U.S. patent application number 12/738580 was filed with the patent office on 2010-08-19 for methods and compositions for detection and enrichment of target small rnas.
Invention is credited to Jingmin Jin, Larry A. McReynolds.
Application Number | 20100209933 12/738580 |
Document ID | / |
Family ID | 40226874 |
Filed Date | 2010-08-19 |
United States Patent
Application |
20100209933 |
Kind Code |
A1 |
McReynolds; Larry A. ; et
al. |
August 19, 2010 |
Methods and Compositions for Detection and Enrichment of Target
Small RNAs
Abstract
Methods and compositions are provided for detecting small target
RNAs where the target RNA may be single-stranded or double-stranded
and may be contained in a mixture of RNAs of different types and
sizes. The methods and compositions utilize a p19 fusion protein
that is capable of binding double-stranded RNA in a size-specific
but sequence-independent manner and is further capable of binding
to a matrix such as beads or plastic microwell plates. By labeling
the p19 fusion protein or the target RNA in a polynucleotide duplex
either directly or indirectly, low levels of target RNA including
microRNAs can be detected from cells. This can be applied to
diagnosis of pathological conditions.
Inventors: |
McReynolds; Larry A.;
(Beverly, MA) ; Jin; Jingmin; (Middleton,
MA) |
Correspondence
Address: |
HARRIET M. STRIMPEL, D. Phil.
New England Biolabs, Inc., 240 COUNTY ROAD
IPSWICH
MA
01938-2723
US
|
Family ID: |
40226874 |
Appl. No.: |
12/738580 |
Filed: |
October 29, 2008 |
PCT Filed: |
October 29, 2008 |
PCT NO: |
PCT/US08/81520 |
371 Date: |
April 16, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60983503 |
Oct 29, 2007 |
|
|
|
Current U.S.
Class: |
435/5 ;
435/320.1; 435/6.12; 530/350; 536/23.72 |
Current CPC
Class: |
G01N 33/5308 20130101;
C12Q 1/6834 20130101; C12Q 1/68 20130101 |
Class at
Publication: |
435/6 ; 530/350;
536/23.72; 435/320.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 14/005 20060101 C07K014/005; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63 |
Claims
1. A recombinant protein having at least 90% sequence homology to
SEQ ID NO:33 and capable of binding a 21 nucleotide double-stranded
RNA (dsRNA).
2. The recombinant protein according to claim 1, further
comprising: a detectable label selected from a fluorescent label, a
radioactive label, a chemiluminescent label, a protein label and a
small molecule label.
3. A DNA encoding the protein of claim 1.
4. A vector, comprising: the DNA of claim 3 for expressing the
recombinant protein in a host cell.
5. A method; comprising: mixing a target RNA with a p19 fusion
protein, wherein either the p19 fusion protein or the target RNA is
labeled, the label being (i) directly linked to the protein or
target RNA, or (ii) indirectly linked by means of a molecule
capable of binding to the p19 fusion protein or target RNA; and
immobilizing on a matrix the p19 fusion protein bound to the target
RNA for detecting the target RNA.
6. A method according to claim 5, wherein the target RNA is a
single-stranded RNA (ssRNA), the ssRNA hybridizing to a
complementary polynucleotide probe to form a double-stranded hybrid
polynucleotide for binding to the p19 fusion protein.
7. A method according to claim 6, wherein the complementary
polynucleotide probe is an RNA, a DNA molecule or a locked nucleic
acid.
8. A method according to claim 6, wherein the complementary
polynucleotide probe extends at the 3' end beyond the target
RNA.
9. A method according to claim 6, wherein the single-stranded
molecule is an miRNA.
10. A method according to claim 5, wherein the target RNA is a
dsRNA.
11. A method according to claim 10, wherein the dsRNA is an
siRNA.
12. A method according to claim 5, wherein the p19 fusion protein
is immobilized prior to binding the target RNA.
13. A method according to claim 5, wherein the target RNA is
immobilized prior to binding the p19 fusion protein.
14. A method according to claim 5, wherein the detectable label is
selected from the group consisting of a fluorescent label, a
radioactive label, a chemiluminescent label, a protein label and a
small molecule label.
15. A method according to claim 5, wherein the p19 fusion protein
is bound to the target RNA to form a complex which is then
immobilized on a matrix.
16. A method according to claim 5, wherein the matrix has a colored
or fluorescent label that differs from the label on the p19 protein
or polynucleotide probe.
17. A method according to claim 5 or 16, wherein the matrix is a
bead.
18. A method according to claim 17, wherein the bead is
magnetic.
19. A method according to claim 17, wherein the bead is coated with
a carbohydrate to which the p19 fusion protein can bind.
20. A method according to claim 5, wherein the immobilized target
RNA is diagnostic for an abnormal condition of a cell.
21. A method according to claim 5, wherein the target RNA is
diagnostic of an abnormal host cell and is present in a mixture
comprising a plurality of types and sizes of RNA.
22. A method for detecting a target RNA in a mixture of RNAs, such
that if the target RNA is (i) single-stranded, adding a
complementary polynucleotide probe for forming a dsRNA and allowing
the dsRNA to bind to a p19 fusion protein; or (ii) double-stranded,
binding the dsRNA directly to the p19 fusion protein, wherein
either the complementary polynucleotide probe or the p19 fusion
protein is linked to a label selected from a fluorescent label, a
radioactive label, a chemiluminescent label, a protein label and a
small molecule label.
23. A method according to claim 20, further comprising:
immobilizing the p19 fusion protein on a matrix for binding labeled
dsRNA and removing unbound RNA.
24. A method according to claim 20, further comprising:
immobilizing the dsRNA on a matrix and binding a labeled p19 fusion
protein to the dsRNA.
25. A kit, comprising: the recombinant protein in claim 1,
instructions for detecting a small RNA, and optionally a matrix for
binding p19 fusion protein or a polynucleotide probe.
26. A kit according to claim 25, wherein the kit contains a matrix
and the p19 fusion protein is bound to the matrix.
27. A kit according to claim 25, wherein the kit contains a matrix
and the matrix further comprises one or more polynucleotide probes;
and the recombinant p19 fusion protein is labeled.
28. A kit according to claim 25, wherein the matrix is a bead.
29. A kit according to claim 25, wherein the bead is colored or
fluorescent.
30. A kit according to claim 27, wherein the bead is magnetic.
31. A small RNA, comprising a 21 nucleotide dsRNA.
Description
BACKGROUND
[0001] Existing methods for detection of small RNAs such as small
interfering RNAs (siRNAs) and micro RNAs (miRNAs) often involve
multiple steps: for example, immobilizing RNA on a filter (Northern
blot), hybridization with a specific probe if the small RNA is a
single-stranded miRNA, washing steps to remove the probe, and
exposure of the filter to a film. Alternatively, small
single-stranded RNAs (ssRNAs) such as micro RNAs (miRNAs) can be
detected using solution hybridization of a probe to the miRNAs,
RNAse treatment and gel electrophoresis to analyze the miRNA/probe
product. Detection of miRNA using a DNA array requires
fluorescent-labeling of total RNA. Labeling of samples adds
complexity and variability to the results. Methods that require DNA
amplification are sensitive but need corrections related to
efficiency of amplification. These methods are not appropriate for
rapid diagnostics or high throughput screening because of the
multiple steps involved in the analysis.
[0002] Evidence is accumulating that small RNAs such as miRNAs are
involved in human disease such as neurological diseases,
cardiomyopathies, and cancers (Alvarez-Garcia et al. Development
132:4653-4662 (2005)). Patterns of altered miRNA expression in
tissue biopsies may serve as diagnostic markers for these diseases.
For example, the use of a reliable quantitative method for
detecting the differential expression of certain miRNAs in various
tumors would be valuable for diagnosis and treatment of cancer.
SUMMARY
[0003] In an embodiment of the invention, a recombinant protein is
provided having at least 90% sequence homology to SEQ ID NO:33, and
being capable of binding a small double-stranded RNA (dsRNA). The
recombinant protein may be additionally labeled by means of a
fluorescent label, a radioactive label, a chemiluminescent label, a
protein label or a small molecule label. In another embodiment of
the invention, a DNA encoding the recombinant protein and a vector
for expressing the recombinant protein in a host cell are
provided.
[0004] In a further embodiment of the invention, a method is
provided that includes mixing a target RNA with a p19 fusion
protein capable of binding small dsRNA, wherein either the p19
fusion protein or the target RNA is labeled, the label being (i)
directly linked to the protein or RNA, or (ii) indirectly linked by
means of a molecule capable of binding to the p19 fusion protein or
the target RNA. The method further includes immobilizing the p19
fusion protein bound to the target RNA on a matrix for detecting
the target RNA. The detectable label is exemplified by a member of
the group consisting of a fluorescent label, a radioactive label, a
chemiluminescent label, a protein label and a small molecule
label.
