claim 49 further including a step prior to said
step of cleaving, the step comprising separating said complexes
from unbound said binding compounds.
51. The method of claim 50 wherein said step of cleaving includes
treating said cleavage linkage with an enzyme to release said eTag
reporters.
52. The method of claim 50 wherein each of said eTag reporters has
a fluorescent label or an electrochemical label.
53. The method of claim 49 wherein said binding compound is an
antibody or fragment thereof.
54. The method of claim 53 wherein said released eTag reporters
have a charge opposite that of said complexes and said binding
compounds, wherein said cleavable linkage is cleaved by oxidation,
and wherein said step of cleaving includes providing an active
species for oxidizing said cleavable linkage.
55. The method of claim 53 wherein said cleavable linkage is
cleaved by oxidation and wherein said step of cleaving includes
providing an active species for oxidizing said cleavable
linkage.
56. The method of claim 55 wherein said active species is selected
from the group consisting of singlet oxygen, hydrogen peroxide,
NADH, and hydroxyl radicals.
57. The method of claim 56 wherein said step of cleaving further
includes providing a second binding compound specific for a protein
of said aggregation of proteins, the second binding compound being
conjugated with an active species producing moiety for generating
said active species for oxidizing said cleavable linkage.
58. The method according to claim 54, 55, 56, or 57 wherein said
active species is singlet oxygen, wherein said second binding
compound is an antibody or fragment thereof, and wherein said
cleavable linkage is an olefin, a thioether, a sulfoxide, or a
selenium analog of the thioether or sulfoxide.
59. The method of claim 58 wherein said binding compound and said
second binding compound are each antibodies.
60. A method of performing a multiplexed assay for the
determination of a plurality of target proteins in an aggregation
of proteins, the method comprising the steps of: providing a
binding compound for each of a plurality of target proteins in the
aggregation, each binding compound having one or more eTag
reporters attached thereto by a cleavable linkage, the one or more
eTag reporters of each different binding compound having a
different mass/charge ratio or adsorption so that eTag reporters of
each different binding compound form distinct peaks upon
electrophoretic or chromatographic separation; providing a second
binding compound for a target protein in the aggregation, the
second binding compound being conjugated with an active species
producing moiety; combining with the aggregation a binding compound
for each of the plurality of target proteins and the second binding
compound such that in the presence of a target protein in the
aggregation a complex is formed between each target protein and the
binding compound or the second binding compound specific therefor,
and such that the active species producing moiety of the second
binding compound causes the generation of an active species and the
cleavage of one or more cleavable linkages to release one or more
eTag reporters; and electrophoretically or chromatographically
separating and identifying the released eTag reporters to determine
the plurality of target proteins in the aggregation.
61. The method of claim 60 wherein said cleavable linkage is
cleaved by oxidation and wherein said active species is singlet
oxygen and wherein said active species producing moiety is a
sensitizer.
62. The method of claim 61 wherein said cleavable linkage is an
olefin, a thioether, a sulfoxide, or a selenium analog of the
thioether or sulfoxide.
63. The method of claim 62 wherein each of said one or more eTag
reporters of each said different binding compound has a different
mass/charge ratio so that said eTag reporters of each said
different binding compound forms a distinct peak upon
electrophoretic separation.
64. The method according to claim 60, 61, 62, or 63 wherein said
binding compound and said second binding compound are each an
antibody or fragment thereof.
65. A method of determining an aggregation of proteins in a sample,
the method comprising the steps of: providing a binding compound
for a protein of the aggregation, the binding compound having one
or more eTag reporters attached thereto by a cleavable linkage, the
eTag reporters having a unique electrophoretic mobility so that a
distinct peak is formed upon electrophoretic separation; combining
with the sample the binding compound such that in the presence of
the aggregation a complex is formed between a protein of the
aggregation and the binding compound specific therefor; cleaving
the cleavable linkage of the binding compound forming such complex
so that eTag reporters are released; and electrophoretically
separating and identifying the released eTag reporters to determine
the presence of the aggregation in the sample.
66. The method of claim 65 further including a step prior to said
step of cleaving, the step comprising separating said complexes
from unbound said binding compounds.
67. The method of claim 66 wherein said step of cleaving includes
treating said cleavage linkage with an enzyme to release said eTag
reporters.
68. The method of claim 66 wherein each of said eTag reporters has
a fluorescent label or an electrochemical label.
69. The method of claim 65 wherein said binding compound is an
antibody or fragment thereof.
70. The method of claim 69 wherein said released eTag reporters
have a charge opposite that of said complexes and said binding
compound, wherein said cleavable linkage is cleaved by oxidation,
and wherein said step of cleaving includes providing an active
species for oxidizing said cleavable linkage.
71. The method of claim 69 wherein said cleavable linkage is
cleaved by oxidation and wherein said step of cleaving includes
providing an active species for oxidizing said cleavable
linkage.
72. The method of claim 71 wherein said active species is selected
from the group consisting of singlet oxygen, hydrogen peroxide,
NADH, and hydroxyl radicals.
73. The method of claim 71 wherein said step of cleaving further
includes providing a second binding compound specific for a protein
of said aggregation of proteins, the second binding compound being
conjugated with an active species producing moiety for generating
said active species for oxidizing said cleavable linkage.
74. The method according to claim 70, 71, 72, or 73 wherein said
active species is singlet oxygen, wherein said second binding
compound is an antibody or fragment thereof, and wherein said
cleavable linkage is an olefin, a thioether, a sulfoxide, or a
selenium analog of the thioether or sulfoxide.
75. The method of claim 74 wherein said binding compound and said
second binding compound are each antibodies.
76. A method of determining an aggregation of proteins in a sample,
the method comprising the steps of: providing a binding compound
for a protein of the aggregation, the binding compound having one
or more eTag reporters attached thereto by a cleavable linkage, the
eTag reporters having a unique electrophoretic mobility or
adsorption so that a distinct peak is formed upon electrophoretic
or chromatographic separation; providing a second binding compound
for a protein of the aggregation, the second binding compound being
conjugated with an active species producing moiety; combining with
the sample the binding compound and the second binding compound
such that in the presence of the aggregation a complex is formed
between proteins of the aggregation and the binding compound and
the second binding compound, and such that the active species
producing moiety of the second binding compound causes the
generation of an active species and the cleavage of one or more
cleavable linkages to release one or more eTag reporters; and
electrophoretically or chromatographically separating and
identifying the released eTag reporters to detect the aggregation
of proteins in the sample.
77. The method of claim 76 wherein said cleavable linkage is
cleaved by oxidation and wherein said active species is singlet
oxygen and wherein said active species producing moiety is a
sensitizer.
78. The method of claim 77 wherein said cleavable linkage is an
olefin, a thioether, a sulfoxide, or a selenium analog of the
thioether or sulfoxide.
79. The method of claim 76 wherein each of said at least one eTag
reporter has a distinct mass/charge ratio so that said eTag
reporter forms a distinct peak upon electrophoretic separation.
80. The method according to claim 76, 77, 78, or 79 wherein said
binding compound and said second binding compound are each an
antibody or fragment thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuing patent application of
application Ser. No. 09/602,586, filed Jun. 21, 2000, which is a
continuing application of application Ser. No. 09/561,579, filed
Apr. 28, 2000.
INTRODUCTION
FIELD OF THE INVENTION
[0002] The field of this invention is separable compositions for
use in multiplexed assay detection.
BACKGROUND OF THE INVENTION
[0003] As the human genome is elucidated, there will be numerous
opportunities for performing assays to determine the presence of
specific sequences, distinguishing between alleles in homozygotes
and heterozygotes, determining the presence of mutations,
evaluating cellular expression patterns, etc. In many of these
cases one will wish to determine in a single reaction, a number of
different characteristics of the same sample. Also, there will be
an interest in determining the presence of one or more pathogens,
their antibiotic resistance genes, genetic subtype and the
like.
[0004] In many assays, there will be an interest in determining the
presence of specific sequences, whether genomic, synthetic or cDNA.
These sequences may be associated particularly with genes,
regulatory sequences, repeats, multimeric regions, expression
patterns, and the like
[0005] There is and will continue to be comparisons of the
sequences of different individuals. It is believed that there will
be about one polymorphism per 1,000 bases, so that one may
anticipate that there will be an extensive number of differences
between individuals. By single nucleotide polymorphism (snp's) is
intended that there will be a prevalent nucleotide at the site,
with one or more of the remaining bases being present in
substantially smaller percent of the population.
[0006] For the most part, the snp's will be in non-coding regions,
primarily between genes, but will also be present in exons and
introns. In addition, the great proportion of the snp's will not
affect the phenotype of the individual, but will clearly affect the
genotype. The snp's have a number of properties of interest. Since
the snp's will be inherited, individual snp's and/or snp patterns
may be related to genetic defects, such as deletions, insertions
and mutations involving one or more bases in genes. Rather than
isolating and sequencing the target gene, it will be sufficient to
identify the snp's involved.
[0007] In addition, the snp's may be used in forensic medicine to
identify individuals. While other genetic markers are available,
the large number of snp's and their extensive distribution in the
chromosomes, make the snp's an attractive target. Also, by
determining a plurality of snp's associated with a specific
phenotype, one may use the snp pattern as an indication of the
phenotype, rather than requiring a determination of the genes
associated with the phenotype.
[0008] The need to determine many analytes or nucleic acid
sequences (for example multiple pathogens or multiple genes or
multiple genetic variants) in blood or other biological fluids has
become increasingly apparent in many branches of medicine. The need
to study differential expression of multiple genes to determine
toxicologically-relevant outcomes or the need to screen transfused
blood for viral contaminants with high sensitivity is clearly
evident.
[0009] Thus most multi-analyte assays or assays which detect
multiple nucleic acid sequences involve mutiple steps, have poor
sensitivity and poor dynamic range (2 to 100-fold differences in
concentration of the analytes is determined) and some require
sophisticated instrumentation.
[0010] Some of the known classical methods for multianalyte assays
include the following:
[0011] a. The use of two different radioisotope labels to
distinguish two different analytes.
[0012] b. The use of two or more different fluorescent labels to
distinguish two or more analytes.
[0013] c. The use of lanthanide chelates where both lifetime and
wavelength are used to distinguish two or more analytes.
[0014] d. The use of fluorescent and chemiluminescent labels to
distinguish two or more analytes.
[0015] e. The use of two different enzymes to distinguish two or
more analytes.
[0016] f. The use of enzyme and acridinium esters to distinguish
two or more analytes.
[0017] g. Spatial resolution of different analytes, for example, on
arrays to identify and quantify multiple analytes.
[0018] h. The use of acridinium ester labels where lifetime or
dioxetane formation is used to quantify two different viral
targets.
[0019] Thus an assay that has higher sensitivity, large dynamic
range (10.sup.3 to 10.sup.4 - fold differences in target levels),
greater degree of multiplexing, and fewer and more stable reagents
would increase the simplicity and reliability of multianalyte
assays.
[0020] The need to identify and quantify a large number of bases or
sequences potentially distributed over centimorgans of DNA offers a
major challenge. Any method should be accurate, reasonably
economical in limiting the amount of reagents required and
providing for a single assay, which allows for differentiation of
the different snp's or differentiation and quantitation of multiple
genes.
[0021] Finally, while nucleic acid sequences provide extreme
diversity for situations that may be of biological or other
interest, there are other types of compounds, such as proteins in
proteomics that may also offer opportunities for multiplexed
determinations.
BRIEF DESCRIPTION OF THE RELATED ART
[0022] holland (Proc. Natl. Acad. Sci. USA (1991) 88:7276)
discloses the exonuclease activity of the thennostable enzyme
Thermus aquaticus DNA polymerase in PCR amplification to generate
specific detectable signal concomitantly with amplification.
[0023] The TaqMan assay is discussed by Lee in Nucleic Acid
Research (1993) 21:16 3761).
[0024] White (Trends Biotechnology (1996) 14(12):478-483) discusses
the problems of multiplexing in the TaqMang assay.
[0025] Marino, Electrophoresis (1996) 17:1499 describes
low-stringency-sequence specific PCR (LSSP-PCR). A PCR amplified
sequence is subjected to single primer amplification under
conditions of low stringency to produce a range of different length
amplicons. Different patterns are obtained when there are
differences in sequence. The patterns are unique to an individual
and of possible value for identity testing.
[0026] Single strand confonnational polymorphism (SSCP) yields
similar results. In this method the PCR amplified DNA is denatured
and sequence dependent conformations of the single strands are
detected by their differing rates of migration during gel
electrophoresis. As with LSSP-PCR above, different patterns are
obtained that signal differences in sequence. However, neither
LSSP-PCR nor SSCP gives specific sequence information and both
depend on the questionable assumption that any base that is changed
in a sequence will give rise to a conformational change that can be
detected. Pastinen, Clin. Chem. (1996) 42:1391 amplifies the target
DNA and immobilizes the amplicons. Multiple primers are then
allowed to hybridize to sites 3' and contiguous to a snp ("single
nucleotide polymorphism") site of interest. Each primer has a
different size that serves as a code. The hybridized primers are
extended by one base using a fluorescently labeled
dideoxynucleoside triphosphate. The size of each of the fluorescent
products that is produced, determined by gel electrophoresis,
indicates the sequence and, thus, the location of the snp. The
identity of the base at the snp site is defined by the triphosphate
that is used. A similar approach is taken by Haff, Nucleic Acids
Res. (1997) 25:3749 except that the sizing is carried out by mass
spectroscopy and thus avoids the need for a label. However, both
methods have the serious limitation that screening for a large
number of sites will require large, very pure primers that can have
troublesome secondary structures and be very expensive to
synthesize.
[0027] Hacia, Nat. Genet. (1996) 14:441 uses a high-density array
of oligonucleotides. Labeled DNA samples are allowed to bind to
96,600 20-base oligonucleotides and the binding patterns produced
from different individuals were compared. The method is attractive
in that SNP's can be directly identified, but the cost of the
arrays is high and non-specific hybridization may confound the
accuracy of the genetic information.
[0028] Fan (Oct. 6-8, 1997, IBC, Annapolis MD) has reported results
of a large scale screening of human sequence-tagged sites. The
accuracy of single nucleotide polymorphism screening was determined
by conventional ABI resequencing.
[0029] Allele specific oligonucleotide hybridization along with
mass spectroscopy has been discussed by Ross in Anal. Chem. (1997)
69:4197.
[0030] Holland, et al., PNAS USA (1991) 88, 7276-7280, describes
use of DNA polymerase 5'-3' exonuclease activity for detection of
PCR products.
[0031] U.S. Pat. No. 5,807,682 describes probe compositions for
detecting a plurality of nucleic acid targets.
SUMMARY OF THE INVENTION
[0032] Compounds and methods are provided for multiplexed
determinations affording convenient separation of released
identifying tags based on individual physical, properties of the
tags. The methods can be performed in a single vessel and may
involve a plurality of reagents added simultaneously or
consecutively. In one group of embodiments, mass will be involved
in the characteristic allowing for separation. One group of
identifying tags for electrokinetic analysis is characterized by
having regions, which serve as (1) a cleavable linking region; (2)
a mass-modifying region; (3) a charge-modifying region: and (4) a
detectable region , the number of different regions depending in
part on the method of separation and identification. Compounds that
have these distinctive regions find use in conjunction with other
compounds where the regions are combined in the same moiety. Of
particular interest is the use of building blocks for forming the
compounds, where the synthesis is performed in a repetitive manner
using the same linking chemistry at a plurality of stages. The
subject compounds are linked to binding compounds for
identification to provide identifying reagents, where binding of an
identifying reagent target in an assay system results in the
release of the identifying tag (hereinafter referred to as an
"eTag.TM. reporter") where the eTag reporters can be
differentiated. Large numbers of eTag reporters can be provided in
kits comprising a linking functionality for bonding to the binding
compounds or kits of building blocks can be provided for
synthesizing eTag reporters in situ in conjunction with the
synthesis of the binding compound. Of particular interest is the
use of the subject eTag reporters in identification of nucleic
acids and proteins.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 is a schematic illustrating exemplary high voltage
configurations utilized in a CE.sup.2 LabCard.TM. device during an
en zme assay.
