U.S. patent application number 16/016086 was filed with the patent office on 2018-12-27 for compositions and methods for detecting viral nucleic acids.
The applicant listed for this patent is ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE UNIVERSITY. Invention is credited to Shengxi Chen, Mingxuan Gao, Sidney Hecht.
Application Number | 20180371526 16/016086 |
Document ID | / |
Family ID | 64692058 |
Filed Date | 2018-12-27 |
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United States Patent
Application |
20180371526 |
Kind Code |
A1 |
Chen; Shengxi ; et
al. |
December 27, 2018 |
COMPOSITIONS AND METHODS FOR DETECTING VIRAL NUCLEIC ACIDS
Abstract
Described herein are compositions that may be used to detect
viral nucleic acid. For example, these compositions may comprise a
DNA-nanostructure, a capture oligonucleotide and a protector
oligonucleotide, wherein the components are designed based on a
duo-toehold-mediated displacement reaction (duo-TMDR) strategy. In
this strategy, a first TMDR can switch off a Foster resonance
energy transfer (FRET) process and a second TMDR can release the
target viral nucleic acid and amplify the signal. Methods of using
such compositions are also provided herein.
Inventors: |
Chen; Shengxi; (Chandler,
AZ) ; Hecht; Sidney; (Phoenix, AZ) ; Gao;
Mingxuan; (Chongqing, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ARIZONA BOARD OF REGENTS ON BEHALF OF ARIZONA STATE
UNIVERSITY |
Scottsdale |
AZ |
US |
|
|
Family ID: |
64692058 |
Appl. No.: |
16/016086 |
Filed: |
June 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62524070 |
Jun 23, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/682 20130101;
C12Q 1/686 20130101; C12Q 2537/1373 20130101; C12Q 2525/30
20130101; C12Q 2565/101 20130101; C12Q 2525/30 20130101; B82Y 5/00
20130101; C12Q 1/6818 20130101; C12Q 2537/155 20130101; C12Q 1/6806
20130101; C12Q 2537/1373 20130101; C12Q 2563/107 20130101; C12Q
2563/107 20130101; C12N 2310/151 20130101; C12Q 1/6818 20130101;
C12Q 1/70 20130101; C12Q 1/682 20130101; C12Q 2537/155
20130101 |
International
Class: |
C12Q 1/6806 20060101
C12Q001/6806; C12Q 1/686 20060101 C12Q001/686; C12Q 1/70 20060101
C12Q001/70 |
Goverment Interests
GOVERNMENT FUNDING
[0002] This invention was made with government support under
W81XWH-16-1-0141 awarded by the ARMY/MRMC. The government has
certain rights in the invention.
Claims
1. A composition for detecting a viral nucleic acid in a sample,
the composition comprising: a DNA-nanostructure, a capture
oligonucleotide and a protector oligonucleotide; wherein the
DNA-nanostructure is operably linked to a fluorophore and the
protector oligonucleotide is operably linked to a quencher or the
DNA-nanostructure is operably linked to a quencher and the
protector oligonucleotide is operably linked to a fluorophore; and
wherein the quencher is capable of quenching the fluorescent light
emitted from the fluorophore; wherein the protector oligonucleotide
is capable of hybridizing to the DNA-nanostructure; wherein the
viral nucleic acid is capable of displacing the protector
oligonucleotide and hybridizing to the DNA-nanostructure; and
wherein the capture oligonucleotide is capable of displacing the
viral nucleic acid and hybridizing to the DNA-nanostructure but is
not capable of displacing the protector oligonucleotide.
2. The composition of claim 1, wherein the DNA-nanostructure
comprises at least one single stranded region.
3. The composition of claim 2, wherein the single stranded region
comprises a nucleic acid sequence that comprises a first toehold
domain, a hybridization region and a second toehold domain.
4. The composition of claim 3, wherein the first toehold domain
comprises a nucleic acid sequence that is complementary to a
portion of the viral nucleic acid.
5. The composition of claim 3, wherein the protector
oligonucleotide is not capable of hybridizing to the first toehold
domain.
6. The composition of claim 3, wherein the second toehold domain
comprises a nucleic acid sequence that is complementary to a
portion of the protector oligonucleotide and a portion of the
capture oligonucleotide.
7. The composition of claim 3, wherein the viral nucleic acid is
not capable of hybridizing to the second toehold domain.
8. The composition of claim 3, wherein the hybridization region
comprises a nucleic acid sequence that is complementary to a
portion of the viral nucleic acid, a portion of the protector
oligonucleotide and a portion of the capture oligonucleotide.
9. The composition of claim 1, wherein the DNA-nanostructure is a
DNA-tetrahedron.
10. The composition of claim 9, wherein the DNA-tetrahedron
comprises five double-stranded edges and one single stranded
edge.
11. The composition of claim 10, wherein the fluorophore or
quencher is operably linked at the tetrahedron vertex, proximal to
the single stranded edge.
12. The composition of claim 9, wherein the DNA-tetrahedron
comprises four oligonucleotides having at least about 90% sequence
identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID
NO:4.
13. The composition of claim 1, wherein the protector
oligonucleotide is between about 15 to about 25 nucleotides in
length.
14. The composition of claim 1, wherein the fluorophore or quencher
is operably linked to the 5' or 3' end of the protector
oligonucleotide.
15. The composition of claim 1, wherein the protector
oligonucleotide comprises a nucleic acid sequence having at least
about 90% sequence identity to SEQ ID NO:5, SEQ ID NO:6 or SEQ ID
NO:7.
16. The composition of claim 1, wherein the capture oligonucleotide
is between about 15 to about 30 nucleotides in length.
17. The composition of claim 1, wherein the capture oligonucleotide
comprises a nucleic acid sequence that is complementary to a
toehold domain in the DNA-nanostructure, and wherein the toehold
domain is linked to a nucleic acid sequence in the
DNA-nanostructure that is capable of hybridizing to the viral
nucleic acid.
18. The composition of claim 1, wherein the capture oligonucleotide
comprises a nucleic acid sequence having at least about 90%
sequence identity to SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10.
19. The composition of claim 1, wherein the viral nucleic acid is
from dengue virus, Ebola virus, human immunodeficiency virus (HIV),
hepatitis B, hepatitis C, Influenza, SARS, measles, Zika, yellow
fever, West Nile fever, smallpox, Marburg viruses, human
papillomavirus, Kaposi's sarcoma-associated herpesvirus or human
T-lymphotropic virus.
20. The composition of claim 1, wherein the viral nucleic acid is
from Dengue virus.
Description
RELATED APPLICATION
[0001] This application claims the benefit of priority of U.S.
Provisional Application Ser. No. 62/524,070 filed on Jun. 23, 2017,
which application is incorporated by reference herein.
BACKGROUND
[0003] RNA viruses, such as human immunodeficiency virus (HIV),
dengue virus and Ebola virus, are some of the most rapidly
spreading viral diseases in the world. For example, dengue
currently threatens more than 2.5 billion people in more than 100
countries, including Africa, Americas, Western Pacific, Southeast
Asia and Eastern Mediterranean, and causes more than 24,000 deaths
annually. For RNA viral diseases, early-stage diagnosis and
treatment is critical, as fatality rates are often high if severe
symptoms occur. For example, a dengue virus infection can cause
severe symptoms and may result in a fatality rate as high as 10% in
the first week if a proper treatment is not performed. Reverse
transcriptase polymerase chain reaction (RT-PCR) is the most
commonly used method to detect viral RNA in a patient's blood,
serum or plasma. However, RT-PCR is expensive and time-consuming
and usually takes 1-2 days for results. Additionally, it is not
suitable for use in remote areas, which may lead to inadequate
treatments.
[0004] Accordingly, new compositions and methods for detecting
viruses are needed (e.g., RNA viruses).
SUMMARY
[0005] Thus, as described herein, a novel duo-toehold-mediated
displacement reaction (duo-TMDR) strategy using a DNA-nanostructure
has been developed to amplify a signal and sensitively detect viral
nucleic acids. In this strategy, a first TMDR can switch off a
Foster resonance energy transfer (FRET) process and a second TMDR
can release the target viral nucleic acid and amplify the signal.
As described in the Example, as low as 6 copies of dengue RNA per
sample could be detected by using a single molecule detecting
technique.
[0006] Accordingly, certain embodiments of the invention provide a
composition for detecting a viral nucleic acid in a sample, the
composition comprising:
[0007] a DNA-nanostructure, a capture oligonucleotide and a
protector oligonucleotide;
[0008] wherein the DNA-nanostructure is operably linked to a
fluorophore and the protector oligonucleotide is operably linked to
a quencher or the DNA-nanostructure is operably linked to a
quencher and the protector oligonucleotide is operably linked to a
fluorophore; and wherein the quencher is capable of quenching the
fluorescent light emitted from the fluorophore;
[0009] wherein the protector oligonucleotide is capable of
hybridizing to the DNA-nanostructure;
[0010] wherein the viral nucleic acid is capable of displacing the
protector oligonucleotide and hybridizing to the DNA-nanostructure;
and
[0011] wherein the capture oligonucleotide is capable of displacing
the viral nucleic acid and hybridizing to the DNA-nanostructure but
is not capable of displacing the protector oligonucleotide.
[0012] Certain embodiments of the invention also provide a method
for detecting a viral nucleic acid in a sample, comprising:
[0013] a) contacting the sample with a detection agent and a
capture oligonucleotide under conditions suitable for strand
displacement,
[0014] wherein the detection agent comprises a protector
oligonucleotide hybridized to a DNA-nanostructure;
[0015] wherein the DNA-nanostructure is operably linked to a
fluorophore and the protector oligonucleotide is operably linked to
a quencher or the DNA-nanostructure is operably linked to a
quencher and the protector oligonucleotide is operably linked to a
fluorophore; and wherein the quencher is capable of quenching the
fluorescent light emitted from the fluorophore;
[0016] wherein the viral nucleic acid is capable of displacing the
protector oligonucleotide and hybridizing to the DNA-nanostructure;
and
[0017] wherein the capture oligonucleotide is capable of displacing
the viral nucleic acid and hybridizing to the DNA-nanostructure but
is not capable of displacing the protector oligonucleotide; and
[0018] b) measuring the fluorescent emission from the fluorophore,
wherein an increase in fluorescent emission as compared to a
control indicates the presence of a viral nucleic acid.
[0019] Certain embodiments of the invention also provide a
DNA-tetrahedron comprising four oligonucleotides, wherein the
oligonucleotides comprise a sequence having at least about 90%
sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ
ID NO:4.
[0020] Certain embodiments of the invention provide a protector
oligonucleotide comprising a nucleic acid sequence having at least
about 90% sequence identity to SEQ ID NO:5, SEQ ID NO:6 or SEQ ID
NO:7.
[0021] Certain embodiments of the invention provide a capture
oligonucleotide comprising a nucleic acid sequence having at least
about 90% sequence identity to SEQ ID NO:8, SEQ ID NO:9 or SEQ ID
NO:10.
[0022] Certain embodiments of the invention provide a kit for
detecting viral nucleic acid in a sample comprising:
[0023] a) a DNA-nanostructure;
[0024] b) a protector oligonucleotide;
[0025] c) a capture oligonucleotide; and
[0026] d) instructions for use;
[0027] wherein the DNA-nanostructure is operably linked to a
fluorophore and the protector oligonucleotide is operably linked to
a quencher or the DNA-nanostructure is operably linked to a
quencher and the protector oligonucleotide is operably linked to a
fluorophore; and wherein the quencher is capable of quenching the
fluorescent light emitted from the fluorophore;
[0028] wherein the protector oligonucleotide is capable of
hybridizing to the DNA-nanostructure;
[0029] wherein the viral nucleic acid is capable of displacing the
protector oligonucleotide and hybridizing to the DNA-nanostructure;
and
[0030] wherein the capture oligonucleotide is capable of displacing
the viral nucleic acid and hybridizing to the DNA-nanostructure but
is not capable of displacing the protector oligonucleotide.
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1. The illustration of the duo-toehold-mediated strand
displacement reaction (duo-TMDR) process for target oligonucleotide
detection.
[0032] FIG. 2. The concept of duo-TMDR on the one side of the DNA
tetrahedron.
[0033] FIGS. 3A-B. Confirmation for the DNA tetrahedron. A) the
native polyacrylamide gel electrophoresis (PAGE) for the DNA
tetrahedron annealed with different length of protector (lane 1 to
3 refer to protector of 17-nt, 18-nt and 19-nt DNA), 100 bps DNA
ladder was on the left; B) the fluorescence spectra for DNA 4, DNA
tetrahedron and different protector annealed DNA tetrahedron. The
concentration of DNA 4 and DNA tetrahedron were 200 nM.
[0034] FIGS. 4A-C. Confirmation of the occurrence of duo-TMDR
process. A) fluorescence spectra of fluorophore (TET) at different
stages of reaction; B) the native PAGE of DNA tetrahedron at
different stages of reaction, lane 1 to 3 represented protector
binding tetrahedron, protector binding tetrahedron with target RNA,
and protector binding tetrahedron with target RNA and capture DNA;
C) fluorescence lifetime of TET at different stages of
reaction.
[0035] FIGS. 5A-B. Single molecule detection of target RNA. A) The
response traces of photon counts by adding different concentration
of target RNA to the solution of protector binding tetrahedron and
capture DNA; B) the average photon counts in 5 s by adding
different concentration of target RNA to the solution of capture
DNA contained protector binding tetrahedron (bottom line of spots)
and only protector binding tetrahedron (top line of spots).
[0036] FIG. 6. The PAGE analysis for the repeatability of synthesis
protector binding tetrahedron. Lane 1 to 8 were different batches
of protector binding tetrahedron. The concentration of protector
binding tetrahedron was 100 nM.