[0005] The target RNA may be an ssRNA having a size in the range of
18 nucleotides to 24 nucleotides where the ssRNA hybridizes to a
complementary polynucleotide probe to form a double-stranded hybrid
polynucleotide for binding to the p19 fusion protein. The
complementary polynucleotide probe may extend at the 3' end beyond
the target RNA.
[0006] The polynucleotide probe may be an RNA, a DNA or a locked
nucleic acid. The target RNA may be a single-stranded molecule such
as a miRNA. The target RNA may be a double-stranded RNA such as an
siRNA.
[0007] In an embodiment of the invention, the p19 fusion protein is
immobilized prior to binding the target RNA. Alternatively, the
target RNA may be immobilized prior to binding the p19 fusion
protein. Alternatively, the p19 fusion protein may be bound to the
target RNA in solution and the p19 fusion protein dsRNA complex
immobilized on a matrix.
[0008] In embodiments of the invention, the matrix is a bead which
may be coated with a carbohydrate or other ligand to which the p19
fusion protein binds. The bead may be magnetic. The bead may be
colored or fluorescent in a manner that differs from the label on
the p19 fusion protein or polynucleotide probe.
[0009] In an embodiment of the invention, immobilization of target
RNA provides a diagnostic test for an abnormal condition in a cell
in which the target RNA is isolated from total RNA obtained from
the cell.
[0010] In an embodiment of the invention, a method is provided for
detecting a target RNA in a mixture of RNAs, such that if the
target RNA is (i) single-stranded, then a complementary
polynucleotide probe is added to the mixture for forming a dsRNA
and allows the dsRNA to bind to a p19 fusion protein; or (ii)
double-stranded, in which case dsRNA binds directly to the p19
fusion protein.
[0011] In either case, one of the complementary polynucleotide
probes or the p19 fusion protein is associated or linked to a label
selected from a fluorescent label, a radioactive label, a
chemiluminescent label, a protein label and a small molecule
label.
[0012] In a further embodiment of the detection method, p19 fusion
protein can be immobilized on a matrix for binding small dsRNA and
removing unbound RNA. Alternatively, small dsRNAs can be
immobilized on the matrix either by hybridizing a target ssRNA to a
matrix bound polynucleotide probe or by directly binding the target
dsRNA. In this case, the p19 fusion protein is preferably
labeled.
[0013] In a further embodiment of the invention, a kit is provided
that contains the recombinant p19 fusion protein described above,
instructions for detecting a small RNA, and optionally a matrix for
binding p19 fusion protein or a polynucleotide probe. The p19
fusion protein may be bound to a matrix in the kit. Alternatively,
a polynucleotide probe may be bound to the matrix, in which case,
the kit may additionally contain a labeled unbound p19 fusion
protein.
[0014] If a matrix is included in the kit, it may be a bead, for
example, a colored or fluorescent bead. The bead may be coated with
a carbohydrate for binding the p19 fusion protein. The bead may be
magnetic.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a summary of the biogenesis of miRNAs
(Esquela-Kerscher & Slack Nature Reviews Cancer 6:259-269
(2006). Transcription (1) of DNA results in the formation of a
primary miRNA (pri-miRNA) (SEQ ID NO:3). This is a dsRNA hairpin
structure that is cleaved by Drosha (2), a nuclear enzyme with
RNase III domains to form a pre-miRNA (3). The cleaved hairpin is
transported to the cytoplasm (4) by exportin 5. Secondary cleavage
occurs with Dicer (5) to generate a dsRNA of about 20 to 22 bases
in length. This RNA then enters the RNA-induced silencing complex
where it is unwound to form ssRNA that hybridizes with the 3'
untranslated region of the miRNA (6). The bound miRNA reduces
protein expression either by blocking translation or causing
cleavage of the miRNA (7).
[0016] FIGS. 2A-2C show properties of the molecule used for
discovery or detection of miRNA.
[0017] FIG. 2A shows a cartoon of a fusion protein consisting of a
maltose-binding protein (MBP) fused to p19 which is fused to a
chitin-binding domain (CBD).
[0018] FIG. 2B shows a gel containing the p19 fusion protein. The
figure demonstrates that this protein can be made in large
quantities.
[0019] FIG. 2C shows the crystal structure of the p19 fusion
protein bound to dsRNA. The view is looking down the center of the
RNA helix.
[0020] FIG. 3 demonstrates that the p19 fusion protein
(MBP-p19-CBD) preferentially binds dsRNA of 21 nucleotides (nt) but
does not bind dsRNA of 25 nucleotides or 17 nucleotides long. 0.5
.mu.g of MBP-p19 fusion protein bound to 30 ng of the 21-mer dsRNA
in a 20 .mu.l reaction. The reaction also contained 30 ng of the
17-mer and 25-mer dsRNA. Lanes 1-9 show use of increasing amounts
of MBP-p19-CBD fusion protein (.mu.g) and lane 10 is a size
marker.
[0021] FIGS. 4A-4B show additional characterization of the p19
fusion protein.
[0022] FIG. 4A shows that 10 ng, 20 ng, 30 ng and 40 ng of dsRNA
can be bound to chitin magnetic beads that contain 3 .mu.g of the
p19 fusion protein. The fusion protein is attached to the beads via
the CBD. When treated with SDS, the dsRNA is released from the
beads and can be detected on the ethidium stained gel.
[0023] FIG. 4B shows that a small amount of dsRNA of 21 nucleotides
can be purified from a large excess of cytoplasmic RNA using p19
fusion protein. Chitin magnetic beads with 5 pg of the bound p19
fusion protein were mixed with 27.5 .mu.g of total rat liver RNA
and 5 ng of 21-mer dsRNA. [0024] Lane 1 shows target dsRNA prior to
mixing with non-target RNA. [0025] Lane 2 shows the RNA that did
not bind to the p19 fusion protein chitin magnetic beads. [0026]
Lane 3 shows 20 .mu.l aliquot of the first 600 .mu.l wash of the
p19 fusion protein chitin magnetic beads. [0027] Lane 4 shows a 20
.mu.l aliquot of the sixth 600 .mu.l wash of the p19 fusion protein
chitin magnetic beads. [0028] Lane 5 shows the dsRNA eluted from
the beads. The top staining band is material trapped in the well.
[0029] Lane 6 contains a 17-mer, 21-mer and 25-mer dsRNA
marker.
[0030] FIGS. 5A and 5B show a competitive gel shift assay to
measure the relative affinity of RNA and DNA to the p19 fusion
protein.
[0031] FIG. 5A shows binding of radiolabeled dsRNA (21 nt) to p19
fusion protein in the presence of increasing amounts of the same
unlabeled dsRNA (21 nt). Each reaction contained 16 .mu.g of p19
fusion protein and 1 ng of radioactive dsRNA.
[0032] FIG. 5B shows binding of radiolabeled dsRNA (21 nt) to
fusion protein in the presence of increasing amounts of unlabeled
double-stranded DNA (dsDNA) (21 nt).
[0033] FIG. 5C shows binding of radiolabeled dsRNA (21 nt) to p19
fusion protein in the presence of increasing amounts of unlabeled
ribosomal RNA.
[0034] FIG. 5D shows binding of radiolabeled dsRNA (21 nt) to p19
fusion protein in the presence of increasing amounts of unlabeled
ssRNA (21 nt).
[0035] FIGS. 6A-6B show the results of eluting 20 ng of 21 nt dsRNA
from 20 .mu.l p19 fusion protein precoated beads (containing 3
.mu.g p19 fusion protein).
[0036] FIG. 6A shows that p19 fusion protein is stable when stored
at 4.degree. C. for at least 8 weeks such that the 21 nt RNA can be
released after the specified time.
[0037] FIG. 6B shows that 21-mer dsRNA is stable when bound to p19
fusion protein stored at 4.degree. C. or -20.degree. C. The absence
of RNA in the supernatent shows efficient binding of RNA. The
elution demonstrates quantitative recovery of dsRNA from p19 fusion
protein beads stored for different times and temperatures.
[0038] FIG. 7 shows the relative binding affinities of p19 fusion
protein to various RNA and DNA substrates. The binding of a labeled
siRNA to MBP-p19-CBD in the presence of increasing concentrations
of nucleic acids described in column 1 was measured by a gel
mobility shift assay. In each competitive assay, a control was
included in which 50% reduction in binding of the radiolabeled
21mer siRNA was observed in the presence of unlabeled 21mer siRNA.
This was assigned a value of one. The identity of the
polynucleotide, its sequence, its structure and its relative
binding affinity to MBP-p19-CBD are listed. The gaps in the
structure of the microRNAs in 13, 14 and 15 denote mismatched base
pairs.
[0039] FIG. 8 describes various approaches to labeling RNA.
Radioactive labeling is very sensitive and gives low background but
is not suitable for high throughput analysis and also has
regulatory issues. Fluorescence and chemiluminescence labeling are
both user friendly labeling methods and can be used in a 96 well
format with or without magnetic beads and are scaleable and can be
automated.
