U.S. patent application number 14/247953 was filed with the patent office on 2014-10-16 for exchange-induced remnant magnetization for label-free detection of dna, micro-rna, and dna/rna-binding biomarkers.
This patent application is currently assigned to University of Houston System. The applicant listed for this patent is University of Houston System. Invention is credited to Qiongzheng HU, Yuhong WANG, Shoujun XU, Haopeng YANG, Li YAO.
Application Number | 20140309134 14/247953 |
Document ID | / |
Family ID | 50933487 |
Filed Date | 2014-10-16 |
United States Patent
Application |
20140309134 |
Kind Code |
A1 |
XU; Shoujun ; et
al. |
October 16, 2014 |
EXCHANGE-INDUCED REMNANT MAGNETIZATION FOR LABEL-FREE DETECTION OF
DNA, MICRO-RNA, AND DNA/RNA-BINDING BIOMARKERS
Abstract
A method of using an exchange-induced remnant magnetization
(EXIRM) technique for label free detection of short strands of
nucleotides and cancer biomarkers, such as DNA and microRNA
strands, DNA/RNA-binding biomarkers, and cancer-specific antigens,
with high sensitivity, high specificity, and broad dynamic range.
The method may provide a label-free approach aimed to facilitate
high reliability, and to require a minimum amount of biochemical
reagents.
Inventors: |
XU; Shoujun; (Houston,
TX) ; YAO; Li; (Houston, TX) ; WANG;
Yuhong; (Houston, TX) ; HU; Qiongzheng;
(Houston, TX) ; YANG; Haopeng; (Houston,
TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Houston System |
Houston |
TX |
US |
|
|
Assignee: |
University of Houston
System
Houston
TX
|
Family ID: |
50933487 |
Appl. No.: |
14/247953 |
Filed: |
April 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61810575 |
Apr 10, 2013 |
|
|
|
Current U.S.
Class: |
506/9 ;
435/6.11 |
Current CPC
Class: |
C12Q 1/6825 20130101;
C12Q 1/6825 20130101; C12Q 2537/113 20130101; C12Q 2537/161
20130101; C12Q 2563/143 20130101; C12Q 2563/149 20130101 |
Class at
Publication: |
506/9 ;
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method of detecting nucleotide sequences, comprising a)
immobilizing a first nucleotide single strand on a surface; b)
adding a second nucleotide single strand to the first nucleotide
single strand to form a hybridized double strand, wherein said
second strand comprises: a first magnetic particle; and a
nucleotide sequence that is less than 100% complementary to the
first nucleotide single stand, and comprises at least a first
mismatched base; c) measuring a first magnetic signal value for
said hybridized double strand; d) incubating a third nucleotide
strand with said hybridized double strand; wherein said third
strand is complementary to said first strand, and wherein said
incubating forms an exchange product; e) measuring a second
magnetic signal value for the exchange product of step d after
applying a weak mechanical force to remove nonspecifically bound
magnetic particles; and f) quantifying the amount of said third
nucleotide strand from the difference in magnetic signal values
measured in step c and step e
2. The method of claim 1, wherein said first nucleotide single
strand is derivatized.
3. The method of claim 1, wherein said first nucleotide strand is
immobilized to said surface through a S--Au covalent bond or by a
streptavidin-biotin covalent bond.
4. The method of claim 1, wherein said magnetic particle is
attached to said second nucleotide strand by a streptavidin-biotin
covalent bond.
5. The method of claim 1, wherein said magnetic particle is about 1
nm to about 10 .mu.m in size.
6. The method of claim 1 wherein said magnetic particle is about 3
.mu.m in size.
7. The method of claim 1, wherein said measuring comprises an
atomic magnetometer.
8. The method of claim 1, wherein said first and said second
magnetic signal comprise magnetic moment measurements.
9. The method of claim 8, wherein step f comprises measuring the
change in magnetic signal (.DELTA.B).
10. The method of claim 9, further comprising calculating the molar
concentration of said third nucleotide strand, wherein said
concentration is linearly related to .DELTA.B.
11. The method of claim 1, wherein said quantifying further
comprises calculating the number of free magnetic particle labels,
wherein the number of said free magnetic particles corresponds to
the number of exchange product molecules.
12. The method of claim 1, wherein said weak mechanical force is
supplied by a shaker, centrifuge, or sonicator.
13. The method of claim 1, wherein said hybridized strand is in a
liquid environment, a cell lysate, blood plasma, or urine.
14. The method of claim 1, wherein said first nucleotide strand is
a RNA or a DNA sequence of about 1-100 nucleotides.
15. The method of claim 1 wherein said third nucleotide strand is a
DNA or microRNA sequence of about 1 to about 100 nucleotides.
16.
17. The method of claim 1, wherein said exchange product is
thermodynamically more stable than said hybridized double
strand.
18. A method of simultaneously detecting an array of heterologous
nucleotide sequences; the method comprising: coating a sample well
comprising an array of compartments; wherein the surface of said
compartments are alternatively: a) coated with a hybridized
nucleotide double strand; and b) uncoated; wherein said uncoated
compartment produces no magnetic signal; and each said coated
compartment comprises a heterologous hybridized double strand
sequence; c) measuring magnetic signals for each compartment; d)
incubating said array with a sample comprising free target
nucleotide sequences, and forming exchange products; d) measuring
magnetic signals for each compartment comprising exchange products;
e) calculating the difference in said signals from step c and d;
and f) quantifying and identifying said target sequence based on
the change in signal calculated in e.