[0037] FIGS. 7A-B. The PAGE analysis for the legitimation of
sequence design. A) the native PAGE for pairwise DNA hybridization;
B) the native PAGE for triple-wise DNA hybridization.
[0038] FIGS. 8A-B. The fluorescence for the optimization of
duo-TMDR. A) Optimizing the concentration of protector annealed DNA
tetrahedron for reaction. Bars 1 to 4 represent protector binding
tetrahedron with target RNA and capture DNA, protector binding
tetrahedron with target RNA, protector binding tetrahedron with
capture DNA and protector binding tetrahedron only, respectively.
Bars 1 to 4 are shown in order from left to right for each
tetrahedron concentration. The concentration of target RNA was 5 nM
and capture DNA was 100 nM; B) Optimizing the pH value for
reaction, the concentration of protector binding tetrahedron was
100 nM and the concentration of target RNA and capture DNA were 5
nM and 100 nM, respectively. The control has no target RNA
presented.
[0039] FIGS. 9A-B. The dynamic fluorescence intensity related to
reaction time by changing the length of protector DNA (A) or the
capture DNA (B). The concentration of protector binding tetrahedron
was 100 nM and the concentration of target RNA and capture DNA were
5 nM and 100 nM respectively.
[0040] FIGS. 10A-B. Variance of fluorescence intensity as a
function of the concentration of target RNA (A) in the range of 40
pM to 20 nM and (B) the linear fitting of the fluorescence
intensity as a function of the concentration of target RNA in the
range of 40 pM to 1 nM. The concentration of protector binding
tetrahedron was 100 nM and the concentration of capture DNA was 100
nM.
[0041] FIG. 11. The photon counts of control group (left) and the
duo-TMDR group (right) in different medium (buffer or human serum).
The concentration of protector binding tetrahedron was 100 nM and
the concentration of target RNA and capture DNA were 10 aM and 100
nM respectively.
DETAILED DESCRIPTION
[0042] Toehold-mediated displacement reaction (TMDR) is a
kinetic-controlled non-enzymatic process. In this process, a single
stranded oligonucleotide (referred to as a toehold), which
neighbors to a double strand helix, mediates a displacement with
another single stranded oligonucleotide. This process can occur
automatically at room temperature without any other assistance.
[0043] As described herein, a novel duo-toehold-mediated strand
displacement method in combination with FRET was developed to
detect the presence of viral nucleic acid in a sample (e.g., dengue
RNA). Specifically, a DNA-nanostructure was developed to amplify
the detection signal of a viral nucleic acid. In the first TMDR
process, a target nucleic acid anneals to a complementary DNA
sequence via a first toehold in the DNA-nanostructure, displaces a
protector DNA and recovers the fluorescence from a quenched
fluorophore. In the second TMDR process, a capture DNA displaces
the target nucleic acid via a second toehold in the
DNA-nanostructure. The target nucleic acid can then be recycled in
the first TMDR process and form an amplifying loop, thereby
enhancing the fluorescence signal. As described in the Example, the
limit of this detection method was as low as 10 pM, which was more
sensitive by 3 orders of magnitude than traditional non-amplified
detecting methods. Using a single molecule detecting technique, the
limit of detection could be as low as 0.1 aM, which means only
about six copies of target RNA presented in the sample.
Accordingly, certain methods and compositions of the invention are
provided below.
Illustrative Methods in Accordance with Certain Embodiments
[0044] Certain embodiments of the invention provide a method for
detecting a viral nucleic acid in a sample, comprising:
[0045] a) contacting the sample with a detection agent and a
capture oligonucleotide under conditions suitable for strand
displacement,
[0046] wherein the detection agent comprises a protector
oligonucleotide hybridized to a DNA-nanostructure;
[0047] wherein the DNA-nanostructure is operably linked to a
fluorophore and the protector oligonucleotide is operably linked to
a quencher or the DNA-nanostructure is operably linked to a
quencher and the protector oligonucleotide is operably linked to a
fluorophore; and wherein the quencher is capable of quenching the
fluorescent light emitted from the fluorophore;
[0048] wherein the viral nucleic acid is capable of displacing the
protector strand and hybridizing to the DNA-nanostructure (i.e.,
and thereby disrupting the quenching between the quencher and the
fluorophore); and
[0049] wherein the capture oligonucleotide is capable of displacing
the viral nucleic acid and hybridizing to the DNA-nanostructure but
is not capable of displacing the protector oligonucleotide; and
[0050] b) measuring the fluorescent emission from the fluorophore,
wherein an increase in fluorescent emission indicates the presence
of a viral nucleic acid (e.g., as compared to a control, such as
the fluorescent emission of the detection agent prior to being
contacted with the sample or a sample comprising no viral nucleic
acid).
[0051] In certain embodiments of the invention, it is desirable to
assay the sample in parallel with a control sample, which comprises
a predetermined amount of the viral nucleic acid.
[0052] Accordingly, certain embodiments of the invention provide a
method for detecting a viral nucleic acid in a test sample,
comprising:
[0053] a) contacting the test sample with a first detection agent
and a first capture oligonucleotide under conditions suitable for
strand displacement;
[0054] b) contacting a control sample comprising a predetermined
amount of viral nucleic acid with a second detection agent and a
second capture oligonucleotide under conditions suitable for strand
displacement;
[0055] wherein each detection agent comprises a protector
oligonucleotide hybridized to a DNA-nanostructure;
[0056] wherein each DNA-nanostructure is operably linked to a
fluorophore and each protector oligonucleotide is operably linked
to a quencher or each DNA-nanostructure is operably linked to a
quencher and each protector oligonucleotide is operably linked to a
fluorophore; and wherein the quencher is capable of quenching the
fluorescent light emitted from the fluorophore;
[0057] wherein the viral nucleic acid is capable of displacing the
protector oligonucleotide and hybridizing to the DNA-nanostructure;
and
[0058] wherein the capture oligonucleotide is capable of displacing
the viral nucleic acid and hybridizing to the DNA-nanostructure but
is not capable of displacing the protector oligonucleotide; and
[0059] c) measuring the fluorescent emission from the fluorophore
in the test sample and in the control sample, wherein the relative
fluorescence in the test sample as compared to the control sample
indicates the presence or absence of the viral nucleic acid. In
certain embodiments, the control sample is a negative control, and
therefore, the predetermined amount of viral nucleic acid in the
control sample is no viral nucleic acid. In such an embodiment, a
fluorescent emission in the test sample that is greater than the
fluorescent emission in the control sample indicates that the test
sample comprises viral nucleic acid.
[0060] In certain embodiments, the fluorescent emission from the
fluorophore in the test sample is at least about 1-100% greater
than the fluorescent emission in the control sample (i.e., a
negative control sample).
[0061] Methods of the invention may also be used to diagnose a
mammal with a viral infection. Thus, certain embodiments of the
invention provide, a method for diagnosing a mammal with a viral
infection comprising:
[0062] a) detecting the presence of a viral nucleic acid in a
sample obtained from the mammal by: [0063] 1) contacting the sample
with a detection agent and a capture oligonucleotide under
conditions suitable for strand displacement, [0064] wherein the
detection agent comprises a protector oligonucleotide hybridized to
a DNA-nanostructure; [0065] wherein the DNA-nanostructure is
operably linked to a fluorophore and the protector oligonucleotide
is operably linked to a quencher or the DNA-nanostructure is
operably linked to a quencher and the protector oligonucleotide is
operably linked to a fluorophore; and wherein the quencher is
capable of quenching the fluorescent light emitted from the
fluorophore; [0066] wherein the viral nucleic acid is capable of
displacing the protector oligonucleotide and hybridizing to the
DNA-nanostructure; and [0067] wherein the capture oligonucleotide
is capable of displacing the viral nucleic acid and hybridizing to
the DNA-nanostructure but is not capable of displacing the
protector oligonucleotide; and [0068] 2) measuring the fluorescent
emission from the fluorophore, wherein an increase in fluorescent
emission as compared to a control indicates the presence of a viral
nucleic acid; and
[0069] b) diagnosing the mammal with a viral infection when the
presence of the viral nucleic acid is detected.
[0070] In certain embodiments, the methods of the invention further
comprise administering a therapeutic agent to the diagnosed mammal.
As used herein, the term "therapeutic agent" includes agents that
provide a therapeutically desirable effect when administered to an
animal (e.g., a mammal, such as a human). The agent may be of
natural or synthetic origin. For example, it may be a nucleic acid,
a polypeptide, a protein, a peptide, or an organic compound, such
as a small molecule. The term "small molecule" includes organic
molecules having a molecular weight of less than about, e.g., 1000
amu. In one embodiment a small molecule can have a molecular weight
of less than about 800 amu. In another embodiment a small molecule
can have a molecular weight of less than about 500 amu.
[0071] In certain embodiments, the therapeutic agent is an
anti-viral agent. In certain embodiments, the viral nucleic acid is
from dengue virus, Ebola virus, human immunodeficiency virus (HIV),
hepatitis B, hepatitis C, Influenza, SARS, measles, Zika, yellow
fever, West Nile fever, smallpox, Marburg viruses, human
papillomavirus, Kaposi's sarcoma-associated herpesvirus or human
T-lymphotropic virus and the anti-viral agent is useful for
treating the particular virus. In certain embodiments, the viral
infection is caused by a dengue virus and the anti-viral agent is
useful for treating dengue virus.
[0072] In certain embodiments, the sample is contacted with a
composition comprising two or more detection agents (e.g., a
plurality of detection agents) and two or more capture
oligonucleotides (e.g., a plurality of capture oligonucleotides).
In such an embodiment, a single viral nucleic acid may sequentially
hybridize to a series of DNA-nanostructures and displace the
protector oligonucleotides hybridized thereto. This recycling of
the viral nucleic acid amplifies fluorescent emission and generates
a stronger signal for detection.
[0073] In certain embodiments, a method of the invention further
comprises incubating the sample, the detection agent and the
capture oligonucleotide for a time sufficient for 1) any viral
nucleic acid in the sample to hybridize to the DNA-nanostructure
and to displace the protector oligonucleotide; 2) the capture
reagent to hybridize to the DNA-nanostructure and to displace the
viral nucleic acid; and 3) optionally, to repeat steps 1-2 one or
more times, so that the displaced viral nucleic acid may hybridize
to an additional DNA-nanostructure and displace an additional
protector oligonucleotide. For example, in certain embodiments, the
sample, the detection agent and the capture oligonucleotide are
incubated for about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60
min. In certain embodiments, the sample, the detection agent and
the capture oligonucleotide are incubated for about 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24 or more hours. In certain embodiments, the sample, the detection
agent and the capture oligonucleotide are incubated for about 3
hours. In certain embodiments, the sample, the detection agent and
the capture oligonucleotide are incubated under a set of conditions
described herein.
[0074] In certain embodiments, the sample, the detection agent and
the capture oligonucleotide are contacted in the presence of a
buffer solution (e.g., Tris-HCl--Mg.sup.2+ buffer). As described
herein, a "buffer solution" refers to an aqueous solution
consisting of a mixture of a weak acid and its conjugate base, or
vice versa, and its pH changes very little when a small amount of
strong acid or base is added to it. Buffer solutions and buffering
agents are known in the art.
[0075] In certain embodiments, the sample, the detection agent and
the capture oligonucleotide are contacted at a pH 8.0.
[0076] In certain embodiments, the sample, the detection agent and
the capture oligonucleotide are contacted at room temperature.
[0077] In certain embodiments, the sample, the detection agent and
the capture oligonucleotide are contacted in the dark.
[0078] In certain embodiments, methods of the invention further
comprise generating the detection agent, comprising contacting the
DNA-nanostructure with the protector oligonucleotide under
conditions suitable for hybridization to occur between the
protector oligonucleotide and the DNA-nanostructure.
[0079] In certain embodiments, the methods further comprise
obtaining a test sample (e.g., a biological sample) from a subject
(e.g., a mammal, e.g., a human).
[0080] In certain embodiments, the methods further comprise
exciting the fluorophore.
[0081] In certain embodiments, the methods further comprise
quantifying the concentration of the viral nucleic acid in the
sample.
Viral Nucleic Acid
[0082] As described herein, methods of the invention may be used to
detect the presence of a viral nucleic acid in a sample. The viral
nucleic acid to be detected should be capable of binding to the
DNA-nanostructure and displacing the protector oligonucleotide, and
as such, should be complementary to a portion of the
DNA-nanostructure (e.g., a single stranded portion of the
nanostructure). In certain embodiments, the viral nucleic acid
comprises a sequence that has at least about 80, 81, 82, 83, 84,
85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%
complementarity with a portion of a single stranded region of the
DNA-nanostructure (i.e., the first toehold and the region of the
DNA-nanostructure to which the protector strand is hybridized).
However, the viral nucleic acid should not hybridize with the
second toehold domain.
[0083] In certain embodiments, the viral nucleic acid is DNA.
[0084] In certain embodiments, the viral nucleic acid is RNA.
[0085] In certain embodiments, the viral nucleic acid is from
dengue virus, Ebola virus, human immunodeficiency virus (HIV),
hepatitis B, hepatitis C, Influenza, SARS, measles, Zika, yellow
fever, West Nile fever, smallpox, Marburg viruses, human
papillomavirus, Kaposi's sarcoma-associated herpesvirus and human
T-lymphotropic virus.
[0086] In certain embodiments, the viral nucleic acid is from
dengue virus. In certain embodiments, the viral nucleic acid is
dengue RNA. In certain embodiments, the dengue RNA comprises SEQ ID
NO:11. In certain embodiments, the dengue RNA consists of SEQ ID
NO:11.
Detection Agent
[0087] As described herein, the detection agent comprises i) a
DNA-nanostructure; and ii) a protector oligonucleotide; wherein the
DNA-nanostructure is operably linked to a fluorophore and the
protector oligonucleotide is operably linked to a quencher or the
DNA-nanostructure is operably linked to a quencher and the
protector oligonucleotide is operably linked to a fluorophore; and
wherein the quencher is capable of quenching the fluorescent light
emitted from the fluorophore.
