U.S. patent application number 11/134851 was filed with the patent office on 2005-12-22 for caged rnas and methods of use thereof.
This patent application is currently assigned to Genospectra, Inc.. Invention is credited to McMaster, Gary, Nguyen, Quan, Witney, Frank.
Application Number | 20050282203 11/134851 |
Document ID | / |
Family ID | 32330203 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050282203 |
Kind Code |
A1 |
Nguyen, Quan ; et
al. |
December 22, 2005 |
Caged RNAs and methods of use thereof
Abstract
Methods of using labeled interfering RNAs to detect and/or
quantitate target mRNAs in cells are provided. Related
compositions, systems, and kits are also provided. Caged
interfering RNAs (e.g., photoactivatable interfering RNAs), methods
of using such caged RNAs, and related systems and kits are also
provided. Caged RNAs capable of repressing translation of a target
mRNA or silencing transcription of a target gene are also provided,
along with related methods, systems, and kits. Methods and
compositions for introducing RNAs into cells, using RNAs covalently
associated with protein transduction domains and/or lipids, are
provided. Also provided are methods and compositions for
selectively attenuating expression of a target mRNA by controlling
expression of an interfering RNA, an RNA capable of initiating
translation repression, or an RNA capable of initiating
transcriptional silencing.
Inventors: |
Nguyen, Quan; (San Ramon,
CA) ; McMaster, Gary; (Ann Arbor, MI) ;
Witney, Frank; (Oakland, CA) |
Correspondence
Address: |
QUINE INTELLECTUAL PROPERTY LAW GROUP, P.C.
P O BOX 458
ALAMEDA
CA
94501
US
|
Assignee: |
Genospectra, Inc.
|
Family ID: |
32330203 |
Appl. No.: |
11/134851 |
Filed: |
May 17, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11134851 |
May 17, 2005 |
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10716393 |
Nov 17, 2003 |
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60572564 |
May 18, 2004 |
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60427664 |
Nov 18, 2002 |
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60436855 |
Dec 26, 2002 |
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60439917 |
Jan 13, 2003 |
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60451177 |
Feb 27, 2003 |
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60456870 |
Mar 21, 2003 |
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60484785 |
Jul 3, 2003 |
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60501599 |
Sep 9, 2003 |
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Current U.S.
Class: |
435/6.12 ;
435/6.1; 514/44A; 536/23.1 |
Current CPC
Class: |
G01N 21/6452
20130101 |
Class at
Publication: |
435/006 ;
514/044; 536/023.1 |
International
Class: |
C12Q 001/68; C07H
021/02; A61K 048/00 |
Claims
What is claimed is:
1. A composition comprising a caged RNA, the caged RNA comprising:
an RNA capable of repressing translation of a target mRNA; and, one
or more first caging groups associated with the RNA, the first
caging groups inhibiting the RNA from repressing translation of the
target mRNA in a cell comprising the caged RNA.
2. The composition of claim 1, wherein the RNA does not initiate
degradation of the target mRNA in a cell comprising the RNA.
3. The composition of claim 1, wherein the RNA is
double-stranded.
4. The composition of claim 1, wherein the RNA comprises at least
an antisense strand, the antisense strand comprising a first region
which is complementary to a second region of the target mRNA, the
first region being interrupted by one or more nucleotides which are
not complementary to the second region.
5. The composition of claim 4, wherein the first region is
interrupted by two, three, four, or more nucleotides which are not
complementary to the second region.
6. The composition of claim 4, wherein the second region is within
the 3'-untranslated region of the target mRNA.
7. The composition of claim 4, wherein the RNA comprises at least
one double-stranded region, the double-stranded region comprising
the antisense strand and a sense strand.
8. The composition of claim 7, wherein the sense strand is
completely complementary to the antisense strand over the
double-stranded region.
9. The composition of claim 7, wherein the sense strand is not
completely complementary to the antisense strand over the
double-stranded region.
10. The composition of claim 7, wherein the RNA comprises a first
polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand.