19. The method of claim 17, wherein said measuring said magnetic
signal from said sample array is by: a scanning single sensor,
scanning the sample well, a two-dimensional sensor array for
simultaneous detection or combinations thereof.
20. An exchange induced remnant magnetization method to detect
cancer biomarkers, the method comprising: (a) immobilizing a first
sequence on a surface; wherein said first sequence comprises N
bases; (b) adding a second sequence to said first sequence, wherein
said second sequence comprises N complementary bases or less; and
wherein said second sequence hybridizes to said first sequence
forming a hybridized double strand; and (c) incubating said
hybridized double strand with a biomarker; and wherein said
biomarker exchanges with said second strand to form an exchange
product; wherein said exchange product is thermodynamically more
stable than said hybridized double strand.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This non provisional application claims the benefit of U.S.
Provisional Application Ser. No. 61/810,575 filed Apr. 10, 2013,
which is fully incorporated herein by reference.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
no. ECCS-1028328 awarded by the National Science Foundation. The
government has certain rights in the invention.
BACKGROUND
[0003] 1. Field of the Invention
[0004] The present disclosure relates to the fields of miRNA
profiling and biomarker detection.
[0005] 2. Background
[0006] Nucleotides are biological molecules that form the building
blocks of nucleic acids (DNA and RNA) and serve to carry packets of
energy within the cell (ATP). In the form of the nucleoside
triphosphates (ATP, GTP, CTP and UTP), nucleotides play central
roles in metabolism. In addition, nucleotides participate in cell
signaling (cGMP and cAMP), and are incorporated into important
cofactors of enzymatic reactions (e.g. coenzyme A, FAD, FMN, NAD,
and NADP.sup.+). Nucleotides may also comprise synthetic sequences,
and comprise chemical modifications to the nucleotide structure to
produce for example nucleotide analogues.
[0007] DNA and RNA are biological molecules that are essential to
life. Genetic information is encoded as specific sequences of DNA
molecules. The information is passed along during transcription and
protein synthesis via messenger RNA. Therefore, they are closely
related, and interplay with various types of diseases, such as
cancers.
[0008] In particular, miRNAs play a significant role in gene
regulation, and are consequently a major category of biomarkers for
cancer diagnostics. Because of their short strands and diverse
expression levels, it remains technically challenging to achieve
precise and quantitative detection. MiRNAs which are short RNA
strands containing 18-25 nucleotides, play numerous important
roles, including those in gene expression, development, and cell
differentiation (1-3). The mature miRNAs incorporate into
RNA-induced silencing complexes that bind with messenger RNAs based
on partial sequence complementarity and consequently cause
inhibition of protein translation. The regulation by miRNAs depends
on their sequence, expression level, and cooperation with other
miRNAs. Therefore, sensitive and specific detection of miRNAs is an
essential step towards understanding their roles in protein
synthesis, cell death, and as biomarkers of disease.
[0009] A range of techniques have been used for miRNA profiling.
Known methods include northern blotting (4), reverse transcriptase
polymerase chain reaction (5), in situ hybridization (6),
microarray (7), bioluminescence (8), surface plasmon resonance (9),
surface-enhanced Raman spectroscopy (10), electrochemical detection
(11), fluorescence (12), and photonic methods (13); however, no
single technique achieves high sensitivity, single-base
specificity, and broad dynamic range. In addition, reproducibility
remains a significant issue when comparing results from different
techniques, due to the many steps and various protocols involved in
analysis (14). Hence there is an unmet need in the field for a
single technique that can detect short nucleotide sequences such as
miRNA, with high sensitivity, high specificity, and broad dynamic
range, which may be a one-step method that facilitates high
reliability, and needs minimum amount of biochemical reagents.
[0010] Thus, the production of a method capable of accurately
detecting short nucleotide sequences, (DNA or RNA sequences such as
miRNA) with high sensitivity, high specificity, and broad dynamic
range would be particularly well received, and embodiments of the
herein presented method are believed to overcome certain above
mentioned limitations by the utilization an exchange-induced
remnant magnetization (EXIRM) technique (15). In addition, many
cancer biomarkers can specifically bind with short DNA strands, for
example prostate specific antigen (16). The EXIRM method can be
directly modified to achieve sensitive and label-free detection of
such cancer biomarkers.
BRIEF SUMMARY OF THE DISCLOSED EMBODIMENTS
[0011] The present disclosure relates to a method of using an
exchange-induced remnant magnetization (EXIRM) technique for
detecting short strands of nucleotides, such as those comprising
deoxyribonucleic acid (DNA); and those comprising ribonucleic acid
(RNA), including microRNA (miRNA), with high sensitivity, high
specificity, and broad dynamic range. Further, the method herein
described may also be a one-step method that facilitates high
reliability, and requires a minimum amount of biochemical
reagents.
[0012] Certain embodiments herein described address such needs, and
uses sequence-specific exchange reactions between label-free
nucleotide sequences (for example miRNA) and magnetically labelled
nucleotide sequences (for example RNA, miRNA, or DNA) with, in some
embodiments a one base difference. In one embodiment, the
exchange-induced remnant magnetization (EXIRM) quantitatively
measures a target miRNA with single-base specificity, and in some
embodiments the detection limit of such target miRNAs reach
zeptomolar levels. In a further embodiment, two miRNAs with only
one base difference may be detected in parallel while showing no
magnetic signal cross-talking, and in still further embodiments,
the EXIRM technique analyses miRNA without any amplification or
washing procedures. In some embodiments the EXIRM method herein
described is suitable for precise miRNA profiling for early
diagnosis and precise prognosis of cancers. The method can also be
extended, wherein some embodiments a sample of interest for which a
quantitative measurement is required may comprise a protein or a
derivative thereof, and in a further embodiment a sample may
comprise an antibody. Further such measurements may also be
performed directly in a biological environment such as but not
limited to blood plasma, urine, or cell lysate, and or other
environments with limited optical detection.