[0034] FIG. 2 is two electropherograms demonstrating eTag reporter
analysis using a CE.sup.2 LabCard. The figure shows the separation
of purified labeled aminodextran with and without sensitizer beads.
The addition of the sensitizer beads lead to the release of the
eTag reporter from the aminodextran using singlet oxygen produced
by sensitizer upon the irradiation at 680 nm. Experimental
conditions: Separation buffer 20.0 mM HEPES pH=7.4, and 0.5% PEO;
voltage configurations as shown in FIG. 1; assay mixture had 29
.mu.g/ml streptavidin coated sensitizer beads and irradiated for 1
min at 680 nm using 680.+-.10 nm filter and a 150 W lamp.
[0035] FIG. 3 is multiple electropherogams demonstrating eTag
reporter analysis using a CE.sup.2 LabCard. The figure shows the
separation of purified labeled aminodextran that has been
irradiated for different lengths of time. Experimental conditions:
Separation buffer 20.0 mM HEPES pH=7.4, and 0.5% PEO; voltage
configurations as shown in FIG. 1; assay mixture had 27 .mu.g/ml
streptavidin coated sensitizer beads and irradiated at 680 nm using
680.+-.10 nm filter and a 150 W lamp.
[0036] FIG. 4 is multiple electropherograms demonstrating eTag
reporter analysis using a CE.sup.2 LabCard. The figure shows the
separation of purified labeled aminodextran using different
concentrations of sensitizer beads. The higher concentration of
sensitizer beads leads to the higher release of eTag reporters from
the labeled aminodextran. Experimental conditions: Separation
buffer 20.0 mM HEPES pH=7.4, and 0.5% PEO; voltage configurations
as shown in
[0037] FIG. 1; assay mixture was irradiated for 1 min at 680 nm
using 680.+-.10 nm filter and a 150 W lamp.
[0038] FIG. 5 depicts the linear calibration curve for the release
of eTag reporters as a function of the sensitizer bead
concentration. Results were obtained using a CE.sup.2 LabCard.
Experimental conditions: Separation buffer 20.0 mM HEPES pH=7.4,
and 0.5% PEO; voltage configurations as shown in FIG. 1 assay
mixture was irradiated for 1 min at 680 nm using 680.+-.10 nm
filter and a 150 W lamp.
[0039] FIG. 6 is a data curve showing the effect of the
concentration of labeled aminodextran on the eTag reporter release
demonstrated in this figure, the lower concentration of labeled
aminodextran for a given concentration of sensitizer beads leads to
more efficient eTag reporter release (or higher ratio of eTag
reporter released to the amount of labeled aminodextran). Results
were obtained using a CE.sup.2 LabCard. Experimental conditions:
Separation buffer 20.0 mM HEPES pH=7.4, and 0.5% PEO; voltage
configurations as shown in FIG 1; assay mixture had 29 .mu.g/ml of
sensitizer beads and was irradiated for 1 min at 680 nm using
680.+-.10 nm filter and a 150 W lamp.
[0040] FIG. 7 is multiple electropherograms showing separation of
individual eTAG reporters. The figure illustrates obtainable
resolution of the reporters which are identified by their ACLA
numbers.
[0041] FIG. 8 is multiple electropherograms showing a separation on
a 310 analyzer that has occurred after an amplification reaction,
in the presence of probe and primer without the addition of
avidin.
[0042] FIG. 9 is multiple electropherograms showing a separation on
a 310 analyzer that has occurred after an amplification reaction,
in the presence of probe and primer with the addition of
avidin.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
[0043] Methods and compounds are provided for multiplexed
determinations, where the compounds can be linked to binding
compounds for detection of reciprocal binding compounds in a
sample. The methods are distinguished by having a plurality of
binding events in a single vessel using a mixture of differentially
eTag receptor conjugated binding compounds, the release of
identifying eTag receptors of those binding compounds bound to
their target compounds in the same vessel, and the detection of the
released identifying tags by separation of the tags in a single
run. The eTag receptors are distinguished by having one or more
physical characteristics that allow them to be separated and
detected.
[0044] The method employs a mixture of binding compounds bound to
eTag reporters, where each eTag reporter has a characteristic that
allows it to be uniquely detected in a single separation run. The
method involves combining the eTag reporter conjugated binding
compound with a sample to determine the presence of a plurality of
targets under conditions where the binding compounds bind to any
reciprocal binding partners to form a binding complex. After
sufficient time for binding to occur, the eTag reporters can be
released from binding complexes in the same vessel. Various
techniques are employed depending upon the nature of the binding
compounds for releasing the eTag reporters bound to the complex.
The released eTag reporters are then separated and identified by
their differentiable characteristics free of interference from the
eTag reporters still bound to the binding compound. The techniques
for differentiating between eTag reporters bound to a complex and
not bound to a complex, include enzymatic reactions that require
the complex to exist for cleavage to occur, modification by using
ligand/receptor binding, where the ligand is part of the binding
compound, so that after cleavage, eTag receptor still bound to the
binding compound is modified, dual binding to the target resulting
in release of the eTag receptor, where optionally eTag receptor
bound to the binding compound is modified, and the like.
[0045] One set of eTag receptors are distinguished by differences,
which include mass as a characteristic. These eTag reporters do not
rely on differentiation based on oligonucleotides of 2 or more,
usually 3 or more nucleotides, but rather on organic chemical
building blocks that are conveniently combined together to provide
for large numbers of differentiable compounds. Therefore, while the
original eTAG reporter or eTag reporter conjugated to the binding
compound can have 2 or more nucleotides, when released from the
binding compound, the released eTag reporter will have not more
than 3, usually not more than 2 nucleotides. Of particular interest
are eTag receptors that are characterized by differences in their
mass/charge ratio. These compounds are distinguished by having
differences in mobility and are characterized by having regions,
which serve as (1) a cleavable linking region; (2) a mass-modifying
region; (3) a charge-modifying region: and (4) a detectable region,
where the regions may be separate and distinct or combined, there
being at least two distinct regions that provide for the
differentiation. These eTag reporters may be combined in kits and
assays with compounds having all of the regions within a single
region to further expand the number of different compounds used as
eTag reporters in a multiplexed determination These compounds find
use with other compounds where the different regions are present in
the same moiety, for example one to two regions, where the
charge-modifying region may also be the detectable region or the
mass-modifying region. By having a plurality of compounds that can
serve as identifying molecules, mixtures of target compounds can be
assayed in a single vessel. By using protocols that result in the
release of eTag.TM. reporters from the binding compound that are
identifiable due to differences in mobility, the analysis is
greatly simplified, since the eTag reporters will be substantially
free of interfering materials and their differences in mobility
will allow for accurate detection and quantitation.
[0046] The eTag reporters will vary depending upon the method of
detection. Groups of at least 10 eTag reporters bound to 10
different binding compounds will be used in the determinations. The
eTag reporters will be characterized by being cleavable from the
binding compound in the same vessel by the same cleavage mechanism,
having a shared characteristic that permits separation and
individual detection, being compatible with the determination
method and being in the molecular weight range of about 30 to 3000
dal, usually in the molecular weight range of about 35 to 1500 dal.
The variation may be mass using a mass spectrometer, where a
magnetic field is used for separation, mass/charge ratio using
electrokinesis, where an electric field is used for separation,
which may also include sieving and/or adsorbing polymers,
adsorption, using chromatography, e.g gas chromatography, high
pressure liquid chromatography, where polar and van der Waal
interactions are used for separation, etc.
[0047] For those eTag reporters that rely on mass as a
characteristic, the mass unit difference in each eTag reporter when
using mass spectrometry for analysis need only be one, preferably
at least about 2. For electrophoresis, one will usually have at
least a 3; usually 5 unit difference as to the mass/charge ratio,
preferably at least about 7, and if one wishes to use shorter
distances for separation, 10 or more. These unit differences are
intended for molecules of similar structure, for as will be
discussed subsequently, structures can affect the mobility without
changing the mass/charge ratio.
[0048] For the most part, the eTag reporters that have independent
regions will have the following formula: 1 * M L * C ( D ) n *
[0049] wherein;
[0050] L is a terminal linking region;
[0051] M is the mass-modifying region;
[0052] C is the charge-modifying region;
[0053] D is the detectable region, being present when the eTAG
reporter is detected using spectrophotometric measurement and is
not present when the eTag reporter is detected using mass
spectrometric measurement;
[0054] n is 0 or 1, being 1 for spectrophotometric measurement and
0 for mass spectrometric measurement; and
[0055] the *intends that M, C and D can be bonded to any of the
other groups at any site, and
[0056] when not independent and distinct regions,
[0057] any of M, C and D may be merged together to provide multiple
functions in a single region and the regions may be bonded directly
to each other or interspersed with linking groups or regions. That
is, parts of one region may be separated by the whole or parts of
another region. Also, as indicated earlier, the different regions
will be free of regions comprising oligonucleotides of 3 or more
nucleotides, usually free of regions comprising oligonucleotides of
2 or more nucleotides.
[0058] Where the eTag reporter is bound to the binding compound,
the eTAG reporter will have the following formula: 2 * M B - L * C
( D ) n *
[0059] wherein:
[0060] B is the binding compound bonded to L';
[0061] L' is a modified linking group as a result of the bonding to
B; and
[0062] the remaining symbols are as defined previously.
[0063] The released eTag reporters will have the following formula:
3 * M L * C * ( D ) n
[0064] wherein:
[0065] L" is the residue of the linking region, which may include
more or less than the original linking group, by including a
portion of the binding compound or retaining only a portion of the
linking region, by cleaving at other than the bond made by joining
the linking region and the binding compound; and
[0066] the remaining symbols are as defined previously.
[0067] Each of the regions may be joined in a variety of ways using
different functionalities and synthetic protocols, where the manner
of linking may serve as one of the regions, for example, having
phosphate links that result in negatively charged links.
[0068] The linking region functions as the link between the
remainder of the eTag reporter and the binding compound. L has
three aspects: a reactive functionality, either inherently or made
so by reacting with an activating moiety; a cleavable linkage,
which may be the linkage formed by joining to the binding compound,
and a group(s) for joining to one or more of the other regions. For
bonding to the binding compound, different reactive functionalities
may be used, depending upon the nature of the binding compound.
[0069] Where the binding compound is an oligonucleotide, that is
DNA, RNA, combinations thereof and analogs thereof, e.g. thio
analogs, groups that react with alcohols will ordinarily be used.
Reactive groups include phosphoramidites, e.g. dialkyl
phosphoramidites, wherein alkyl is of from 1-6 carbon atoms; alkyl,
cyanoethyl phosphoramidites, wherein alkyl is of from 1-6 carbon
atoms, etc.; trialkyl phosphites or phosphates, where alkyl is of
from 1-6 carbon atoms; carboxylic acids or derivatives thereof,
such as acyl halides, anhydrides and active esters, e.g.
dinitrophenyl ester; active halides, such as .alpha.-halomethyloxo-
and non-oxo, where the halo will be of atomic number 17-53, chloro,
bromo and iodo; and the like. The products will be esters, both
inorganic and organic acid esters, and ethers. Alternatively, in
some cases, one may use other than phosphate derivatives as the
linking unit, using amino acids instead, such as glycine and
substituted glycines. In this instance, the units of the eTag
reporter would use analogous chemistry to synthesize the eTag
reporter in situ. The exemplary linkers are only illustrative and
not intended to be exhaustive.
[0070] For the most part for oligonucleotides, cleavage will be at
a phosphate bond between two nucleosides cleaved by an enzyme
having nuclease activity e.g. 5'-3' nuclease activity. Therefore,
the linking region will usually include a phosphoric acid
derivative for coupling to the terminal hydroxy of an
oligonucleotide having an appropriate base, such as adenine,
cytosine, guanosine, thymidine and uracil. As will be discussed
subsequently other available hydroxyl groups of the sugar, ribose
or deoxyribose, may be substituted with one of the other regions.
Where other methods than nuclease activity are used for release of
the eTag reporter, then any of the other functionalities may be
used for linking to the oligonucleotide. The linking region will
then include a functional entity that allows for specific
cleavage.
[0071] One need not use oligonucleotides for detection of specific
nucleic acid sequences. By employing binding compounds that
recognize a particular sequence, either as ssDNA or dsDNA, one may
attach a different eTag reporter to each of the different binding
compounds. Combining the nucleic acid sample with the eTag reporter
labeled binding compounds results in the binding of the binding
compounds to sequences that are present in the sample. Various
protocols can be used depending on the nature of the binding
compound. For example, oligomers of heterocyclic compounds,
particularly azole compounds, e.g. pyrrole, imidazole,
hydroxyimidazole, joined by two atom chains, particularly having
--NH-- groups, and amino acids, e.g. glycine, alanine,
.beta.-alanine, .gamma.-aminobutyric acid, etc. are employed. The
azoles are normally connected by a two atom bridge containing an
--NH-- group, desirably from the 2 to the 4 or 5 position. These
compounds form hairpins that bind in the minor groove of dsDNA with
high affinity and specificity for the sequence. See, for example,
U.S. Pat. Nos. 6,090,947 and 5,998,140, which are specifically
incorporated by reference herein for the disclosure of binding
sequences.
[0072] By adding the appropriate oligomers to a dsDNA sample, which
may include intact or fragmented dsDNA, sequestering the bound
oligomers from unbound oligomers and releasing the eTag reporters
bound to the dsDNA, one can rapidly determine the presence of dsDNA
sequences in the sample. Sequestering can be achieved with proteins
that bind dsDNA, by having ligands bound to the dsDNA, e.g. using
PCR with primers carrying a ligand, etc. Alternatively, by having a
biotin or other ligand bonded to the eTag reporter conjugated to
the binding compound that is retained with the binding compound on
release of the eTag reporter, one can add the ligand receptor
having a charge opposite to the released eTag reporter, so that in
electrophoresis the eTag reporter would migrate in the opposite
direction. The methods can find particular use where the
sensitivity of the system is adequate to avoid amplification and
directly determine the presence of a sequence without denaturation.
This approach can find use with detecting infectious organisms,
e.g. bacteria, viruses and protista, identifying specific chiasmas,
identifying genomes, and the like.
[0073] There are a large number of different functional entities
that are stable under the conditions used for the binding event
with the binding compound and may then be cleaved without affecting
adversely the eTag reporter. Functional entities may be cleaved by
chemical or physical methods, involving oxidation, reduction,
solvolysis, e.g. hydrolysis, photolysis, thermolysis, electrolysis,
chemical substitution, etc. Specific functional entities include
thio ethers that may be cleaved with singlet oxygen, disulfide that
may be cleaved with a thiol, diketones that may be cleaved by
permanganate or osmium tetroxide, .beta.-sulfones,
tetralkylammonium, trialkylsulfonium, tetralkylphosphonium, etc.
where the .alpha.-carbon is activated with carbonyl, nitro, etc.,
that may be cleaved with base, quinones where elimination occurs
with reduction, substituted benzyl ethers that can be cleaved
photolytically, carbonates that can be cleaved thermally, metal
chelates, where the ligands can be displaced with a higher affinity
ligand, as well as many other functional entities that are known in
the literature. Cleavage protocols are described in U.S. Pat. Nos.
5,789,172, 6,001,579, and references cited therein.
[0074] The eTag reporters find use in determinations involving a
plurality of target entities. Usually, one will be interested in at
least about 3 target entities, more usually at least 5, frequently
at least about 10 or more, and may be interested in at least about
20 or more, even about 100 or more. The number of eTAG reporters
will usually be equal to the number of target entities, although in
some situations, the same eTag reporter may be used to identify a
plurality of related target entities and one may then deconvolute
the results as to individual target entities. The eTag reporters
bound to the binding members can be added individually or in
combination to the sample and then processed to determine the
presence of the target entities.