11. The composition of claim 10, wherein the first
polyribonucleotide comprises between 17 and 29 nucleotides, the
second polyribonucleotide comprises between 17 and 29 nucleotides,
and the double-stranded region comprises between 17 and 29 base
pairs.
12. The composition of claim 11, wherein the first
polyribonucleotide comprises between 18 and 25 nucleotides, the
second polyribonucleotide comprises between 18 and 25 nucleotides,
and the double-stranded region comprises between 18 and 25 base
pairs.
13. The composition of claim 11, wherein the first
polyribonucleotide and the second polyribonucleotide each comprise
a two nucleotide TT 3' overhang.
14. The composition of claim 10, wherein at least one of the one or
more first caging groups is covalently attached to a 5' hydroxyl or
a 5' phosphate of the second polyribonucleotide.
15. The composition of claim 10, wherein the first caging group is
covalently attached to the first polyribonucleotide and to the
second polyribonucleotide.
16. The composition of claim 15, wherein the first caging group is
attached to the 3' end of the first polyribonucleotide and to the
5' end of the second polyribonucleotide.
17. The composition of claim 7, wherein the RNA comprises a
self-complementary polyribonucleotide.
18. The composition of claim 1, comprising the target mRNA, a cell,
a cell comprising the target mRNA, or a cell comprising the caged
RNA.
19. The composition of claim 1, wherein the first caging groups
inhibit the RNA from repressing translation of the target mRNA by
at least about 30%, at least about 50%, at least about 75%, at
least about 90%, at least about 95%, or at least about 98%, as
compared to the RNA in the absence of the first caging groups.
20. The composition of claim 1, wherein the first caging groups
prevent the RNA from repressing translation of the target mRNA.
21. The composition of claim 1, wherein removal of or an induced
conformational change in the first caging groups permits the RNA to
repress translation of the target mRNA.
22. The composition of claim 1, wherein the one or more first
caging groups associated with the RNA are covalently attached to
the RNA.
23. The composition of claim 1, wherein the one or more first
caging groups are removable by sonication, photoactivatable, or
photolabile.
24. The composition of claim 1, wherein the one or more first
caging groups each comprises a first binding moiety; the
composition comprising a second binding moiety that can bind at
least one of the first binding moieties.
25. The composition of claim 1, wherein the RNA comprises at least
one label.
26. The composition of claim 1, wherein the RNA is associated with
a cellular delivery module that can mediate introduction of the RNA
into a cell.
27. The composition of claim 26, wherein the cellular delivery
module comprises a polypeptide, a PEP-1 pep tide, an amphipathic
peptide, an MPG.sup..DELTA.NLS peptide, a cationic peptide, a
homopolymer of D-arginine, a homopolymer of histidine, a
homopolymer of lysine, a protein transduction domain, a protein
transduction domain derived from an HIV-1 Tat protein, from a
herpes simplex virus VP22 protein, or from a Drosophila
antennapedia protein, a model protein transduction domain, or a
model protein transduction domain comprising a homopolymer of
D-arginine.
28. The composition of claim 26, wherein the cellular delivery
module is covalently attached to the RNA.
29. The composition of claim 28, wherein the cellular delivery
module is attached to the RNA through a disulfide bond, or wherein
the covalent attachment is reversible by exposure to light of a
preselected wavelength.
30. The composition of claim 28, wherein the cellular delivery
module comprises a lipid or one or more myristoyl groups.
31. The composition of claim 26, wherein the cellular delivery
module is associated with one or more second caging groups which
inhibit the cellular delivery module from mediating introduction of
the RNA into a cell.
32. The composition of claim 1, wherein the RNA comprises a first
polyribonucleotide comprising a sense strand and a second
polyribonucleotide comprising an antisense strand, and wherein a
cellular delivery module is covalently attached to the second
polyribonucleotide.
33. The composition of claim 1, wherein the first caging group is a
cellular delivery module.
34. The composition of claim 1, wherein the caged RNA is bound to a
matrix.