[0013] In one embodiment, a method of detecting nucleotide
sequences comprises: (a) immobilizing a first nucleotide single
strand on a surface; (b) adding a second nucleotide single strand
to the first nucleotide single strand to form a hybridized double
strand, where the second strand comprises a first magnetic
particle; and a nucleotide sequence that is less than 100%
complementary to the first nucleotide single strand, and comprises
at least a first mismatched base; (c) measuring a first magnetic
signal value for the hybridized double strand; (d) incubating a
third nucleotide strand with the hybridized double strand; wherein
the third strand is complementary to the first strand, and wherein
incubating forms an exchange product; (e) measuring a second
magnetic signal value for the exchange product of step d; and (f)
quantifying the amount of the third nucleotide strand from the
difference in magnetic signal values measured in step c and step
e.
[0014] In some embodiments of the method of detecting nucleotide
sequences, the first nucleotide single strand is derivatized; in
some other embodiments the first nucleotide strand may be
biotinylated or thiol captured. In another embodiment of the method
of detecting nucleotide sequences the first nucleotide strand is
immobilized to a surface through a S--Au covalent bond. In a
further embodiment of the method of detecting nucleotide sequences,
a magnetic particle is attached to the second nucleotide strand by
a streptavidin-biotin covalent bond. In another embodiment the
magnetic particle is about 1 nm to about 10 .mu.m in size (for
example, diameter of spherical magnetic particles). in a further
embodiment about 10 nm to about 5 .mu.m in size, and in a further
embodiment the magnetic particle is about 3 .mu.m in size.
[0015] In another embodiment of the method of detecting nucleotide
sequences, measuring comprises an atomic magnetometer; in a further
embodiment, the first and the second magnetic signal values
comprise magnetic moment measurements (17), and in another
embodiment of the method of detecting nucleotide sequences, step
(f) comprises measuring the change in magnetic signal (AB), and in
some further embodiments the molar concentration of the third
nucleotide strand may be calculated wherein the molar concentration
is linearly related to AB or the change in magnetic moment
measurements. In some embodiments of the method of detecting
nucleotide sequences, quantifying further comprises calculating the
number of free magnetic particle labels, wherein the number of said
free magnetic particles corresponds to the number of exchange
product molecules.
[0016] In some embodiments of the method of detecting nucleotide
sequences, the surface is in a sample holder. In one embodiment,
the hybridized double stranded sequence is in a liquid environment.
In further embodiment the environment is a cell lysate, and in a
still further embodiment the environment is blood plasma, and in a
further embodiment, the environment is urine.
[0017] In some embodiments of the method of detecting nucleotide
sequences, the first nucleotide strand is a RNA or a DNA sequence,
in another embodiment the third nucleotide strand is a DNA or
microRNA sequence. In some embodiments, the first nucleotide strand
is about 1-100 nucleotides in length, in some further embodiments,
the second nucleotide strand is about 1-100 nucleotides in length,
and in some still further embodiments the third nucleotide strand
is about 1-100 nucleotides in length.
[0018] In some embodiments, the first nucleotide strand is about
10-50 nucleotides in length, in some further embodiments, the
second nucleotide strand is about 10-50 nucleotides in length, and
in some still further embodiments the third nucleotide strand is
about 10-50 nucleotides in length.
[0019] In some embodiments, the first nucleotide strand is about
18-25 nucleotides in length, in some further embodiments, the
second nucleotide strand is about 18-25 nucleotides in length, and
in some still further embodiments the third nucleotide strand is
about 18-25 nucleotides in length.
In some embodiments, the first nucleotide strand is about 18-25
nucleotides in length, in some further embodiments, the second
nucleotide strand is replaced by the DNA/RNA-binding biomarker, and
in some still further embodiments the third nucleotide strand is
about 18-25 nucleotides in length.
[0020] In other embodiments of the method of detecting nucleotide
sequences, the exchange product is thermodynamically more stable
than the hybridized double strand, in some embodiments the double
strand is 12 pN (pN: 10.sup.-12 N) less stable than said exchange
product.
[0021] In another embodiment, a method of simultaneously detecting
an array of heterologous nucleotide sequences is provided wherein
the method comprises: (a) coating a sample well comprising an array
of compartment; wherein the surface of adjacent compartments are
alternatively coated with i) a hybridized nucleotide double strand;
and ii) are uncoated; wherein the uncoated compartment produces no
magnetic signal; and each coated compartment comprises a
heterologous hybridized double strand sequence; (b) measuring
magnetic signals for each compartment; (c) incubating the array
with a sample comprising free target nucleotide sequences, and
forming exchange products; (d) measuring magnetic signals for each
compartment comprising exchange products after applying a weak
mechanical force to remove nonspecifically bound magnetic
particles; (e) calculating the difference in said signals from step
b and d; and (f) quantifying and identifying said target sequence
based on the change in signal calculated in step (e). In a further
embodiment of the method of simultaneously detecting an array of
heterologous nucleotide sequences measuring the magnetic signal
from the sample array is by: a scanning single sensor, scanning the
sample well, a two-dimensional sensor array for simultaneous
detection or combinations thereof.