[0075] Of interest is to have two eTag reporters that are closely
similar in mobility, usually closer in mobility to each other than
to unrelated eTag reporters. Where there are paired situations to
be analyzed, such as alleles, MHC antigens, single nucleotide
polymorphisms, etc., by having the eTag reporters in proximity in
the electropherogram, particularly where they have distinguishable
detectable regions, e.g. fluorescers fluorescing at different
wavelengths, one obtains a quick determination if none, one or both
of the pairs are present in the sample.
[0076] Genetic analyses may take many forms and involve
determinations of different information. Genetic analyses are
involved with sequencing, detection of specific sequences as
related to the presence of specific genes or regulatory sequences,
identification of organisms, identification of transcription events
as related to different cells, different cell stages and external
stimuli, identification of single nucleotide polymorphisms,
alleles, repetitive sequences, plastid DNA, mitochondrial DNA,
etc., forensic medicine, and the like. In each case one has a
complex sample to be assayed, where one is interested in numerous
binding events. By providing for a unique eTAG reporter for each
event, one can perform simultaneously a number of assays in the
same flask and with a single sample or a few aliquots of the
sample. For example, where an assay involves a single nucleotide in
each vessel, one would use four vessels, one for each nucleotide.
In most cases, the eTag reporters can be separated from other
components of the assay mixture to substantially reduce
interference from these other components when assaying for the eTag
reporters.
[0077] There are a number of genetic analyses that involve cleavage
of a phosphate bond of a nucleic acid sequence as a result of
hybridization. For the most part, the initial step will be in
solution, although one may have one or more reagents bound to a
solid support in the first and succeeding stages of the
determination. One technique is described in U. S. Pat. Nos.
5,876,930 and 5,723,591, where a primer and a probe are bound to a
target sequence and by extending the primer with a DNA polymerase
having 5'-3' nuclease activity, the terminal nucleotides are
cleaved as the polymerase processes along the target DNA. By having
an eTag reporter bonded to the terminal and/or internal
nucleotide(s), the eTag reporter will be released when the target
nucleic acid is present. Another technique employs an enzyme
referred to as a cleavase, which recognizes a three member complex
of the target nucleic acid, a primer and a probe. See, U.S. Pat.
No. 5,719,028. Attached to the terminus of the probe is an eTag
reporter that is released by the cleavase, where the three membered
complex is formed.
[0078] For detecting single nucleotide polymorphisms ("snps"),
various techniques can be employed of varying complexity. In one
technique, a primer is employed that terminates at the nucleotide
immediately preceding the snp. One can have the eTag reporter bound
to the primer and a ligand bound to the nucleotide reciprocal to
the snp. One can either have 4 vessels, each with a different
labeled nucleotide or one vessel with each of the labeled
nucleotides having a different label. Various polymerases having
3'-5' editing can be used to ensure that mismatches are rare. The
extended primers may then be captured, for example, by having a
ligand, e.g. biotin, and contacting the extension mixture with the
reciprocal receptor, e.g. streptavidin, bound to a support and the
eTag reporter released and analyzed. By grouping targets of
interest having the same nucleotide for the snp, the assay may be
multiplexed for a plurality of targets. Other techniques include
having probes where the snp is mismatched. The mismatching
nucleotide is labeled with the eTAG reporter. When the snp is
present, the eTag reporter labeled nucleotide will be released for
detection. See U.S. Pat. No. 5,811,239.
[0079] In another variation, one may ligate a primer and a probe,
where one is 3' of the other when hybridized to a target nucleic
acid. By having one of the pair of primer and probe with an eTag
reporter with a cleavable linkage and the other of the pair with an
agent capable of causing cleavage of the cleavable linkage in
conjunction with another agent, the primer and probe may be ligated
together when bound to the target. One can release the ligated pair
from the target, e.g. heat, and recycle by cooling the mixture to
allow for hybridization of the primer and probe, ligating primer
and probe bound to target and then denaturing to release the
ligated primer and probe, amplifying the number of ligated primers
and probes. Once the desired degree of amplification has been
achieved, one may provide the additional reagent resulting in
release of the eTag reporters.
[0080] Where PCR or other amplification reaction is used involving
a primer, the primer can be labeled with a ligand that allows for
sequestering of the amplified DNA, one can then sequester the DNA
by means of a receptor reciprocal to the ligand, which receptor is
bound to a support and add probes labeled with eTag reporters
specific for the probe sequence. After hybridization and washing to
remove non-specifically bound and unbound nucleic acid, the eTag
reporters are released and analyzed.
[0081] Instead of nucleic acid assays, one may be interested in
protein assays. For determining a mixture of proteins, one may use
intact cells, intact viruses, viral infected cells, lysates,
plastids, mitochondria or other organelles, fractionated samples,
or other aggregation of proteins, by themselves or in conjunction
with other compounds. Any source of a mixture of proteins can be
used, where there is an interest in identifying a plurality of
proteins.
[0082] Proteomics has come to the fore, where one is interested in
cellular expression during metabolism, mitosis, meiosis, in
response to an external stimulus, e.g. drug, virus, change in
physical or chemical condition, involving excess or deficient
nutrients and cofactors, stress, aging, presence of particular
strains of an organism and identifying the organism and strain,
multiple drug resistance, and the like. It is necessary to have a
means for identifying a large number of proteins in a single
sample, as well as providing some quantitation of the different
proteins being detected. In one assay one may use binding proteins
specific for the target proteins. One group of binding proteins is
bound to a support, such as a vessel or channel wall, particles,
magnetic or non-magnetic, e.g. latex particles, dextrose,
sepharose, cellulose, etc., where the support permits sequestering
the target proteins to the support. Most commonly, antibodies,
particularly monoclonal antibodies rather than antisera, will be
used, although the latter may also find use. In some situations
other receptors may find use, such as lectins, enzymes, surface
membrane proteins, etc. and in some situations, ligands for the
proteins may be employed. The reciprocal-binding members, receptors
and ligands, may be bound to the support through covalent or
non-covalent bonding. Activated surfaces find use, where the
surface has an active functional group that will react with the
reciprocal-binding member to provide for stable binding to the
surface, e.g. silyl chloride modified glass, cyanogen bromide
modified polysaccharides, etc. Proteins bind tightly to some
plastic surfaces, so that no covalent bonding is required. Ligands
have or can be provided with active functional groups for bonding
to the surface. If desired the binding to the surface can be
accomplished in two steps by bonding a ligand to the reciprocal
binding member and binding a ligand binding member to the support,
for example, biotin as the ligand and strept/avidin as the ligand
binding member, or one may have anti-Ig bound to the surface to
bind to antibodies bound to the target protein. In addition, where
a change in environment is localized, one may have a large
concentration of a counteracting agent, e.g. a large amount of
buffer at pH 7, for example, .quadrature.200 mM phosphate, where
ammonia is produced that creates a localized basic environment.
[0083] The sample is combined with the reciprocal binding member,
which may be bound to the support or subsequently bound to the
support. After washing away the other components of the mixture,
receptor for the target protein labeled with eTag reporter
molecules specific for the particular receptor are added to the
bound target protein, so as to become bound to the support through
the target protein. One or more eTag reporter molecules will be
bound to the receptor, usually not more than about 20, frequently
not more than about 10. The number will be limited by the degree of
loss of the binding affinity as the number of eTag reporter
molecules is increased. Normally, the support bound receptor and
the eTag reporter labeled receptor will bind to different epitopes
of the target protein, although in some situations where the target
has a plurality of the same epitope, the receptors may be specific
for the same epitope. After washing away all eTag reporter labeled
receptor that is not specifically bound to the target protein(s),
the eTag reporter molecules are released and assayed.
[0084] Where the target permits binding of two reciprocal binding
members or where an additional reagent is provided which permits
this event, one can use determinations involving "channeling" or
energy transfer. See, for example, U.S. Pat. Nos. 5,843,666 and
5,573,906. There are numerous methodologies involving channeling in
the literature, where for the most part, the channeling was
involved in producing a directly detectable signal, usually a
change in absorption or emission of light. Channeling involves
having two reagents, where the first reagent, when in proximity to
the second reagent, produces a detectable signal. For the eTag
reporter, the detectable signal is the release of the eTag reporter
from the binding component. The release will usually be a function
of the production of a short-lived entity, such as a chemical
species or a photoactivated excited species, but may be the result
of changing the local environment as compared to the bulk solution.
So far as the chemical species, illustrative species include
singlet oxygen, hydrogen peroxide, NADH, and hydroxyl radicals. Two
entities are employed that have reciprocal binding members that
bind to the same target moiety. One of the entities generates an
active species. The other entity has a susceptible functionality
that interacts with the active species resulting in release of the
eTag reporter or responds to the changed local environment to
release the eTag reporter. Either the active species is short
lived, so that it will not create significant background because
beyond its vicinity, the active species becomes inactive or a
scavenger is employed that efficiently scavenges the active
species, so that it is not available to react with the susceptible
functionality that is not bound to the target. of reactive species
include enzymes, such as oxidases, such as glucose oxidase,
xanthene oxidase, D-amino acid oxidase, NADH-FMN oxidoreductase,
galactose oxidase, glyceryl phosphate oxidase, sarcosine oxidase,
choline oxidase and alcohol oxidase, that produce hydrogen
peroxide, horse radish peroxidase, that produces hydroxyl radical,
various dehydrogenases that produce NADH or NADPH, urease that
produces ammonia to create a high local pH. One cleavable link can
be based on the oxidation of sulfur or selenium, where a thioether,
sulfoxide, or selenium analog thereof, is present at the .alpha.-
or .beta.-position in relation to an activating group, which makes
the hydrogen a to the activating group acidic and capable of being
removed by base, so as to release the oxidized functionality to
which is attached the eTag reporter or to be subject to oxidation
with release of the eTag reporter. Alternatively, one may use metal
chelates that are stable at one oxidation state and unstable at
another oxidation state.
[0085] Other compounds include .alpha.-substituted methylquinones,
which have an eTag reporter bonded through a leaving group, such as
sulfonyl, oxy, amino, etc.
[0086] By using a heterogeneous system, a first agent for causing
cleavage may be bound to a surface to provide an environment for
release of the eTag reporter when bound to the surface. Where a
second agent is required to cause the release of the eTag reporter,
the second agent is added after sufficient time for the eTag
reporter conjugated binding compound to become bound to the
surface. Where the target is a nucleic acid, the nucleic acid may
be bound to the first agent containing surface by having ssDNA
binding proteins bound to the surface or other convenient means
known in the art. Once the target is bound to the surface, the eTag
reporter conjugated oligonucleotides homologous the target nucleic
acid sequences are added, followed by the second agent. With
ligands and proteins, one can have receptors, which bind at one
site, on the surface and eTag reporter binding compounds that bind
at a different site forming what is referred to in the art as a
"sandwich."
[0087] For singlet oxygen, one may use various sensitizers, such as
squarate derivatives. See. for example, Ullman, et al., Proc. Natl.
Acad. Sci. USA 91, 5426-5430 (1994). Examples of combinations that
find use in this invention may be found in U.S. Pat. Nos.
5,536,498; 5,536,834; references cited therein; H. H. Wasserman and
R. W. Murray. Singlet Oxygen. Academic Press, New York (1979); A.
L. Baumstark, Singlet Oxygen, Vol. 2, CRC Press Inc., Boca Raton,
Fla. 1983. Other cleavage mechanisms may be found in WO99/64519;
WO99/13108; WO98/01533 and WO097/28275.
[0088] Singlet oxygen reacts with a wide variety of double bonds,
with cleavage of the double bond to an oxo group with separation of
the eTag reporter. Illustrative olefins include vinyl sulfides,
vinyl ethers, enamines, imines substituted at the carbon atoms with
an .alpha.-methine (CH, a carbon atom having at least one hydrogen
atom), where the vinyl group may be in a ring, the heteroatom may
be in a ring, or substituted on the cyclic olefinic carbon atom,
and there will be at least one and up to four heteroatoms bonded to
the olefinic carbon atoms. The resulting dioxetane may decompose
spontaneously, by heating above ambient temperature, usually below
about 75.degree. C., reaction with acid or base, or photolytically
in the absence or presence of a sensitizer. Numerous articles
describe a variety of compounds that can be decomposed with singlet
oxygen, where the articles are frequently interested in light
emission, so that the compounds have more complicated structures
than are required for the subject purposes, where only cleavage is
required for release of the eTag reporter from the binding
compound. Therefore, for the most part, synthetic convenience,
stability under the conditions of the linking to the binding
compound and conditions of the binding, and efficiency of release
will be the primary factors in selecting a particular
structure.
[0089] Articles of interest which are illustrative of a much larger
literature include: Adam and Liu, J. Amer. Chem. Soc. 94,
1206-1209, 1972, Ando, et al., J.C.S. Chem. Comm. 1972, 477-8,
Ando, et al., Tetrahedron 29, 1507-13, 1973, Ando, et al., J. Amer.
Chem. Soc. 96, 6766-8, 1974, Ando and Migita, ibid 97, 5028-9,
1975, Wasserman and Terao, Tetra. Lett. 21, 1735-38, 1975, Ando and
Watanabe, ibid 47, 4127-30, 1975, Zaklika, et al., Photochemistsry
and Photobiology 30, 35-44, 1979, and Adam, et al., Tetra. Lett.
36, 7853-4, 1995. See also, U.S. Pat. No. 5,756,726.
[0090] The formation of dioxetanes is obtained by the reaction of
singlet oxygen with an activated olefin substituted with an eTag
reporter at one carbon atom and the binding compound at the other
carbon atom of the olefin. See, for example, U.S. Pat. No.
5,807,675. These compounds may be depicted by the following
formula:
(eTag reporter --W)(X).sub.nC.sub..alpha.=C.sub..beta.(Y)(Z)
[0091] wherein:
[0092] W may be a bond, a heteroatom, e.g. O, S, N, P, M (intending
a metal that forms a stable covalent bond), or a functionality,
such as carbonyl, imino, etc., and may be bonded to X or C.sub.60
;
[0093] at least one X will be aliphatic, aromatic, alicyclic or
heterocyclic and bonded to C.sub..alpha. through a hetero atom,
e.g. N, O, or S and the other X may be the same or different and
may in addition be hydrogen, aliphatic, aromatic, alicyclic or
heterocyclic, usually being aromatic or aromatic heterocyclic
wherein one X may be taken together with Y to form a ring, usually
a heterocyclic ring, with the carbon atoms to which they are
attached, generally when other than hydrogen being from about 1 to
20, usually 1 to 12, more usually 1 to 8 carbon atoms and one X
will have 0 to 6, usually 0 to 4 heteroatoms, while the other X
will have at least one heteroatom and up to 6 heteroatoms, usually
1 to 4 heteroatoms;
[0094] Y will come within the definition of X, usually being bonded
to C.sub..beta. through a heteroatom and as indicated may be taken
together with X to form a heterocyclic ring;
[0095] Z will usually be aromatic, including heterocyclic aromatic,
of from about 4 to 12, usually 4 to 10 carbon atoms and 0 to 4
heteroatoms, as described above, being bonded directly to
C.sub..beta. or through a heteroatom, as described above;
[0096] n is 1 or 2, depending upon whether the eTag reporter is
bonded to C.sub..alpha. or X;
[0097] wherein one of Y and Z will have a functionality for binding
to the binding member or be bound to the binding member.
[0098] While not depicted in the formula, one may have a plurality
of eTag reporters in a single molecule, by having one or more eTag
reporters joined to one or both Xs.
[0099] Illustrative compounds include S-(eTag reporter)
3-thiolacrylic acid, N-(eTag reporter), N-methyl
4-arnino-4-butenoic acid, O-(eTag reporter 3hydroxyacrolein,
N-(4-carboxyphenyl) 2-(eTag reporter) imidazole, oxazole, and
thiazole.
[0100] Also of interest are N-alkyl acridinyl derivatives,
substituted at the 9 position with a divalent group of the formula:
--(CO) X.sup.1(A)--
[0101] wherein:
[0102] X.sup.1 is a heteroatom selected from the group consisting
of O, S, N, and Se, usually one of the first three; and
[0103] A is a chain of at least 2 carbon atoms and usually not more
than 6 carbon atoms substituted with an eTag reporter, where
preferably the other valences of A are satisfied by hydrogen,
although the chain may be substituted with other groups, such as
alkyl, aryl, heterocyclic, etc. groups, A generally being not more
than 10 carbon atoms.