35. The composition of claim 34, wherein the matrix is a surface,
and the RNA is bound to the surface at a predetermined location
within an array comprising other RNAs.
36. A kit for making the caged RNA of claim 1, comprising an RNA,
one or more first caging groups, and instructions for assembling
the RNA and the first caging groups to form the caged RNA, packaged
in one or more containers; or comprising one or more first caging
groups and instructions for assembling the first caging groups and
an RNA supplied by a user of the kit to form the caged RNA,
packaged in one or more containers.
37. A method of selectively attenuating expression of a target gene
in a cell, the method comprising: introducing a caged RNA into the
cell, the caged RNA comprising (a) an RNA capable of repressing
translation of a target mRNA transcribed from the target gene, and
(b) one or more first caging groups associated with the RNA, the
first caging groups inhibiting the RNA from repressing translation
of the target mRNA in the cell; and, initiating repression of
translation of the target mRNA by exposing the cell to uncaging
energy, whereby exposure to the uncaging energy frees the RNA from
inhibition by the caging groups.
38. The method of claim 37, wherein the amount of the target mRNA
present in the cell is not affected by the presence of the RNA in
the cell.
39. The method of claim 37, wherein exposing the cell to uncaging
energy comprises exposing the cell to light of a first
wavelength.
40. The method of claim 39, wherein exposing the cell to light of
the first wavelength comprises exposing the cell to light wherein
intensity of the light and duration of exposure of the cell to the
light are controlled such that a first portion of the caged RNA is
uncaged and a second portion of the caged RNA remains caged.
41. The method of claim 40, comprising exposing the cell to light
of the first wavelength again.
42. The method of claim 40, wherein the first portion is a selected
amount.
43. The method of claim 37, comprising contacting the cell and a
test compound, and wherein the cell is exposed to the uncaging
energy at a preselected time point with respect to a time at which
the cell and the test compound are contacted.
44. The method of claim 37, wherein the uncaging energy is directed
at a preselected subset of a cell population comprising the
cell.
45. The method of claim 37, wherein the caged RNA comprises a
cellular delivery module that can mediate introduction of the caged
RNA into the cell, the cellular delivery module being associated
with the RNA, and wherein introducing the caged RNA into the cell
comprises contacting the cell with the caged RNA associated with
the cellular delivery module.
46. The method of claim 37, wherein the RNA comprises at least one
label, the method comprising detecting a signal from the label.
47. A composition comprising a caged RNA, the caged RNA comprising:
an RNA capable of silencing transcription of a target gene; and,
one or more first caging groups associated with the RNA, the first
caging groups inhibiting the RNA from silencing transcription of
the target gene in a cell comprising the caged RNA.
48. A method of selectively attenuating expression of a target gene
in a cell, the method comprising: introducing a caged RNA into the
cell, the caged RNA comprising (a) an RNA capable of silencing
transcription of the target gene, and (b) one or more first caging
groups associated with the RNA, the first caging groups inhibiting
the RNA from silencing transcription of the target gene in the
cell; and, initiating silencing of transcription of the target gene
by exposing the cell to uncaging energy, whereby exposure to the
uncaging energy frees the RNA from inhibition by the caging
groups.
49. A method of selectively attenuating expression of a target gene
in a cell, the method comprising: introducing a first caged DNA and
a second caged DNA into the cell, the first caged DNA comprising a
first DNA encoding an RNA sense strand and one or more caging
groups associated with the first DNA, the second caged DNA
comprising a second DNA encoding an RNA antisense strand and one or
more caging groups associated with the second DNA, the caging
groups inhibiting transcription of the first and second DNAs, the
first and second DNAs each comprising at least a portion of the
target gene, and the sense and antisense strands being at least
partially complementary and able to form a duplex over at least a
portion of their lengths; and, initiating translational repression
by generating double-stranded RNA by exposing the cell to uncaging
energy, whereby exposure to the uncaging energy frees the first and
second DNAs from inhibition by the caging groups and permits
transcription of the first and second DNAs to occur.