[0022] In another embodiment, an exchange induced remnant
magnetization method to detect specific nucleotide sequences is
herein described, the method comprising: (a) immobilizing a first
single stranded sequence on a surface; wherein the first sequence
comprises N bases; (b) adding a second single stranded sequence to
the first single stranded sequence, wherein the second single
stranded sequence comprises N-1 complementary bases; wherein said
complementary bases are complementary to the sequence of the first
single strand sequence, and wherein the second single stranded
sequence hybridizes to the first single stranded sequence forming a
hybridized double stranded sequence with N-1 base pairs; and (c)
incubating the hybridized double stranded sequence with a third
single stranded sequence, wherein the third single stranded
sequence comprises N complementary bases, wherein said
complementary bases are complementary to the sequence of the first
single strand sequence; and wherein said third single stranded
sequence exchanges with said second single stranded sequence to
form an exchange product comprising a double strand with N
complementary base pairs; wherein said exchange product is
thermodynamically more stable than said hybridized double stranded
sequence. Further embodiments may include species wherein the
second single stranded sequence is mismatched by greater that one
complementary base.
In another embodiment, an exchange induced remnant magnetization
method to detect specific biomarkers is herein described, the
method comprising: (a) immobilizing a first single stranded
sequence on a surface; wherein the first sequence comprises N
bases; (b) adding a second single stranded sequence to the first
single stranded sequence, wherein the second single stranded
sequence comprises N complementary bases; wherein said
complementary bases are complementary to the sequence of the first
single strand sequence, and wherein the second single stranded
sequence hybridizes to the first single stranded sequence forming a
hybridized double stranded sequence with N base pairs; and (c)
incubating the hybridized double stranded sequence with a
biomarker, wherein the biomarker exchanges with said second single
stranded sequence to form an exchange product comprising a
DNA-biomarker complex; wherein said exchange product is
thermodynamically more stable than said hybridized double stranded
sequence. Further embodiments may include species wherein the
second single stranded sequence is mismatched by one complementary
base or more.
[0023] Thus, embodiments described herein comprise a combination of
features and characteristics intended to address various
shortcomings associated with certain methods of detecting
nucleotide sequences such as DNA and microRNA sequences, wherein
the exchange-induced remnant magnetization technique described
detects such sequences with high sensitivity, high specificity, and
over a broad dynamic range as compared to some techniques known in
the art. The various features and characteristics described above,
as well as others, will be readily apparent to those skilled in the
art upon reading the following detailed description, and by
referring to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] For a detailed description of the disclosed embodiments,
reference will now be made to the accompanying drawings,
wherein:
[0025] FIG. 1 depicts an embodiment of the EXIRM technique
described herein, where three RNA strands are involved: immobilized
Strand 1, hybridized Strand 2 with one mismatching base, and Strand
3 which is the target miRNA. Strand 2 is labelled by magnetic
particles (the approach may also be applied to DNA);
[0026] FIG. 2 depicts a plot of a magnetic signal changes (AB (in
pT; 10.sup.-12 T)) for various concentrations (femto molar,
10.sup.-15 M) of the target DNA, wherein the linear correlation
shows good quantification of the DNA molecules in accordance with
principles described herein. The diameter of the magnetic particles
is 2.8 .mu.m in accordance with principles described herein;
[0027] FIG. 3 depicts a graphic representation of normalized
measurements with 1 second signal averaging for the 12-base DNA
exchange reaction at five different concentrations; wherein the
normalized profiles were obtained in this embodiment by averaging
33 adjacent data points in the raw data, where each data point was
measured for 30 ms, the X-axis represents the relative position of
the sample to the atomic sensor during scanning the sample, and
performed in accordance with principles described herein;
[0028] FIG. 4 depicts a plot of magneto-optical resonance to show
the maximum measurable magnetic fields by the atomic magnetometer,
wherein the highlighted rectangular area shows the responsive range
of the atomic magnetometer to the samples magnetic signal, in
accordance with principles described herein;
[0029] FIG. 5 depicts a plot of a magnetic signal changes AB (pT)
for various concentrations of the target DNA using smaller magnetic
particles, with diameter of 1 .mu.m, and shows the quantitative
relationship between the change in B with concentration of magnetic
particle labelled DNA;
[0030] FIG. 6 depicts an illustration of multiplexed detection, in
accordance with principles described herein and wherein (A) is a
Schematic of an array of sample wells. Each golden square is for a
specific target of RNA or DNA. Each blank separates two adjacent
sample wells; and (B) is an example of detecting multiple targeted
RNA or DNA. The atomic magnetometer is composed of an atomic sensor
housed in a multi-layered magnetic shield, the sample cell
containing an array of sample wells is introduced through a sample
inlet channel through the magnetic shield, and an optical fiber may
be used to deliver the laser beam to the atomic sensor. The signal
comes out through a signal cable;
[0031] FIG. 7 depicts a plot of a magnetic signal as a function of
reaction time for an EXIRM experiment; wherein a 12-base DNA was
the target strand, which replaced another DNA with one-base
difference that was pre-hybridized with the complementary strand to
the former (in control, the target DNA was absent) performed in
accordance with principles described herein;
[0032] FIG. 8 (A-E) depicts EXIRM for multiplexed miRNA analysis
with single-base specificity; (A) shows the MiRNA sequences let-7a,
1a;2; 1b; 2; let-7c respectively; which are used in the
corresponding experiment; and bases that are used for the analysis
are in red (fourth line) with the mismatching bases underlined; (B)
shows an image depicting a sample holder with two sample wells;
(C); (D); and (E) depict the magnetic signal change for adding
let-7c (C), let-7a (D), and no miRNA (E) into both of the sample
wells shown in B; The dashed lines in C,D, and E, show the
positions of the two sample wells.