[0088] DNA-Nanostructure
[0089] DNA-nanostructures are nanoscale structures made of DNA,
wherein the DNA acts both as a structural and function element.
DNA-nanostructures can serve as a scaffold for the formation of
other structures. DNA-nanostructures may be prepared by methods
known in the art using nucleic acid oligonucleotides. For example,
such nanostructures may be assembled based on the concept of
base-pairing, and while no specific sequence is required, the
sequences of each oligonucleotide must be partially complementary
to certain other oligonucleotides to enable hybridization of all
strands.
[0090] The length of each oligonucleotide or DNA strand is variable
and depends on, for example, the type of nanostructure. In certain
embodiments, the oligonucleotide or DNA strand is about 15
nucleotides in length to about 3000 nucleotides in length, about 15
to about 1500 nucleotides in length, about 15 to about 1000
nucleotides in length, about 15 to about 500 nucleotides in length,
about 15 to about 250 nucleotides in length, about 15 to about 100
nucleotides in length, about 15 to about 80 nucleotides in length,
or about 30 to about 80 nucleotides in length.
[0091] For use in the present invention, the nucleic acids can be
synthesized de novo using any of a number of procedures well known
in the art. For example, the cyanoethyl phosphoramidite method
(Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981);
nucleoside H-phosphonate method (Garegg et al., Tet. Let.
27:4051-4054, 1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407,
1986; Garegg et al., Tet. Let. 27:4055-4058, 1986, Gaffney et al.,
Tet. Let. 29:2619-2622, 1988). These chemistries can be performed
by a variety of automated oligonucleotide synthesizers available in
the market.
[0092] As described herein, the methods of the invention
incorporate the use of TMDR, and as such, the nanostructure should
comprise at least one single stranded region, comprising two
toehold domains. Portions of this single stranded region should
also be complementary to the protector oligonucleotide, the viral
nucleic acid and the capture oligonucleotide.
[0093] In certain embodiments, the first toehold domain may be used
by the viral nucleic acid to displace the protector oligonucleotide
and the second toehold domain may be used by the capture
oligonucleotide to displace the viral nucleic acid. The toehold
domain should comprise a nucleic acid sequence that is
complementary to a region of the displacing strand (e.g., the viral
nucleic acid or the capture oligonucleotide) and should be located
adjacent to a double stranded region comprising the strand to be
displaced (e.g., the protector strand bound to the
DNA-nanostructure or the viral nucleic acid bound to the DIN
A-nanostructure). The toehold domain should be long enough to
enable sufficient hybridization for strand displacement to occur.
While the toehold domain may be longer or shorter, such a domain
typically includes between about 4 to about 15 nucleotides, or
about 5 to about 8 nucleotides.
[0094] Accordingly, in certain embodiments, the DNA-nanostructure
comprises a single stranded nucleic acid sequence that comprises a
first toehold domain, a hybridization region and a second toehold
domain. In certain embodiments, the first toehold domain comprises
a nucleic acid sequence that is complementary to a portion of the
viral nucleic acid. In certain embodiments, the hybridization
region comprises a nucleic acid sequence that is complementary to a
portion of the viral nucleic acid, the protector oligonucleotide
and the capture oligonucleotide. In certain embodiments, the second
toehold domain comprises a nucleic acid sequence that is
complementary to a portion of the protector oligonucleotide and a
portion of the capture oligonucleotide. In certain embodiments, the
viral nucleic acid does not hybridize to the second toehold domain.
In certain embodiments, the protector oligonucleotide does not
hybridize to the first toehold domain. In certain embodiments, the
first toehold domain is linked to the 5' end of the hybridization
region and the second toehold domain is linked to the 3' end of the
hybridization region (e.g., linked through a phosphodiester bond).
In certain embodiments, the first toehold domain is linked to the
3' end of the hybridization region and the second toehold domain is
linked to the 5' end of the hybridization region (e.g., linked
through a phosphodiester bond).
[0095] In certain embodiments, the DNA-nanostructure comprises a
single stranded nucleic acid sequence of formula I:
A-B-C (I)
[0096] wherein:
[0097] A is a first toehold domain;
[0098] B is a hybridization region; and
[0099] C is a second toehold domain;
[0100] wherein, the hybridization region and the second toehold
domain comprise nucleic acid sequences that are complementary to
the protector oligonucleotide and the capture oligonucleotide; and
wherein the first toehold domain and hybridization region comprise
sequences that are complementary to the viral nucleic acid.
[0101] As described herein, the DNA-nanostructure is operably
linked to a fluorophore/quencher. The fluorophore/quencher should
be operably linked in proximity to the single stranded region of
the DNA-nanostructure, such that quenching may occur between
fluorophore/quencher linked to the DNA-nanostructure and the
fluorophore/quencher operably linked to the protector
oligonucleotide. The linkage between the DNA-nanostructure and the
fluorophore/quencher is not critical, and may be any group that can
connect the DNA-nanostructure and the fluorophore/quencher using
known chemistry, provided that is does not interfere with the
quenching or with the strand displacement. Certain embodiments of
various fluorophores and quenchers are discussed below.
[0102] In certain embodiments, the quencher and fluorophore are
separated by between about 1 to about 60 base pairs, about 1 to
about 50 base pairs, about 1 to about 40 base pairs, about 1 to
about 30 base pairs, about 1 to about 20 base pairs, about 1 to
about 15 base pairs or about 1 to about 10 base pairs. In certain
embodiments, the quencher and fluorophore are separated by between
about 9, 8, 7, 6, 5, 4, 3, 2 or about 1 base pair(s).
[0103] In certain embodiments, a fluorophore is operably linked to
the DNA-nanostructure and a quencher is operably linked to the
protector oligonucleotide.
[0104] In certain embodiments, a quencher is operably linked to the
DNA-nanostructure and a fluorophore is operably linked to the
protector oligonucleotide.
[0105] In certain embodiments, the DNA-nanostructure is a
DNA-tetrahedron. In certain embodiments, the DNA-tetrahedrons may
be prepared by methods described in Zhang, et al., Chem Commun, 46,
6792-6794 (2010) and He et al., Nature, 2008, 452, 198, which are
herein incorporated by reference.
[0106] In certain embodiments, the DNA-tetrahedron comprises five
double-stranded edges (e.g., 20 bps) and 1 single stranded edges
(e.g., 28 bps).
[0107] In certain embodiments, the fluorophore/quencher is operably
linked at the vertex of the tetrahedron proximal to the single
stranded edge.
[0108] In certain embodiments, the DNA-tetrahedron is comprised of
four DNA oligonucleotides.
[0109] In certain embodiments, the DNA-tetrahedron comprises four
DNA oligonucleotides, wherein three of the oligonucleotides
comprise at least about 75% sequence identity to SEQ ID NO:2, SEQ
ID NO:3 and SEQ ID NO:4 and the fourth oligonucleotide comprises a
nucleic acid sequence that is complementary to the viral nucleic
acid to be detected. In certain embodiments, the fourth
oligonucleotide comprises two nucleic acid sequences that can
function as toehold domains. In certain embodiments, the fourth
oligonucleotide comprises a nucleic acid sequence of formula I. In
certain embodiments, the three DNA oligonucleotides comprise
nucleic acid sequences independently having at least about 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In certain
embodiments, the three DNA oligonucleotides consist of a nucleic
acid sequence independently having at least about 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% sequence identity
to SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In certain
embodiments, the fluorophore/quencher is operably linked to SEQ ID
NO:4. In certain embodiments, the fluorophore/quencher is operably
linked to the 5' end of SEQ ID NO:4. In certain embodiments, the
fluorophore/quencher is operably linked to the 3' end of SEQ ID
NO:4. In certain embodiments, a fluorophore (e.g., TET) is operably
linked to the 5' end of SEQ ID NO:4.
[0110] In certain embodiments, the DNA-tetrahedron is used to
detect a dengue nucleic acid (e.g., RNA) and comprises four DNA
oligonucleotides comprising at least about 75% sequence identity to
SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In certain
embodiments, the four DNA oligonucleotides comprise nucleic acid
sequences independently having at least about 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In certain
embodiments, the four DNA oligonucleotides consist of a nucleic
acid sequence independently having at least about 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% sequence identity
to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In
certain embodiments, the fluorophore/quencher is operably linked to
SEQ ID NO:4. In certain embodiments, the fluorophore/quencher is
operably linked to the 5' end of SEQ ID NO:4. In certain
embodiments, the fluorophore/quencher is operably linked to the 3'
end of SEQ ID NO:4. In certain embodiments, a fluorophore (e.g.,
TET) is operably linked to the 5' end of SEQ ID NO:4.
[0111] Protector Oligonucleotide
[0112] As described herein, the protector oligonucleotide is
operably linked to a quencher (DNA-nanostructure operably linked to
a fluorophore) or a fluorophore (DNA-nanostructure operably linked
to a quencher) and is capable of hybridizing to a single stranded
region of the DNA-nanostructure, in a position that is suitable for
quenching to occur between the fluorophore and the quencher.
[0113] The linkage between the protector oligonucleotide and the
fluorophore/quencher is not critical, and may be any group that can
connect the protector oligonucleotide and the fluorophore/quencher
using known chemistry, provided that is does not interfere with
quenching or with the strand displacement. Certain embodiments of
various fluorophores and quenchers are discussed below.
[0114] In certain embodiments, a fluorophore is operably linked to
the DNA-nanostructure and a quencher is operably linked to the
protector oligonucleotide.
[0115] In certain embodiments, a quencher is operably linked to the
DNA-nanostructure and a fluorophore is operably linked to the
protector oligonucleotide.
[0116] In certain embodiments, the fluorophore/quencher is operably
linked to the 3'-end of the protector oligonucleotide. In certain
embodiments, the fluorophore/quencher is operably linked to the
5'-end of the protector oligonucleotide.
[0117] The protector oligonucleotide should be capable of being
displaced by the viral nucleic acid and should not be capable of
being displaced by the capture oligonucleotide. Accordingly, in
certain embodiments, the protector oligonucleotide is complementary
to a single stranded region of the DNA-nanostructure and hybridizes
to the second toehold but not the first toehold. In certain
embodiments, the protector oligonucleotide comprises a sequence
that has at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% complementarity with a
portion of the single stranded region of the DNA-nanostructure
(i.e., the second toehold and an adjacent hybridization
region).
[0118] In certain embodiments, the protector oligonucleotide is
hybridized to a single-stranded region of the DNA-nanostructure,
wherein the region of hybridization is linked to a toehold domain,
and wherein the toehold domain is complementary to the viral
nucleic acid. In certain embodiments, the region of hybridization
includes a second toehold domain, and wherein the second toehold
domain is complementary to the capture oligonucleotide.
[0119] The length of the protector oligonucleotide will depend on a
variety of factors, including the size of the DNA-nanostructure and
the sequence of the viral nucleic acid to be detected. In certain
embodiments, the protector oligonucleotide is between about 10 to
about 50 nucleotides in length. In certain embodiments, the
protector oligonucleotide is between about 10 to about 40
nucleotides in length. In certain embodiments, the protector
oligonucleotide is between about 10 to about 30 nucleotides in
length. In certain embodiments, the protector oligonucleotide is
between about 10 to about 25 nucleotides in length. In certain
embodiments, the protector oligonucleotide is between about 15 to
about 25 nucleotides in length. In certain embodiments, the
protector oligonucleotide is between about 17 nucleotides in
length. In certain embodiments, the protector oligonucleotide is
between about 18 nucleotides in length. In certain embodiments, the
protector oligonucleotide is between about 19 nucleotides in
length.
[0120] In certain embodiments, a method of the invention is used to
detect a dengue nucleic acid. In certain embodiments, the protector
oligonucleotide comprises a nucleic acid sequence having at least
about 75% sequence identity to SEQ ID NO:5, SEQ ID NO:6 or SEQ ID
NO:7. In certain embodiments, the protector oligonucleotide
comprises a nucleic acid sequence having at least about 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to SEQ ID NO:5. In certain embodiments, the protector
oligonucleotide consists of a nucleic acid sequence having at least
about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO:5. In certain embodiments, the
protector oligonucleotide comprises a nucleic acid sequence having
at least about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or 100% sequence identity to SEQ ID NO:6. In certain
embodiments, the protector oligonucleotide consists of a nucleic
acid sequence having at least about 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6.
In certain embodiments, the protector oligonucleotide comprises a
nucleic acid sequence having at least about 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ
ID NO:7. In certain embodiments, the protector oligonucleotide
consists of a nucleic acid sequence having at least about 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to SEQ ID NO:7.
[0121] In certain embodiments, a quencher is operably linked to the
3' end of the protector oligonucleotide (e.g., comprising SEQ ID
NO:5, SEQ ID NO:6 or SEQ ID NO:7).
[0122] Fluorophore & Quencher
[0123] As described herein, the DNA-nanostructure is operably
linked to a fluorophore and the protector oligonucleotide is
operably linked to a quencher or the DNA-nanostructure is operably
linked to a quencher and the protector oligonucleotide is operably
linked to a fluorophore; and the quencher is capable of quenching
the fluorescent light emitted from the fluorophore.