[0104] Also of interest are heterocyclic compounds, such as
diheterocyclopentadienes, as exemplified by substituted imidazoles,
thiazoles, oxazoles, etc., where the rings will usually be
substituted with at least one aromatic group and in some instances
hydrolysis will be necessary to release the eTag reporter.
[0105] Also of interest are tellurium (Te) derivatives, where the
Te is bonded to an ethylene group having a hydrogen atom .beta. to
the Te atom, wherein the ethylene group is part of an alicyclic or
heterocyclic ring, that may have an oxo group, preferably fused to
an aromatic ring and the other valence of the Te is bonded to the
eTag reporter. The rings may be coumarin, benzoxazine, tetralin,
etc.
[0106] The mass-modifying region, when not including the
charge-modifying region or the detectable label, will usually be a
neutral organic group, aliphatic, alicyclic, aromatic or
heterocyclic, where the heteroatoms will be neutral under the
conditions employed for the assay protocol. The heteroatoms may be
oxygen as oxy or non-oxo- or oxo-carbonyl, sulfur as thio or
thiono, halo, nitrogen as amide, nitro or cyano, phosphorous as
phosphite or phosphate triester, etc. Conveniently, the region may
be methylene, including polymethyene, alkyleneoxy, including
polyalkyleneoxy, particularly alkylene of 2-3 carbon atoms, aryl or
substituted aryl, such as phenylene, diphenylene, cyanophenylene,
nitrophenylene, thiophenylene, chlorophenylene, furanylene, amino
acids, such as N-acyl glycinamide and polyglycinamide, including
substituted glycinamides, cyclopentylene, bis-biphenylene-E, where
E is carbonyl, oxy, thio, ureido, methylene, isopropylene, and the
like, etc. The mass-modifying region will generally be from about 1
to 100, more usually 1 to 60 atoms other than hydrogen, generally
having at least one carbon atom and up to 60 carbon atoms and from
about 0 to 40 heteroatoms, usually about 0 to 30 heteroatoms.
[0107] The charge-modifying region will vary depending upon the
other groups present and whether one wishes to reduce the number of
unneutralized charges in the molecule or increase the number of
unneutralized charges. Charges in the molecule may come from other
than the charge-modifying group, such as the label, connecting
groups between regions may be included in the charge modifying
region, the linking region, and any residue of the binding compound
that is retained with the eTag reporter. For the most part, the
eTag reporter will have an overall negative charge, although in
some instances, there may be an overall positive charge,
particularly if positive and negative eTag reporters are to be
determined in the same electrophoretic separation. Negative charges
can be provided by phosphate, including phosphonate, phosphinate,
thiophosphate, etc., borate, carboxylate, sulfonate, enolate,
phenoxide, etc. Positive charges can be provided by amines and
substituted amines, e.g. ammonium, sulfonium, hydrazine, imine,
amidine, metal ions, particularly as chelates and metallocenes,
etc. The charge-modifying region may have from 1 to 60 atoms other
than hydrogen, usually from about 1 to 30 atoms, where there will
be at least one heteroatom, which may be oxygen, nitrogen, sulfur,
boron, phosphorous, metal ion, etc.
[0108] One may combine the mass-modifying and charge-modifying
functions in a single region in a convenient manner using
poly(amino acids), where the naturally occurring aspartate and
glutamate may serve to provide negative charges, and the naturally
occurring lysine, arginine and histidine may serve to provide
positive charges. However, one may wish to use unnatural amino
acids, such as sulfonic, phosphonic, and boronic acid substituted
amino acids. By appropriate choice in conjunction with the other
regions, a large number of different mobilities can be achieved.
When used in combination with mass-modifying regions, the number of
eTAG reporters having different mobilities is greatly expanded.
[0109] One may use combinations of substituted diols or diamines
and dibasic acids, where the substituents are charged, to form
diesters and diamides. Illustrative of such oligomers are the
combination of diols or diamino, such as 2,3-dihydroxypropionic
acid, 2,3-dihydroxysuccinic acid, 2,3-diaminosuccinic acid,
2,4-dihydroxyglutaric acid, etc. The diols or diamino compounds can
be linked by dibasic acids, which dibasic acids include the
inorganic dibasic acids indicated above, as well as dibasic acids,
such as oxalic acid, malonic acid, succinic acid, maleic acid,
furmaric acid, carbonic acid, citric acid, tartaric acid, etc.
Alternatively, one may link the hydroxyls or amines with alkylene
or arylene groups, dicarbonyls, activated dihalo compounds, etc.
Other combinations include substituted dithiols, that can be
copolymerized with dienes, activated dihalo compounds, etc. Thus,
by appropriate selection of the different monomers, low order
oligomers can be produced that may then be separated by molecular
weight.
[0110] The detection region may include any label that can be
detected spectrophotometrically and/or electrochemically. A wide
variety of labels are available for detection in an electrophoretic
device. Conmnonly used fluorescers include, fluorescein and
fluorescein derivatives, lanthanide dyes, rhodamine and rhodamine
derivatives, Cy-5, Cy-3, HEX, TET, squarates, and cyanine dyes. The
dyes may be charged or uncharged, so as to add or diminish the
overall charge of the molecule. Electrochemical labels also find
use, such as ferrocene and ruthenium complexes.
[0111] For economic and operational reasons, it is generally
desirable to use as few lasers for excitation as feasible.
Therefore, it will be desirable to use combinations of energy
absorbers/transmitters, frequently a fluorescer, and energy
receivers/ emitters, usually a fluorescer, keeping the energy
absorber constant for excitation where energy exchange between the
two entities allows for variation in the emission wavelength due to
changes in the Stokes shift. Combinations of dyes include
fluorescein and HEX, (ex.sub.488 nm, em.sub.560 nm), and
phthalocyanine (ex.sub.488nm, em.sub.690nm). One can provide for
various combinations of fluorescers to be bound in proper proximity
for energy transfer. A ribosyl group in the linking region or the
mass-modifying region provides for one hydroxyl group for linkage
of a member of an energy transfer pair and two hydroxyls for
insertion into the chain, while deoxyribose substituted with two
fluorescers can react with an hydroxyl group as a side chain. The
particular unit used to which the members of the energy transfer
pair are bonded can be selected to provide mass-modification and/or
charge-modification.
[0112] The mobility of the eTag reporter will not only depend on
the mass/charge ratio according to the formula (M/z).sup.2/3, but
will also depend on structure. Entities within the eTag reporter
that are rigid and extend the molecule enhance the drag and
therefore reduce the mobility. Therefore by using rigid groups,
such as aromatics, 5- and 6-membered heterocyclics, e.g.
tetrahydrofuran, polyenes and polyacetylenes, one can enhance
differences in mobility even while the ratio of mass to charge is
not significantly different.
[0113] Synthesis of eTags comprising nucleotides can be easily and
effectively achieved via assembly on a solid phase support during
probe synthesis using standard phosphoramidite chemistries. The
eTag reporters are assembled at the 5'-end of probes after coupling
of a final nucleosidic residue, which becomes part of the eTag
reporter during the assay. One may have a nucleotide triphosphate
bonded to one of the termini of the building blocks of the eTag
reporter. In one approach, the eTag reporter is constructed
sequentially from a single or several monomeric phosphoramidite
building blocks (one containing a detectable region, e.g. dye),
which are chosen to generate eTag reporters with unique
electrophoretic mobilities based on their mass to charge ratio. The
eTag reporter is thus composed of monomeric units of variable
charge to mass ratios bridged by phosphate linkers (Figure A). The
separation of eTag reporters, which differ by 9 mass units (Table
1) has been demonstrated. The nucleosidic phosphoramidites employed
for eTag reporter synthesis are initially either modified or
natural residues. Fluorescein has been the initial dye employed but
other dyes can be used as well, as 1
[0114] Figure A. The design and synthesis of eTag reporters on
solid phase support using standard phosphoramidite coupling
chemistry described previously. Some of the combinations of
phosphoramidite building blocks with their predicted elution times
are presented in Table 2. As shown in Figure B, eTag reporters are
synthesized to generate a continuous spectrum of signals, one
eluting after another with none of them coeluting (Figure B)
1TABLE 1 eTag reporters that have been separated on a LabCard (See
experimental section for description.) (detection: 4.7 cm; 200
V/cm). Elution Time E-Tag on CE (sec) Mass 2 385 778 3 428 925 4
438 901 5 462 994 6 480 985 7 555 961
[0115]
2TABLE 2 Predicted and experimental (*) elution times of eTag
reporters. Elution Etag Charge Time 8 -9 41.12 9 -8 43.72 10 -9
45.66 11 -8 48.14 12 -7 51.21 13 -6 53.53 14 -6 55.13 15 -5 57.66
16 -5 60.00 17 -5 62.86 18 -6 65.00* 19 -5 67.50* 20 -4 69.61 21 -4
72.00* C.sub.3, C.sub.6, C.sub.9C.sub.18, are commercially
available phosphoramidite spacers from Glen Research, Sterling VA.
The units are derivatives of N,N-diisopropyl, O-cyanoethyl
phosphoramidite, which is indicated by "Q". C.sub.3 is DMT
(dimethoxytrityl)oxypropyl Q; C.sub.6 is DMToxyhexyl Q; C.sub.9 is
DMToxy(triethyleneoxy) Q; C.sub.12 is DMToxydodecyl Q; C.sub.18 is
DMToxy(hexaethyleneoxy) Q.
[0116] All of the above eTag reporters work well and are easily
separable and elute after 40 minutes. To generate eTag reporters
that elute faster, highly charged low molecular weight eTag
reporters are required. Several types of phosphoramidite monomers
allow for the synthesis of highly charged eTag reporters with early
elution times. Use of dicarboxylate phosphoramidites (Figure C),
allows for the addition of 3 negative charges per coupling of
monomer. Polyhydroxylated phosphoramidites (Figure D) in
combination with a common phosphorylation reagent enable the
synthesis of highly phosphorylated eTag reporters. Combinations of
these reagents with other mass modifier linker phosphoramidites
allow for the synthesis of eTag reporters with early elution times.
22
[0117] Figure C. Charge modifier phosphoramidites. (EC or CE is
cyanoethyl) 23
[0118] Figure D. Polyhydroxylated charge modifier
phosphoramidites.
[0119] The aforementioned label conjugates with different
electrophoretic mobility permit a multiplexed amplification and
detection of multiple targets, e.g. nucleic acid targets. The label
conjugates are linked to oligonucleotides in a manner similar to
that for labels in general, by means of linkages that are
enzymatically cleavable. It is, of course, within the purview of
the present invention to prepare any number of label conjugates for
performing multiplexed determinations. Accordingly, for example,
with 40 to 50 different label conjugates separated in a single
separation channel and 96 different amplification reactions with 96
separation channels on a single microfluidic chip, one can detect
4000 to 5000 single nucleotide polymorphisms.
[0120] The separation of eTag reporters, which differ by 9 mass
units (Table 1) has been demonstrated as shown in FIG. 7. The
penultimate coupling during probe synthesis is initially carried
out using commercially available modified (and unmodified)
phosphoramidites (Table 2). This residue is able to form hydrogen
bonds to its partner in the target strand and is considered a mass
modifier but could potentially be a charge modifier as well. The
phosphate bridge formed during this coupling is the linkage severed
during a 5'-nuclease assay. The final coupling is done using a
phosphoramidite analogue of a dye. Fluorescein is conveniently
employed, but other dyes can be used as well.
[0121] One synthetic approach is outlined in Scheme 1. Starting
with commercially available 6-carboxyfluorescein, the phenolic
hydroxyl groups are protected using an anhydride. Isobutyric
anhydride in pyridine was employed but other variants are equally
suitable. It is important to note the significance of choosing an
ester functionality as the protecting group. This species remains
intact though the phosphoramidite monomer synthesis as well as
during oligonucleotide construction. These groups are not removed
until the synthesized oligo is deprotected using ammonia. After
protection, the crude material is then activated in situ via
formation of an N-hydroxy succinimide ester (NHS-ester) using DCC
as a coupling agent. The DCU byproduct is filtered away and an
amino alcohol is added. Many amino alcohols are commercially
available some of which are derived from reduction of amino acids.
Only the amine is reactive enough to displace N-hydroxy
succinimide. 24
[0122] Upon standard extractive workup, a 95% yield of product is
obtained. This material is phosphitylated to generate the
phosphoramidite monomer (Scheme 1). For the synthesis of additional
eTag reporters, a symmetrical bisamino alcohol linker is used as
the amino alcohol (Scheme 2). As such the second amine is then
coupled with a multitude of carbdxylic acid derivatives (Table 1)
prior to the phosphitylation reaction. Using this methodology
hundreds even thousands of eTag reporters with varying charge to
mass ratios can easily be assembled during probe synthesis on a DNA
synthesizer using standard chemistries. 25
[0123] Additional eTag reporters are accessed via an alternative
strategy which uses 5-aminofluorescein as starting material (Scheme
3). Addition of 5-aminofluorescein to a great excess of a diacid
dichloride in a large volume of solvent allows for the predominant
formation of the monoacylated product over dimer formation. The
phenolic groups are not reactive under these conditions. Aqueous
workup converts the terminal acid chloride to a carboxylic acid.
This product is analogous to 6-carboxy fluorescein and using the
same series of steps is converted to its protected phosphoramidite
monomer (Scheme 3). There are many commercially available diacid
dichorides and diacids, which can be converted to diacid chlorides
using SOCl.sub.2 or acetyl chloride. This methodology is highly
attractive in that a second mass modifier is used. As such, if one
has access to 10 commercial modified phosphoramidites and 10 diacid
dichlorides and 10 amino alcohols there is a potential for 1000
different eTag reporters. There are many commercial diacid
dichlorides and amino alcohols (Table 3). These synthetic
approaches are ideally suited for combinatorial chemistry.
3TABLE 3 Mass and charge modifiers that can be used for conversion
of amino dyes into eTag reporter phosphoramidite monomers. 26 27 28
29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50
51 52 53 54
[0124] 55
[0125] Substituted aryl groups can serve as both mass- and
charge-modifying regions. (Table 4) Various functionalities may be
substituted onto the aromatic group, e.g. phenyl, to provide mass
as well as charges to the eTag reporter. The aryl group may be a
terminal group, where only one linking functionality is required,
so that a free hydroxyl group may be acylated, may be attached as a
side chain to an hydroxyl present on the eTag reporter chain, or
may have two functionalities, e.g. phenolic hydroxyls, that may
serve for phophite ester formation and othe substitutients, such as
halo, haloalkyl, nitro, cyano, alkoxycarbonyl, alkylthio, etc.
where the groups may be charged or uncharged.
4TABLE 4 Benzoic acid derivatives as mass and charge modifiers.
(Mass is written below each modifier) 56 57 58 59 60 61 62 63 64 65
66 67
[0126] A variety of maleimide derivatized eTag reporters have also
been synthesized. These compounds were subsequently bioconjugated
to 5'-thiol adorned DNA sequences and subjected to the 5'-nuclease
assay. The species formed upon cleavage are depicted in Table
5.
5TABLE 5 eTag reporters derived from maleimide-linked precursors.
68 1 69 2 70 3 71 4 72 5 73 6 74 7 75 8 76 9 77 10
[0127] The eTag reporter may be assembled having an appropriate
functionality at one end for linking to the binding compound. Thus
for oligonucleotides, one would have a phosphoramidite or phosphate
ester at the linking site to bond to an oligonucleotide chain,
either 5' or 3', particularly after the oligonucleotide has been
synthesized, while still on a solid support and before the blocking
groups have been removed. While other techniques exist for linking
the oligonucleotide to the eTag reporter, such as having a
functionality at the oligonucleotide terminus that specifically
reacts with a functionality on the eTag reporter, such as maleimide
and thiol, or amino and carboxy, or amino and keto under reductive
amination conditions, the phosphoramidite addition is preferred.