DETAILED DESCRIPTION OF DISCLOSED EMBODIMENTS
[0033] Embodiments herein addressed are intended to overcome
certain above mentioned limitations by using a method for an
exchange-induced remnant magnetization (EXIRM) technique for
detecting DNA, microRNA, and DNA/RNA-binding proteins with high
sensitivity, high specificity, and broad dynamic range (15). Herein
and throughout the application, the term "strand" and "sequence"
may be used interchangeably to describe sequences of nucleotides
which are single stranded. Similarly, "hybridized strand,"
"hybridized double strand," "hybridized double stranded sequence"
or "hybridized sequence" may be used interchangeably. As used
herein, the term "about," when used in conjunction with a
percentage or other numerical amount, means plus or minus 10% of
that percentage or other numerical amount. For example, the term
"about 80%," would encompass 80% plus or minus 8%. Further, all
references cited herein are incorporated in their entirety.
[0034] General Principle
[0035] In some embodiments, an exchange induced remnant
magnetization method to detect specific nucleotide sequences is
herein described, the method comprises: (a) immobilizing a first
single stranded sequence on a surface; wherein the first sequence
comprises N bases; (b) adding a second single stranded sequence to
the first single stranded sequence, wherein the second single
stranded sequence comprises N-1 complementary bases; wherein said
complementary bases are complementary to the sequence of the first
single strand sequence, and wherein the second single stranded
sequence hybridizes to the first single stranded sequence forming a
hybridized double stranded sequence with N-1 base pairs; and (c)
incubating the hybridized double stranded sequence with a third
single stranded sequence, wherein the third single stranded
sequence comprises N complementary bases; and wherein said third
single stranded sequence exchanges with said second single stranded
sequence to form an exchange product comprising a double strand
with N complementary base pairs; wherein said exchange product is
thermodynamically more stable than said hybridized double stranded
sequence.
[0036] In some embodiments, a method of detecting nucleotide
sequences is herein described, comprising (a) immobilizing a first
nucleotide single strand on a surface; (b) adding a second
nucleotide single strand to the first nucleotide single strand to
form a hybridized double strand, where the second strand comprises
a first magnetic particle; and a nucleotide sequence that is less
than 100% complementary to the first nucleotide single stand, and
comprises at least a first mismatched base; (c) measuring a first
magnetic signal value for the hybridized double strand; (d)
incubating a third nucleotide strand with the hybridized double
strand; wherein the third strand is complementary to the first
strand, and wherein incubating forms an exchange product; (e)
measuring a second magnetic signal value for the exchange product
of step d after applying a weak mechanical force to remove
nonspecifically bound magnetic particles; and (f) quantifying the
amount of the third nucleotide strand from the difference in
magnetization value measured in step c and step e.
[0037] Thus, in one embodiment of the invention herein described, a
specific nucleotide sequence such as (but not limited to) a miRNA
sequence can be detected by magnetic signal changes caused by
exchange reactions between a target miRNA sequence and a
magnetically labelled RNA sequence with a single nucleotide base
difference; as illustrated in the embodiment of FIG. 1, a
hybridized RNA double strand is first prepared, a strand composed
of nucleotide bases that compliments the target miRNA (Strand 1) is
immobilized on the surface of a sample well/plate, and a second
strand (Strand 2) composed of nucleotide bases that differ (are
mismatched) by (at least) one base from Strand 1, and the target
miRNA (Strand 3) is further labelled with a magnetic particle.
Strand 1 and Strand 2 form a hybridized double strand of RNA,
immobilized on the surface through Strand 1 and magnetically
labelled through Strand 2.
[0038] The target miRNA is then incubated with the hybridized
double strand in the sample well. An exchange reaction then takes
place, in which the target miRNA replaces the mismatching strand
because the former has thermodynamically stronger binding with the
immobilized RNA than the latter. When the strands with one
mismatching base (hybridized pair) which are immobilized and
magnetically labelled undergo magnetization by a strong magnet
(>0.1 Tesla), the magnetic dipoles of the particles are aligned
and produce a strong magnetic signal; hence when the mismatching
RNA undergoes dissociation from the immobilized strand due to the
thermodynamically favoured binding of the complementary target
strand, randomization of the magnetic dipoles of the magnetic
labels occurs due to Brownian motion of the now free magnetically
labelled strands which is induced by a weak mechanical force
provided by a shaker or a centrifuge. The exchange reaction thus
produces a decrease in the magnetic signal (.DELTA.B in pT),
because of the randomization of the magnetic particles, which is
measured by an atomic magnetometer (17), the decreasing amplitude
of the signal thus represents the quantity of the target miRNA
molecules.
[0039] Thus, in some embodiments of the method of detecting
nucleotide sequences, the first nucleotide strand is a RNA or a DNA
sequence, in another embodiment the third nucleotide strand is a
DNA or microRNA sequence. In some embodiments, the first nucleotide
strand is about 1-100 nucleotides in length, in some further
embodiments, the second nucleotide strand is about 1-100
nucleotides in length, and in some still further embodiments the
third nucleotide strand is about 1-100 nucleotides in length.