[0124] Chemical moieties that quench fluorescent light operate
through a variety of mechanisms, including fluorescence resonance
energy transfer (FRET) processes and ground state quenching. FRET
is one of the most common mechanisms of fluorescent quenching and
can occur when the emission spectrum of the fluorescent donor
overlaps the absorbance spectrum of the quencher and when the donor
and quencher are within a sufficient distance known as the Forster
distance. The energy absorbed by a quencher can subsequently be
released through a variety of mechanisms depending upon the
chemical nature of the quencher. Captured energy can be released
through fluorescence or through non-fluorescent mechanisms,
including charge transfer and collisional mechanisms, or a
combination of such mechanisms. When a quencher releases captured
energy through non-fluorescent mechanisms FRET is simply observed
as a reduction in the fluorescent emission of the fluorescent
donor. Although FRET is the most common mechanism for quenching,
any combination of molecular orientation and spectral coincidence
that results in quenching is a useful mechanism for quenching. For
example, ground-state quenching can occur in the absence of
spectral overlap if the fluorophore and quencher are sufficiently
close together to form a ground state complex.
[0125] Accordingly, the term "quenching" as used herein refers to
the process wherein the quencher molecule absorbs energy from an
excited fluorophore and then releases the captured energy through
either fluorescent or non-fluorescent mechanisms. As used herein,
the term "quencher" includes both molecules that do not emit any
fluorescence signal ("dark quenchers"), as well as molecules that
are themselves fluorophores and emit a signal ("fluorescent
quenchers").
[0126] As discussed above, for quenching to occur, the fluorophore
and quencher must be in physical proximity. When the fluorophore
and quencher are separated (i.e., when the protector
oligonucleotide is not hybridized to the DNA-nanostructure), energy
absorbed by the fluorophore is no longer transferred to the
quencher and is instead emitted as light at the wavelength
characteristic of the fluorophore. Appearance/increase of a
fluorescent signal from the fluorophore following removal of
quenching is a detectable event and constitutes a "positive signal"
in the assay of the present invention, and indicates the presence
of a viral nucleic acid in a sample.
[0127] Specifically, detection agents that employ a fluorescent
quencher will emit light both when the protector oligonucleotide is
hybridized and unhybridized to the DNA-nanostructure; however, the
wavelength of the light will differ depending on the hybridization
state. In the hybridized state, energy captured by the fluorophore
is transferred to the fluorescent quencher via FRET and is emitted
as light at a wavelength characteristic of the fluorescent
quencher. In the unhybridized state, the fluorophore and quencher
are separated and energy absorbed by the fluorophore is no longer
transferred to the quencher and is instead emitted as light at a
wavelength characteristic of the fluorophore. In contrast, when the
detection agent employs a dark quencher, a variation in the amount
of fluorescent emission from the fluorophore will be observed
depending on the hybridization state. In particular, when protector
oligonucleotide is not hybridized to the DNA-nanostructure, energy
absorbed by the fluorophore is emitted as light at a wavelength
characteristic of the fluorophore. However, when the protector
oligonucleotide is hybridized, energy captured by the dark quencher
is released by non-fluorescent mechanisms, which appears as a
reduction in the fluorescent emission from the fluorophore.
[0128] As discussed herein, quenching processes that rely on the
interaction of two dyes as their spatial relationship changes can
be used conveniently to detect the presence of a viral nucleic
acids using a method described herein. As noted previously, the
energy transfer process requires overlap between the emission
spectrum of the fluorescent donor and the absorbance spectrum of
the quencher. Therefore, quencher/fluorophore pairs may be selected
by one skilled in the art based on their emission and absorbance
spectrums to ensure sufficient quenching. For example, the quencher
BHQ-1, which maximally absorbs light in the wavelength range of
about 500-550 nm, can quench the fluorescent light emitted from the
fluorophore fluorescein, which has a wavelength of about 520 nm. In
contrast, the quencher BHQ-3, which maximally absorbs light in the
wavelength range of about 650-700 nm would be less effective at
quenching the fluorescence of fluorescein but would be quite
effective at quenching the fluorescence of the fluorophore Cy5
which fluoresces at about 670 nm.
[0129] A fluorophore is a molecule that absorbs light (i.e.,
excites) at a characteristic wavelength and emits light (i.e.,
fluoresces) at a second lower-energy wavelength. Fluorescence
reporter groups that can be operably linked to the
DNA-nanostructure/protector oligonucleotide include, but are not
limited to, fluorescein, tetrachlorofluorescein (TET),
hexachlorofluorescein, tetramethylrhodamine, rhodamine,
cyanine-derivative dyes, Texas Red, Bodipy, and Alexa dyes. In
certain embodiments, the fluorophore is TET. Characteristic
absorption and emission wavelengths for each of these are well
known to those of skill in the art.
[0130] In certain embodiments, the fluorophore is selected from the
fluorophores listed in Table A below.
[0131] Additionally, as discussed above, a fluorophore may also be
a fluorescent quencher, provided its absorbance spectrum overlaps
with emission spectrum of the selected fluorophore donor (i.e., the
fluorophore and fluorescent quencher are a FRET donor/acceptor
pair).
[0132] Accordingly, in certain embodiments, the quencher is a
fluorescent quencher. In certain embodiments, the fluorescent
quencher is selected from the fluorophores listed in Table A.
TABLE-US-00001 TABLE A Probe Excitation (nm) Emission (nm)
Hydroxycoumarin 325 386 Alexa fluor 325 442 Aminocoumarin 350 445
Methoxycoumarin 360 410 Cascade Blue (375); 401 423 Pacific Blue
403 455 Pacific Orange 403 551 Lucifer yellow 425 528 Alexa fluor
430 430 545 NBD 466 539 R-Phycoerythrin (PE) 480; 565 578 PE-Cy5
conjugates 480; 565; 650 670 PE-Cy7 conjugates 480; 565; 743 767
Red 613 480; 565 613 PerCP 490 675 Cy2 490 510 TruRed 490, 675 695
FluorX 494 520 Fluorescein 495 519 FAM 495 515 BODIPY-FL 503 512
TET 526 540 Alexa fluor 532 530 555 HEX 535 555 TRITC 547 572 Cy3
550 570 TMR 555 575 Alexa fluor 546 556 573 Alexa fluor 555 556 573
Tamara 565 580 X-Rhodamine 570 576 Lissamine Rhodamine B 570 590
ROX 575 605 Alexa fluor 568 578 603 Cy3.5 581 581 596 Texas Red 589
615 Alexa fluor 594 590 617 Alexa fluor 633 621 639 LC red 640 625
640 Allophycocyanin (APC) 650 660 Alexa fluor 633 650 688 APC-Cy7
conjugates 650; 755 767 Cy5 650 670 Alexa fluor 660 663 690 Cy5.5
675 694 LC red 705 680 710 Alexa fluor 680 679 702 Cy7 743 770
IRDye 800 CW 774 789
[0133] Thus, in certain embodiments, the fluorophore is selected
from the group consisting of fluorescein, tetrachlorofluorescein
(TET), hexachlorofluorescein, tetramethylrhodamine, rhodamine,
cyanine-derivative dyes, Texas Red, Bodipy, Alexa dyes and the
fluorophores listed in Table A.
[0134] In certain in vivo embodiments, the fluorophore emits in the
near infrared range, such as in the 650-900 nm range. (Weissleder
et al., "Shedding light onto live molecular targets, Nature
Medicine, 9:123-128 (2003)).
[0135] In one embodiment of the invention, the quencher does not
itself emit a fluorescence signal, i.e. is a "dark quencher". "Dark
quenchers" useful in compositions of the invention include, but are
not limited to, dabcyl, QSY.TM.-7, QSY-33 (4',5-dinitrofluorescein,
pipecolic acid amide) and Black-Hole Quenchers.TM., 1, 2, and 3
(Biosearch Technologies, Novato, Calif.). In certain embodiments,
the quencher is BHQ-1.
[0136] In certain embodiments, the quencher is one or more of the
quenchers listed in Table B.
TABLE-US-00002 TABLE B Quencher Absorption Maximum (nm) DDQ-I 430
Dabcyl 475 Eclipse 530 Iowa Black FQ 532 BHQ-1 534 QSY-7 571 BHQ-2
580 DDQ-II 630 Iowa Black RQ 645 QSY-21 660 BHQ-3 670 IRDye QC-1
737
[0137] Thus, in certain embodiments, the quencher is selected from
dabcyl, QSY.TM.-7, QSY-33 (4',5-dinitrofluorescein, pipecolic acid
amide) Black-Hole Quenchers (BHQ-) -1, -2, and -3 and the quenchers
listed in Table B.
[0138] Additional quenchers are described in U.S. Pat. No.
7,439,341, which is incorporated by reference herein.
[0139] In certain embodiments, the fluorophore is TET and the
quencher is BHQ-1.
[0140] When compositions that employ fluorescent quenchers are used
in a FRET assay, detection may be done using a fluorometer,
fluorescence spectrometer or time-correlated single photon counting
(TCSPC). In certain embodiments, detection agents that employ a
"dark quencher" will emit light only when the protector group is
not hybridized to DNA-nanostructure, thereby enabling signal
detection to be performed visually (detection may also be done
using a fluorometer, fluorescence spectrometer or TCSPC). Visual
detection is rapid, convenient, and does not require the
availability of any specialized equipment. Thus, as used herein,
the term "measuring" also includes visual detection and comparison
(e.g., as compared to a negative control or as compared to the
fluorescence of the detection agent prior to contact with the
sample). Accordingly, it may be possible to detect the presence of
the viral nucleic acid with unassisted visual inspection of the
sample after being contacted with the detection agent and capture
oligonucleotide. However, the fluorescent emission in the test and
control samples may also be measured spectrophotometrically using a
spectrophotometer, fluorometrically using a fluorometer or using
TCSPC to measure the intensity, or by using any other devices
capable of detecting absorbance/fluorescent light emission in a
quantitative or qualitative fashion.
[0141] Linkers
[0142] As described herein, the fluorophore/quencher is operably
linked to the DNA-nanostructure/protector oligonucleotide. In
certain embodiments, the fluorophore and/or quencher is operably
linked to the DNA-nanostructure/protector oligonucleotide by means
of a linker.
[0143] Chemistries that can be used to link the fluorophores and
quencher to an oligonucleotide are known in the art, such as
disulfide linkages, amino linkages, covalent linkages, etc. In
certain embodiments, aliphatic or ethylene glycol linkers that are
well known to those with skill in the art can be used. In certain
embodiments phosphodiester, phosphorothioate and/or other modified
linkages are used.
[0144] In certain embodiments, the linker is a binding pair. In
certain embodiments, the "binding pair" refers to two molecules
which interact with each other through any of a variety of
molecular forces including, for example, ionic, covalent,
hydrophobic, van der Waals, and hydrogen bonding, so that the pair
have the property of binding specifically to each other. Specific
binding means that the binding pair members exhibit binding to each
other under conditions where they do not bind to another molecule.
Examples of binding pairs are biotin-avidin, hormone-receptor,
receptor-ligand, enzyme-substrate probe, IgG-protein A,
antigen-antibody, and the like. In certain embodiments, a first
member of the binding pair comprises avidin or streptavidin and a
second member of the binding pair comprises biotin.
Capture Oligonucleotide
[0145] As described herein, the capture oligonucleotide should be
capable of displacing the viral nucleic acid and hybridizing to the
DNA-nanostructure but should not be capable of displacing the
protector oligonucleotide. Accordingly, in certain embodiments, the
capture oligonucleotide is complementary to a single stranded
region of the DNA-nanostructure and is capable of hybridizing to
the second toehold (i.e., the viral nucleic acid is bound and the
second toehold domain is accessible). In certain embodiments, the
capture oligonucleotide comprises a sequence that has at least
about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99 or 100% complementarity with a portion of the
single stranded region of the DNA-nanostructure (i.e., the second
toehold and the adjacent region wherein the viral nucleic acid is
capable of hybridizing).
[0146] In certain embodiments, the capture oligonucleotide
comprises a nucleic acid sequence that is complementary to a
toehold domain in the DNA-nanostructure, wherein the toehold domain
is linked to a nucleic acid sequence in the DNA-nanostructure that
is capable of hybridizing to the viral nucleic acid.
[0147] The length of the capture oligonucleotide will depend on a
variety of factors, including the size of the DNA-nanostructure and
the sequence of the viral nucleic acid to be detected. In certain
embodiments, the capture oligonucleotide is between about 10 to
about 50 nucleotides in length. In certain embodiments, the capture
oligonucleotide is between about 10 to about 40 nucleotides in
length. In certain embodiments, the capture oligonucleotide is
between about 10 to about 30 nucleotides in length. In certain
embodiments, the capture oligonucleotide is between about 15 to
about 30 nucleotides in length. In certain embodiments, the capture
oligonucleotide is between about 20 to about 27 nucleotides in
length. In certain embodiments, the capture oligonucleotide is
about 23 nucleotides in length. In certain embodiments, the capture
oligonucleotide is about 24 nucleotides in length. In certain
embodiments, the capture oligonucleotide is about 25 nucleotides in
length.
[0148] In certain embodiments, a method of the invention is used to
detect a dengue nucleic acid. In certain embodiments, the capture
oligonucleotide comprises a nucleic acid sequence having at least
about 75% sequence identity to SEQ ID NO:8, SEQ ID NO:9 or SEQ ID
NO:10. In certain embodiments, the capture oligonucleotide
comprises a nucleic acid sequence having at least about 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to SEQ ID NO:8. In certain embodiments, the capture
oligonucleotide consists of a nucleic acid sequence having at least
about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO:8. In certain embodiments, the
capture oligonucleotide comprises a nucleic acid sequence having at
least about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity to SEQ ID NO:9. In certain embodiments, the
capture oligonucleotide consists of a nucleic acid sequence having
at least about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or 100% sequence identity to SEQ ID NO:9. In certain
embodiments, the capture oligonucleotide comprises a nucleic acid
sequence having at least about 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10. In
certain embodiments, the capture oligonucleotide consists of a
nucleic acid sequence having at least about 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ
ID NO:10.
Sample
[0149] The methods described herein may be used to detect the
presence of viral nucleic acid in a sample, such as a biological
fluid (e.g., present in molar, millimolar, micromolar, nanomolar,
picomolar, femtomolar, attomolar or sub-attomolar concentrations).