50. The method of claim 49, wherein the sense strand comprises a
first polyribonucleotide and the antisense strand comprises a
second polyribonucleotide, or wherein the sense and antisense
strands comprise a single, self-complementary
polyribonucleotide.
51. The method of claim 49, wherein exposing the cell to uncaging
energy comprises exposing the cell to light of a first
wavelength.
52. A composition, comprising: a protein transduction domain
covalently attached to an RNA; and, the RNA, which RNA comprises:
(a) at least one double-stranded region, the double-stranded region
comprising a sense strand and an antisense strand, the antisense
strand comprising a region which is substantially complementary to
a region of a target mRNA, or (b) a single polyribonucleotide
strand comprising an antisense strand, the antisense strand
comprising a region which is substantially complementary to a
region of a target mRNA corresponding to the target gene.
53. The composition of claim 52, wherein the region of the
antisense strand is completely complementary to the region of the
target mRNA.
54. The composition of claim 52, wherein the region of the
antisense strand which is substantially complementary to the region
of the target mRNA comprises at least a first and a second
subregion, each of which is completely complementary to the target
mRNA, flanking one or more nucleotides which are not complementary
to the target mRNA.
55. The composition of claim 54, wherein the first and second
subregions flank two, three, four, or more nucleotides which are
not complementary to the target mRNA.
56. The composition of claim 52, comprising one or more first
caging groups associated with the RNA, the first caging groups
inhibiting the RNA from repressing translation of the target mRNA
in a cell.
57. A method of introducing an RNA into a cell, the method
comprising: (a) providing a composition comprising (i) an RNA
comprising at least one double-stranded region, the double-stranded
region comprising a sense strand and an antisense strand, the
antisense strand comprising a region which is substantially
complementary to a region of a target mRNA; or an RNA comprising a
single polyribonucleotide strand comprising an antisense strand,
the antisense strand comprising a region which is substantially
complementary to a region of a target mRNA, and (ii) a protein
transduction domain covalently attached to the RNA; and, (b)
contacting the composition and the cell, whereby the protein
transduction domain mediates introduction of the RNA into the
cell.
58. The method of claim 57, wherein the composition comprises one
or more first caging groups associated with the RNA, the first
caging groups inhibiting the RNA from repressing translation of the
target mRNA in the cell; the method comprising initiating
translational repression of the target mRNA by exposing the cell to
uncaging energy of a first type, whereby exposure to the uncaging
energy frees the RNA from inhibition by the first caging groups.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and benefit of U.S.
Provisional Patent Application No. 60/572,564, filed May 18, 2004,
entitled "RNAi-based Sensors, Caged RNAs, and Methods of Use
Thereof" by Nguyen et al., and is a continuation-in-part of U.S.
patent application Ser. No. 10/716,393, filed Nov. 17, 2003,
entitled "RNAi-based Sensors, Caged Interfering RNAs, and Methods
of Use Thereof" by Nguyen and McMaster, which claims priority to
and benefit of the following prior provisional patent applications:
U.S. Ser. No. 60/427,664, filed Nov. 18, 2002, entitled "Photo
Activated Sensors, Regulators and Compounds" by Nguyen and
McMaster, U.S. Ser. No. 60/436,855, filed Dec. 26, 2002, entitled
"Caged Sensors, Regulators and Compounds and Uses Thereof" by
Nguyen and McMaster, U.S. Ser. No. 60/439,917, filed Jan. 13, 2003,
entitled "Caged Sensors, Regulators and Compounds and Uses Thereof"
by Nguyen and McMaster, U.S. Ser. No. 60/451,177, filed Feb. 27,
2003, entitled "Caged Sensors, Regulators and Compounds and Uses
Thereof" by Nguyen et al., U.S. Ser. No. 60/456,870, filed Mar. 21,
2003, entitled "Caged Sensors, Regulators and Compounds and Uses
Thereof" by Nguyen et al.; U.S. Ser. No. 60/484,785, filed Jul. 3,
2003, entitled "RNAi-based Sensors and Methods of Use Thereof" by
Nguyen and McMaster, and U.S. Ser. No. 60/501,599 filed Sep. 9,
2003, entitled "Caged Sensors, Regulators and Compounds and Uses
Thereof" by Nguyen et al., each of which is incorporated herein by
reference in its entirety for all purposes.