[0040] FIG. 9 shows how MBP-p19-CBD (measured in ng) when bound to
fluorescent dsRNA increases flourescence polarization which can be
quantitatively measured (mP). Fluorescence polarization is defined
by the following equation: P=(V-H)/(V+H) where P equals
polarization, V equals the vertical component of the emitted light,
and H equals the horizontal component of the emitted light of a
fluorophore when excited by vertical plan polarized light. The term
mP stands for 1/1000 of the polarization P. It is not dependent
upon concentration (Lundblad et al. Mol. Endocrinol 10:607-612
(1996)).
[0041] FIGS. 10A-10B show how the p19 fusion protein can be used
for discovery of novel endogenous siRNAs.
[0042] FIG. 10A shows the results of loading 350 .mu.g of total
unc-22 RNA extracted from C. elegans onto a 20% acrylamide TBE gel.
Extraction of RNA from the gel in the 15 to 30 base pair range
yields 6.1 .mu.g of small RNA.
[0043] FIG. 10B shows the results from binding the gel purified
small RNA to the p19 fusion protein chitin magnetic beads, washing
the beads and then eluting the RNA. The mobility of the eluted RNA
is indicated by an arrow at the right of the figure.
[0044] FIG. 11 shows the isolation of endogenous siRNA from a
filiarial parasite D. immitis using p19 fusion protein chitin
magnetic beads. 50,000 fold enrichment was obtained. The mobility
of the eluted RNA, in lane 9, is indicated by an arrow. [0045] Lane
1 is double-stranded siRNA marker. [0046] Lane 2 is 10 ng of a
21mer dsRNA. [0047] Lane 3 is gel purified D. immitis small RNA.
[0048] Lane 4 is gel purified RNA plus p19 fusion protein. [0049]
Lane 5 is RNA not bound to chitin magnetic beads with p19 fusion
protein. [0050] Lane 6 is the first wash of the p19 chitin magnetic
beads. [0051] Lane 7 is the result of wash 2. [0052] Lane 8 is the
result of wash 3. [0053] Lane 9 is the first elution from the
beads. [0054] Lane 10 is the second elution from the beads.
[0055] FIG. 12 shows a protocol for detecting dsRNA using
biotin-labeled p19 fusion protein. A polynucleotide probe (9) is
covalently linked to beads (8). Small ssRNAs (tRNAs, rRNAs etc.)
(10) hybridize to the polynucleotide probe (9) to form a stable RNA
hybrid on the bead (8). A biotin-labeled p19 fusion protein (12)
binds to RNA hybrids on the beads (8). The p19 fusion protein can
be labeled using biotin, which can bind tightly to streptavidin
(13). The streptavidin can be detected by linkage via a second
biotin molecule to an enzyme, like alkaline phosphatase or
peroxidase, or by means of a fluorescent-labeled protein.
Substrates for the enzyme will give a colored or fluorescent
product that can be detected with a laser. The beads to which p19
fusion protein attaches can be identified with a laser which
detects the bar code signature of dyes. The amount of miRNA bound
to a specific bead can be measured by the amount of p19 fusion
protein bound to the beads.
[0056] FIG. 13 shows p19 fusion protein capture of dsRNA hybrid and
detection with a miRNA specific probe. An ssRNA polynucleotide (9)
labeled with
(5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacet-
amido]fluorescein (FAM) binds specifically to miRNA (10) in a
background of total RNA including tRNA, rRNA and mRNA (11) to form
a dsRNA that binds to a p19 fusion protein (12) which is attached
to a solid support, like an ELISA plate (14) or a bead for
detection.
[0057] FIG. 14 shows a p19 fusion protein-based miRNA detection
method. Total cellular RNA, which includes miRNA, rRNA and mRNA
(11), is hybridized to a specific probe complementary to a miRNA
(9). The double-stranded miRNA/RNA probe is then selectively and
tightly bound to p19 fusion protein (12) chitin magnetic chitin
beads (8). The unbound probe can be removed rapidly by washing the
p19 fusion protein beads and then isolating them with the aid of a
magnetic rack (15). The dsRNA is then eluted from the beads in the
presence of a protein denaturing agent.
[0058] FIGS. 15A and 15B show a diagnostic test for miRNAs.
[0059] FIG. 15A shows p19 fusion protein (12) attached to a bead
(8) and binding dsRNA where a single strand of the duplex is
labeled with biotin (9). Streptavidin (13) is bound to the biotin
and with the help of a suitable enzyme label (15) and a substrate
(16), a chemiluminescent or fluorescent reaction can be
initiated.
[0060] FIG. 15B shows a standard curve assay for quantifying the
results from the detection method described in FIG. 15A. The
biotin-labeled RNA probe is linked, via a streptavidin bridge, to
alkaline phosphatase. The enzyme is detected using the substrate
CDP-Star, #N7001S from New England Biolabs (NEB, Ipswich, Mass.),
which generates light.
[0061] FIG. 16 shows a biotin-labeled ssRNA (9) immobilized on a
streptavidin (13) coated solid substrate (14). Target miRNA (10) is
hybridized to the immobilized polynucleotide (9). The dsRNA is
subsequently recognized and bound by a labeled p19 fusion protein
(12) for detection.
[0062] FIGS. 17A-17D show the quantitative measurement of the liver
specific miRNA (miRNA122a) from rat liver total RNA.
[0063] FIG. 17A shows a non-denaturing gel with increasing amounts
of synthetic miRNA122a in picograms (pg), hybridized to 1 ng of a
radioactive RNA probe complementary to miR-122a. The dsRNA is bound
to p19 fusion protein chitin magnetic beads, washed, eluted and
analyzed on a 20% non-denaturing acrylamide gel. The radioactivity
is associated with the miRNA/RNA probe.
[0064] FIG. 17B shows a standard curve for miR122 that was
calculated from radioactivity eluted from the p19 fusion protein
beads.
[0065] FIG. 17C shows a non-denaturing gel of miRNA/RNA probe
eluted from p19 fusion protein beads using a miR122 specific probe
and different amounts of rat liver RNA.
[0066] FIG. 17D gives the results for 2, 5 and 10 .mu.g of rat
liver total RNA containing between 38 and 68 pg of miRNA122a/.mu.g
of total RNA.
[0067] FIG. 18 shows detection of Let-7 miRNA in total JURKAT cell
RNA using an autoradioagraph of an acrylamide gel in which the
labeled RNA corresponds to the miRNA/RNA probe hybrid, which is
shown to increase according to the total RNA. A radioactive RNA
probe complementary to Let-7 was hybridized to different amounts of
JURKAT cell RNA, bound to p19 fusion protein magnetic beads, washed
and eluted. Lanes 2-4 show 2 .mu.g, 5 .mu.g and 10 .mu.g of total
JURKAT RNA respectively.
[0068] FIG. 19 shows the DNA and amino acid sequences (SEQ ID
NOS:32 and 33) for CBD-p19-MBP where MBP corresponds with amino
acid 1 to 394 and nucleotide 1 to 1183, p19 corresponds with amino
acid 395 to 565 and nucleotide 1184 to 1698, a polylinker
corresponds with amino acid 566 to 575 and nucleotide 1698 to 1726
and CBD corresponds with amino acid 576 to 626 and nucleotide 1727
to 1878.
DETAILED DESCRIPTION OF THE EMBODIMENTS
[0069] Existing methods for detection of small RNAs are often
complex and require ligation and amplification or gel
electrophoresis steps. A fusion protein is described here that can
be used in a simplified, sensitive and quantitative assay to detect
and/or isolate small RNAs.
[0070] The fusion protein described here is exemplified by a p19
fusion protein which binds a dsRNA regardless of the sequence but
in a size-specific manner, is capable of being immobilized on a
substrate, and can be readily purified.
[0071] RNAs of interest can be isolated from biological samples
using the p19 fusion protein. This protein may also be used to
detect specific endogenous RNAs of a specific size in physiological
samples that contain a wide variety of RNAs normally associated
with cells. The amounts of the RNAs of interest can be determined
using quantitative assays (see for example, FIGS. 9, 15 and
17).
[0072] Definitions
[0073] The term "small" RNA as used here and in the claims refers
to RNA fragments containing or capable of forming a double-stranded
region of a size of greater than about 17 nucleotides and less than
about 25 nucleotides, for example 21-23 nucleotides. The dsRNA may
result from two complementary strands in a linear duplex or a
single-stranded molecule that is folded to form a hairpin.
[0074] Where a small RNA is single-stranded, it can be hybridized
to a complementary polynucleotide probe to create either a
completely dsRNA molecule or RNA/DNA hybrid or a partially dsRNA.
The RNA/DNA or RNA/DNA hybrids may include a single-stranded
polynucleotide tail at one end of the polynucleotide probe that
extends beyond the duplex region containing the target RNA.
[0075] Examples of small RNAs include miRNAs, siRNAs, repeats
associated RNAs (rasiRNAs). rasiRNAs are found for example in C.
elegans and may prevent migration of transposons.