Thus, in certain embodiments, the concentration of the viral
nucleic acid in the sample is less than about, e.g., 10 mole, 1
mole, 100 millimole, 10 millimole, 1 millimole, 100 micromole, 10
micromole, 1 micromole, 100 nanomole, 10 nanomole, 1 nanomole, 100
picomole, 10 picomole, 1 picomole, 100 femtomole, 10 femtomole, 1
femtomole, 100 attomole, 10 attomole, 1 attomole or 0.1
attomole.
[0150] As used herein, a "sample" may be any sample potentially
comprising a viral nucleic acid. In certain embodiments, the sample
is a liquid sample. In certain embodiments, the sample is a
biological sample obtained from a subject, such as a mammal. In
certain embodiments, the sample is derived from a biological sample
obtained from a subject, such as a mammal. Thus, certain
embodiments of the invention, further comprise obtaining a
biological sample from a subject. As described herein, the term
"biological fluid" refers to any bio-organic fluid produced by an
organism and includes, but is not limited to, e.g., amniotic fluid,
aqueous humour, vitreous humour, bile, blood or components of blood
(e.g., serum or plasma), milk, cerebrospinal fluid (CSF),
endolymph, perilymph, feces, lymph, mucus, pericardial fluid,
peritoneal fluid, pleural fluid, pus, serous fluid, semen, sputum,
synovial fluid, sweat, urine, saliva, tears, vaginal secretions and
vomit. In certain embodiments, the biological fluid is blood or a
blood component, such as serum. In certain embodiments, a
biological fluid is processed prior to performing an assay
described herein. In certain embodiments, a biological fluid is not
processed prior to performing an assay described herein.
Illustrative Compositions and Kits in Accordance with Certain
Embodiments
[0151] Certain embodiments of the invention provide a
DNA-nanostructure described herein (e.g., a DNA tetrahedron
described herein). In certain embodiments, the DNA-nanostructure is
a DNA-tetrahedron that comprises a fluorophore operably linked to
one of the oligonucleotides. Certain embodiments of the invention
provide a protector oligonucleotide described herein. Certain
embodiments of the invention provide a detector agent described
herein. Certain embodiments of the invention provide a capture
oligonucleotide described herein.
[0152] Certain embodiments of the invention provide a composition
comprising a detection agent described herein and a capture
oligonucleotide described herein, and optionally, a buffer. In
certain embodiments, the composition comprises a plurality of each
of the components.
[0153] Certain embodiments of the invention provide a composition
comprising a DNA-nanostructure described herein, a protector
oligonucleotide described herein, and/or a capture oligonucleotide
described herein. Certain embodiments of the invention provide a
composition comprising a DNA-nanostructure described herein, a
protector oligonucleotide described herein, and optionally, a
capture oligonucleotide described herein. In certain embodiments,
the composition further comprises a carrier. In certain
embodiments, the composition comprises a plurality of each of the
components.
[0154] Accordingly, certain embodiments of the invention provide a
composition for detecting a viral nucleic acid in a sample,
comprising:
[0155] a DNA-nanostructure, a capture oligonucleotide and a
protector oligonucleotide;
[0156] wherein the DNA-nanostructure is operably linked to a
fluorophore and the protector oligonucleotide is operably linked to
a quencher or the DNA-nanostructure is operably linked to a
quencher and the protector oligonucleotide is operably linked to a
fluorophore; and wherein the quencher is capable of quenching the
fluorescent light emitted from the fluorophore;
[0157] wherein the protector oligonucleotide is capable of
hybridizing to the DNA-nanostructure;
[0158] wherein the viral nucleic acid is capable of displacing the
protector oligonucleotide and hybridizing to the DNA-nanostructure;
and
[0159] wherein the capture oligonucleotide is capable of displacing
the viral nucleic acid and hybridizing to the DNA-nanostructure but
is not capable of displacing the protector oligonucleotide.
[0160] In certain embodiments, the DNA-nanostructure comprises at
least one single stranded region.
[0161] In certain embodiments, the single stranded region comprises
a nucleic acid sequence that comprises a first toehold domain, a
hybridization region and a second toehold domain. In certain
embodiments, the first toehold domain comprises a nucleic acid
sequence that is complementary to a portion of the viral nucleic
acid. In certain embodiments, the protector oligonucleotide is not
capable of hybridizing to the first toehold domain. In certain
embodiments, the second toehold domain comprises a nucleic acid
sequence that is complementary to a portion of the protector
oligonucleotide and a portion of the capture oligonucleotide. In
certain embodiments, the viral nucleic acid is not capable of
hybridizing to the second toehold domain. In certain embodiments,
the hybridization region comprises a nucleic acid sequence that is
complementary to a portion of the viral nucleic acid, a portion of
the protector oligonucleotide and a portion of the capture
oligonucleotide.
[0162] In certain embodiments, the DNA-nanostructure is a
DNA-tetrahedron. In certain embodiments, the DNA-tetrahedron
comprises five double-stranded edges and one single stranded edge.
In certain embodiments, the fluorophore/quencher is operably linked
at the tetrahedron vertex, proximal to the single stranded edge. In
certain embodiments, the DNA-tetrahedron comprises four
oligonucleotides having at least about 90% sequence identity to SEQ
ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
[0163] In certain embodiments, the protector oligonucleotide is
between about 15 to about 25 nucleotides in length. In certain
embodiments, the fluorophore/quencher is operably linked to the 5'
or 3' end of the protector oligonucleotide. In certain embodiments,
the protector oligonucleotide comprises a nucleic acid sequence
having at least about 90% sequence identity to SEQ ID NO:5, SEQ ID
NO:6 or SEQ ID NO:7.
[0164] In certain embodiments, the capture oligonucleotide is
between about 15 to about 30 nucleotides in length. In certain
embodiments, the capture oligonucleotide comprises a nucleic acid
sequence that is complementary to a toehold domain in the
DNA-nanostructure, and wherein the toehold domain is linked to a
nucleic acid sequence in the DNA-nanostructure that is capable of
hybridizing to the viral nucleic acid. In certain embodiments, the
capture oligonucleotide comprises a nucleic acid sequence having at
least about 90% sequence identity to SEQ ID NO:8, SEQ ID NO:9 or
SEQ ID NO:10.
[0165] In certain embodiments, the viral nucleic acid is from
dengue virus, Ebola virus, human immunodeficiency virus (HIV),
hepatitis B, hepatitis C, Influenza, SARS, measles, Zika, yellow
fever, West Nile fever, smallpox, Marburg viruses, human
papillomavirus, Kaposi's sarcoma-associated herpesvirus or human
T-lymphotropic virus. In certain embodiments, viral nucleic acid is
from Dengue virus.
[0166] Certain embodiments of the invention provide a
DNA-tetrahedron comprising four DNA oligonucleotides, wherein three
of the oligonucleotides comprise at least about 75% sequence
identity to SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4 and the fourth
oligonucleotide comprises a nucleic acid sequence that is
complementary to the viral nucleic acid to be detected. In certain
embodiments, the fourth oligonucleotide comprises two nucleic acid
sequences that can function as toehold domains. In certain
embodiments, the fourth oligonucleotide comprises a nucleic acid
sequence of formula I. In certain embodiments, the three DNA
oligonucleotides independently comprise at least about 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In certain
embodiments, the three DNA oligonucleotides consist of a nucleic
acid sequences independently having at least about 76%, 77%, 78%,
79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94% , 95%, 96%, 97%, 98%, 99% or 100% sequence identity
to SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In certain
embodiments, the fluorophore/quencher is operably linked to SEQ ID
NO:4. In certain embodiments, the fluorophore/quencher is operably
linked to the 5' end of SEQ ID NO:4. In certain embodiments, the
fluorophore/quencher is operably linked to the 3' end of SEQ ID
NO:4. In certain embodiments, a fluorophore (e.g., TET) is operably
linked to the 5'end of SEQ ID NO:4.
[0167] As described herein, methods of the invention may be used to
detect viral nucleic acid in a sample. In certain embodiments, the
viral nucleic acid is from dengue virus. The following embodiments
describe DNA-nanostructures, protector oligonucleotides and capture
oligonucleotides, which may be used to detect a dengue RNA using
methods described herein (e.g., to detect SEQ ID NO:11).
[0168] Certain embodiments of the invention provide a
DNA-tetrahedron comprising four oligonucleotides, wherein the
oligonucleotides comprise a sequence having at least about 75%
sequence identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ
ID NO:4. In certain embodiments, the four DNA oligonucleotides
independently comprise at least about 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In certain embodiments,
the four DNA oligonucleotides consist of a nucleic acid sequences
independently having at least about 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% ,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4. In certain embodiments, a
fluorophore/quencher is operably linked to one of the
oligonucleotides (e.g., a fluorophore or quencher described
herein). In certain embodiments, a fluorophore/quencher is operably
linked to SEQ ID NO:4. In certain embodiments, a
fluorophore/quencher is operably linked to the 5' end of SEQ ID
NO:4. In certain embodiments, a fluorophore/quencher is operably
linked to the 3' end of SEQ ID NO:4. In certain embodiments, a
fluorophore (e.g., TET) is operably linked to the 5' end of SEQ ID
NO:4.
[0169] Certain embodiments of the invention provide a protector
oligonucleotide comprising nucleic acid sequence having at least
about 75% sequence identity to SEQ ID NO:5, SEQ ID NO:6 or SEQ ID
NO:7. In certain embodiments, the protector oligonucleotide
comprises a nucleic acid sequence having at least about 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to SEQ ID NO:5. In certain embodiments, the protector
oligonucleotide consists of a nucleic acid sequence having at least
about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO:5. In certain embodiments, the
protector oligonucleotide comprises a nucleic acid sequence having
at least about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or 100% sequence identity to SEQ ID NO:6. In certain
embodiments, the protector oligonucleotide consists of a nucleic
acid sequence having at least about 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:6.
In certain embodiments, the protector oligonucleotide comprises a
nucleic acid sequence having at least about 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ
ID NO:7. In certain embodiments, the protector oligonucleotide
consists of a nucleic acid sequence having at least about 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to SEQ ID NO:7. In certain embodiments, a
fluorophore/quencher is operably linked to the 3'-end of the
protector oligonucleotide (e.g., a fluorophore or quencher
described herein). In certain embodiments, the fluorophore/quencher
is operably linked to the 5'-end of the protector oligonucleotide
(e.g., a fluorophore or quencher described herein). In certain
embodiments, a quencher is operably linked to the 3' end of the
protector oligonucleotide (e.g., comprising SEQ ID NO:5, SEQ ID
NO:6 or SEQ ID NO:7). In certain embodiments, the quencher is
BHQ-1.
[0170] Certain embodiments of the invention provide a capture
oligonucleotide comprising a nucleic acid sequence having at least
about 75% sequence identity to SEQ ID NO:8, SEQ ID NO:9 or SEQ ID
NO:10. In certain embodiments, the capture oligonucleotide
comprises a nucleic acid sequence having at least about 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence
identity to SEQ ID NO:8. In certain embodiments, the capture
oligonucleotide consists of a nucleic acid sequence having at least
about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity to SEQ ID NO:8. In certain embodiments, the
capture oligonucleotide comprises a nucleic acid sequence having at
least about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% sequence identity to SEQ ID NO:9. In certain embodiments, the
capture oligonucleotide consists of a nucleic acid sequence having
at least about 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or 100% sequence identity to SEQ ID NO:9. In certain
embodiments, the capture oligonucleotide comprises a nucleic acid
sequence having at least about 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:10. In
certain embodiments, the capture oligonucleotide consists of a
nucleic acid sequence having at least about 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ
ID NO:10.
[0171] The present invention further provides kits for practicing
the present methods. Accordingly, certain embodiments of the
invention provide a kit for detecting viral nucleic acid in a
sample comprising:
[0172] a) a DNA-nanostructure;
[0173] b) a protector oligonucleotide;
[0174] b) a capture oligonucleotide; and
[0175] c) instructions for use;
[0176] wherein the DNA-nanostructure is operably linked to a
fluorophore and the protector oligonucleotide is operably linked to
a quencher or the DNA-nanostructure is operably linked to a
quencher and the protector oligonucleotide is operably linked to a
fluorophore; and wherein the quencher is capable of quenching the
fluorescent light emitted from the fluorophore;
[0177] wherein the protector oligonucleotide is capable of
hybridizing to the DNA-nanostructure;
[0178] wherein the viral nucleic acid is capable of displacing the
protector oligonucleotide and hybridizing to the DNA-nanostructure;
and
[0179] wherein the capture oligonucleotide is capable of displacing
the viral nucleic acid and hybridizing to the DNA-nanostructure but
is not capable of displacing the protector oligonucleotide.
[0180] In certain embodiments, the kit comprises a
DNA-nanostructure described herein (e.g., a DNA-tetrahedron
described herein). In certain embodiments, the kit comprises a
protector oligonucleotide as described herein. In certain
embodiments, the kit comprises a capture oligonucleotide as
described herein. In certain embodiments, the kit comprises a
quencher described herein (e.g., a dark quencher or a fluorescent
quencher). Such kits may optionally contain one or more of: a
positive and/or negative control, RNase-free water, and one or more
buffers. In certain embodiments, a kit may further include
RNase-free laboratory plasticware (e.g., a plate(s), such a
multi-well plate(s), such as a 96 well plate(s), a petri dish(es),
a test tube(s), a cuvette(s), a plate(s) for fluorescence or
luminescence etc.).
Administration
[0181] As described herein, methods of the invention may further
comprise administering a therapeutic agent to a mammal (e.g., a
mammal diagnosed with a particular disease, disorder or condition
using a method described herein). Such a therapeutic agent may be
formulated as pharmaceutical composition and administered to a
mammalian host, such as a human patient in a variety of forms
adapted to the chosen route of administration, i.e., orally or
parenterally, by intravenous, intramuscular, topical or
subcutaneous routes.