FIELD OF THE INVENTION
[0002] This invention is in the field of caged RNA, e.g.,
photoactivatable caged RNA. The invention relates to caged RNAs
capable of repressing translation of a target mRNA and/or silencing
transcription of a target gene and use of such caged RNAs to
precisely control, spatially and/or temporally, expression of the
target mRNA or gene. Methods and compositions for selectively
attenuating expression of a target mRNA by controlling expression
of an interfering RNA or an RNA that mediates translational
repression are also provided. The invention also relates to
introduction of single- or double-stranded RNAs into cells.
BACKGROUND OF THE INVENTION
[0003] A number of experimental designs in basic research, clinical
diagnosis, drug discovery, and the like involve the detection, and
frequently also the quantitation, of a particular mRNA. However,
current methods for detecting and/or measuring mRNA transcripts
from cells (such as Northern blot, quantitative rt-PCR, microarray,
branched DNA, and in situ hybridization assays) generally require
the cells to be lysed or fixed. In addition, most current methods
require that mRNA purification and/or reverse transcription be
performed. Furthermore, current methods typically involve
multi-step processing that contributes to high intra- and
inter-assay variation. Consequently, current methods do not provide
live, dynamic, and location-specific imaging and measurements of
mRNA, because the cells are exposed to environmental changes.
[0004] Cellular assays are critical tools in the drug discovery
process and in basic research. In the future, these assays will
play a major role in systems biology, permitting the examination of
cell structure and function and the determination of a drug
compound's ability to enter a cell, the compound's toxicity and its
overall efficacy. Advances in imaging technologies, fluorescent
probes, and assay automation are predicted to drive the worldwide
cellular assays market from an estimated $300 million in 2002 to
$500 million in 2007. The most common application for cellular
assay technology in drug discovery is target validation and lead
identification and optimization. However, the complexity and
richness in cellular assay data sets, compared to genomics and
proteomics studies, will provide scientists with unparalleled tools
to aid discovery efforts throughout the discovery process and for
basic research applications.
[0005] To achieve the goal of measuring the spectrum of molecular
events in cells, there is definite need for "in cell sensor probes"
that quantitatively measure protein (or other) activities, mRNA
levels, or the like, directly in cells in a regulated fashion to
give real time functional data, without using expression vectors.
These "in cell sensor probes" could be used to define pathways in a
Parallel Quantitative Biology (PQB) format for systems biology,
providing novel regulated cell-based functional screening in a high
throughput mode. Such probes, termed PAC probes (PhotoActivated
Cell probes), are described herein, for example, probes that
comprise interfering RNAs. The invention also provides other
benefits which will become apparent upon review of the disclosure.
A complete understanding of the invention will be obtained upon
review of the following.
SUMMARY OF THE INVENTION
[0006] The present invention provides methods, compositions, and
kits including caged RNAs (e.g., photoactivatable caged RNAs). For
example, the invention provides caged RNAs capable of repressing
translation of a target mRNA or silencing transcription of a target
gene, as well as methods of using such caged RNAs to selectively
attenuate expression of target genes. In addition, the invention
provides methods and compositions for introducing RNAs into cells.
Methods and compositions for selectively attenuating expression of
a target mRNA by controlling expression of an RNA capable of
repressing translation of the target mRNA are also provided.
[0007] One general class of embodiments provides a composition
comprising a caged RNA. The caged RNA includes an RNA capable of
repressing translation of a target mRNA. The caged RNA also
includes one or more first caging groups associated with the RNA.