[0076] The "p19 fusion protein" refers to a member of the p19
family of RNA binding proteins (Silhavy et al. Embo J. 21:3070-80
(2002) fused to one or more additional proteins which surprisingly
retains the binding properties of the native enzyme where the
fusion protein binds small dsRNA in a sequence-unspecific but
size-specific manner (see for example, FIGS. 2-6). The binding
properties of the p19 fusion protein was determined for various
substrates using competitive gel shift analysis. The results
confirmed that the p19 fusion protein does not bind ssRNAs or dsDNA
but does bind dsRNA in a size specific manner (FIG. 7).
[0077] P19 proteins are highly conserved in Tombus plant viruses.
Related proteins have been isolated from many plant viruses such as
Carnation Italian ringspot virus p19 (NP 612584), Tomato bushy
stunt virus (CAC01278), Artichoke mottled crinkle virus (NP
039812), Lettuce necrotic stunt virus (CAC01267), Lisianthus
necrosis virus (CAM98056), Grapevine Algerian necrosis virus
(AAX76895), Cucumber necrosis virus (CAC01089), Pelargonium
necrotic spot virus (NP 945118), Cucumber Bulgarian virus
(AA033943), Maize necrotic streak virus (AAG21219), Pear latent
virus (AAM49806), Grapevine Algerian latent virus (AAX76895), and
Cymbidium ringspot virus (CAA33535) (accession numbers in
parenthesis).
[0078] The p19 proteins described above includes a fusion to one or
more proteins where at least one of the proteins has a size greater
than 10 amino acids. Examples of proteins for fusion to p19 include
carbohydrate-binding proteins exemplified by MBP, CBD and
cellulose-binding domain; enzymes such as O6-alkylguanine-DNA
alkyltransferase (U.S. Patent Applications 2006/0292651;
2006/0024775; 2004/0115130; 2007/0082336; 2007/0207532;
2007/0243568) or luciferase that are capable of responding to a
substrate to produce fluorescence or a detectable color signal;
enzyme substrates such as biotin; antibodies; and protein
epitopes.
[0079] A "polynucleotide probe" refers to a ssDNA, ssRNA or a
locked nucleic acid that may be synthetic and is complementary at
least in part to the target RNA if the target RNA is
single-stranded (Vester & Wengel Biochemistry 43(42):13233-41
(2004)). The probe may be labeled. Alternatively, the probe is not
labeled and is used in an assay in which the p19 fusion protein is
or becomes labeled during the assay.
[0080] Methods of Making p19 Fusion Proteins
[0081] The formation of fusion proteins can be readily achieved
using DNA vectors available in the art such as those described in
the New England Biolabs (NEB, Ipswich, Mass.) catalog. Example 1
which is not intended to be limiting describes a method of making a
p19 fusion protein from Carnation Italian ring spot virus in which
the protein is fused at the amino end to an MBP, which permits
purification of the protein, and at the carboxy end to the CBD, for
tightly binding the magnetic chitin beads (FIG. 2).
[0082] Labeling the p19 Fusion Protein or Polynucleotide Probe
[0083] Labeling a target molecule may be achieved either directly
or indirectly by labeling a molecule capable of binding the target
molecule. These labels may be attached to a reagent polynucleotide
probe for binding the target RNA, or to the p19 fusion protein. Any
suitable label known in the art can be used such as a radioactive
label (for example, .sup.32P), a fluorescent label, a chromogenic
label such as phycoerythrin, an enzyme label or a modified base for
reacting with an enzyme (biotin-strepavidin).
[0084] Examples of methods of detection using fluorescent labels
include FAM. Another method is Fluorescence Resonance Energy
Transfer (FRET). This involves two different fluorescent molecules
(or proteins), one for linking to the p19 fusion protein and the
other for linking to the RNA probe. When the dsRNA binds to the p19
fusion protein, the two molecules are in close enough proximity for
efficient energy transfer to generate a fluorescent signal. This
method does not require removal of the unbound RNA probe and is
therefore suitable for large scale screening. Another method is
Fluorescent Polarization (FP) where the two subunits of the
MBP-p19-CBD protein are about 10.times. the molecular weight of the
17-25 base-paired dsRNA. This difference in size between the bound
and unbound RNA can be detected by FP. Another method is quantum
dot analysis such as described by Yezhelyev et al. J Am Chem Soc.
130(28):9006-12 (2008)), which may also be used for detection of
dsRNAs.
[0085] Examples of enzyme labels include methods in which for
example a luciferase, green fluorescent protein or alkyl guanine
DNA alkyl transferase are used. Small molecules such as biotin may
be used. Biotin is linked to the polynucleotide probe, and
avidin-bound enzymes, like horseradish peroxidase or alkaline
phosphatase react with biotin through streptavidin to signal the
presence of a molecule of interest. This is a standard method for
ELISA detection of antibodies.
[0086] Immobilization of Reagents
[0087] The target RNA may be immobilized either directly or
indirectly on or in a matrix. Indirect immobilization of the target
RNA may occur by means of (a) hybridizing a target ssRNA to a
matrix bound polynucleotide probe to form a small dsRNA which is
subsequently recognized by a p19 fusion protein in solution; or (b)
binding small dsRNA to p19 fusion protein bound to a matrix; or (c)
binding dsRNA with p19 fusion protein in solution and then binding
the complex to a matrix.
[0088] The p19 fusion protein can be readily immobilized on a
matrix if that solid substrate is coated with a molecule with which
the fusion protein binds, for example, chitin for binding
chitin-binding domain or amylose for binding MBP. A polynucleotide
probe can be bound to a matrix by means of a ligand such as
biotin.
[0089] Examples of matrices include beads, columns, microtiter
plates, a chip, or other 2-dimensional or three-dimensional formats
known in the art. In addition, channels coated with chitin in
microfluidic devices may be used to immobilize RNAs of
interest.
[0090] Sensitivity of the Assay
[0091] Levels of enrichment of dsRNA of any sequence having a size
preferably of greater than 17 nucleotides and less than 25
nucleotides can be achieved of greater than 20,000 fold and as much
as 100,000 fold from total RNA. Moreover, as little as 10 pg of
miRNA can be detected in a million-fold excess of total RNA and 50
pg of miRNA can be measured in total cell RNA from a tissue.
Competitive gel shift data demonstrate that neither unbound
single-stranded probe nor cytoplasmic RNA blocks binding of a miRNA
and RNA probe in a p19 fusion protein detection assay.
[0092] Uses of the Assay
[0093] A specific polynucleotide probe may be attached to a
substrate such as a plate or beads using standard methodology (for
example biotin-steptavidin labeling) and exposed to a cell lysate
from, for example, a biopsy. Only ssRNA of a specific sequence
contained in the cell lysate will bind the immobilized
polynucleotide probe. Labeled p19 fusion protein may then bind to
the dsRNA or RNA/DNA hybrid and can be detected. FIGS. 17 and 18
demonstrate the feasibility of this approach for the quantitative
detection of mir122a miRNA in total RNA extract from rat liver.
Examples 4 and 5, FIGS. 17 and 18 illustrate how radioactive
labeling or fluorescent-labeling is effective in detecting small
amounts of the target miRNA in total lysate.
[0094] The labeled p19 fusion protein may either be in solution and
optionally subsequently immobilized or already immobilized on a
coated matrix (for example, chitin) where for example, the matrix
is magnetic beads or the well of a microtiter plate. Accordingly,
unbound material can be removed and the target RNA can be
subsequently eluted from the matrix for analysis.
[0095] This approach can be adapted for high throughput screening
of samples for target miRNAs (FIG. 14). Automation of detection can
be facilitated using magnetic beads. For example, a biotinylated
probe, previously hybridized to a miRNA target, can be linked to a
plate coated with streptavidin. In this way, a labeled p19 fusion
protein provides a signal for detecting miRNA (FIG. 16).
[0096] In an embodiment, automation of detection for high
throughput processing of samples is facilitated by scanning an
ELISA plate for binding of modified p19 fusion protein with labeled
double-stranded miRNA. The ELISA plate can be coated with chitin to
which p19-CBD fusion proteins bind. The unbound RNA can be removed
by washing the plate and the remaining bound RNA can be detected by
means of the attached probe. If the probe contains biotin, then it
can be linked to a chromogenic readout using avidin conjugated to
alkaline phosphatase or horseradish peroxidase (FIG. 15). An ELISA
plate format for miRNA detection has many advantages. There exists
an extensive amount of instrumentation for washing, reading and
handling of ELISA plates. High throughput analysis is an important
feature of analyzing clinical samples.
[0097] The methods and compositions described herein may be used to
quantitatively determine the presence and amounts of endogenous
miRNAs for purposes that include diagnosis or analyzing a wide
range of pathologies such as cancers including determining the
tissue of origin of the metastasized cancer, neuropathologies, and
pathologies in other organs. These methods provide rapid, cost
effective, efficient and scaleable detection of miRNAs in
vitro.