[0182] Thus, the therapeutic agents may be systemically
administered, e.g., orally, in combination with a pharmaceutically
acceptable vehicle such as an inert diluent or an assimilable
edible carrier. They may be enclosed in hard or soft shell gelatin
capsules, may be compressed into tablets, or may be incorporated
directly with the food of the patient's diet. For oral therapeutic
administration, the active compound may be combined with one or
more excipients and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. Such compositions and preparations should contain at
least 0.1% of active compound. The percentage of the compositions
and preparations may, of course, be varied and may conveniently be
between about 2 to about 60% of the weight of a given unit dosage
form. The amount of active compound in such therapeutically useful
compositions is such that an effective dosage level will be
obtained. The tablets, troches, pills, capsules, and the like may
also contain the following: binders such as gum tragacanth, acacia,
corn starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, fructose, lactose or aspartame or
a flavoring agent such as peppermint, oil of wintergreen, or cherry
flavoring may be added. When the unit dosage form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a vegetable oil or a polyethylene glycol. Various
other materials may be present as coatings or to otherwise modify
the physical form of the solid unit dosage form. For instance,
tablets, pills, or capsules may be coated with gelatin, wax,
shellac or sugar and the like. A syrup or elixir may contain the
active compound, sucrose or fructose as a sweetening agent, methyl
and propylparabens as preservatives, a dye and flavoring such as
cherry or orange flavor. Of course, any material used in preparing
any unit dosage form should be pharmaceutically acceptable and
substantially non-toxic in the amounts employed. In addition, the
active compound may be incorporated into sustained-release
preparations and devices.
[0183] The active compound may also be administered intravenously
or intraperitoneally by infusion or injection. Solutions of the
active compound or its salts can be prepared in water, optionally
mixed with a nontoxic surfactant. Dispersions can also be prepared
in glycerol, liquid polyethylene glycols, triacetin, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0184] The pharmaceutical dosage forms suitable for injection or
infusion can include sterile aqueous solutions or dispersions or
sterile powders comprising the active ingredient which are adapted
for the extemporaneous preparation of sterile injectable or
infusible solutions or dispersions, optionally encapsulated in
liposomes. In all cases, the ultimate dosage form should be
sterile, fluid and stable under the conditions of manufacture and
storage. The liquid carrier or vehicle can be a solvent or liquid
dispersion medium comprising, for example, water, ethanol, a polyol
(for example, glycerol, propylene glycol, liquid polyethylene
glycols, and the like), vegetable oils, nontoxic glyceryl esters,
and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the formation of liposomes, by the
maintenance of the required particle size in the case of
dispersions or by the use of surfactants. The prevention of the
action of microorganisms can be brought about by various
antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will include isotonic agents, for example, sugars,
buffers or sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0185] Sterile injectable solutions are prepared by incorporating
the active compound in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filter sterilization. In the case of sterile
powders for the preparation of sterile injectable solutions, the
specific methods of preparation include vacuum drying and the
freeze drying techniques, which yield a powder of the active
ingredient plus any additional desired ingredient present in the
previously sterile-filtered solutions.
[0186] For topical administration, the present compounds may be
applied in pure form, i.e., when they are liquids. However, it will
generally be desirable to administer them to the skin as
compositions or formulations, in combination with a
dermatologically acceptable carrier, which may be a solid or a
liquid.
[0187] Useful solid carriers include finely divided solids such as
talc, clay, microcrystalline cellulose, silica, alumina and the
like. Useful liquid carriers include water, alcohols or glycols or
water-alcohol/glycol blends, in which the present compounds can be
dissolved or dispersed at effective levels, optionally with the aid
of non-toxic surfactants. Adjuvants such as fragrances and
additional antimicrobial agents can be added to optimize the
properties for a given use. The resultant liquid compositions can
be applied from absorbent pads, used to impregnate bandages and
other dressings, or sprayed onto the affected area using pump-type
or aerosol sprayers.
[0188] Thickeners such as synthetic polymers, fatty acids, fatty
acid salts and esters, fatty alcohols, modified celluloses or
modified mineral materials can also be employed with liquid
carriers to form spreadable pastes, gels, ointments, soaps, and the
like, for application directly to the skin of the user.
[0189] Examples of useful dermatological compositions which can be
used to deliver a therapeutic agent to the skin are known to the
art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392),
Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No.
4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).
[0190] Useful dosages of therapeutic agents can be determined by
comparing their in vitro activity, and in vivo activity in animal
models. Methods for the extrapolation of effective dosages in mice,
and other animals, to humans are known to the art; for example, see
U.S. Pat. No. 4,938,949.
[0191] The amount of the therapeutic agent, or an active salt or
derivative thereof, required for use in treatment will vary not
only with the particular salt selected but also with the route of
administration, the nature of the condition being treated and the
age and condition of the patient and will be ultimately at the
discretion of the attendant physician or clinician.
[0192] The therapeutic agent is conveniently formulated in unit
dosage form. In one embodiment, the invention provides a
composition comprising a therapeutic agent formulated in such a
unit dosage form. The desired dose may conveniently be presented in
a single dose or as divided doses administered at appropriate
intervals, for example, as two, three, four or more sub-doses per
day. The sub-dose itself may be further divided, e.g., into a
number of discrete loosely spaced administrations; such as multiple
inhalations from an insufflator or by application of a plurality of
drops into the eye.
Certain Definitions
[0193] As used herein, the term "about" means .+-.10%.
[0194] "Operably-linked" refers to the association two chemical
moieties so that the function of one is affected by the other,
e.g., an arrangement of elements wherein the components so
described are configured so as to perform their usual function.
[0195] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form, made of monomers (nucleotides) containing a
sugar, phosphate and a base that is either a purine or pyrimidine.
Unless specifically limited, the term encompasses nucleic acids
containing known analogs of natural nucleotides that have similar
binding properties as the reference nucleic acid and are
metabolized in a manner similar to naturally occurring nucleotides.
Unless otherwise indicated, a particular nucleic acid sequence also
encompasses conservatively modified variants thereof (e.g.,
degenerate codon substitutions) and complementary sequences, as
well as the sequence explicitly indicated. Specifically, degenerate
codon substitutions may be achieved by generating sequences in
which the third position of one or more selected (or all) codons is
substituted with mixed-base and/or deoxyinosine residues.
[0196] The terms "nucleotide sequence" and "nucleic acid sequence"
refer to a sequence of bases (purines and/or pyrimidines) in a
polymer of DNA or RNA, which can be single-stranded or
double-stranded, optionally containing synthetic, non-natural or
altered nucleotide bases capable of incorporation into DNA or RNA
polymers, and/or backbone modifications (e.g., a modified oligomer,
such as a morpholino oligomer, phosphorodiamate morpholino oligomer
or vivo-mopholino). The terms "oligo", "oligonucleotide" and
"oligomer" may be used interchangeably and refer to such sequences
of purines and/or pyrimidines. The terms "modified oligos",
"modified oligonucleotides" or "modified oligomers" may be
similarly used interchangeably, and refer to such sequences that
contain synthetic, non-natural or altered bases and/or backbone
modifications (e.g., chemical modifications to the internucleotide
phosphate linkages and/or to the backbone sugar).
[0197] Modified nucleotides are known in the art and include, by
example and not by way of limitation, alkylated purines and/or
pyrimidines; acylated purines and/or pyrimidines; or other
heterocycles. These classes of pyrimidines and purines are known in
the art and include, pseudoisocytosine; N4, N4-ethanocytosine;
8-hydroxy-N6-methyladenine; 4-acetylcytosine,
5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil;
5-carboxymethylaminomethyl-2-thiouracil; 5-carboxymethylaminomethyl
uracil; dihydrouracil; inosine; N6-isopentyl-adenine;
1-methyladenine; 1-methylpseudouracil; 1-methylguanine;
2,2-dimethylguanine; 2-methyladenine; 2-methylguanine;
3-methylcytosine; 5-methylcytosine; N6-methyladenine;
7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino
methyl-2-thiouracil; .beta.-D-mannosylqueosine;
5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2-methylthio-N6-i
sopentenyladenine; uracil-5-oxyacetic acid methyl ester;
psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil;
4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid
methylester; uracil 5-oxyacetic acid; queosine; 2-thiocytosine;
5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine;
5-butyluracil; 5-pentyluracil; 5-pentylcytosine; and
2,6,-diaminopurine; methylpsuedouracil; 1-methylguanine;
1-methylcytosine. Backbone modifications are similarly known in the
art, and include, chemical modifications to the phosphate linkage
(e.g., phosphorodiamidate, phosphorothioate (PS), N3'
phosphoramidate (NP), boranophosphate, 2',5' phosphodiester,
amide-linked, phosphonoacetate (PACE), morpholino, peptide nucleic
acid (PNA) and inverted linkages (5'-5' and 3'-3' linkages)) and
sugar modifications (e.g., 2' -O-Me, UNA, LNA).
[0198] The oligonucleotides described herein may be synthesized
using standard solid or solution phase synthesis techniques which
are known in the art. In certain embodiments, the oligonucleotides
are synthesized using solid-phase phosphoramidite chemistry (U.S.
Pat. No. 6,773,885) with automated synthesizers. Chemical synthesis
of nucleic acids allows for the production of various forms of the
nucleic acids with modified linkages, chimeric compositions, and
nonstandard bases or modifying groups attached in chosen places
through the nucleic acid's entire length.
[0199] Certain embodiments of the invention encompass isolated or
substantially purified nucleic acid compositions. In the context of
the present invention, an "isolated" or "purified" DNA molecule or
RNA molecule is a DNA molecule or RNA molecule that exists apart
from its native environment and is therefore not a product of
nature. An isolated DNA molecule or RNA molecule may exist in a
purified form or may exist in a non-native environment such as, for
example, a transgenic host cell. For example, an "isolated" or
"purified" nucleic acid molecule is substantially free of other
cellular material or culture medium when produced by recombinant
techniques, or substantially free of chemical precursors or other
chemicals when chemically synthesized. In one embodiment, an
"isolated" nucleic acid is free of sequences that naturally flank
the nucleic acid (i.e., sequences located at the 5' and 3' ends of
the nucleic acid) in the genomic DNA of the organism from which the
nucleic acid is derived.
[0200] By "portion" or "fragment," as it relates to a nucleic acid
molecule, sequence or segment of the invention, when it is linked
to other sequences for expression, is meant a sequence having at
least, e.g., about 80 nucleotides, 150 nucleotides, or 400
nucleotides. If not employed for expressing, a "portion" or
"fragment" means at least, e.g., about 9, 12, 15, or 20 consecutive
nucleotides, e.g., probes and primers (oligonucleotides),
corresponding to the nucleotide sequence of the nucleic acid
molecules of the invention.
[0201] "Recombinant DNA molecule" is a combination of DNA sequences
that are joined together using recombinant DNA technology and
procedures used to join together DNA sequences as described, for
example, in Sambrook and Russell, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
Press (3.sup.rd edition, 2001).
[0202] "Homology" refers to the percent identity between two
polynucleotides or two polypeptide sequences. Two DNA or
polypeptide sequences are "homologous" to each other when the
sequences exhibit at least about 75% to 85% (including 75%, 76%,
77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, and 85%), at least about
90%, or at least about 95% to 99% (including 95%, 96%, 97%, 98%,
99%) contiguous sequence identity over a defined length of the
sequences.
[0203] The following terms are used to describe the sequence
relationships between two or more nucleotide sequences: (a)
"reference sequence," (b) "comparison window," (c) "sequence
identity" (d) "percentage of sequence identity," (e) "substantial
identity" and (f) "complementarity".
[0204] (a) As used herein, "reference sequence" is a defined
sequence used as a basis for sequence comparison. A reference
sequence may be a subset or the entirety of a specified sequence;
for example, as a segment of a full-length cDNA or gene sequence,
or the complete cDNA or gene sequence.
[0205] (b) As used herein, "comparison window" makes reference to a
contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. Generally, the
comparison window is at least 20 contiguous nucleotides in length,
and optionally can be 30, 40, 50, 100, or longer. Those of skill in
the art understand that to avoid a high similarity to a reference
sequence due to inclusion of gaps in the polynucleotide sequence a
gap penalty is typically introduced and is subtracted from the
number of matches.
[0206] Methods of alignment of sequences for comparison are
well-known in the art. Thus, the determination of percent identity,
including sequence complementarity, between any two sequences can
be accomplished using a mathematical algorithm. Non-limiting
examples of such mathematical algorithms are the algorithm of Myers
and Miller (Myers and Miller, CABIOS, 4, 11 (1988)); the local
homology algorithm of Smith et al. (Smith et al., Adv. Appl. Math.,
2, 482 (1981)); the homology alignment algorithm of Needleman and
Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)); the
search-for-similarity-method of Pearson and Lipman (Pearson and
Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444 (1988)); the algorithm
of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci.
USA, 87, 2264 (1990)), modified as in Karlin and Altschul (Karlin
and Altschul, Proc. Natl. Acad. Sci. USA 90, 5873 (1993)).
[0207] Computer implementations of these mathematical algorithms
can be utilized for comparison of sequences to determine sequence
identity or complementarity. Such implementations include, but are
not limited to: CLUSTAL in the PC/Gene program (available from
Intelligenetics, Mountain View, Calif.); the ALIGN program (Version
2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Version 8 (available from Genetics
Computer Group (GCG), 575 Science Drive, Madison, Wis., USA).
Alignments using these programs can be performed using the default
parameters. The CLUSTAL program is well described by Higgins et al.