For a peptide-binding compound a variety of functionalities can be
employed, much as with the oligonucleotide functionality, although
phosphoramidite chemistry may only occasionally be appropriate
Thus, the functionalities normally present in a peptide, such as
carboxy, amino, hydroxy and thiol may be the targets of a reactive
functionality for forming a covalent bond.
[0128] Of particular interest in preparing eTag reporter labeled
nucleic acid binding compounds is using the solid support
phosphoramidite chemistry to build the eTag reporter as part of the
oligonucleotide synthesis. Using this procedure, one attaches the
next succeeding phosphate at the 5' or 3' position, usually the 5'
position of the oligonucleotide chain. The added phosphoramidite
may have a natural nucleotide or an unnatural nucleotide. Instead
of phosphoramidite chemistry, one may use other types of linkers,
such as thio analogs, amino acid analogs, etc. Also, one may use
substituted nucleotides, where the mass-modifying region and/or the
charge-modifying region may be attached to the nucleotide, or a
ligand may be attached to the nucleotide. In this way,
phosphoramidite links are added comprising the regions of the eTag
reporter, whereby when the synthesis of the oligonucleotide chain
is completed, one continues the addition of the regions of the eTag
reporter to complete the molecule. Conveniently, one would provide
each of the building blocks of the different regions with a
phosphoramidite or phosphate ester at one end and a blocked
functionality, where the free functionality can react with a
phosphoramidite, mainly a hydroxyl. By using molecules for the
different regions that have a phosphoramidite at one site and a
protected hydroxyl at another site, the eTag reporter can be built
up until the terminal region, which does not require the protected
hydroxyl.
[0129] Illustrative of the synthesis would be to employ a diol,
such as an alkylene diol, polyalkylene diol, with alkylene of from
2 to 3 carbon atoms, alkylene amine or poly(alkylene amine) diol,
where the alkylenes are of from 2 to 3 carbon atoms and the
nitrogens are substituted, for example with blocking groups or
alkyl groups of from 1-6 carbon atoms, where one diol is blocked
with a conventional protecting group, such as a dimethyltrityl
group. This group can serve as the mass-modifying region and with
the amino groups as the charge-modifying region as well. If
desired, the mass modifier can be assembled by using building
blocks that are joined through phosphoramidite chemistry. In this
way the charge modifier can be interspersed between the mass
modifier. For example, one could prepare a series of polyethylene
oxide molecules having 1, 2, 3, n units. Where one wished to
introduce a number of negative charges, one could use a small
polyethylene oxide unit and build up the mass and charge-modifying
region by having a plurality of the polyethylene oxide units joined
by phosphate units. Alternatively, by employing a large spacer,
fewer phosphate groups would be present, so that without large mass
differences, one would have large differences in mass-to-charge
ratios.
[0130] The chemistry that is employed is the conventional chemistry
used in oligonucleotide synthesis, where building blocks other than
nucleotides are used, but the reaction is the conventional
phosphoramidite chemistry and the blocking group is the
conventional dimethoxyltrityl group. Of course, other chemistries
compatible with automated synthesizers can also be used, but there
is no reason to add additional complexity to the process.
[0131] For the peptides, the eTag reporters will be linked in
accordance with the chemistry of the linking group and the
availability of functionalities on the peptide binding compound.
For example, with Fab fragments specific for a target compound, a
thiol group will be available for using an active olefin, e.g.
maleimide, for thioether formation. Where lysines are available,
one may use activated esters capable of reacting in water, such as
nitrophenyl esters or pentafluorophenyl esters, or mixed anhydrides
as with carboduimide and half-ester carbonic acid. There is ample
chemistry for conjugation in the literature, so that for each
specific situation, there is ample precedent in the literature for
the conjugation.
[0132] Once the binding compound conjugated with the eTag reporter
has been prepared, it may find use in a number of different assays,
many of which have already been discussed. The samples may be
processed using lysing, nucleic acid separation from proteins and
lipids and vice versa, and enrichment of different fractions. For
nucleic acid related determinations, the source of the DNA may be
any organism, prokaryotic and eukaryotic cells, tissue,
environmental samples, etc. The DNA or RNA may be isolated by
conventional means, RNA may be reverse transcribed, DNA may be
amplified, as with PCR, primers may be used with ligands for use in
subsequent processing, the DNA may be fragmented using restriction
enzymes, specific sequences may be concentrated or removed using
homologous sequences bound to a support, or the like. Proteins may
be isolated using precipitation, extraction, and chromatography.
The proteins may be present as individual proteins or combined in
various aggregations, such as organelles, cells, viruses, etc. Once
the target components have been preliminarily treated, the sample
may then be combined with the eTag reporter targeted binding
proteins.
[0133] For a nucleic acid sample, after processing, the probe
mixture of eTag reporters for the target sequences will be combined
with the sample under hybridization conditions, in conjunction with
other reagents, as necessary. Where the reaction is heterogeneous,
the target sequence will have a ligand for binding to a reciprocal
binding member for sequestering hybrids to which the eTag reporter
is bound. In this case, all of the DNA sample carrying the ligand
will be sequestered, both with and without eTag reporter labeled
probe. After sequestering the sample, and removing non-specific
binding eTag reporter labeled probe under a predetermined
stringency based on the probe sequence, using washing at an
elevated temperature, salt concentration, organic solvent, etc.,
the eTag reporter is released into an electrophoretic buffer
solution for analysis.
[0134] For a homogeneous assay, the sample, eTag reporter labeled
probe mixture and ancillary reagents are combined in a reaction
mixture supporting the cleavage of the linking region. The mixture
may be processed to separate the eTag reporters from the other
components of the mixture. The mixture, with or without eTag
reporter enrichment, may then be transferred to an electrophoresis
device, usually a microfluidic or capillary electrophoresis device
and the medium modified as required for the electrophoretic
separation. Where one wishes to remove from the separation channel
intact eTag reporter molecules, a ligand is bound to the eTag
reporter that is not released when the eTag reporter is released.
Alternatively, by adding a reciprocal binding member that has the
opposite charge of the eTag reporter, so that the overall charge is
opposite to the charge of the eTag reporter, these molecules will
migrate toward the opposite electrode from the released eTag
reporter molecules. For example, one could use biotin and
streptavidin, where streptavidin carries a positive charge. In the
case of an oligonucleotide, the eTag reporter label would be bonded
to at least two nucleotides, where cleavage occurs between the two
nucleotides with release of the eTag reporter, with the terminal
nucleotide of the dinucleotide labeled with a biotin (the eTag
reporter would be released without the biotinylated nucleotide). In
the case of a peptide analyte, one would have cleavage at a site,
where the ligand remains with the peptide analyte. For example, one
could have the eTag reporter substituted for the methyl group of
methionine. Using the pyrazolone of the modified methionine, one
could bond to an available lysine. The amino group of the
pyrazolone would be substituted with biotin. Cleavage would then be
achieved with cyanogen bromide, releasing the eTag reporter, but
the biotin would remain with the peptide and any eTag reporter that
was not released from the binding member. Avidin is then used to
change the polarity or sequester the eTag reporter conjugated to
the binding compound.
[0135] The separation of the eTag reporters by electrophoresis can
be performed in conventional ways. See, for example, U.S. Pat. Nos.
5,750,015; 5,866,345; 5,935,401; 6,103,199, and 6,110,343 and
WO098/5269, and references cited therein. Also, the sample can be
prepared for mass spectrometry in conventional ways. See, for
example, U.S. Pat. Nos. 5,965,363; 6,043,031; 6,057,543 and
6,111,251.
[0136] For convenience, kits can be provided comprising building
blocks for preparation of eTag reporters in situ or have assembled
eTag reporters for direct bonding to the binding compound. For
preparing the eTag reporter in situ during the synthesis of
oligonucleotides, one would provide phosphoramidites or phosphates,
where the esters would include alkyl groups, particularly of from 1
to 3 carbonatoms, and cyanoethyl groups, while for the
phosphoramidite, dialkylamino, where the alkyl groups are of from
1-4 carbon atoms, while the other group would be a protected
hydroxy, where the protecting group would be common to
oligonucleotide synthesis, e.g. dimethoxytrityl. For large numbers
of eTag reporters, that is, 20 or more, one kit would supply at
least 3 each of mass-modifying regions and charge-modifying
regions, each having at least the phosphate linking group and a
protected hydroxyl. The two functional groups may be separated by 2
or more atoms, usually not more than about 60 atoms, and may be
vicinal (.alpha.,.beta.to .alpha.,.omega.) The nature of the
compounds has been discussed previously. In the simplest case, the
phosphorous acid derivative would serve as the charge-modifying
region, so that the mass-modifying region and the charge-modifying
region would be added as a single group. In addition, one would
have at least 2 detectable regions, which would be a fluorescer
having the phosphate linker and other functionalities protected for
purposes of the synthesis. Alternatively, instead of having the
detection region the terminal region, where the detectable region
allows for the presence of two functionalities that can be used for
linking, one of the other regions may serve as the terminal region.
Also, one of the regions may be conveniently linked to a mono- or
dinucleotide for direct linking to the oligonucleotide chain, where
cleavage will occur at the 3' site of the nucleotide attached to
the eTag reporter. By using tri- or tetrasubstituted groups, one
can provide a detectable region that provides the pair for energy
transfer. One need only have one or two different energy transfer
agents, while having a plurality of emitting agents to greatly
expand the number of different eTag reporters.
[0137] Other reagents that are useful include a ligand-modified
nucleotide and its receptor. Ligands and receptors include biotin
and strept/avidin, ligand and antiligand, e.g. digoxin or
derivative thereof and antidigoxin, etc. By having a ligand
conjugated to the oligonucleotide, one can sequester the eTag
conjugated oligonucleotide probe and its target with the receptor,
remove unhybridized eTag reporter conjugated oligonucleotide and
then release the bound eTAG reporters or bind an oppositely charged
receptor, so that the ligand-receptor complex with the eTag
reporter migrates in the opposite direction.
[0138] Where one prepares the eTag reporter, there will be the
additional linking region, which in the above description is served
by the phosphorous acid derivative or the mono- or dinucleotide
unit phosphorous acid derivative. For these eTag reporters, one
need not be restricted to phosphate links, but may use other
convenient chemistries, particularly chemistries that are
automated. Thus, instead of phosphorous acid and protected alcohol,
one can use carboxy and alcohol or amino, activated olefin and
thiol, amino and oxo-carbonyl, particularly with reductive
amination, an hydroxy with an active halide or another hydroxy to
form an ether, and the like. One may employ compounds that are
difunctional with the same or different functionalities, where one
could have a diacid and a diol or an hydroxyacid or cyclic ester
for producing the eTag reporter. Numerous examples of these types
of compounds have already been described and are well known in the
literature. By appropriate selection of the monomers and
conditions, one can select a particular order of reaction, namely
the number of monomers that react or one may separate the mixture
by the different mobilities.
[0139] For separations based on sorption, adsorption and/or
absorption, the nature of the eTag reporters to provide for
differentiation can be relatively simple. By using differences in
composition, such as aliphatic compounds, aromatic compounds and
halo derivatives thereof, one may make the determinations with gas
chromatography, with electron capture or negative ion mass
spectrometry, when electronegative atoms are present. In this way
one may use hydrocarbons or halo-substituted hydrocarbons as the
eTag reporters bonded to a releasable linker. See, U.S. Pat. Nos.
5,565,324 and 6,001,579, which are specifically incorporated by
reference as to the relevant disclosure concerning cleavable groups
and detectable groups.
[0140] The kits will include at least two detectable regions and
sufficient reagents to have at least 10, usually at least 20 and
frequently at least 50 or more different eTag reporters that can be
separated by their mobility.
[0141] For 20 different eTag reporters, one only requires 5
different mass-modifying regions, one phosphate link and four
different detectable regions. For 120 eTag reporters, one need only
have 10 different mass-modifying regions,3 different
charge-modifying regions and 4 different detectable regions. For
500 different eTag reporters, one need only have 25 different
mass-modifying regions, 5 different charge-modifying regions and 4
different detectable regions.
[0142] For an inclusive but not exclusive listing of the various
manners in which the subject invention may be used, the following
table is provided.
[0143] Recognition Event Leads to Generation or Modification of
eTag Reporters.
6 eTag reporter Recognition Event Activation Amplification Mode
Format Binding Assays (solution Multiplexed assays (2-1000) Phase
eTag reporter leading to release of generation followed by library
of eTag reporters. separation by CE, HPLC Every eTag reporter or
Mass Spectra) codes for a unique binding event or assay.
Hybridization followed 5' Nuclease assay PCR, Invader Sequence
recognition for by enzyme recognition example for multiplexed gene
expression, SNP's scoring etc... 3' Nuclease assay Multiplexed
assays Sequence recognition Restriction Multiplexed assays enzymes
Sequence recognition Ribonuclease H Multiplexed assays Sequence
recognition Hybridization followed by Singlet Oxygen Single eTag
reporter release Multiplexed assays channeling per binding event
Sequence recognition Hybridization followed by Singlet Oxygen
Amplification due to Multiplexed assays channeling turnover of eTag
reporter Sequence recognition binding moiety Amplification due to
release Multiplexed assays of multiple eTag reporters Sequence
recognition (10 to 100,000) per binding event Hydrogen peroxide
Amplification due to Multiplexed assays turnover of eTag reporter
Sequence recognition binding moiety Amplification due to release
Multiplexed assays of multiple eTag reporters Sequence recognition
(10 to 100,000) per binding event Light; Energy Amplification due
to Multiplexed assays Transfer turnover (Photocleavage)of Sequence
recognition eTag reporter binding moiety Amplification due to
release Multiplexed assays of multiple eTag reporters Sequence
recognition (10 to 100,000) per binding event IMMUNOASSAYS Sandwich
assays Singlet Oxygen A few (2-10) eTag reporters Proteomics
Antibody-1 decorated release per binding event Multiplexed
Immunoassays with Sensitizer while antibody-2 Is decorated with
singlet oxygen cleavable eTag reporters Singlet Oxygen
Amplification due to release Proteomics of multiple eTag reporters
Multiplexed Immunoassays (10 to 100,000) per binding event Sandwich
assays Hydrogen Peroxide A few (2-10) eTag reporters Proteomics
Antibody-1 decorated release per binding event Multiplexed
Immunoassays with Glucose oxidase while antibody-2 Is decorated
with hydrogen peroxide cleavable eTag reporters Hydrogen Peroxide
Amplification due to release Proteomics of multiple eTag reporters
Multiplexed Immunoassays (10 to 100,000) per binding event
Competition assays Singlet Oxygen A few (2-10) eTag reporters
Antibody-1 decorated release per binding event with Sensitizer
while Antigen Is decorated with singlet oxygen cleavable eTag
reporters
[0144] The following examples are offered by way of illustration
and not by way of limitation.
EXPERIMENTAL
Synthetic Preparation of Modified Fluorescein Phosphoramidites
[0145] Pivaloyl protected carboxyfluorescein: Into a 50 mL round
bottom flask was placed 5(6)-carboxyfluorescein (0.94 g, 2.5 mmol),
potassium carbonate (1.0 g, 7.5 mmol) and 20 mL of dry DMF. The
reaction was stirred under nitrogen for 10 min, after which
trimethylacetic
[0146] anhydride (1.1 mL, 5.5 mmol) was added via syringe. The
reaction was stirred at room temperature overnight, and then
filtered to remove excess potassium carbonate and finally poured
into 50 mL of 10% HCI. A sticky yellow solid precipitated out of
solution. The aqueous solution was decanted off and the residual
solid was dissolved in 10 mL of methanol. Dropwise addition of this
solution to 10% HCl yielded a fine yellow precipitate, which was
filtered and air dried to yield an off white solid (0.88 g, 62%).
TLC (45:45:10 Hxn,EtOAc,MeOH) NHS ester of protected pivaloyl
carboxyfluorescein. Into a 200 mL round bottom flask was placed the
protected carboxyfluorescein (2.77 g, 5.1 mmol) and 50 mL of
dichloromethane. N-hydroxysuccinimide (0.88 g, 7.6 mmol) and
dicyclohexylcarbodiimide (1.57 g, 7.6 mmol) were added and the
reaction was stirred at room temperature for 3 hours. The reaction
was then filtered to remove the precipitated dicyclohexyl urea
byproduct and reduced to approx. 10 mL of solvent in vacuo.