[0098] All references cited above and below, as well as U.S.
provisional application Ser. No. 60/983,503 filed Oct. 29, 2007,
are incorporated by reference.
Examples
Example 1
Cloning of the D19 Fusion Protein and Binding to Size-Specific
dsRNA
[0099] The p19 protein from the Carnation Italian ringspot virus
codes for a 19 kDa protein and has a binding preference for 17-25
by RNAs (Vargason et al. Cell 115:799-811) (2003)).
[0100] Sequence of the p19 protein from the Carnation Italian
ringspot virus is as follows:
TABLE-US-00001 (SEQ ID NO: 1) 1 MERAIQGNDT REQANGERWD GGSGGITSPF
KLPDESPSWT EWRLYNDETN SNQDNPLGFK 61 ESWGFGKVVF KRYLRYDRTE
ASLHRVLGSW TGDSVNYAAS RFLGANQVGC TYSIRFRGVS 121 VTISGGSRTL
QHLCEMAIRS KQELLQLTPV EVESNVSRGC PEGIETFKKE SE
[0101] The plasmid vector pMAL-c2G from New England Biolabs (NEB,
Ipswich, Mass.), was cleaved with PstI and HindIII, within the
multiple cloning site. The following two PCR primers were used to
amplify the chitin-binding domain, CBD, from the plasmid pTYB1 from
New England Biolabs (NEB, Ipswich, Mass.): 5' G ACT CTG CAG ACG ACA
AAT CCT GGT GTA TCC GCT 3' (SEQ ID NO:34) CBD (PstI) forward primer
and 5' T AGG AAG CTT TCA TTG AAG CTG CCA CAA GGC AGG AAC 3' (SEQ ID
NO:35) CBD (HindIII) reverse primer. After amplification the PCR
product was cleaved with Pst I and Hind III and cloned into the
plasmid pMAL-c2G. The new vector was then cleaved with BamHI. The
p19 fusion protein coding sequencing, accession number NC 003500,
was amplified with two primers containing BamHI sites. The PCR
products was cleaved with BamHI and then cloned into the MBP CBD
vector described above. The resulting plasmid construct coded for a
fusion protein that contained an amino terminal MBP and a carboxy
terminal CBD.
[0102] The MBP-p19-CBD fusion protein was isolated in high yields
by binding and elution from amylose resin. The fusion protein was
shown to be functionally active as described below.
[0103] The MBP-p19-CBD fusion protein (FIG. 19) was shown to bind
an siRNA in a size-dependent sequence-independent manner where the
size was preferably greater than 17 nucleotides and smaller than 25
nucleotides. (FIG. 3) It was concluded that the presence of a
large, 42 kDa, fusion partner like MBP does not have a major effect
on siRNA binding to p19 fusion protein (FIG. 2).
[0104] The affinity of the p19 fusion protein for different
substrates was determined by gel shift analysis (FIGS. 5 and 7).
The affinity of p19 fusion protein was greater for siRNAs than for
miRNAs which contained mismatched base pairs. The p19 fusion
protein bound to 21-mer dsRNA/DNA hybrid but not to dsDNA of the
same size or ssRNA, single-stranded DNA or ribosomal RNA. Binding
of RNA to p19 was also detected by fluorescence-polarization
studies that used fluorescent-labeled RNA (FIGS. 5, 7 and 9).
[0105] The MBP-p19-CBD fusion protein was used for siRNA isolation
(FIGS. 12 and 13). Small RNAs that bound to the p19 fusion protein
were purified with chitin magnetic beads. The CBD portion of the
p19 fusion protein attached the dsRNA:protein complex to the beads
(FIGS. 14 to 16). Background binding could be reduced with the
addition of bovine serum albumin (BSA). The small RNAs were eluted
from the chitin beads by denaturing the protein with 0.5% SDS.
[0106] In a reconstruction experiment, a defined amount of siRNA
was added to total rat liver RNA, bound to p19 fusion protein,
concentrated with chitin magnetic beads and eluted. Greater then
5,000.times. enrichment was obtained using this approach (FIGS. 3
and 4).
Example 2
Determination of the Sensitivity and Quantification of the
Detection Method for RNAs using p19 Fusion Protein and Liquid
Scintillation
[0107] A method was developed using .sup.32P-labeled polynucleotide
probes to detect and quantify the abundance of endogenous miRNA in
a total RNA sample. The p19 fusion protein specifically detected a
hybrid between a miRNA and a labeled radioactive RNA probe in a one
million-fold excess of cytoplasmic RNA. This was demonstrated using
the abundant liverspecific miRNA, miR122a. A standard curve was
made using increasing amounts of synthetic miR122a mixed with a
large excess of JURKAT total RNA (T lymphocyte cell) to mimic assay
conditions. JURKAT cell total RNA did not contain any detectable
miR122a by this method. Variable amounts of miR122a oligo were
hybridized to a constant amount or radioactive RNA probe, which was
complementary to miR122a. A background standard lacking any added
miR122a oligo was processed in the same as the samples (FIG.
17).
[0108] A quantitative measurement of endogenous miR122a was made by
incubating rat liver total RNA with the miR122a specific probe.
Three assays with different amounts of rat liver total RNA were
performed to ensure concordance of the results. After
hybridization, each sample was incubated with p19 fusion
protein-coated magnetic beads to allow for binding of the
miR122a-probe duplex. To remove the unbound RNA, p19 fusion protein
beads were washed 5 times and the miR122a duplex was eluted from
the beads by denaturing p19 fusion protein with an elution buffer
containing 0.5% SDS. The eluted radioactive duplex was counted and
the background control was subtracted from each sample. To
demonstrate that the radioactivity corresponded to the
miR122a/probe, the eluent was loaded on an acrylamide gel (FIG. 5A)
and exposed to X-ray film. As observed on the autoradiograph, the
eluted RNA was double-stranded, not single-stranded probe.
[0109] After subtracting the control radioactive count from each
result, the standard counts corresponding to the miR122a duplex
were plotted as a function of synthetic miR122a amount (FIG. 5B). A
linear curve demonstrated the proportionality between the
radioactive signal and the amount of miR122a complex. Comparison to
the standard curve provides a relative measurement of the miR122a
abundance in a physiological sample (FIGS. 5C and 5D). A value of
50 pg +/-12 pg of miR122a per .mu.g of rat liver total RNA was
obtained for the three concentrations of RNA. The detection is
linear over two orders of magnitude and has a sensitivity of 2-5 pg
of miRNA.
(a) Labeling miR122a Probe using .gamma.-.sup.32P-ATP
[0110] In a microfuge tube, 300 ng of miR122a probe (5'
OH-aacaccauugucacacuccaua) was added to 2 .mu.L of T4
polynucleotide kinase reaction buffer (70 mM Tris-HCl (pH7.6), 10
mM MgCl2, 5 mM dithiothreitol from New England Biolabs (NEB,
Ipswich, Mass.)) and 3 .mu.L of miliQ water. Then, 10 .mu.L of
.gamma.-.sup.32P- ATP (PerkinElmer, Waltham, Mass.) at 6,000
Ci/mmol and 2 .mu.L of T4 polynucleotide kinase (10,000 units/mL)
from New England Biolabs (NEB, Ipswich, Mass.) were added to the
labeling reaction. The reaction tube was placed in a 37.degree. C.
heat block for 40 to 60 minutes, and then the reaction was stopped
by inactivating the enzyme at 65.degree. C. for 20 minutes. After
stopping the reaction, the entire labeling reaction was loaded onto
a CentriSep column (Princeton Separation, Freehold, N.J.) and
centrifuged for 2 minutes at 3,000 rpm to remove the excess
.gamma.-.sup.32P-ATP. The specific activity of labeled probe was
determined by counting 1 .mu.L of a ten-fold dilution of the
purified labeling reaction in a scintillation counter.
(b) Total RNA Extraction from JURKAT Cells
[0111] JURKAT total RNA was obtained using Trizol (Invitrogen,
Carlsbad, Calif.) as recommended by the manufacturer. 10 mL of
Trizol were added to the JURKAT cell pellet. The sample was
transferred in a 30 mL tube and cells were disrupted by
homogenization using a syringe. After complete homogenization, the
tube was incubated 5 minutes at room temperature to permit the
complete dissociation of nucleoprotein complex. 2 mL of chloroform
was added to the sample, vortexed for 15 seconds and incubated at
room temperature for 2 to 3 minutes and centrifuged at 4.degree. C.
for 15 minutes at 12,000 g. To precipitate the RNA sample, the
aqueous phase was transferred to a new tube containing 5 mL of
isopropanol, placed at room temperature for 10 minutes and
centrifuged at 4.degree. C. for 10 minutes at 12,000 g. The RNA
pellet was then washed with 10 mL 75% ethanol and thoroughly
drying. The pellet was resuspended with 200 .mu.L of sterile
Tris-EDTA. The total RNA concentration was estimated via optical
density using the nanodrop spectrophotometer and the purity was
evaluated by the ratio A260 nm/A280 nm.