The first caging groups inhibit (e.g., prevent) the RNA from
repressing translation of the target mRNA in a cell comprising the
caged RNA. Typically, removal of or an induced conformational
change in the first caging groups permits the RNA to repress
translation of the target mRNA. In a preferred class of
embodiments, the RNA does not initiate degradation of the target
mRNA in a cell comprising the RNA.
[0008] The RNA can have any of a variety of structures, lengths,
and/or the like. For example, the RNA can be single-stranded, or,
preferably, double-stranded. The RNA typically comprises at least
an antisense strand (e.g., only the antisense strand if the RNA is
single-stranded, or the antisense strand and a complementary or
partially complementary sense strand if the RNA is
double-stranded). In one class of embodiments, the antisense strand
of the RNA comprises a first region which is complementary to a
second region of the target mRNA. The first region is interrupted
by one or more nucleotides which are not complementary to the
second region; for example, one or more nucleotides which form a
bulge when the antisense strand binds the target mRNA. The first
region is optionally interrupted by two, three, four, or more
nucleotides which are not complementary to the second region.
[0009] The second region, the region of the mRNA to which the
antisense strand binds, can be located essentially anywhere within
the mRNA, e.g., the 5' UTR, an exon, an exon, an exon-intron
boundary, or the like. In one class of embodiments, the second
region is within the 3' UTR of the target mRNA. The target mRNA
optionally includes a plurality of repeats of the second region,
e.g., tandem repeats, and/or a region complementary to a different
RNA capable of repressing translation of the target.
[0010] The RNA optionally comprises at least one double-stranded
region that includes the antisense strand and a sense strand. The
sense strand can be completely complementary to the antisense
strand over the double-stranded region. Alternatively, in some
embodiments, the sense strand is not completely complementary to
the antisense strand over the double-stranded region. For example,
the double-stranded region can include one or more mismatches,
bulges, loops, and/or the like. The mismatched nucleotides can be
the same or different nucleotides than those mismatched to the
target mRNA.
[0011] In certain embodiments, the RNA comprises a first
polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand. For example,
the first polyribonucleotide can comprise between 17 and 29
nucleotides, the second polyribonucleotide can comprise between 17
and 29 nucleotides, and the double-stranded region can comprise
between 17 and 29 base pairs. The first and second
polyribonucleotides can form a duplex over their entire length, or
they can have overhangs (e.g., 5' or 3' overhangs). For example, in
some embodiments, the first polyribonucleotide and the second
polyribonucleotide each comprise a two nucleotide TT 3' overhang
(where T is 2'-deoxythymidine). In one class of embodiments, at
least one of the one or more first caging groups is optionally
covalently attached to a 5' hydroxyl or a 5' phosphate of the
second polyribonucleotide. The first caging group is optionally
covalently attached to the first polyribonucleotide and to the
second polyribonucleotide, e.g., to the 3' end of the first
polyribonucleotide and to the 5' end of the second
polyribonucleotide. Instead of comprising two polyribonucleotides,
in some embodiments, the RNA comprises a self-complementary
polyribonucleotide (e.g., a shRNA).
[0012] The composition optionally also includes the target mRNA
and/or a cell. For example, the composition can include a cell
comprising the target mRNA and/or the caged RNA.
[0013] The one or more first caging groups associated with the RNA
can be covalently or non-covalently attached to the RNA. In a
preferred aspect, the one or more first caging groups are
photoactivatable (e.g., photolabile). Other caging groups are
removable via input of different uncaging energies; e.g., the one
or more caging groups can be removable by sonication or application
of heat, or can be removed by a chemical or enzyme. In some
embodiments, the one or more first caging groups each comprises a
first binding moiety, and the composition includes a second binding
moiety that can bind at least one of the first binding
moieties.
[0014] In some embodiments, the RNA also includes at least one
label, e.g., a fluorescent label. Optionally, binding and/or
repression of translation of the target mRNA by the RNA results in
a binding and/or repression-dependent change in a signal output of
the label.