[0040] In some embodiments, the first nucleotide strand is about
10-50 nucleotides in length, in some further embodiments, the
second nucleotide strand is about 10-50 nucleotides in length, and
in some still further embodiments the third nucleotide strand is
about 10-50 nucleotides in length. In some embodiments, the first
nucleotide strand is about 18-25 nucleotides in length, in some
further embodiments, the second nucleotide strand is about 18-25
nucleotides in length, and in some still further embodiments the
third nucleotide strand is about 18-25 nucleotides in length. In
some embodiments the second nucleotide single strand comprises
1-100 mismatched bases, in another embodiment the second nucleotide
single strand comprises 1-50 mismatched bases, in another
embodiment the second nucleotide single strand comprises 1-10
mismatched bases; and in a preferred embodiment the second
nucleotide single strand comprises 1 mismatched based, wherein the
definition of mismatched is that the base bonds with a second
molecule or base that is not it's natural Watson and Crick base
pair interaction i.e. guanine/cytosine bonding, adenine/thymine
bonding and adenine/uracil bonding.
[0041] In other embodiments of the method of detecting nucleotide
sequences, the exchange product is thermodynamically more stable
than said hybridized double strand, in some embodiments the double
strand is at least 12 pN less stable than said exchange product,
wherein the stability declines based on the loss of hydrogen
bonding between base pairs.
In some embodiments of the method of detecting cancer biomarkers,
the exchange product of the DNA-biomarker complex is
thermodynamically more stable than said hybridized double
strand.
[0042] In some embodiments of the method of detecting nucleotide
sequences, the first nucleotide single strand is derivatized; in
some other embodiments the first nucleotide strand may be
biotinylated or thiol captured. In another embodiment of the method
of detecting nucleotide sequences, the first nucleotide strand is
immobilized to a surface through a S--Au covalent bond. In a
further embodiment of the method of detecting nucleotide sequences,
a magnetic particle is attached to the second nucleotide strand by
a streptavidin-biotin covalent bond. In another embodiment the
magnetic particle is about 1 nm to about 10 .mu.m in size, about 10
nm to about 5 .mu.m in size, and in a further embodiment the
magnetic particle is about 3 .mu.m in size.
[0043] Detection Level and Sensitivity of the Method
[0044] The level of detection for the embodiments of the method
herein described is obtained by varying the concentration of the
target nucleotide sequences, for example the magnetic signal
decrease (.DELTA.B) is plotted against the concentration of target
nucleotides, in one such embodiment illustrated in FIG. 2, five
different concentrations of a DNA target sequence were used, and
EXIRM performed. The five measurements of .DELTA.B were linearly
correlated, and the linear fit goes through the origin, which
indicates the high sensitivity of the method for measuring changes
in magnetic signal at these concentrations, and that the
quantification is highly reliable.
[0045] In some embodiments, given a sample well with a known volume
of 8 .mu.L, the total number of the DNA sequences being replaced
can be calculated to be 660 zeptomole, or 4.times.10.sup.5
molecules, for the concentration of 83 fM. The error bars were
obtained by normalizing the measuring time to 1 second, which was
nearly 1 pT (FIG. 3). From the .DELTA.B value of 12 pT for the 83
fM sample, the detection limit has already reached
3.3.times.10.sup.4 molecules. Therefore, the detection limit is
substantially better than other current techniques for DNA/RNA
oligomers (18). In a further embodiment, based on the sensitivity
of 150 fT of the apparatus (19), the detection level of the method
can allow measurements of 6.times.10.sup.3 molecules with 1 second
signal averaging time.
Dynamic Range
[0046] The dynamic range of EXIRM is defined as the span between
the number of target molecules that give the lowest detectable
magnetic signal and the number of target molecules that give the
highest detectable magnetic signal.
[0047] The dynamic range of embodiments of the method described
herein, can be derived from the sensitivity of the magnetometer and
the width of magneto-optical resonance. The latter represents the
optical response by the magnetometer to the magnetic field to be
measured. Therefore, it provides the range for the magnetic signal
that the magnetometer is sensitive to. In some embodiments, a
sensitivity of about 150 fT defines the lower end of the dynamic
range. In some embodiments, given a resonance width of .about.70 Hz
for the magnetometer (FIG. 4, highlighted area) a frequency range
(the horizontal axis) is the frequency change is almost linear to
the magnetic signal. This width gives an upper measurement limit of
about 10 nT, because B=.omega./2.gamma., where .omega. is the
modulation frequency of the laser and .gamma. of 3.5 Hz/nT is the
gyromagnetic ratio for Cs (cesium, the atom for the atomic
magnetometer shown here). Hence in one such embodiment, the dynamic
range is about 5 orders of magnitude for a selected magnetic
particle.
[0048] In another embodiment, a broad dynamic range is preferred
for miRNA profiling, because it is well known that the expression
levels may be drastically different for different miRNAs. Therefore
in some embodiment's large numbers of miRNA are available for
exchange wherein the signal change will be greater, while in other
embodiments the number of miRNA expressed and available for
exchange will be small. In one embodiment of the method herein
described, the atomic magnetometer has an upper detection limit of
approximately 10 nT; and the lower limit is determined by the
sensitivity, which is 150 fT. Therefore, with a selected type of
magnetic particles for labelling, the dynamic range is about five
orders of magnitude as described above.