(c) Assay for Small RNAs
[0112] RNA samples were added to loading buffer and loaded into
pre-rinsed wells of a TBE Gel 20% acrylamide (Invitrogen, Carlsbad,
Calif.) as well as the RNA size marker (siRNA Marker, New England
Biolabs (NEB, Ipswich, Mass.)). After running at 100V for 2 hours,
the gel was stained with SybrGold (Invitrogen, Carlsbad, Calif.)
and visualized on a fluorimager. In the competitive binding assays
of RNA/DNA to p19 fusion protein, the gels were exposed to X-ray
film and the bands scanned to determine relative binding
affinities.
(d) Quantification of Endogenous miR122a using p19 Fusion
Protein-Based Liquid Scintillation miRNA Detection Method
[0113] Quantification of sensitivity was determined by (1) varying
amounts of total rat liver RNA (2, 5 and 10 .mu.g) incubated with 1
ng of specific probe; and (2) varying amounts of miR122a RNA
(target RNA) (500 pg to 0.32 pg).
[0114] A miR122a complementary sequence was synthesized and labeled
with a radioactive label. Specifically, 5' .gamma.-.sup.32P
aacaccauugucacacuccaua (SEQ ID NO:2) was labeled at the 5' end
using .gamma.-.sup.32P-ATP. Incubation was performed 2 hours at
65.degree. C. in 1.times. p19 fusion protein binding buffer (20 mM
Tris HCl, 100 mM NaCl, 1 mM EDTA and 1 mM TCEP, pH 7 at 25.degree.
C.). The labeled probe was mixed with miR122a RNA and total JURKAT
RNA and incubated for 2 hours at 65.degree. C. to allow
hybridization between the target miR122a and the probe.
[0115] Hybridized dsRNA was allowed to bind to p19 fusion protein
coated beads. These beads were made as follows: Chitin magnetic
beads (NEB#E8036) were pretreated with BSA by washing the beads in
1.times. BSA buffer (20 mM Tris HCl, 100 mM NaCl, 1 mM EDTA, 1 mM
TCEP and BSA at 1 mg/mL pH 7 at 25.degree. C.) twice using a
magnetic rack (New England Biolabs #S1506S (NEB, Ipswich, Mass.))
and resuspended in the same buffer and incubated overnight at
4.degree. C. p19 fusion protein-bound magnetic beads were made by
mixing p19-CBD fusion protein with pretreated chitin beads (30
.mu.g p19 fusion protein for 200 .mu.l beads suspension) in 200
.mu.L 1.times. p19 fusion protein binding buffer with 1 mg/mL BSA
and incubated at 4.degree. C. overnight. The subsequent protein
beads were stored at 4.degree. C.
[0116] Binding of miR122a-probe duplex to p19 fusion protein
occurred when 10 .mu.L of the p19 fusion protein-coated magnetic
beads were incubated for 1.5 hours at room temperature in an
orbital shaker in 1.times. p19-binding buffer containing RNAse
inhibitor and BSA at 1 mg/mL. Unbound RNA was eliminated by washing
the beads 5 times with 500 .mu.L 1.times. p19 fusion protein wash
buffer prewarmed at 37.degree. C. (20 mM Tris HCl, 100 mM NaCl, 1
mM EDTA and 1 mM TCEP, pH 7 at 25.degree. C.) shaking for 5 minutes
at room temperature.
[0117] miR122a-probe duplex was eluted from the beads with 20 .mu.L
of 1.times. p19 fusion protein elution buffer (20 mM Tris HCl, 100
mM NaCl, 1 mM EDTA and 0.5% SDS, pH 7 at 25.degree. C.) after
incubation 10 minutes at 37.degree. C. followed by mixing 10
minutes at room temperature. The beads were spun down and the
supernatant was removed. 18 .mu.L of the elution were loaded on 20%
acrylamide gel (Invitrogen, Carlsbad, Calif.) using TBE buffer and
2 .mu.L of the eluted RNA were used for radioactive counting. The
results are shown in FIG. 17.
(e) Qualitative Detection of Let-7a using p19 Fusion Protein-Based
Liquid Scintillation miRNA Detection Method
[0118] miRNA Let-7a was detected in JURKAT total RNA as follows:
Three samples were prepared containing different amounts of JURKAT
total RNA. JURKAT total RNA was incubated with the Let-7a specific
probe to allow hybridization between the target and probe. The
probe was a synthetic Let-7a complementary sequence labeled at the
5'end using .gamma.-32P-ATP. After hybridization, the three samples
containing the potential Let-7a complex were incubated with p19
fusion protein-coated beads.
[0119] To remove the unbound RNA and the excess single-stranded
probe, p19 fusion protein coated beads were washed 5 times and the
Let-7a complex was eluted from the beads by denaturing p19 fusion
protein with an elution buffer containing 0.5% SDS. The
radioactivity in each sample was measured using liquid
scintillation counting. To ensure that the radioactive count
corresponded to the signal of the Let-7a duplex, the rest of the
elution was separated and autoradiographed (FIG. 7).
[0120] The results confirmed that Let-7a miRNA was present in the
JURKAT cell line at detectable levels by gel electrophoresis and
scintillation counting.
[0121] Different samples containing various amount of JURKAT total
RNA were incubated with 600 pg of a specific RNA probe (synthetic
Let-7a complementary sequence, 5'
.gamma.-.sup.32P-cuauacaaccuacuaccucaaa) (SEQ ID NO:2) labeled at
the 5'end using .gamma.-.sup.32P-ATP. Incubation was performed for
2 hours at 65.degree. C. in 1.times. p19 fusion protein binding
buffer (20 mM Tris HCl, 100 mM NaCl, 1 mM EDTA and 1 mM TCEP, pH 7
at 25.degree. C.) to allow hybridization between the target Let-7a
and the RNA probe. In order for the p19 fusion protein-coated beads
to recognize the Let-7a-probe duplex, the solution containing miRNA
duplex was incubated with p19 fusion protein beads (from 10 .mu.L
of a suspension of p19 fusion protein coated beads) for 1.5 hours
at room temperature in 1.times. p19 fusion protein binding buffer
containing RNAse inhibitor and BSA at 1 mg/mL. Unbound RNA was
eliminated by washing the beads 5 times with 500 .mu.L 1.times. p19
fusion protein wash buffer pre-warmed at 37.degree. C. (20 mM Tris
HCl, 100 mM NaCl, 1 mM EDTA and 1 mM TCEP, pH 7 at 25.degree. C.)
shaking for 5 minutes at room temperature. The Let-7a-probe duplex
was eluted from the beads with 20 .mu.L of 1.times. p19 fusion
protein elution buffer (20 mM Tris HCl, 100 mM NaCl, 1 mM EDTA and
0.5% SDS, pH 7 at 25.degree. C.) after incubation 10 minutes at
37.degree. C. followed by mixing 10 minutes at room temperature.
The beads were spun down and the supernatant was removed. 18 .mu.L
of the elution were loaded on TBE 20% acrylamide gel (Invitrogen,
Carlsbad, Calif.) and 2 .mu.L were used for radioactive counting.
The results are shown in FIG. 18.
(f) Binding of p19 Fusion Protein to miRNA/RNA PolyA-Tail Probe
[0122] To improve the sensitivity of detection, p19 fusion protein
binding to a miRNA hybridized to a longer RNA probe was tested.
Synthetic miR122a was hybridized to a FAM-labeled probe containing
a 20 to 30 base poly A-tail. The hybrid obtained was incubated with
p19 fusion protein and separated by PAGE electrophoresis and
stained by SybrGold (Invitrogen, Carlsbad, Calif.). A mobility
shift was observed when the hybrid was incubated with the p19
fusion protein. No shift was observed in the sample without p19
fusion protein. This result demonstrated that p19 fusion protein
binds a miRNA/RNA polyA-tail probe (FIG. 8) and may be used in
future detection methods. An increase in the length of the
polynucleotide probe allows for increased sensitivity of
detection.
[0123] 10 ng of synthetic miR122a was hybridized to 10 ng of probe
containing a non-radioactive 3' polyA-tail (5'
P-aacaccauugucacacuccaua-polyA tail) (SEQ ID NO:2) at 65.degree. C.
for 10 minutes. The hybrid obtained was incubated with 1.5 .mu.g of
p19 fusion protein (New England Biolabs (NEB, Ipswich, Mass.)) at
room temperature for 1.5 hours in binding buffer (20 mM Tris HCl,
100 mM NaCl, 1 mM EDTA, 1 mM TCEP, pH 7 at 25.degree. C.), to allow
binding. A sample without p19 fusion protein was performed in
identical condition as a control. The two samples were loaded on
20% acrylamide gel in TBE buffer (Invitrogen, Carlsbad, Calif.) and
stained by SybrGold (Invitrogen, Carlsbad, Calif.).