Dropwise addition of hexanes with cooling produced a yellow-orange
colored solid, which was triturated with hexanes, filtered and air
dried to yield 3.17 g (95%) of product. TLC (45:45:10
Hxn,EtOAc,MeOH)
[0147] Alcohol. Into a 100 mL round bottom flask was placed the NHS
ester (0.86 g, 1.34 mmol) and 25 mL of dichloromethane. The
solution was stirred under nitrogen after which aminoethanol (81
.mu.L, leq) was added via syringe. The reaction was monitored by
TLC (45:45:10 Hxn,EtOAc,MeOH) and was found to be complete after 10
min. The dichloromethane was then removed in vacuo and the residue
dissolved in EtOAc, filtered and absorbed onto 1 g of silica gel.
This was bedded onto a 50 g silica column and eluted with
Hxn:EtOAc:MeOH (9:9:1) to give 125 mg (20%) of clean product.
[0148] Phosphoramidite. Into a 10 mL round bottom flask containing
125 mg of the alcohol was added 5 mL of dichloromethane.
Diisopropyl ethylamine (139 .mu.l, 0.8 mmol) was added via syringe.
The colorless solution turned bright yellow. 2-cyanoethyl
diisopropylchlorophosphoramidite (81 .mu.l, 0.34 mmol) was added
via syringe and the solution immediately went colorless. After 1
hour TLC (45:45:10 Hxn:EtOAc:TEA) showed the reaction was complete
with the formation of two closely eluting isomers. Material was
purified on a silica column (45:45:10 Hxn:EtOAc:TEA) isolating both
isomers together and yielding 130 mg (85%).
[0149] Carboxylic acid. Into a 4 mL vial was placed
12-aminododecanoic acid (0.1 g, 0.5 mmol) and 2 mL of pyridine. To
this suspension was added chlorotrimethyl silane (69.mu.L, 1.1 eq)
via syringe. After all material dissolved (10 min) NHS ester (210
mg, 0.66 eq) was added. The reaction was stirred at room
temperature overnight and then poured into water to precipitate a
yellow solid, which was filtered, washed with water, and air dried.
TLC (45:45:10 Hxn:EtOAc:MeOH) shows a mixture of two isomers.
[0150] General Procedure for Remaining Syntheses. The carboxylic
acid formed described above is to be activated by NHS ester
formation with 1.5 eq each of N-hydroxysuccinimide and
dicyclohexylcarbodiimide in dichloromethane. After filtration of
the resulting dicyclohexylurea, treatment with leq of varying amino
alcohols will effect amide bond formation and result in a terminal
alcohol. Phosphitylation using standard conditions described above
will provide the phosphoramidite. 78
Synthesis of Biotinylated 2'-Deoxycytosine Phosphoramidite; Scheme
# 1
Synthesis of 3',5'-O-di-t-butyldimethylsilyl-2'-Deoxyuridine(1)
[0151] 2'-Deoxyuridine (4 gm, 17.5 mmol) and imidazole (3.47 gm,
52.5 mmol) were dissolved in 30 ml of dry DMF and
t-butyldimethyl-silyl chloride (7.87 gm, 52.5 mmol) added to the
stirring solution at room temperature. After 3 hrs, TLC on silica
gel (10% MeOH+90% CH.sub.2Cl.sub.2) showed that all starting
material had been converted to a new compound with higher R.sub.f.
The solution was concentrated into a small volume, then about 200
ml of ether was added and washed three times with saturated aqueous
NaCl solution. The organic layer was dried over anhydrous
Na.sub.2SO.sub.4, and the filtrate was evaporated to give a
colorless gummy material which converted to a white solid product
(eight gm, 100%). This product was identified with HNMR and
ES-MS.
Synthesis of
3',5'-O-di-t-butyldimethylsilyl-N.sup.4-(1,2,4-triazolo)-2'-D-
eoxycytidine(2)
[0152] 1,2,4-Triazole (19.45 gm, 282 mmol) was suspended in 300 ml
of anhydrous CH.sub.3CN at 0.degree. C., 8 ml of POCl.sub.3, then
50 ml of triethylamine was added slowly in 5 min. After an hour,
3',5'-O-Di-t-butyldimethylsilyl-2'-Deoxyuridine (1) (9 gm, 19.7
mmol) was dissolved in 200 ml of dry CH.sub.3CN and added to the
reaction over 20 min. After stirring the reaction for 16 hours at
RT, TLC (100% ether) showed that all starting material was
converted to a new compound with lower R.sub.f. The reaction
mixture was filtered, reduced the volume of CH.sub.3CN, diluted
with ethyl acetate and washed with saturated aqueous NaHCO.sub.3
then twice with saturated aqueous NaCl. The organic layer was dried
over anhydrous Na.sub.2SO.sub.4 and the solvent was evaporated,
co-evaporated from toluene to give a yellow solid product (10 gm.
100%). This product was identified with HNMR and ES-MS.
Synthesis of
3',5'-O-di-t-butyldimethylsilyl-N.sup.4-(4,7,10-trioxa-1-trid-
ecaneamino)-2'-Deoxycytidine(3)
[0153] 4,7,10-Trioxa-1,13-tridecanediamine (10.44 gm, 47.4 mmol)
was dissolved in 100 ml dioxane, then
3',5'-O-di-t-butyldimethylsilyl-4-(1,2,-
4-triazolo)-2'-deoxycytidine (2) (8.03 gm, 15.8 mmol) was dissolved
in 200 ml of dioxane (heated to about 50 C. and cooling it dawn to
RT) and added dropwise in 10 min., to the solution of
4,7,10-Trioxa-1,13-tridecanediami- ne with vigorous stirring at RT.
After 5 hrs, TLC on silica gel showed that all starting material
was converted to a new product with lower Rf, the resulting mixture
was evaporated to dryness. The residue was dissolved in
dichloromethane and washed twice with 5% sodium bicarbonate
solution and saturated sodium chloride solution. The organic layer
was dried over sodium sulphate, filtered and evaporated to dryness
to give a yellow gummy product (7.87 gm). The product was purified
on a silica gel column eluted with a gradient of 0 to 10% methanol
in dichloromethane with 1% triethylamine. The product was obtained
as a yellowish gum (5.66 gm, 54%%). This product was identified
with FNMR and ES-MS.
Synthesis of
3',5'-O-di-t-butyldimethylsilyl-4-N-(4,7,10-trioxa-1-tridecan-
eaminobiotin)-2,-Deoxycytidine(4)
[0154]
3',5'-O-di-t-butyldimethylsilyl-4-N-(4,7,10-trioxa-1-tridecaneamino-
)-2'-deoxycytidine(3) (2.65 ) gm, 4.43 mmol) and Biotin-NHS ester
(1.814 gm, 5.316 mmol) were dissolved in 20 ml of dry DMF and about
1 ml of triethylamine was added. After stirring the reaction
mixture for 4 hrs at RT, the reaction was stopped by evaporating
all DMF to give a yellow gum material (4.36 gm). This material was
dissolved in dichloromethane and washed three times with saturated
solution of NaCl, dried over sodium sulphate and evaporated to
dryness. TLC on silica gel (5% MeOH+1% TEA+94% CH.sub.2Cl.sub.2)
indicated the formation of a new product which was higher R.sub.f.
This product was purified with column chromatography on silica gel
using (99% CH.sub.2Cl.sub.2+1% TEA) to (1% MeOH+1%
TEA+98%CH.sub.2Cl.sub.2) to yield a yellow foamy product (2.13 gm,
60%). This product was identified with HNMR and ES-MS.
Synthesis of
4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2'-Deoxycytidine(5-
)
[0155]
3',5'-O-di-t-butyldimethylsilyl-4-N-(4,7,10-trioxa-1-tridecaneamino-
biotin)-2'-deoxycytidine(4) (1.6 gm, 1.8 mmol) was dissolved in 50
ml of dry THF, then about 5.5 ml of tetrabutylammonium fluoride in
THF was added in 2 min. while stirring at RT. After 3 hrs, TLC on
silica gel (10% MeOH+1% TEA+89% CH.sub.2Cl.sub.2) showed that a new
product with lower R.sub.f formed. The solvent was evaporated to
give a yellow oily product. Column chromatography on silica gel
eluted with (99% CH.sub.2C.sub.2+1% TEA) to (7% MeOH+1%
TEA+92%CH.sub.2Cl.sub.2) permitted the purification of the product
as a gummy colorless product (1.14 gm, 97%). This product was
identified with HNMR and ES-MS.
t-Butylbenzoylation of the biotin of
4-N-(4,7,10-trioxa-1-tridecaneaminobi-
otin)-2'-deoxycytidine(6)
[0156] 4-N-(4,7,10-trioxa-1-tridecaneaminobiotin)-2'-Deoxycytidine
(5) (14.14 gm, 21.5 mmol ) was dissolved in 100 ml of dry pyridine.
Chlorotrimethyl silane (11.62 gm, , 107.6 mmol) was added and the
mixture was stirred for 2 hrs at RT. 4-t-butylbenzoyl chloride
(5.07 gm, 25.8 mmol) was added and the mixture was stirred for
another 2 hrs at RT. The reaction mixture was cooled with ice-bath
and the reaction stopped by adding 50 ml of water and 50 ml of 28%
aqueous ammonia solution. The solution kept stirring at RT for 20
min., then evaporated to dryness in high vacuum and finally
co-evaporated twice from toluene. The material was dissolved in
dichloromethane and extracted twice with 5% aqueous sodium
bicarbonate solution. The organic layer was dried over sodium
sulphate, evaporated to dryness, re-dissolved in dichloromethane
and applied to a silica gel column. The column was eluted with
gradient from 0 to 10% of methanol in dichloromethane and obtained
a product as a white foam (9.4 gm, 53.5% ). This product was
identified with HNMR and ES-MS.
Synthesis of
5'-O-(4,4'-dimethoxytriphenylmethyl)-4-N-(4,7,10-trioxa-1-tri-
decaneaminobiotin)-2'-Deoxycytidine (7)
[0157] Compound (6)(10.82 gm, 13.3 mmol) was co-evaporated twice
from dry pyridine, then dissolved in pyridine (100 ml) and
4,4'-dimethoxytritylchl- oride(DMT-Cl) (6.76 gm 19.95 mmol) was
added and the resulting mixture stirred for 3 hrs. TLC (10% MeOH+1%
TEA+89% CH.sub.2Cl.sub.2) showed the formation of new product with
higher Rf, and some starting material remained unreacted, then
another amount of DMTCl (2 gm ) was added and kept stirring for 2
hrs. The reaction stopped by adding ethanol and the mixture was
stirred for 15 min. After evaporation to dryness and co-evaporation
from toluene, the material was dissolved in dichloromethane. The
organic layer washed twice with 5% aqueous sodium bicarbonate
solution, dried over sodium sulphate, evaporated to dryness. The
product was purified on a silica column using a gradient of
methanol from 0 to 5% in dichloromethane/1% TEA. The product was
obtained as a white foam (4.55 gm, 31%). This product was
identified with HNMR and ES-MS.
Synthesis of 3'-O-[ (diisopropylamine)(2-cyanoethoxy)
phosphino)]-5'-O-(4,4'-dimethoxytriphenylmethyl)-4-N-(4,7,10-trioxa-1-tri-
decaneaminobiotin)-2'-Deoxycytidine (8)
[0158] The 5'-DMT-Biotin-dC (7) (507 mg, 0.453 mmol) was dissolved
in dry acetonitrile (30 ml) and dichloromethane (5 ml), then
diisopropylamine (73 mg, 0.56 mmol), tetrazole (1.15 ml, 0.52 mmol)
and 2-cyanoethyl N,N,N'N'-tetraisopropylphosphane 214 mg, 234 ul,
0.7 mmol) were added and the mixture stirred under nitrogen at RT.
After 2 hrs, TLC on silica gel (45%/ 45%/ 5%/ 5%: Ethyl
acetate/dichloromethane/triethylamine/methanol) showed that only
about 30% of product was formed and about 70% of starting material
was unreacted. More reagents were added until most of starting
material was converted, only about 5% left unreacted. The solvent
was evaporated to dryness, dissolved in dry dichloromethane, washed
with sodium bicarbonate solution (5%), saturated brine solution,
then the organic layer dried over sodium sulphate, evaporated to
dryness. Column chromatography on silica gel using (48%/ 48%/ 4%:
Ethyl acetate/dichloromethane/triethylamine) to (47%/ 47%/ 5% 1%:
Ethyl acetate/dichloromethane/triethylamine/methanol). The desired
product was obtained as a colorless gummy product (406 mg, 70% ).
This material was co-evaporated three times from a mixture of dry
benzene and dichloromethane, then was kept in desiccated containing
P.sub.2O.sub.5 and NaOH pellets under vacuum for 26 hrs before used
in DNA synthesis.
Synthesis of Biotinylated 2'-Deoxyadenosine Phosphoramidite;
Scheme#2
Synthesis of 8-Bromo-2'-Deoxyadenosine
[0159] 2'-Deoxyadenosine (7 gm. 25.9 mmol) was dissolved in sodium
acetate buffer (150, 1 M, pH5.0) by worming it to about 50 C., then
was cooled dawn to 30 C., then 3 ml of bromine in 100 ml of the
same buffer was added dropwise at RT for 15 min., to the reaction.
After, 6 hrs the TLC on silica gel (20% MeOH in CH2Cl2) showed that
all starting material was converted to a new product. The reaction
was discolored by adding some sodium metabisulfite
(Na.sub.2S.sub.2O.sub.5) while it was stirring, the color changed
to a white solution, the pH of the reaction was neutralized by
adding NaOH (1 M solution). The reaction mixture was kept at
4.degree. C. (refrigerator) for 16 hrs. Next day the solid material
was filtered, washed with cold water, then acetone to give a solid
yellow powder product (5.75 gm. 64%). The structure of this product
was confirmed by H NMR and ES-MS.
Synthesis of
N.sup.6-Benzoyl-8-bromo-5'-O-(4,4'-dimethoxytrityl)-2'-Deoxya-
denosine (1)
[0160] 8-Bromo-2'-Deoxyadenosine (7.7 gm. 22.17 mmol) was dried by
co-evaporation with dry pyridine and the solid was suspended in 200
ml of dry pyridine followed by the addition of
4,4'-dimethoxytriphenylmethyl chloride (DMT-Cl) (9 gm, 26.6 mmol).
After stirring for 4 hrs at RT, TLC on silica gel showed that a new
product was formed and some starting material was unreacted.
Another amount of DMT-Cl (3 gm) was added and stirred at RT for 2
hrs. When TLC showed that all starting material was converted to
new product with higher Rf, the reaction mixture was cooled to 0 C
and trimethylchlorosilane (12.042 gm., 14 ml, 110.85 mmol) was
added dropwise while cooling and after 40 min. while stirring
benzoyl chloride (15.58 gm, 12.88 ml, 110.85 mmol) was similarly
added. The reaction was allowed to react at RT over 2 hrs. The
reaction was quenched by slow addition of cold water (50 ml),
followed by addition of concentrated ammonia (30%, 50 ml). After 30
min. the reaction mixture was evaporated to dryness. The residue
was dissolved in water, and the solution was extracted with ethyl
acetate three times, the organic layer washed with saturated sodium
bicarbonate solution, and then brine. The organic phase was dried
over sodium sulphate, evaporated to dryness. The product was
purified on a silica column chromatography, to give a yellowish
solid product (6.79 gm, 41.6%). The structure of this product was
confirmed by H NMR and ES-MS.
Synthesis of
N.sup.6-benzoyl-8-bromo-3'-O-t-butyldimethylsilyl-5'-O-(4,4'--
dimethoxytrityl)-2'-deoxyadenosine
[0161]
6N-Benzoyl-8-bromo-5'-O-(4,4'-dimethoxytrityl)-2'-Deoxyadenosine(1)
(14 gm. 19 mmol) and imidazole (1.94 gm, 28.5 mmol) were dissolved
in 100 ml of dry DMF and t-butyldimethyl-silyl chloride (4.3 gm,
28.5 mmol) added to the stirring solution at room temperature.