[0049] Furthermore, in some embodiments, the dynamic range in terms
of number of miRNA molecules can be adjusted by tuning the magnetic
property of the particles. This is because for magnetically weaker
particles, a larger number of particles will be needed to reach the
upper limit of the detection range. Hence more target molecules can
be detected. While for magnetically stronger particles, a fewer
number of particles will provide sufficient magnetic signal so that
a fewer number of target molecules will be detected. Potentially
single-molecule detection is achievable when the magnetic particle
gives sufficiently strong signal (20). Therefore, again in some
embodiments the dynamic range of EXIRM may be greater than five
orders of magnitude. An example of using different sized magnetic
particles to adjust the dynamic range is shown in FIG. 5.
[0050] Multiplexed Detection
[0051] In another embodiment, a method of simultaneously detecting
an array of heterologous nucleotide sequences is provided wherein
the method comprises: (a) coating a sample well comprising an array
of squares; wherein the surface of adjacent squares are
alternatively coated with i) a hybridized nucleotide double strand;
and ii) remain uncoated; wherein the uncoated square produces no
magnetic signal; and each coated square comprises a heterologous
hybridized double strand sequence; (b) measuring magnetic signals
for each square; (c) incubating the array with a sample comprising
free target nucleotide sequences, and forming exchange products;
(d) measuring magnetic signals for each square comprising exchange
products; (e) calculating the difference in said signals from step
b and d; and (f) quantifying and identifying said target sequence
based on the change in signal calculated in step (e). In a further
embodiment of the method of simultaneously detecting an array of
heterologous nucleotide sequences measuring the magnetic signal
from the sample array is by: a scanning single sensor, scanning the
sample well, a two-dimensional sensor array for simultaneous
detection or combinations thereof.
[0052] Thus, multiplexed detection is a simultaneous measurement or
a method of identifying multiple species in a single experimental
run. In some embodiments, multiplexed detection assays are
experiments that endeavour to detect or to assay the state of all
biomolecules of a given class (e.g., miRNAs) within a biological
sample, to determine the effect of an experimental treatment or the
effect of a DNA mutation over all of the biomolecules or pathways
in the sample.
[0053] In some embodiments of the method herein described,
multiplexed detection of arrayed samples is needed because miRNAs
often do not function alone. It has been reported that groups of
miRNAs play important roles cooperatively (21). In addition, miRNA
expression is highly heterogeneous (22). Therefore, monitoring a
group of miRNAs that may be closely related in sequence in a single
sample is required. FIG. 6 shows an embodiment of a one-dimensional
multiplexed detection. The bottom of a single sample well is
patterned into an array of squares, which are alternatively coated
with RNA double strands which have sequences that vary cell to
cell, and a blank. Each coated square aims at targeting one type of
miRNA sequence. In some embodiments, a blank square is needed in
between two coated squares to avoid signal cross-talking that
arises from the overlap of the magnetic signals. In one embodiment,
2 mm atomic sensors are used, thus each sample square is 2.times.2
mm.sup.2, the distance between two adjacent samples is 4 mm center
to center, and the area of the sample being 4.times.1 mm.sup.2,
thus in one such embodiment, a sample well with 2.6.times.2.6
cm.sup.2 bottom area can analyze 49 different types of miRNAs.
Smaller atomic sensors may be used for multiplexed detection of a
greater number of different miRNAs if needed.
[0054] As such there are two ways to detect the magnetic signals
from the sample array: one embodiment comprises a scanning single
sensor, and another embodiment comprises using a two-dimensional
sensor array for simultaneous detection. FIG. 6B shows a schematic
of using the single sensor approach. The approach of using an array
of sensors can be carried out similarly.
[0055] In some embodiments, no amplification is needed, and no
washing or sample transfer is used. This simplifies the analysis
procedure and improves the reliability of the measurements as
compared to known techniques which often involve multiple steps of
sample preparation, amplification, and multiple washing steps.
[0056] In some embodiments, the EXIRM technique described herein,
provides a new avenue for miRNA analysis. In some embodiments, the
high sensitivity of atomic magnetometers allows detection of about
10.sup.4 molecules; in further embodiments detection may be in the
order of 10.sup.3 molecules. In other embodiments single-base
specificity is achieved from the sequence-specific exchange
reactions; and in further embodiments, cross-talking is not
observed between miRNAs wherein in some embodiments, there is only
a one base difference between sequences.
EXAMPLES
Example 1(A)
EXIRM's Single-Base Specificity in DNA Detection
[0057] In one embodiment, to demonstrate EXIRM, three 12-base DNA
strands were chosen. The nucleotide strand was a thiolated GGG AAA
AAA GGG (Strand 1), which was loaded into a sample well (of
4.times.2.times.1 mm.sup.3 in size) and subsequently immobilized on
the bottom surface of the well via S--Au covalent bonds (15).
[0058] The second strand was then added for hybridization, using
biotinylated oligonucleotide sequences, CCC AAA AAT CCC (Strand 2;
11 base pair match to strand 1) and was labelled with magnetic
particles (examples of such magnetic particles include
Streptavidin-coated 2.8 .mu.m sized magnetic particles (Invitrogen,
M280)). The target strand (CCC AAA AAA CCC (Strand 3)) which was
fully complimentary to the immobilized strand (12 base pair match),
was thus added to the sample well. This system was chosen because
the force of Strand 1 binding to Strand 2 (11-base pair match) is
12 pN weaker than that of Strand 1 binding to Strand 3 (12 base
pair match). The magnetic signal showed a decrease when Strand 3
was added into the sample well containing Strand 1-Strand 2 double
helix (lower trace in FIG. 7). The decrease signal indicated the
occurrence of the exchange reaction, in which Strand 3 replaced
Strand 2. In contrast, when the target Strand 3 was absent, no
magnetic signal decrease was observed (upper trace in FIG. 7). The
magnetic signal profile also showed that the exchange reaction took
less than one hour to complete at 37.degree. C.