Example 3
A D19 Fusion Protein-Based RNA Detection Method using a
Fluorescent-Labeled Polynucleotide Probe
[0124] A standard curve using a synthetic FAM-labeled RNA probe
(FIG. 9) complementary to miR31 was created. A background standard
without synthetic miR31 was processed in a similar way. The
hybridization step was performed in presence of a large excess of
JURKAT total RNA. JURKAT cells do not have miR31. Each sample was
processed in triplicate to ensure concordance of the results. After
hybridization, each sample was incubated with p19 fusion
protein-coated magnetic beads to allow recognition between
miR31-probe duplex and p19 fusion protein protein. To remove
unbound RNA, p19 fusion protein beads were washed twice and the
miR31 duplex was eluted from the beads by denaturing p19 fusion
protein with an elution buffer containing 0.5% SDS. Fluorescence of
the samples was read with an excitation wavelength of 485 nm, an
emission wavelength of 520 nm and a cutoff of 495 nm. The
background signal was subtracted from each standard to remove the
signal due to non-specific binding to the beads. For each
triplicate, we calculated the average and standard deviation. The
average of standard Relative Fluorescence Unit (RFU) corresponding
to the miR31 duplex was plotted as a function of synthetic miR31. A
linear graph demonstrated the proportionality between the
fluorescent signal and the amount of the miR31 complex.
[0125] To increase the sensitivity and reduce the variability, each
component of the buffer was analyzed to determine which reagents
might cause a shift or a quenching of the specific fluorescence
signal. To reduce the fluorescent background, each buffer and
sample were maintained in the absence of UV absorbing compounds and
dust. Excitation scans obtained demonstrated that the
[tris(2-carboxyethyl)phosphine] (TCEP) contained in each buffer
quenched the fluorescent signal (FIG. 11A). Unmodified binding
buffer absent TCEP and BSA (20 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA,
pH 7.0 at 25.degree. C.) was used. Sensitivity tests were performed
to define the best conditions for optimal sensitivity. The
sensitivity was higher when the miRNA was in an elution buffer
containing 0.1M NaOH (detect 5 pg) compared with an elution buffer
containing SDS 0.5% (detect 40 pg) (FIG. 12). Elution of the miRNA
complex from the p19 fusion protein beads was performed using an
alkaline buffer containing 0.1 M NaOH.
[0126] Fluorescence of the samples was read with an excitation
wavelength of 492 nm, an emission wavelength of 520 nm and a cutoff
of 515 nm. The background signal was subtracted from each sample.
Reactions were in triplicate. The average of standard Relative
fluorescence units (RFU) corresponding to the miR31 duplex was
plotted as a function of synthetic miR31 amount. The detection is
linear. The variability of the detection protocol was markedly
reduced in alkaline buffer with a detection limit of 10 pg compare
to 5 ng for the previous experiment suggesting reduced fluorescence
background and a higher sensitivity of detection.
Sequence CWU 1
1
351172PRTCarnation Italian ringspot virus 1Met Glu Arg Ala Ile Gln
Gly Asn Asp Thr Arg Glu Gln Ala Asn Gly1 5 10 15Glu Arg Trp Asp Gly
Gly Ser Gly Gly Ile Thr Ser Pro Phe Lys Leu 20 25 30Pro Asp Glu Ser
Pro Ser Trp Thr Glu Trp Arg Leu Tyr Asn Asp Glu 35 40 45Thr Asn Ser
Asn Gln Asp Asn Pro Leu Gly Phe Lys Glu Ser Trp Gly 50 55 60Phe Gly
Lys Val Val Phe Lys Arg Tyr Leu Arg Tyr Asp Arg Thr Glu65 70 75
80Ala Ser Leu His Arg Val Leu Gly Ser Trp Thr Gly Asp Ser Val Asn
85 90 95Tyr Ala Ala Ser Arg Phe Leu Gly Ala Asn Gln Val Gly Cys Thr
Tyr 100 105 110Ser Ile Arg Phe Arg Gly Val Ser Val Thr Ile Ser Gly
Gly Ser Arg 115 120 125Thr Leu Gln His Leu Cys Glu Met Ala Ile Arg
Ser Lys Gln Glu Leu 130 135 140Leu Gln Leu Thr Pro Val Glu Val Glu
Ser Asn Val Ser Arg Gly Cys145 150 155 160Pro Glu Gly Ile Glu Thr
Phe Lys Lys Glu Ser Glu 165 170222RNAunknowncompletely synthetic
2aacaccauug ucacacucca ua 22380RNAHomo sapiens 3ugggaugagg
uaguagguug uauaguuuua gggucacacc caccacuggg agauaacuau 60acaaucuacu
gucuuuccua 80421RNAunknownsynthetic oligonucleotide 4cguacgcgga
auacuucgau u 21521RNAunknownsynthetic oligonucleotide 5ucgaaguauu
ccgcguacgu u 21617RNAunknownsynthetic oligonucleotide 6cucaaccagc
cacugcu 17717RNAunknownsynthetic oligonucleotide 7caguggcugg
uugagau 17825RNAunknownsynthetic oligonucleotide 8cguacgcgga
auacuucgaa auguu 25925RNAunknownsynthetic oligonucleotide
9cauuucgaag uauuccgcgu acguu 251021RNAunknownsynthetic
oligonucleotide 10cguacgcgga auacuucgau u 211121RNAunknownsynthetic
oligonucleotide 11ucgaaguauu ccgcguacgu u 211221RNAunknownsynthetic
oligonucleotide 12cguacgcgga auacuucgau u 211319RNAunknownsynthetic
oligonucleotide 13cguacgcgga auacuucga 191419RNAunknownsynthetic
oligonucleotide 14cguacgcgga auacuucga 191521RNAunknownsynthetic
oligonucleotide 15acguacgcgg aauacuucga u 211621RNAunknownsynthetic
oligonucleotide 16cguacgcgga auacuucgau u 211721DNAunknownsynthetic
oligonucleotide 17tcgaagtatt ccgcgtacgt t 211821DNAunknownsynthetic
oligonucleotide 18cgtacgcgga atacttcgat t 211921DNAunknownsynthetic
oligonucleotide 19tcgaagtatt ccgcgtacgt t 212021RNAunknownsynthetic
oligonucleotide 20cguacgcgga auacuucgau u 212121DNAunknownsynthetic
oligonucleotide 21ucgaagtauu ccgcguacgu u 212221RNAunknownsynthetic
oligonucleotide 22cguacgcgga auacuucgau u 212322RNACaenorhabditis
elegans 23ugagguagua gguuguauag uu 222422RNACaenorhabditis elegans
24cuaugcaauu uucuaccuua cc 222521RNAGallus gallus 25agagguagug
gguugcauag u 212621RNAGallus gallus 26uauacaaccu gcugccuuuc u
212721RNACaenorhabditis elegans 27ugacuagaga cacauucagc u
212822RNACaenorhabditis elegans 28cuggaugugc ucguuaguca ua
222922RNAunknownsynthetic 29uggaguguga caaugguguu ug
223022DNAunknownsynthetic 30aacactauug ucacacucca ua
223121RNAunknownsynthetic 31ucgaaguauu ccgcguacgu u
21321881DNACarnation Italian ringspot
virusCDS(1)..(1878)1184CDS(1)..(1698) 32atg aaa act gaa gaa ggt aaa
ctg gta atc tgg att aac ggc gat aaa 48Met Lys Thr Glu Glu Gly Lys
Leu Val Ile Trp Ile Asn Gly Asp Lys1 5 10 15ggc tat aac ggt ctc gct
gaa gtc ggt aag aaa ttc gag aaa gat acc 96Gly Tyr Asn Gly Leu Ala
Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30gga att aaa gtc acc
gtt gag cat ccg gat aaa ctg gaa gag aaa ttc 144Gly Ile Lys Val Thr
Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45cca cag gtt gcg
gca act ggc gat ggc cct gac att atc ttc tgg gca 192Pro Gln Val Ala
Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55 60cac gac cgc
ttt ggt ggc tac gct caa tct ggc ctg ttg gct gaa atc 240His Asp Arg
Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile65 70 75 80acc
ccg gac aaa gcg ttc cag gac aag ctg tat ccg ttt acc tgg gat 288Thr
Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp 85 90
95gcc gta cgt tac aac ggc aag ctg att gct tac ccg atc gct gtt gaa
336Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu
100 105 110gcg tta tcg ctg att tat aac aaa gat ctg ctg ccg aac ccg
cca aaa 384Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro
Pro Lys 115 120 125acc tgg gaa gag atc ccg gcg ctg gat aaa gaa ctg
aaa gcg aaa ggt 432Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu
Lys Ala Lys Gly 130 135 140aag agc gcg ctg atg ttc aac ctg caa gaa
ccg tac ttc acc tgg ccg 480Lys Ser Ala Leu Met Phe Asn Leu Gln Glu
Pro Tyr Phe Thr Trp Pro145 150 155 160ctg att gct gct gac ggg ggt