(Higgins et al., CABIOS, 5, 151 (1989)); Corpet et al. (Corpet et
al., Nucl. Acids Res., 16, 10881 (1988)); Huang et al. (Huang et
al., CABIOS, 8, 155 (1992)); and Pearson et al. (Pearson et al.,
Meth. Mol. Biol., 24, 307 (1994)). The ALIGN program is based on
the algorithm of Myers and Miller, supra. The BLAST programs of
Altschul et al. (Altschul et al., JMB, 215, 403 (1990)) are based
on the algorithm of Karlin and Altschul supra.
[0208] Software for performing BLAST analyses is publicly available
through the National Center for Biotechnology Information. This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold. These initial neighborhood word hits act as seeds for
initiating searches to find longer HSPs containing them. The word
hits are then extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Cumulative scores are calculated using, for nucleotide sequences,
the parameters M (reward score for a pair of matching residues;
always >0) and N (penalty score for mismatching residues; always
<0). For amino acid sequences, a scoring matrix is used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when the cumulative alignment score falls off
by the quantity X from its maximum achieved value, the cumulative
score goes to zero or below due to the accumulation of one or more
negative-scoring residue alignments, or the end of either sequence
is reached.
[0209] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences. One measure of similarity
provided by the BLAST algorithm is the smallest sum probability
(P(N)), which provides an indication of the probability by which a
match between two nucleotide or amino acid sequences would occur by
chance. For example, a test nucleic acid sequence is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid sequence to the reference
nucleic acid sequence is less than about 0.1, less than about 0.01,
or even less than about 0.001.
[0210] To obtain gapped alignments for comparison purposes, Gapped
BLAST (in BLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in
BLAST 2.0) can be used to perform an iterated search that detects
distant relationships between molecules. When utilizing BLAST,
Gapped BLAST, PSI-BLAST, the default parameters of the respective
programs (e.g., BLASTN for nucleotide sequences, BLASTX for
proteins) can be used. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSUM62 scoring matrix. Alignment may also be performed manually
by inspection.
[0211] For purposes of the present invention, comparison of
nucleotide sequences for determination of percent sequence identity
may be made using the BlastN program (version 1.4.7 or later) with
its default parameters or any equivalent program. By "equivalent
program" is intended any sequence comparison program that, for any
two sequences in question, generates an alignment having identical
nucleotide or amino acid residue matches and an identical percent
sequence identity when compared to the corresponding alignment
generated by the program.
[0212] (c) As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences makes
reference to a specified percentage of residues in the two
sequences that are the same when aligned for maximum correspondence
over a specified comparison window, as measured by sequence
comparison algorithms or by visual inspection.
[0213] (d) As used herein, "percentage of sequence identity" means
the value determined by comparing two optimally aligned sequences
over a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison, and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0214] (e)(i) The term "substantial identity" of polynucleotide
sequences means that a polynucleotide comprises a sequence that has
at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or
94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity,
compared to a reference sequence using one of the alignment
programs described using standard parameters.
[0215] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0216] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. Generally, stringent conditions are
selected to be about 5.degree. C. lower than the thermal melting
point (T.sub.m) for the specific sequence at a defined ionic
strength and pH. However, stringent conditions encompass
temperatures in the range of about 1.degree. C. to about 20.degree.
C., depending upon the desired degree of stringency as otherwise
qualified herein. Nucleic acids that do not hybridize to each other
under stringent conditions are still substantially identical if the
polypeptides they encode are substantially identical. This may
occur, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code. One
indication that two nucleic acid sequences are substantially
identical is when the polypeptide encoded by the first nucleic acid
is immunologically cross reactive with the polypeptide encoded by
the second nucleic acid.
[0217] The phrase "hybridizing specifically to" refers to the
binding, duplexing, or hybridizing of a molecule only to a
particular nucleotide sequence under stringent conditions when that
sequence is present in a complex mixture (e.g., total cellular) DNA
or RNA. "Bind(s) substantially" refers to complementary
hybridization between a probe nucleic acid and a target nucleic
acid and embraces minor mismatches that can be accommodated by
reducing the stringency of the hybridization media to achieve the
desired detection of the target nucleic acid sequence.
[0218] (f) The term "complementary" as used herein refers to the
broad concept of complementary base pairing between two nucleic
acids aligned in an antisense position in relation to each other.
When a nucleotide position in both of the molecules is occupied by
nucleotides normally capable of base pairing with each other, then
the nucleic acids are considered to be complementary to each other
at this position. Thus, two nucleic acids are substantially
complementary to each other when at least, e.g., about 50%, at
least about 60% or at least about 80% of corresponding positions in
each of the molecules are occupied by nucleotides which normally
base pair with each other (e.g., A:T (A:U for RNA) and G:C
nucleotide pairs).
[0219] As used herein, the term "derived" or "directed to" with
respect to a nucleotide molecule means that the molecule has
complementary sequence identity to a particular molecule of
interest.
[0220] The term "mammal" as used herein refers to humans, higher
non-human primates, rodents, domestic, cows, horses, pigs, sheep,
dogs and cats. In one embodiment, the mammal is a human.
[0221] The term "therapeutically effective amount," in reference to
treating a disease state/condition, refers to an amount of a
therapeutic agent that is capable of having any detectable,
positive effect on any symptom, aspect, or characteristics of a
disease state/condition when administered as a single dose or in
multiple doses. Such effect need not be absolute to be
beneficial.
[0222] The terms "treat" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or decrease an undesired physiological change
or disorder. For purposes of this invention, beneficial or desired
clinical results include, but are not limited to, alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not
worsening) state of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. Those in
need of treatment include those already with the condition or
disorder as well as those prone to have the condition or disorder
or those in which the condition or disorder is to be prevented.
[0223] The invention will now be illustrated by the following
non-limiting Examples.
EXAMPLE 1
A Duo-Toehold-Mediated Displacement Reaction on DNA Tetrahedron for
RNA Detection of Dengue Virus
[0224] Since Rothemund established the first DNA origami in 2006,
this unique technique has garnered worldwide interest amongst
researchers. Simply by using the complementary property of base
pairs, highly selective double helixes can be formed in different
spatial forms. For example, DNA tetrahedrons, which are one of the
simplest spatial DNA forms, can be used to provide a rigid
structure for further application. The stability of the tetrahedron
form strongly restricts the distance between each position on the
tetrahedron, making it an optimal optical probe for target
detection using Foster resonance energy transfer (FRET). The only
problem is that the concentration of virus RNA is very low at the
early stage, and therefore, the signal needs to be amplified for
proper detection. Toehold-mediated displacement reaction (TMDR) is
an ideal approach to achieve this goal under moderate conditions.
TMDR is a kinetic-controlled non-enzymatic process. In this
process, a single stranded oligonucleotide (referred to as
toehold), which neighbors to a double strand helix, mediates a
displacement with another single stranded oligonucleotide. This
process can occur automatically at room temperature without any
other assistance and has been applied to many fields, such as logic
gates, catalysts or self-assembly. Now, a duo-TMDR amplification on
a DNA tetrahedron for sensitively detecting RNA sequence of dengue
virus is described herein (FIG. 1). In the first TMDR process,
target RNA annealed to a complementary DNA sequence via a first
toehold and displaced a protector DNA and recovered fluorescence.
In the second TMDR process, capture DNA displaced the target RNA
via a second toehold. The target RNA can then be recycled in the
first TMDR process and form an amplifying loop, thereby enhancing
the fluorescence signal. Moreover, with the help of single molecule
detection technique, 0.1 attomolar of target RNA could be
detected.
Results and Discussion
[0225] To ensure the stability and efficiency for the detection,
DNA tetrahedron was designed to maintain its rigid structure. Four
DNAs (DNA1-DNA 4, see Table 1 for sequence details) were used to
construct the frame of the tetrahedron. The tetrahedron had five 20
bp double stranded edges and one edge with a 28-base single strand.
Fluorescent organic dye tetrachlorofluorescein (TET) was labeled on
the 5' end of DNA 4, which was at the vertex of the tetrahedron
neighboring to the single strand. Protector DNA, which was modified
with black hole quencher 1 (BHQ-1) on its 3' end, was also annealed
to the tetrahedron on the single strand side and left a few bases
beyond its 3' end as the first toehold. At this stage, the
fluorescence of TET could be quenched by BHQ-1 due to the FRET
process. Once the target RNA was added to the solution, the first
TMDR process could be triggered automatically. Protector DNA could
be displaced and recover the fluorescence of TET. Once the
protector DNA was displaced, the second toehold beyond the 5' end
of target RNA could be exposed. If capture DNA was also in the
solution, the second TMDR process could be triggered and target RNA
could be displaced. The displaced target RNA could be recycled in
this process, displace more protector DNA and amplify the signal
(FIG. 1). There were two purposes of designing the protector DNA,
the first was to quench the fluorescence of TET and form the
"FRET-on" status; the second was to prevent the single strand on
the tetrahedron to anneal with capture DNA directly.
TABLE-US-00003 TABLE 1 Sequence detail of the nucleic acid used in
this study. Nucleic Acid Name Type Sequence (5' to 3') Modification
Length DNA 1 DNA TGC TCT TCC CGA AGG TCG CAT N/A 71 ATG AGC AAC TCC
CAC TCA ACT GCC TGG TGA TAC GAG GAT GGG CA (SEQ ID NO: 1) DNA 2 DNA
GGT GAT AAA ACG TGT AGC AAG CTG TAA N/A 63 TCG ACG GGA AGA GCA TGC
CCA TCC ACT ACT ATG GCG (SEQ ID NO: 2) DNA 3 DNA AGG CAG TTG AGA
CGA ACA TTC CTA AGT N/A 63 CTG AAA TTT ATC ACC CGC CAT AGT AGA CGT
ATC ACC (SEQ ID NO: 3) DNA 4 DNA TCG ATT ACA GCT TGC TAC ACG ATT
CAG 5'-TET 43 ACT TAG GAA TGT TCG T (SEQ ID NO: 4) Protector 17 DNA
AGT TGC TCA TAT GCG AC (SEQ ID NO: 5) 3'-BHQ1 17 Protector 18 DNA
AGT TGC TCA TAT GCG ACC (SEQ ID NO: 6) 3'-BHQ1 18 Protector 19 DNA
AGT TGC TCA TAT GCG ACC T (SEQ ID NO: 7) 3'-BHQ1 19 Capture 22 DNA
GGG AGT TGC TCA TAT GCG ACC T (SEQ ID N/A 23 NO: 8) Capture 24 DNA
GGG AGT TGC TCA TAT GCG ACC TTG (SEQ N/A 24 ID NO: 9) Capture 25
DNA GGG AGT TGC TCA TAT GCG ACC TTG C (SEQ N/A 25 ID NO: 10) Dengue
RNA CUC AUA UGC GAC CUU GCA UC (SEQ ID N/A 20 Target NO: 11) The
toeholds are shown in italic. Recognition regions are shown in
bold. Amplification regions are underlined.
[0226] The formation of DNA tetrahedron could be clearly verified
by the native polyacrylamide gel electrophoresis (PAGE) (FIG. 3a).
Different lengths of protector DNAs were used to form the protected
tetrahedron (p-TETRA). A single strand indicated the tetrahedron
(TETRA) and P-TETRA with different protector DNA could be formed
with high efficiency. High repeatability could also be achieved
since the sequence of DNA was carefully designed (FIG. 6). The
fluorescence also indicated the formation of the P-TETRA (FIG. 3b).
In the absence of protector DNA, the fluorescence of TET could be
slightly quenched due to the heating process. However, the addition
of protector DNAs could severely quench the fluorescence with an
efficiency of 59.6%. The length of protector DNA could barely
influence the quenching efficiency since the distance between the
organic fluorescent dye and quencher were only two bases
difference. For the FRET model, the interaction between dipole and
dipole followed the power law distance dependence and the FRET
distance R.sub.0 (50% quenching distance) could be described
as:
R.sub.0=9.78.times.10.sup.2(.kappa..sup.2n.sup.-4Q.sub.dyeJ).sup.1/6
(1)
in which, .kappa..sup.2 is the dipole orientation factor and is
always 2/3, n is the refractive index of the medium (1.333 for
water medium), Q.sub.dye is the quantum yield of TET and J is the
overlap integral between the emission of TET and the absorption of
BHQ-1. The theoretical quenching efficiency of FRET
(.eta..sub.FRET) follows the 6.sup.th-power law and is described
as:
.eta. FRET = 1 1 + ( r R 0 ) 6 ( 2 ) ##EQU00001##
where the r is the actual distance between TET and BHQ-1. The
calculated R.sub.0 was 4.41 nm. The value of r could be longer than
the 8 bases (3.6 nm) considering the twist angle and the diameter
of DNA helix structure. In this circumstance, the r was 3.95 nm and
the calculated .eta..sub.FRET was 0.659, which agreed with the
experimental result. Pairwise and triple-wise hybridization was
also performed to verify the design of the sequences were
legitimate (FIGS. 7a and 7b).
[0227] The feasibility of this duo-toehold-mediated displacement
reaction was first tested (FIG. 4a). By adding target RNA or
capture DNA separately, the fluorescence of the mixture could
hardly be recovered. This is because the addition of only target
could just trigger the first toehold-mediated displacement
reaction. Displacing the protector can switch off the FRET process
between TET and BHQ-1 and restore the fluorescence, but the
difference was too small and could not be told by the spectrum,
which meant that the traditional FRET process could not distinguish
the existence of the target at this level. Meanwhile, the reacting
site for the second toehold-mediated displacement reaction was
blocked by protector DNA, and therefore, the addition of only
capture DNA could not restore the fluorescence either. Only when
both the target and capture were added, the two toehold-mediated
displacement reactions could be activated simultaneously and the
fluorescence could be restored with a recovering efficiency of
142.8%, which was almost fully recovered to the fluorescence
intensity of TETRA. The displacement reaction could also be
observed from the PAGE (FIG. 4b). By only adding the target RNA
(lane 2), the migrating rate was not changed from the P-TETRA (lane
1). By adding both target RNA and capture DNA, the protector DNA on
the P-TETRA would eventually be displaced by capture DNA. The
additional 8 bases were manifested by slower migrating rate on the
gel (lane 3). The average fluorescence lifetime was also measured
could indicate the occurrence of this duo-TMDR process more clearly
(FIG. 4c and Table 2).