Example 1(B)
EXIRM's Single-Base Specificity in Multiplexed MiRNA Analysis
[0059] In one embodiment of the method herein described, two sample
wells were placed in parallel along the sample holder (FIG. 8B),
the two selected target miRNAs were let-7a and let-7c, which are
biomarkers for lung cancer (21) and colorectal cancer (22)
respectively, and the two miRNA only differ by one base as can be
seen in FIG. 8A which shows the experimental sequences. In this
embodiment, nucleotide Strand 1a is entirely complimentary (base by
base) with let-7a and nucleotide Strand 1b is entirely
complimentary (base by base match) with let-7c. The two sample
wells used a common Strand 2, which had one mismatching base for
both Strand 1a and Strand 1b. When let-7c was added to both sample
wells, only the right sample well (located at 182 mm) gave a
magnetic signal decrease, as depicted by the magnetic signal change
(.DELTA.B(pT)) seen in FIG. 8C). This is because let-7c compliments
Strand 1b and therefore replaces Strand 2 only in the right sample
well. Let-7c is one-base mismatched for Strand 1a, thus no exchange
reaction occurred in the left sample well (at 168 mm). Similarly,
when let-7a was added to both sample wells, only the left sample
well produced a magnetic signal decrease as depicted by the
magnetic signal change (.DELTA.B(pT)) seen in FIG. 8D). As a
control experiment, when no miRNA was added to the sample wells, no
magnetic signal change was observed in either sample well as
depicted in FIG. 8E. This confirms the responses were due to the
presence of appropriate miRNAs as they exchanged with Strand 2 in
the sample well containing their respective complimentary strands,
and that no cross talking of magnetic signal is exhibited between
let-7a and let-7c, thus the EXIRM method herein described has
single-base specificity. In some embodiments, this capability is
required for miRNA profiling because miRNAs in the same family
often differ by only a few nucleotides; conventional techniques are
often unable to obtain single base specificity especially in light
of the short sequences often associated with miRNA's.
[0060] Materials and Methods.
[0061] In some embodiments, the biotinylated or thiol-derivatized
first nucleotide strands were immobilized on the bottom surface of
sample wells wherein the surface is streptavidin- or gold-coated.
After hybridization with their corresponding biotinylated strand 2,
with at least one mismatching base, the samples are incubated in on
embodiment with streptavidin-conjugated magnetic particles
(Invitrogen M280) at room temperature in tris-buffered saline (TBS)
solution with 1% (w/v) bovine serum albumin (BSA) and 0.05%
detergent Tween 20, the magnetic particles were then magnetized by
a permanent magnet. The M280 particles are uniform,
superparamagnetic beads of 2.8 mm in diameter with a streptavidin
monolayer covalently bound to the surface. They are supplied as a
suspension.
[0062] In some embodiments, to initiate the exchange reaction, the
target DNA or miRNA with an entirely complementary sequence (to the
capture probe) to strand, was then added and incubated in TE buffer
(10 mM tris, 1 mM EDTA, 1 M NaCl, pH 8.0) at 37.degree. C. The
samples' magnetic signal was measured by an atomic magnetometer
after applying a weak centrifugal force to eliminate physisorption
of the magnetic particles. For the DNA exchange reaction, reaction
time was varied between 20-220 min. For miRNA targets (let-7a and
let-7c), two capture probes (1a and 1b) were located in two sample
wells were placed on a sample holder, one with the double helix of
Strand 1a and Strand 2, and the other with Strand 1b and Strand 2.
The center-to-center distance between the sample wells was 14 mm.
The reaction time was 6 hrs for miRNAs exchange.
[0063] In some embodiments, using miniature atomic magnetometers
(23), EXIRM is capable of sensitive and precise miRNA profiling,
and in some further embodiments will be used in cancer
diagnostics.
[0064] While certain embodiments of the invention described herein
specifically focus on a novel method to detect DNA and miRNA
sequences of interest based on their specific binding pairs and
specificity, one of ordinary skills in the art, with the benefit of
this disclosure, will recognize the extension of the approach to
other systems.
[0065] Other and further embodiments, versions and examples of the
invention may be devised without departing from the basic scope
thereof and the scope thereof is determined by the claims that
follow. References cited herein are incorporated by reference in
their entirety.
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Sequence CWU 1
1
8122DNAArtificialLET-7A 1uugauauguu ggaugaugga gu
22222DNAArtificialStrand-1a 2aactatacaa cctactacct ca
22322DNAArtificialStrand-2 (Figure-8) 3uuguuauguu ggaugaugga gu
22422DNAArtificialStrand-1b 4aaccatacaa cctactacct ca
22522DNAArtificialLET-7C 5uugguauguu ggaugaugga gu
22622DNAArtificialStrand 1(EXAMPLE 1(A)) 6uugguauguu ggaugaugga gu
22712DNAArtificialStrand 2 (EXAMPLE 1(A)) 7cccaaaaatc cc
12812DNAArtificialStrand 3 (EXAMPLE 1(A)) 8cccaaaaaac cc 12
* * * * *