tat gcg ttc aag tat gaa aac ggc aag 528Leu Ile Ala Ala Asp Gly Gly
Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175tac gac att aaa gac
gtg ggc gtg gat aac gct ggc gcg aaa gcg ggt 576Tyr Asp Ile Lys Asp
Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190ctg acc ttc
ctg gtt gac ctg att aaa aac aaa cac atg aat gca gac 624Leu Thr Phe
Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200 205acc
gat tac tcc atc gca gaa gct gcc ttt aat aaa ggc gaa aca gcg 672Thr
Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala 210 215
220atg acc atc aac ggc ccg tgg gca tgg tcc aac atc gac acc agc aaa
720Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser
Lys225 230 235 240gtg aat tat ggt gta acg gta ctg ccg acc ttc aag
ggt caa cca tcc 768Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys
Gly Gln Pro Ser 245 250 255aaa ccg ttc gtt ggc gtg ctg agc gca ggt
att aac gcc gcc agt ccg 816Lys Pro Phe Val Gly Val Leu Ser Ala Gly
Ile Asn Ala Ala Ser Pro 260 265 270aac aaa gag ctg gca aaa gag ttc
ctc gaa aac tat ctg ctg act gat 864Asn Lys Glu Leu Ala Lys Glu Phe
Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285gaa ggt ctg gaa gcg gtt
aat aaa gac aaa ccg ctg ggt gcc gta gcg 912Glu Gly Leu Glu Ala Val
Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300ctg aag tct tac
gag gaa gag ttg gcg aaa gat cca cgt att gcc gcc 960Leu Lys Ser Tyr
Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala305 310 315 320act
atg gaa aac gcc cag aaa ggt gaa atc atg ccg aac atc ccg cag 1008Thr
Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln 325 330
335atg tcc gct ttc tgg tat gcc gtg cgt act gcg gtg atc aac gcc gcc
1056Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala
340 345 350agc ggt cgt cag act gtc gat gaa gcc ctg aaa gac gcg cag
act aat 1104Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln
Thr Asn 355 360 365tcg agc tcg aac aac aac aac aat aac aat aac aac
aac ctc ggg ccg 1152Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn
Asn Leu Gly Pro 370 375 380ggt gcg gca cac tac gta gaa ttc gga tcc
atg gaa cga gct ata caa 1200Gly Ala Ala His Tyr Val Glu Phe Gly Ser
Met Glu Arg Ala Ile Gln385 390 395 400gga aac gac act agg gaa caa
gct aac ggt gaa cgt tgg gat gga gga 1248Gly Asn Asp Thr Arg Glu Gln
Ala Asn Gly Glu Arg Trp Asp Gly Gly 405 410 415tca gga ggt atc act
tct ccc ttc aaa ctt cct gac gaa agt ccg agt 1296Ser Gly Gly Ile Thr
Ser Pro Phe Lys Leu Pro Asp Glu Ser Pro Ser 420 425 430tgg act gag
tgg cgg cta tat aac gat gag acg aat tcg aat caa gat 1344Trp Thr Glu
Trp Arg Leu Tyr Asn Asp Glu Thr Asn Ser Asn Gln Asp 435 440 445aat
ccc ctt ggt ttc aag gaa agc tgg ggt ttc ggg aaa gtt gta ttt 1392Asn
Pro Leu Gly Phe Lys Glu Ser Trp Gly Phe Gly Lys Val Val Phe 450 455
460aag aga tat ctc aga tac gac agg acg gaa gct tca ctg cac aga gtc
1440Lys Arg Tyr Leu Arg Tyr Asp Arg Thr Glu Ala Ser Leu His Arg
Val465 470 475 480ctt gga tct tgg acg gga gat tcg gtt aac tat gca
gca tct cga ttt 1488Leu Gly Ser Trp Thr Gly Asp Ser Val Asn Tyr Ala
Ala Ser Arg Phe 485 490 495ctc ggt gcc aac cag gtc gga tgt acc tat
agt att cgg ttt cga gga 1536Leu Gly Ala Asn Gln Val Gly Cys Thr Tyr
Ser Ile Arg Phe Arg Gly 500 505 510gtt agt gtc acc att tct gga ggg
tcg aga act ctt cag cat ctc tgt 1584Val Ser Val Thr Ile Ser Gly Gly
Ser Arg Thr Leu Gln His Leu Cys 515 520 525gag atg gca att cgg tct
aag caa gaa ctg tta cag ctt acc cca gtc 1632Glu Met Ala Ile Arg Ser
Lys Gln Glu Leu Leu Gln Leu Thr Pro Val 530 535 540gaa gtg gaa agt
aat gta tca aga gga tgc cct gaa ggt att gaa acc 1680Glu Val Glu Ser
Asn Val Ser Arg Gly Cys Pro Glu Gly Ile Glu Thr545 550 555 560ttc
aag aaa gaa agc gag gga tcc tct aga gtc gac ctg cag acg aca 1728Phe
Lys Lys Glu Ser Glu Gly Ser Ser Arg Val Asp Leu Gln Thr Thr 565 570
575aat cct ggt gta tcc gct tgg cag gtc aac aca gct tat act gcg gga
1776Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr Ala Gly
580 585 590caa ttg gtc aca tat aac ggc aag acg tat aaa tgt ttg cag
ccc cac 1824Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys Leu Gln
Pro His 595 600 605acc tcc ttg gca gga tgg gaa cca tcc aac gtt cct
gcc ttg tgg cag 1872Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn Val Pro
Ala Leu Trp Gln 610 615 620ctt caa tga 1881Leu
Gln62533626PRTCarnation Italian ringspot virus 33Met Lys Thr Glu
Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys1 5 10 15Gly Tyr Asn
Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr 20 25 30Gly Ile
Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe 35 40 45Pro
Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala 50 55
60His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile65
70 75 80Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp
Asp 85 90 95Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala
Val Glu 100 105 110Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro
Asn Pro Pro Lys 115 120 125Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys
Glu Leu Lys Ala Lys Gly 130 135 140Lys Ser Ala Leu Met Phe Asn Leu
Gln Glu Pro Tyr Phe Thr Trp Pro145 150 155 160Leu Ile Ala Ala Asp
Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys 165 170 175Tyr Asp Ile
Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly 180 185 190Leu
Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp 195 200
205Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala
210 215 220Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr
Ser Lys225 230 235 240Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe
Lys Gly Gln Pro Ser 245 250 255Lys Pro Phe Val Gly Val Leu Ser Ala
Gly Ile Asn Ala Ala Ser Pro 260 265 270Asn Lys Glu Leu Ala Lys Glu
Phe Leu Glu Asn Tyr Leu Leu Thr Asp 275 280 285Glu Gly Leu Glu Ala
Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala 290 295 300Leu Lys Ser
Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala305 310 315
320Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln
325 330 335Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn
Ala Ala 340 345 350Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp
Ala Gln Thr Asn 355 360 365Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn
Asn Asn Asn Leu Gly Pro 370 375 380Gly Ala Ala His Tyr Val Glu Phe
Gly Ser Met Glu Arg Ala Ile Gln385 390 395 400Gly Asn Asp Thr Arg
Glu Gln Ala Asn Gly Glu Arg Trp Asp Gly Gly 405 410 415Ser Gly Gly
Ile Thr Ser Pro Phe Lys Leu Pro Asp Glu Ser Pro Ser 420 425 430Trp
Thr Glu Trp Arg Leu Tyr Asn Asp Glu Thr Asn Ser Asn Gln Asp 435 440
445Asn Pro Leu Gly Phe Lys Glu Ser Trp Gly Phe Gly Lys Val Val Phe
450 455 460Lys Arg Tyr Leu Arg Tyr Asp Arg Thr Glu Ala Ser Leu His
Arg Val465 470 475 480Leu Gly Ser Trp Thr Gly Asp Ser Val Asn Tyr
Ala Ala Ser Arg Phe 485 490 495Leu Gly Ala Asn Gln Val Gly Cys Thr
Tyr Ser Ile Arg Phe Arg Gly 500 505 510Val Ser Val Thr Ile Ser Gly
Gly Ser Arg Thr Leu Gln His Leu Cys 515 520 525Glu Met Ala Ile Arg
Ser Lys Gln Glu Leu Leu Gln Leu Thr Pro Val 530 535 540Glu Val Glu
Ser Asn Val Ser Arg Gly Cys Pro Glu Gly Ile Glu Thr545 550 555
560Phe Lys Lys Glu Ser Glu Gly Ser Ser Arg Val Asp Leu Gln Thr Thr
565 570 575Asn Pro Gly Val Ser Ala Trp Gln Val Asn Thr Ala Tyr Thr
Ala Gly 580 585 590Gln Leu Val Thr Tyr Asn Gly Lys Thr Tyr Lys Cys
Leu Gln Pro His 595 600 605Thr Ser Leu Ala Gly Trp Glu Pro Ser Asn
Val Pro Ala Leu Trp Gln 610 615 620Leu Gln6253434DNAunknownprimer
34gactctgcag acgacaaatc ctggtgtatc cgct 343537DNAunknownprimer
35taggaagctt tcattgaagc tgccacaagg caggaac 37
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