After 4 hrs, TLC on silica gel (2.5% MeOH in CH.sub.2Cl.sub.2)
showed that all starting material had been converted to a new
product with higher R.sub.f. The solution was concentrated into a
small volume, then about 400 ml of ether was added and washed three
times with saturated aqueous NaCl solution. The organic layer was
dried over anhydrous Na.sub.2SO.sub.4, and the filtrate was
evaporated to give an off-white foamy product (16.18 gm, 100%). H
NMR and ES-MS confirmed the structure.
Synthesis of
N.sup.6-benzoyl-8-(4,7,10-trioxa-1-tridecaneamino)-3'-O-t-but-
yldimethylsilyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine
(2)
[0162]
N.sup.6-Benzoyl-8-bromo-3'-O-t-butyldimethylsilyl-5'-O-(4,4'-dimeth-
oxytrityl)-2'-deoxyadenosine (8.31 gm. 9.7 mmol) was dissolved in
200 ml of ethanol then 4,7,10-trioxa-1,13-tridecanediamine (6.75
gm. 6.7 ml. 30 mmol) was added at once and kept stirring at 50 C.
After 16 hrs TLC showed that all starting material was converted to
a one major product with lower Rf and other minor products. The
solvent was evaporated to dryness, dissolved in dichloromethane,
washed three times with solution of brine, dried over anhydrous
Na.sub.2SO.sub.4, evaporated to give a yellow gummy material.
Column chromatography (1% TEA+CH.sub.2Cl.sub.2) to (1% TEA+5%
MeOH+CH.sub.2Cl.sub.2) permitted the purification of the major
product as an off-white gummy material (4.53 gm. 47%). This product
was identified with HNMR and ES-MS.
Synthesis of
N.sup.6-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-3'-O-
-t-butyldimethylsilyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine
[0163] N.sup.6-benzoyl-8-(4,7,10-trioxa-1-tridecaneamino)-3
'-O-t-butyldimethylsilyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine
(4.53 gm. 4.57 mmol) and biotin-NHS ester (3.12 gm. 9.13 mmol) were
dissolved in 75 ml of DMF and few drops of TEA were added and the
reaction was stirred at RT. After, 2 hrs TLC on silica gel (5%
MeOH+1% TEA+94 CH.sub.2C.sub.12) showed the formation of one major
product less polar than starting material and another minor spot
has lower Rf. The solvent was evaporated to dryness, then dissolved
in CH.sub.2Cl.sub.2 and washed three times with a saturated
solution of NaCl, dried the organic layer, evaporated to dryness to
leave a yellow gummy material. This material was purified with
column chromatography on silica gel by using (1%
TEA+CH.sub.2Cl.sub.2) to (1% TEA+2.5% MeOH+CH.sub.2Cl.sub.2) as
eluant. After evaporating the fractions containing the product,
gave a yellowish solid material (3.16 g, 78%). HNMR and ES-MS
confirmed the structure.
Synthesis of
N.sup.6-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-5'-O-
-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (3)
[0164]
N.sup.6-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-3'-O-t-but-
yldimethylsilyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine (3.16
gm, 2.6 mmol) was dissolved in 100 ml of dry THF, and then about
(3.25 ml, 3.25 mmol) of tetrabutylammonium fluoride in THF was
added in 5 min. while stirring at RT. After 8 hrs, TLC on silica
gel (10% MeOH+1% TEA+89% CH.sub.2Cl.sub.2) showed that a new
product with lower R.sub.f formed. The solvent was evaporated to
give a yellow oily material. Column chromatography on silica gel
eluted with (99% CH.sub.2Cl.sub.2+1% TEA) to (5% MeOH+1%
TEA+94%CH.sub.2Cl.sub.2) permitted the purification of the product
as a white foamy product (2.86 gm, 100%). HNMR and ES-MS confirmed
the structure.
Synthesis of
N.sup.6-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-3'-O- -[
(diisopropylamine)(2-cyanoethoxy)
phosphino)]-5'-O-(4,4'-dimethoxytrity- l)-2'-deoxyadenosine (4)
[0165]
N.sup.6-benzoyl-8-(4,7,10-trioxa-1-tridecaneaminobiotin)-5'-O-(4,4
'-dimethoxytrityl)-2'-deoxyadenosine (0.959 gm, 0.86 mmol) was
dissolved in a mixture of dry acetonitrile (200 ml) and
dichloromethane (50 ml ), and diisopropylamine (224 .mu.l, 1.29
mmol) followed by the addition of 2-cyanoethyl
N,N,N',N'-tetraisopropylphosphane (404 ul, 1.29 mmol) and tetrazole
(2.6 ml, 1.2 mmol, 0.45 M solution in dry acetonitrile). The
addition and subsequent reaction are performed under argon while
stirring at RT. After 1.5 h, TLC on silica gel (5% MeO+5% TEA+45%
EA+45%CH.sub.2Cl.sub.2) showed that only about 50% of starting
material (SM) was converted to a new product. The same above amount
of reagents were added to the reaction and kept stirring for
another 2 hrs at RT. TLC showed that about 95% of SM was converted
to a new product with higher R.sub.f. The solvent was evaporated to
dryness then was dissolved in dichloromethane, extracted once with
5% solution of bicarbonate, followed by saturated brine solution
and then dried over anhydrous sodium sulfate and evaporated to
dryness. Column chromatography on silica gel (10% TEA+45%EA+45%
CH.sub.2Cl.sub.2) first then (5% TEA+5% MeOH+45%EA+45%
CH.sub.2Cl.sub.2). After evaporating the fractions containing the
product, gave a yellow gummy material (774 mg). This material was
co-evaporated three times from a mixture of dry benzene and
dichloromethane, then was kept in desiccated containing
P.sub.2O.sub.5 and NaOH pellets under vacuum for 24 hrs before used
in DNA synthesis.
Synthesis of oligonucleotides containing biotin-dC and
Biotin-dA
[0166] The syntheses of oligonucleotides containing biotin-dC and
Biotin-dA, site-specifically located, were performed on a CPG
support using a fully automated DNA synthesizer and the
commercially available fully protected deoxynucleosides
phosphoramidites. Syntheses of all these oligonucleotides were
carried out at 1.0 and 0.4 .mu.mol scale. The coupling time for the
biotin-dC and dA were extended to 900 seconds. The coupling
efficiency of the biotin-dC and dA phosphoramidites was found
greater than 96%. After coupling of the biotinylated
phosphoramidites, the remaining residues comprising the eTAG
reporter of interest were added. Upon completion of the synthesis
of the oligonucleotides, they were deprotected with concentrated
ammonia at 65.degree. C. for 1 hour. These oligonucleotides were
purified by reverse-phase HPLC and desalted by OPC column, then
used as such.
Synthetic Preparation of ACLA1 on an ABI 394 DNA Synthesizer
[0167] 6-Carboxyfluorescein (6-FAM) phosphoramidite is prepared by
the addition of 2.96 ml of anhydrous acetonitrile to a 0.25 gram
bottle of the fluorescein phosphoramidite, to give a 0.1M solution.
The bottle is then loaded onto the ABI 394 DNA synthesizer at
position 8 using the standard bottle change protocol. The other
natural (dA.sup.bz (0.1M: 0.25 g/2.91 mL anhydrous acetonitrile),
dC.sup.Ac(0.1M: 0.25 g/3.24 mL anhydrous acetonitrile), dT(0.1M:
0.25 g/ 3.36 mL anhydrous acetonitrile), dG.sup.dmf(0.1M:
0.25g/2.81 mL anhydrous acetonitrile) phosphoramidite monomers are
loaded in a similar fashion to ports 1-4. Acetonitrile is loaded
onto side port 18, standard tetrazole activator is loaded onto port
9, CAP A is loaded onto port 11, CAP B is loaded onto port 12,
oxidant is loaded onto port 15, and deblock solution is loaded onto
port 14 all using standard bottle change protocols.
[0168] Standard Reagents Employed for DNA Synthesis:
[0169] Oxidizer: 0.02 M Iodine (0.01 5 for MGB Probes)
[0170] DeBlock: 3% Trichloracetic Acid in Dichloromethane
[0171] Activator: 1 H-Tetrazole in Anhydrous Acetonitrile
[0172] HPLC Grade Acetonitrile (0.002% water)
[0173] Cap A: Acetic Anhydride
[0174] Cap B: N-Methyl Imidazole.
[0175] The target sequence of interest is then input with a
terminal coupling from port 8 to attach ACLAl to the 5'-end of the
sequence. A modified cycle is then chosen such that the desired
scale 0.2(mol, 1.0 (mol, . . . etc) of DNA is synthesized. The
modified cycle contains an additional wait step of 800 seconds
after any addition of 6-FAM. A standard DNA synthesis column
containing the support upon which the DNA will be assembled is then
loaded onto one of four positions of the DNA synthesizer. DNA
containing eTag reporters have been synthesized on various standard
500.ANG. CPG supports (Pac-dA-CPG, dmf-dG-CPG, Ac-dC-CPG, dT-CPG )
as well as specialty supports containing 3'-biotin, 3'-amino
linker, and minor grove binding species.
[0176] Upon completion of the synthesis, the column is removed from
the synthesizer and either dried under vacuum or by blowing air or
nitrogen through the column to remove residual acetonitrile. The
column is then opened and the CPG is removed and placed in a 1-dram
vial. Concentrated ammonia is added (2.0 mL) and the vial is sealed
and placed into a heat block set at 65.degree. C. for a minimum of
two hours. After two hours the vial is allowed to cool to room
temperature after which the ammonia solution is removed using a
Pasteur pipette and placed into a 1.5 mL Eppendorf tube. The
solution is concentrated in vacuo and submitted for HIPLC
purification.
Synthetic Preparation of ACLA 2 on an ABI 394 DNA Synthesizer
[0177] 6-Carboxyfluorescein (6-FAM) phosphoramidite is prepared by
the addition of 2.96 ml of anhydrous acetonitrile to a 0.25 gram
bottle of the fluorescein phosphoramidite, to give a 0.1M solution.
The bottle is then loaded onto the ABI 394 DNA synthesizer at
position 8 using the standard bottle change protocol. The other
natural (dA.sup.bz (0.1M: 0.25 g/2.91 mL anhydrous acetonitrile),
dC.sup.AC(0.1M: 0.25 g/3.24 mL anhydrous acetonitrile), dT(0.1M:
0.25 g/3.36 mL anhydrous acetonitrile), dG.sup.dmf(0.1M: 0.25
g/2.81 mL anhydrous acetonitrile) phosphoramidite monomers are
loaded in a similar fashion to ports 1-4. Acetonitrile is loaded
onto side port 18, standard tetrazole activator is loaded onto port
9, CAP A is loaded onto port 11, CAP B is loaded onto port 12,
oxidant is loaded onto port 15, and deblock solution is loaded onto
port 14 all using standard bottle change protocols. The target
sequence of interest is then input with a terminal coupling from
port 8 and a penultimate coupling of thymidine to the 5'-end of the
sequence to assemble ACLA2. A modified cycle is then chosen such
that the desired scale (0.2.mu.mol, 1.0 .mu.mol, . . . etc) of DNA
is synthesized. The modified cycle contains an additional wait step
of 800 seconds after any addition of 6-FAM. A standard DNA
synthesis column containing the support upon which the DNA will be
assembled is then loaded onto one of four positions of the DNA
synthesizer. DNA containing eTag reporters have been synthesized on
various standard 500.ANG. CPG supports (Pac-dA-CPG, dmf-dG-CPG,
Ac-dC-CPG, dT-CPG ) as well as specialty supports containing
3'-biotin, 3'-amino linker, and minor grove binding species.
[0178] Upon completion of the synthesis the column is removed from
the synthesizer and either dried under vacuum or by blowing air or
nitrogen through the column to remove residual acetonitrile. The
column is then opened and the CPG is removed and placed in a 1-dram
vial. Concentrated ammonia is added (2.0 mL) and the vial is sealed
and placed into a heat block set at 65.degree. C. for a minimum of
two hours. After two hours the vial is allowed to cool to room
temperature after which the ammonia solution is removed using a
Pasteur pipet and placed into a 1.5 mL Eppendorf tube. The solution
is concentrated in vacuo and submitted for HPLC purification.
Synthetic Preparation of ACLA 3 on an ABI 394 DNA Synthesizer
[0179] 6-Carboxyfluorescein (6-FAM) phosphoramidite is prepared by
the addition of 2.96 ml of anhydrous acetonitrile to a 0.25 gram
bottle of the fluorescein phosphoramidite, to give a 0.1M solution.
The bottle is then loaded onto the ABI 394 DNA synthesizer at
position 8 using the standard bottle change protocol. The other
natural (dA.sup.bz (0.1M: 0.25 g/2.91 mL anhydrous acetonitrile),
dC.sup.AC(0.1M: 0.25 g/3.24 mL anhydrous acetonitrile), dT(0.1M:
0.25 g/3.36 mL anhydrous acetonitrile), dG.sup.dmf (0.1M: 0.25
g/2.81 mL anhydrous acetonitrile) phosphoramidite monomers are
loaded in a similar fashion to ports 1-4. Acetonitrile is loaded
onto side port 18, standard tetrazole activator is loaded onto port
9, CAP A is loaded onto port 11, CAP B is loaded onto port 12,
oxidant is loaded onto port 15, and deblock solution is loaded onto
port 14 all using standard bottle change protocols. The target
sequence of interest is then input with a terminal coupling from
port 8 and two penultimate couplings of thymidine to the 5'-end of
the sequence to assemble ACLA 3. A modified cycle is then chosen
such that the desired scale (0.2 umol, 1.0 (mol, . . . etc) of DNA
is synthesized. The modified cycle contains an additional wait step
of 800 seconds after any addition of 6-FAM. A standard DNA
synthesis column containing the support upon which the DNA will be
assembled is then loaded onto one of four positions of the DNA
synthesizer. DNA containing eTags have been synthesized on various
standard 500.ANG. CPG supports (Pac-dA-CPG, dmf-dG-CPG , Ac-dC-CPG,
dT-CPG ) as well as specialty supports containing 3'-biotin,
3'-amino linker, and minor grove binding species.
[0180] Upon completion of the synthesis, the column is removed from
the synthesizer and either dried under vacuum or by blowing air or
nitrogen through the column to remove residual acetonitrile. The
column is then opened and the CPG is removed and placed in a 1-dram
vial. Concentrated ammonia is added (2.0 mL) and the vial is sealed
and placed into a heat block set at 65.degree. C. for a minimum of
two hours. After two hours the vial is allowed to cool to room
temperature after which the ammonia solution is removed using a
Pasteur pipet and placed into a 1.5 mL Eppendorf tube. The solution
is concentrated in vacuo and submitted for HPLC purification.
Synthetic Preparation of ACLA 16 on an ABI 394 DNA Synthesizer
[0181] 6-Carboxyfluorescein (6-FAM) phosphoramidite is prepared by
the addition of 2.96 ml of anhydrous acetonitrile to a 0.25 gram
bottle of the fluorescein phosphoramidite, to give a 0.1M solution.