[0228] The initial lifetime of TET on DNA 4 was 3.550 ns. A small
proportion of fast component was contributed by the rotation
restriction of DNA. However, by forming the p-TETRA, the
fluorescence lifetime decreased to 1.552 ns. The quenching
efficiency of lifetime was contributed by both radiative,
non-radiative decay and FRET, which referred as total quenching
efficiency (.eta..sub.total) and can be described as:
.eta. total = 1 - .tau. N .tau. 0 ( 3 ) ##EQU00002##
where the .tau..sub.N is the lifetime of p-TETRA and .tau..sub.0 is
the lifetime of TET-labelled DNA-4. The .eta..sub.total was 0.563,
which was identical with the quenching efficiency in fluorescence
spectra and the .eta..sub.FRET. That was to say, the quenching of
fluorescence was mostly contributed to the occurrence of FRET. The
addition of only target RNA could hardly recover the lifetime of
TET. However, by adding both target RNA and capture DNA, the
fluorescence lifetime could be recovered to 3.168 ns, which proved
the displacement quencher labeled protector DNA and switched off
the FRET process. The fast component in recovered p-TETRA was a
little higher than DNA-4, this was because the tetrahedron DNA
could handicap the free rotation of TET more severely than single
strand DNA.
TABLE-US-00004 TABLE 2 Fluorescence lifetime for TET at different
stage of reaction. .tau..sub.1 .tau..sub.2 .tau..sub.3 .tau. (ns)
.alpha..sub.1 (ns) .alpha..sub.2 (ns) .alpha..sub.3 (ns).sup.a DNA
4 0.5584 0.1005 3.8847 0.8995 n/a n/a 3.550 p- 0.1065 0.4197 0.8412
0.2228 3.694 0.3574 1.552 TETRA p- 0.08724 0.3995 0.7935 0.2119
3.677 0.3885 1.632 TETRA + target RNA p- 0.4582 0.1774 3.752 0.8226
n/a n/a 3.168 TETRA + target RNA + capture DNA .sup.athe average
fluorescent lifetime was calculated from the following equation:
.tau. = .SIGMA..sub.i=1.sup.n(.tau..sub.i .times.
.alpha..sub.i)
[0229] For sensitive and rapid detection of target RNA, the length
of protector DNA was studied since it was critical to the
displacing rate in the reaction. Therefore, the influence of the
length of the protector DNA to the dynamic fluorescence changes of
p-TETRA was measured. Three oligonucleotides with different length
were annealed to the tetrahedron as previously described and served
as a protector. The same target RNA and capture DNA were used to
trigger the displacement reaction. The fluorescence signal was
constantly measured for 150 min and the dynamic changes were
recorded (FIG. 9a). When the protector with 17 bases was used to
form the tetrahedron, the fastest recovering rate could be
achieved, which manifested as the largest slope at the early stage.
By using a protector with 17 bases, the length of the first toehold
increased to 8 bases, and therefore, it was much easier for the
target to displace the protector and recover the fluorescence
signal. The influence of DNA length on the second toehold-mediated
strand displacement reaction was also investigated. Protector 17
DNA was used to form the p-TETRA and capture DNA with different
lengths were used along with the same concentration of target RNA
(FIG. 9b). By using capture DNA with 25 bases, over 80% of
fluorescence could be recovered within 60 min, which was much
faster than the other two samples. This was because only 6 binding
spots were left for target RNA by using capture 25 DNA, which is
shorter than using other capture DNAs. Since the target RNA could
be displaced easier and faster, the duo-TMDR process could be
accelerated and manifested as a faster fluorescence recovering
rate.
[0230] The concentration of dengue virus RNA was further quantified
via two methods, the common spectrometer detection and the
single-molecule detection. FIG. 10 shows the fluorescence intensity
increased with the concentration of target RNA of dengue virus. A
good linear relationship (R.sup.2>0.99) could be found in the
range of 40 pM to 1 nM and the limit of detection (LOD) was 15 pM
(3.sigma./k). The sensitivity by using this duo-TMDR was much
higher than the reported methods. FIG. 5a showed the response
traces of photon counts with 5 s by using the single molecule
detection. The photons, which generated from the emission of TET,
associated with the concentration of target RNA. As the
concentration of target RNA increased, more protector DNA could be
displaced and switch off the FRET. By departing from the BHQ-1,
more excited electrons could release the energy by radiatively
emitting photons rather than in a non-radiative manner. Therefore,
the higher concentration of target, the higher photon counts could
be observed. As shown in FIG. 5b, by adding only target RNA to the
p-TETRA, no significant changes could be observed comparing to the
p-TETRA. By adding capture DNA and different concentration of
target RNA to the p-TETRA, the photon counts increased gradually.
For 0.1 M (10.sup.-19 M) of target RNA to trigger the duo-TMDR, the
photon counts could increase from 346.+-.33 ms.sup.-1 to 387.+-.28
ms.sup.-1. In our experiments, the volume was set to 100 .mu.L,
which meant that only about six copies of target RNA were in the
system. It should be noted that simply by changing the sequence of
DNA 1, this method could be used for the other targets
detection.
[0231] To demonstrate the stability of this method, the recovery of
fluorescence was also performed in the present of 10% human serum
by using the single molecule detection (FIG. 11). The presence of
serum could not influence the recovery of fluorescence signal which
indicated this method has potential usage in the practical
application.
[0232] In conclusion, a noble approach for sensitively detecting
RNA of dengue virus using the duo-TMDR process was developed. The
protector DNA ensured the reaction could only occur when target
presented. The presence of target RNA could displace the protector
DNA and recover the fluorescence signal. Meanwhile, the exposed
second toehold could anneal to capture DNA and displace target RNA.
The displaced RNA could be recycled in the process and amplify the
fluorescence signal. The detection limit of this method could reach
sub-attomolar, where only about six copies of RNA was in the
system. Moreover, this method could be compatible to detect other
types of nucleic acids simply by changing the sequence on the
p-TETRA. This method is rapid and sensitive and might be extended
to the early-stage diagnosis.
Materials and Methods
[0233] Materials and Reagents. All DNA and RNA samples were
purchased from Sangon Biotech Co., Ltd. (Shanghai, China). The
sequences were listed in the Table 1. Magnesium chloride 6-hydrate
was purchased from Mallinckrodt Pharmaceuticals (St. Louis, Mo.),
Tris base was purchased from Geno Technology, Inc. (St. Louis,
Mo.), ammonium persulfate (APS) and
N,N,N',N'-tetramethylethylenediamine (TEMED) were purchased from
Sigma-Aldrich Co. LLC (St. Louis, Mo.), 40%
acrylamide/bis-acrylamide solution was purchased from Thermo Fisher
Scientific Inc. (Ward Hill, Mass.). All reagents are of analytical
grade unless otherwise statements. Analog vortex mixer (VWR,
Radnor, Pa.) was used to mix the solutions and 18.2 M.OMEGA.cm
H.sub.2O was used for all experiments.
[0234] DNA stock solutions. The purchased oligonucleotides were
dissolved in 10 mM pH 8.0 Tris-HCl buffer with 10 mM Mg.sup.2+
(referred to as TH--Mg buffer) and stored at -4.degree. C. The
as-mentioned buffer was used as reaction buffer throughout.
[0235] Annealing. The annealing processes were all performed on the
dry bath incubator (Boekel Scientific, Feasterville, Pa.). The
solution of mixed DNAs was heated to 95.degree. C. for 15 min and
gradually cooled down to room temperature for a period over 2 h.
The annealed DNA complex solution was stored at -4.degree. C.
[0236] Optimization of magnesium concentration for synthesizing DNA
tetrahedron. 200 nM DNA 1, 2, 3, 4 and protector was added to 10 mM
pH 8.0 Tris-HCl buffer with different concentrations of magnesium
chloride and proceeded the annealing process, followed by further
characterization.
[0237] Characterization of DNA tetrahedron. DNA tetrahedron was
synthesized with DNA1, 2, 3, 4 and protector by an annealing
process. 5% native polyacrylamide gel electrophoresis (PAGE) was
involved in characterizing the formation of DNA tetrahedron. DNA
sequencing system (model 4200, Fotodyne, Hartland, Wis.) was used
to supply the constant direct current. The voltage was set at 80 V
while the power should be less than 3 W for reducing the
temperature rising. Normally, the electrophoresis could finish in
90 min.
[0238] Characterization of toehold-mediated displacement reaction
with the fluorescence spectrometer. To 100 nM DNA tetrahedron, 10
nM target RNA and 100 .mu.M capture DNA was added. TH--Mg buffer
was used to set the volume to 100 .mu.L. The reaction was kept at
room temperature for 3 h in dark and the fluorescence was
measured.
[0239] Optimization of the concentration of DNA tetrahedron in the
reaction. To different concentration of DNA tetrahedron, 10 nM
target RNA and 100 .mu.M capture DNA was added. TH--Mg buffer was
used to set the volume to 100 .mu.L. The reaction was kept at room
temperature for 3 h in dark place and the fluorescence was
measured. Solution without target DNA or capture DNA or both were
also measured as a control.
[0240] Optimization of the pH value in the reaction. To 100 nM DNA
tetrahedron, 10 nM target RNA and 100 .mu.M capture DNA was added.
10 mM Tris-HCl and 10 mM Mg.sup.2+ buffer with different pH value
were used to set the volume to 100 .mu.L. The reaction was kept at
room temperature for 3 h in dark and the fluorescence was measured.
Analogous solution without target DNA was measured as a
control.
[0241] Experimental setup for the fluorescence lifetime
measurement. Fluorescence lifetime was measured using the
time-correlated single-photon counting (TCSPC) technique. The
excitation source was a fiber supercontinuum laser based on a
passive modelocked fiber laser and a high-nonlinearity photonic
crystal fiber supercontinuum generator (Fianium SC450-PP). The
laser provides 6-ps pulses at a repetition rate variable between
0.1-40 MHz. The laser output was sent through an Acousto-Optical
Tunable Filer (Fianium AOTF) to obtain excitation pulses at desired
wavelength of 500 nm. Fluorescence emission was collected at a
90.degree. geometry setting and detected using a double-grating
monochromator (Jobin-Yvon, Gemini-180) and a microchannel plate
photomultiplier tube (Hamamatsu R3809U-50). The polarization of the
emission was set at 54.7.degree. relative to that of the
excitation. Data acquisition was done using a single photon
counting card (Becker-Hickl, SPC-830). The typical IRF had a FWHM
of 40 ps, measured from the scattering of sample at the excitation
wavelength. The excitation power was kept at the repetition rate of
20 MHz. The data was fitted with a sum of exponential decay model
globally or at a single wavelength using a home-written program
ASUFIT.
[0242] Experimental setup for the single molecule detection. Single
molecule detection was performed on the Nikon inverted TE2000-U
microscope (Nikon Instruments Inc., Melville, N.Y.). Krypton/argon
laser (Melles Griot 35-KAP-431-208, IDEX Health & Science LLC.,
Carlsbad, Calif.) was used as excitation source for all
experiments. The laser beam was reflected by a double dichroic
mirror (514 nm/647 nm, Chroma Tech. Co., Bellows Falls, Vt.) and
focused by a water immersion 60.times./1.20 Plan-Apo objective lens
(Nikon Instruments Inc., Melville, N.Y.) to excite the samples on
the cover glasses (Fisher Scientific International, Inc.,
Asheville, N.C.). Emitted Photons were collected by the same
objective lens. The collected photons were then focused through a
100 micron confocal pinhole and filtered through a 525 nm long-pass
emission filter. A single photon counting module (.tau.-SPAD,
PicoQuant, Germany) detected the signal which was subsequently
processed by a 6602 counter/timer module (National Instruments,
Austin, Tex.). The power of the laser was set at 0.1 mW and the
time of signal acquiring was 5 s to prevent from severe
photobleaching of fluorescent organic dyes.
[0243] All publications, patents, and patent documents are
incorporated by reference herein, as though individually
incorporated by reference. The invention has been described with
reference to various specific and preferred embodiments and
techniques. However, it should be understood that many variations
and modifications may be made while remaining within the spirit and
scope of the invention.
Sequence CWU 1
1
13171DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 1tgctcttccc gagatgcaag gtcgcatatg
agcaactccc actcaactgc ctggtgatac 60gaggatgggc a 71263DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 2ggtgataaaa cgtgtagcaa gctgtaatcg acgggaagag
catgcccatc cactactatg 60gcg 63363DNAArtificial SequenceDescription
of Artificial Sequence Synthetic oligonucleotide 3aggcagttga
gacgaacatt cctaagtctg aaatttatca cccgccatag tagacgtatc 60acc
63443DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 4tcgattacag cttgctacac gattcagact
taggaatgtt cgt 43517DNAArtificial SequenceDescription of Artificial
Sequence Synthetic oligonucleotide 5agttgctcat atgcgac
17618DNAArtificial SequenceDescription of Artificial Sequence
Synthetic oligonucleotide 6agttgctcat atgcgacc 18719DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7agttgctcat atgcgacct 19822DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8gggagttgct catatgcgac ct 22924DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9gggagttgct catatgcgac cttg 241025DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10gggagttgct catatgcgac cttgc 251120RNADengue virus
11cucauaugcg accuugcauc 201230DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 12agatgcaagg
tcgcatatga gcaactccca 301320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 13ctcatatgcg
accttgcatc 20
* * * * *