The bottle is then loaded onto the ABI 394 DNA synthesizer at
position 8 using the standard bottle change protocol. Spacer
phosphoramidite C3 (0.25 g) is dissolved in 5.0 mL of anhydrous
acetonitrile and loaded onto position 5 of the synthesizer. The
other natural (dA.sup.bz (0.1M: 0.25 g/2.91 mL anhydrous
acetonitrile), dC.sup.AC(0.1M: 0.25 g/3.24 mL anhydrous
acetonitrile), dT(0.1M: 0.25 g/3.36 mL anhydrous acetonitrile),
dG.sup.dmf (0.1M: 0.25 g/2.81 mL anhydrous acetonitrile)
phosphoramidite monomers are loaded in a similar fashion to ports
1-4. Acetonitrile is loaded onto side port 18, standard tetrazole
activator is loaded onto port 9, CAP A is loaded onto port 11, CAP
B is loaded onto port 12, oxidant is loaded onto port 15, and
deblock solution is loaded onto port 14 all using standard bottle
change protocols. The target sequence of interest is then input
with a terminal coupling from port 8 and a penultimate coupling of
the C3 spacer from port 5 to assemble ACLA16. A modified cycle is
then chosen such that the desired scale (0.2 .mu.mol, 1.0 .mu.mol,
. . . etc) of DNA is synthesized. The modified cycle contains an
additional wait step of 800 seconds after any addition of 6-FAM. A
standard DNA synthesis column containing the support upon which the
DNA will be assembled is then loaded onto one of four positions of
the DNA synthesizer. DNA containing eTag reporters have been
synthesized on various standard 500.ANG. CPG supports (Pac-dA-CPG,
dmf-dG-CPG, Ac-dC-CPG, dT-CPG ) as well as specialty supports
containing 3'-biotin, 3'-amino linker, and minor grove binding
species.
[0182] Upon completion of the synthesis the column is removed from
the synthesizer and either dried under vacuum or by blowing air or
nitrogen through the column to remove residual acetonitrile. The
column is then opened and the CPG is removed and placed in a 1-dram
vial. Concentrated ammonia is added (2.0 mL) and the vial is sealed
and placed into a heat block set at 65.degree. C. for a minimum of
two hours. After two hours the vial is allowed to cool to room
temperature after which the ammonia solution is removed using a
Pasteur pipette and placed into a 1.5 mL Eppendorf tube. The
solution is concentrated in vacuo and submitted for HPLC
purification.
[0183] All other eTag reporters are synthesized in a similar manner
to that described above.
[0184] The following Table 6 provides a list of different eTag
reporters with their structures, where the symbols are as defined
in Table 2 and are repeated here for convenience. C.sub.3, C.sub.6,
C.sub.9 and C.sub.18 are commercially available phosphoramidite
spacers from Glen Research, Sterling, Va. The units are derivatives
of N,N-diisopropyl, O-cyanoethyl phosphoramidite, which is
indicated by Q. The subscripts indicate the number of atoms in the
chain, which comprises units of ethyleneoxy terminating in Q with
the other terminus protected with DMT. The letters without
subscripts A, T, C and G indicate the conventional nucleotides,
while .sub.T.sup.NH.sup..sub.2 intends amino thymidine and
CB.sup.Br intends bromocytidine. In FIG. 7, the numbers indicate
the eTag reporter as numbered in Table 6 below:
7TABLE 6 eTAG Reporters HC,1 Etags 79 ACLA001 80 ACLA002 81 ACLA003
82 ACLA004 83 ACLA005 84 ACLA006 85 ACLA007 86 ACLA008 87 ACLA009
88 ACLA010 89 ACLA011 90 ACLA012 91 ACLA013 92 ACLA014 93 ACLA015
94 ACLA016 95 ACLA017 96 ACLA018 97 ACLA019 98 ACLA020 99 ACLA021
100 ACLA022 101 ACLA023 102 ACLA024 103 ACLA025 104 ACLA026 105
ACLA027 106 ACLA028 107 ACLA029 108 ACLA030 109 ACLA031 110 ACLA032
111 ACLA033 112 ACLA034 113 ACLA035 114 ACLA036 115 ACLA037 116
ACLA038 117 ACLA039 118 ACLA040 119 ACLA041 120 ACLA042 121 ACLA043
122 ACLA044 123 ACLA045 124 ACLA046 125 ACLA047 126 ACLA048 127
ACLA049 128 ACLA050 129 ACLA051 130 ACLA052 131 ACLA053 132 ACLA054
133 ACLA055 134 ACLA056 135 ACLA057 136 ACLA058 137 ACLA059 138
ACLA060 139 ACLA061 140 ACLA062 141 ACLA063 142 ACLA064 143 ACLA065
144 ACLA066 145 ACLA067 146 ACLA068 147 ACLA069 148 ACLA070 149
ACLA071 150 ACLA072 151 ACLA073 152 ACLA074 153 ACLA075 154 ACLA076
155 ACLA077 156 ACLA078 157 ACLA079 158 ACLA080 159 ACLA081 160
ACLA082 161 ACLA083 162 ACLA084 163 ACLA085 164 ACLA086 165 ACLA087
166 ACLA088 167 ACLA089 168 ACLA090 169 ACLA091 170 ACLA092 171
ACLA093 172 ACLA094 173 ACLA095 174 ACLA096 175 ACLA097
S1 Nuclease Digestion of eTag reporter Probes
[0185] In a 1.5 ml tube, add 10 .mu.l of eTag reporter probe at a
concentration of 10 .mu.M, add 1.5 .mu.l of 10.times.S1 nuclease
reaction buffer, add 0.5 .mu.l of S1 nuclease (Promega, Cat# M5761,
20-100 unit/.mu.l), and add 3 .mu.l of Tris-EDTA buffer to bring
the final volume to 15 .mu.l. Incubate the reaction at 37.degree.
C. for 20 min followed by 25 min at 96.degree. C. to inactivate the
nuclease.
8TABLE 7 Elution Time on 3100 E-Tag POP 4 (min) 176 6.85 177 8.06
178 8.05 179 6.43 180 6.57 181 7.02 182 6.90 183 7.49 184 5.81 185
9.15 186 6.43 187 4.72 188 6.15 189 3.82 190 4.55 191 4.26 192 4.45
193 3.51 194 2.98 195 4.50 196 8.45
5' Nuclease Assays for Monitoring Specific mRNA Expression in Cell
Lysates
[0186] THP-1 cells (American Type Culture Collection, Manassas,
Va.) were cultured in the presence or absence of 10 nM phorbol
12-myristate 13-acetate (Sigma-Aldrich, St. Louis, Mo.) in RPMI
1640 medium with 10% fetal bovine serum (v/v), 2 mM L-glutamine, 10
mM HEPES, 0.05 mM 2-mercaptoethanl. Twenty-four hours after the
induction, cells were harvested and washed twice with PBS before
lysed with lysis buffer (20 mM Tris pH 7.5, 0.5% Nonidet P-40,5 mM
MgCl 2, 20 ng/ul tRNA) at 25.degree. C., for 5 min. The lysate was
heated at 75.degree. C. for 15 min before tested in 5' nuclease
assay.
[0187] Ten microliter cell lysate was combined with a single
stranded upstream invader DNA oligo, (5'CTC-TCA-GTT-CT), a single
stranded downstream biotinylated signal DNA oligo (eTag-labeled, ),
and 2 ng/ul 5' nuclease (Cleavase IX) in 20 ul of buffer (10 mM
MOPS pH 7.5, 0.05% Tween-20 and 0.05% Nonidet P-40, 12.5 mM MgCl 2,
100 uM ATP, 2 U/ul Rnase inhibitor). The reactions were carried out
at 60.degree. C. for 4 hours before analyzed by capillary
electrophoresis. To eliminate background signal, due to the
non-specific activity of the enzyme, 1 ul of 1 mg/ml avidin was
added to the reactions to remove all the eTag-labeled uncleaved
oligo, or eTag-labeled non-specifically cleaved oligos. FIGS. 8 and
9 show separations that were conducted both with and without the
addition of avidin.
PCR Amplification with 5' Nuclease Activity Using eTag
Reporters
[0188] The eTag reporters are described in Table 6. The eTag
reporters that were prepared were screened to provide 20 candidates
that provided sharp separations. 31 eTag reporters were generated
with synthetic targets using the TaqMan (reagents under conditions
as shown in the following tabular format. There were 62 reactions
with the synthetic targets (1 reaction and one negative control for
eTag reporter). The master mix involves preparing a solution of
TaqMan master mix, primer (both reverse and forward) and water.
This mix is then aliquoted into individual PCR tubes followed by
the addition of probe and template.
9 Volume (I Stock Stock Conc. (25(.mu.l/reax) Final conc. Master
mix (Vol * 64) TaqMan mix 2X 6.25 0.5X 400 Probe (eTag 4 .mu.M 1.25
200 nM reporter) Primer 5 .mu.M 2.5 500 nM 160 Template 100 fM 1.25
5 fM Water 13.75 880 Total 25 .mu.l 1440/64 = 22.5 (+1.25 .mu.l
(probe) + 1.25 .mu.l (template) = 25 .mu.l.reax
[0189] All the individual reactions were then run on an ABI 3100
using POP4 as the separation matrix. The samples were diluted 1:20
in 0.5.times. TaqMan buffer and 1 (l of avidin (10 mg/ml) was added
to bind to any intact probe. The sample was further diluted 1:2
with formamide before injecting the sample into the ABI 3100
capillaries. The following on the conditions used with the ABI 3100
for the separation.
10 Temperature 60(C. Pre-run voltage 15 KV Pre-run time 180 sec
Matrix POP4 Injection voltage 3 KV Injection time 10 sec Run
voltage 15 KV Run time 900 sec Run module eTag reporter POP4 Dye
set D
[0190] Subsequent separation of multiple eTAG reporters in a single
run were accomplished as shown in FIG. 7, the structures of which
are identified in Table 6 above.
eTag Reporter Proteomic Analog Assay
1-Labeling of Aminodextran (MW .about.500,000) with eTag Reporter
and Biotin
[0191] Aminodextran was used as a model for demonstrating eTag
reporter release in relation to a high molecular weight molecule,
which also serves as a model for proteins. The number of amino
groups for 10 mg aminodextran was calculated as 2.times.10.sup.-8
moles. For a ratio of 1:4 biotin to eTag reporter, the number of
moles of biotin NHS ester employed was 1.85.times.10.sup.-6 and the
number of moles of maleimide NHS ester was 7.4.times.10.sup.-6.
10.9 mg of aminodextran was dissolved in 6 ml of 0.1% PBS buffer.
Then, 10 mg of Biotin-x-x NHS ester and 23.7 mg of EMCS were
dissolved together in 1 ml of DMF. This DMF solution was added in
50 .mu.l portion (30 min interval) to the aminodextran solution
while it was stirring and keeping away from the light. After final
addition of the DMF solution, the mixtured was kept overnight
(while stirring and away from the light). Then, the mixture was
dialyzed using membrane with cut off molecular weight of 10,000.
The membrane immersed in a beaker containing 2 l of water while
stirring. This water was changed four times (2 h interval). The
membrane was kept in the water overnight (while stirring and
keeping away from the light). Then the solution was lyophilized and
the lyophilized powder was used for eTag reporter labeling.
2- Reaction of Biotin and Maleimide Labeled Aminodextran with the
eTag Reporter, SAMSA
[0192] SAMSA
[5-((2-(and-3)-S-acetylmercapto)succinoyl)amino)fluorescein]w- as
employed as an eTag reporter to react with maleimide in the
aminodextran molecule. For this purpose 0.3 mg
(.about.5.3.times.10.sup.-- 9 moles) of biotin and EMCS labeled
with aminodextran were dissolved in 10 .mu.l of water and then
reacted with 10 times the mol ratio of SAMSA, for the complete
conversion of the maleimide to the eTag reporter. Therefore, 1.1 mg
of SAMSA (.about.1.2.times.10.sup.-6 moles) is dissolved in 120
.mu.l of 0.1 M NaOH and incubated at room temperature for 15 min
(for the activation of the thiol group). Then the excess of NaOH
was neutralized by the addition of 2 .mu.l of 6M HCl, and the pH of
the solution was adjusted to 7.0 by the addition of 30 .mu.l of
phosphate buffer (200 mM, pH=7.0). The activated SAMSA solution was
added to the 10 .mu.l solution of the labeled aminodextran and
incubated for 1 h. The eTag reporter labeled aminodextran was
purified with gel filtration using Sephadex G-25 (Amersham), and
purified samples were collected.
3- The Release of eTag From Labeled Aminodextran
[0193] 2 .mu.l of streptavidin coated sensitizer beads (100
.mu.g/ml) were added carefully in the dark to the 5 .mu.l of
purified labeled aminodextran and incubated in the dark for 15 min.
Then the solution was irradiated for I min at 680 nm. The release
of the eTag reporter was examined be CE using CE.sup.2 LabCard.TM.
device. As shown in FIG. 1, the CE.sup.2 LabCard consists of two
parts; evaporation control and injection/separation. The
evaporation control incorporates a channel (450 .mu.m wide and 50
.mu.m deep) with two buffer reservoirs (2 mm in diameter) and the
evaporation control well (1 mm diameter) right in the center of the
channel. The volume of the side wells (replenishment wells) are 4.7
.mu.l while the volume of the middle well is only 1.2 .mu.l and the
volume of the channel beneath the middle well is about 40 nl. The
second part of the CE.sup.2 device which is the
injection/separation part consists of injection and separation
channels with dimensions of 120 .mu.m wide and 50 .mu.m deep. The
injection channel is connected directly to the evaporation control
well. The channels are closed by laminating a film (MT40) on the
LabCard.TM.. After filling the CE.sup.2 LabCard device with the
separation buffer (20 mM HEPES, pH=7.4 and 0.5% PEO), 300 nl of the
assay mixture was added to the middle well (sample well) and
separated by CE as is shown in FIG. 1.
[0194] FIG. 2 shows the electropherograms of purified labeled
aminodextran with and without sensitizer beads. As shown, the
addition of the sensitizer beads lead to the release of the eTag
reporter from the aminodextran using singlet oxygen produced by
sensitizer upon the irradiation at 680 nm. In order to optimize the
irradiation time, different tubes containing the same mixture of
beads and sensitizer were irradiated for different lengths of time
ranging from 1 to 10 min. There is no significant increase in the
eTag reporter release for irradiation longer than 1 min. FIG. 4,
shows the effect of sensitizer bead concentration on the eTag
reporter release. As depicted in FIG. 4, the higher concentration
of sensitizer beads leads to the higher release of eTag reporters
from the labeled aminodextran. FIG. 5 depicts the linear
calibration curve for the release of eTag reporters as a function
of the sensitizer bead concentration. In addition, the effect of
the concentration of labeled aminodextran on the eTag reporter
release was also examined and the result is shown in FIG. 6. As can
be seen, the lower concentration of labeled aminodextran for a
given concentration of sensitizer beads leads to more efficient
eTag reporter release (or higher ratio of eTag reporter released to
the amount of labeled aminodextran).
[0195] It is evident from the above results that the subject
inventions provide powerful ways of preparing compositions for use
in multiplexed determinations and for performing multiplexed
determinations. The methods provide for homogeneous and
heterogeneous protocols, both with nucleic acids and proteins, as
exemplary of other classes of compounds. In the nucleic acid
determinations, snp determinations are greatly simplified where the
protocol can be performed in only one to four vessels and a large
number of snps readily determined within a short period of time
with great efficiency and accuracy. For other sequences, genomes
can be investigated from both prokaryotes and eukaryotes, including
for the prokaryotes, drug resistance, species, strain, etc. and for
the eukaryotes, species, cell type, response to external stimuli,
e.g. drugs, physical changes in environment, etc., mutations,
chiasmas, etc. With proteins, one can determine the response of the
host cell, organelles or the like to changes in the chemical and
physical environments in relation to a plurality of pathways,
changes in the surface protein population, changes due to aging,
neoplasia, activation, or other naturally occurring phenomenon,
where the amount of protein can be quantitated.
[0196] Particularly as to nucleic acid determinations, the subject
eTag reporters can be synthesized conveniently along with the
synthesis of the oligonucleotides used as probes, primers, etc.,
where the eTag reporter is released in the presence of the
homologous target sequence. Kits of building blocks or eTag
reporters are provided for use in the different determinations.
[0197] All publications and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains. All publications and
patent applications set forth herein are incorporated by reference
to the same extent as if each individual publication or patent
application was specifically and individually indicated to be
incorporate by reference.
[0198] The invention now having been fully described, it will be
apparent to one of ordinary skill in the art that many changes and
modifications can be made thereto without departing from the spirit
or scope of the appended claims.
Sequence CWU 1
1
1 1 11 DNA Human 1 ctctcagttc t 11
* * * * *