[0015] In one class of embodiments, the RNA is associated with a
cellular delivery module that can mediate introduction of the RNA
into a cell. The cellular delivery module can comprise, e.g., a
polypeptide or a lipid. The cellular delivery module is optionally
covalently attached to the RNA, e.g., through a disulfide bond or a
covalent attachment which is reversible by exposure to light of a
preselected wavelength. The cellular delivery module is optionally
associated with one or more second caging groups which inhibit the
cellular delivery module from mediating introduction of the RNA
into a cell. The cellular delivery module optionally serves as the
first caging group. In one class of embodiments, the RNA comprises
a first polyribonucleotide comprising a sense strand and a second
polyribonucleotide comprising an antisense strand, and a cellular
delivery module is covalently attached to the second
polyribonucleotide.
[0016] Optionally, in the embodiments herein, the caged RNA is
bound to a matrix (e.g., electrostatically, covalently, directly or
via a linker). In one aspect, the matrix is a surface and the RNA
is bound to the surface at a predetermined location within an array
comprising other RNAs. In other embodiments, the matrix comprises a
bead (e.g., color-coded or otherwise addressable).
[0017] Kits for making the caged RNA are also a feature of the
invention. Thus, one class of embodiments provides a kit including
an RNA, one or more first caging groups, and instructions for
assembling the RNA and the first caging groups to form the caged
RNA, packaged in one or more containers. Another class of
embodiments provides a kit comprising one or more first caging
groups and instructions for assembling the first caging groups and
an RNA supplied by a user of the kit to form the caged RNA,
packaged in one or more containers.
[0018] Another general class embodiments provides methods of
selectively attenuating expression of a target gene in a cell. In
the methods, a caged RNA is introduced into the cell. The caged RNA
includes an RNA capable of repressing translation of a target mRNA
transcribed from the target gene. The caged RNA also comprises one
or more caging groups associated with the RNA, the caging groups
inhibiting (e.g., preventing) the RNA from repressing translation
of the target mRNA in the cell. Repression of translation is
initiated by exposing the cell to uncaging energy (e.g., light of a
predetermined wavelength), freeing the RNA from inhibition by the
caging groups. In a preferred class of embodiments, the amount of
the target mRNA present in the cell is not affected by the presence
of the RNA in the cell; i.e., uncaging the RNA does not initiate
RNAi.
[0019] Exposing the cell to uncaging energy optionally includes
exposing the cell to light of a first wavelength. This exposure can
be addressable; e.g., the caged RNA can be exposed to light of the
first wavelength by exposing one or more preselected areas (e.g.,
wells of a microtiter plate or portions thereof, or the like) to
the light. As another example, the uncaging energy can be directed
at a preselected subset of a cell population comprising the
cell.
[0020] Exposing the cell to light of the first wavelength
optionally comprises exposing the cell to light such that the
intensity of the light and the duration of exposure to the light
are controlled such that a first portion (which can be a selected
amount) of the caged RNA is uncaged and a second portion of the
caged RNA remains caged. Furthermore, the uncaging step can be
repeated until the caged RNA is depleted.
[0021] Caging the RNA permits temporal control over initiation of
translational repression. For example, the method can include
contacting the cell and a test compound and exposing the cell to
the uncaging energy at a preselected time point with respect to a
time at which the cell and the test compound are contacted.
[0022] Essentially all of the features noted for the compositions
above apply to these methods as well, as relevant. For example, in
one class of embodiments, the caged RNA comprises a cellular
delivery module that can mediate introduction of the caged RNA into
the cell, the cellular delivery module being associated with the
RNA. In this class of embodiments, the caged RNA is introduced into
the cell by contacting the cell with the caged RNA associated with
the cellular delivery module. As another example, in certain
embodiments, the RNA comprises at least one label, and the methods
include detecting a signal from the label.
[0023] Another aspect of the invention relates to RNAs capable of
inducing histone methylation and chromatin silencing. Thus, one
general class of embodiments provides a caged RNA that includes an
RNA capable of silencing transcription of a target gene and one or
more first caging groups associated with the RNA. The first caging
groups inhibit (e.g., prevent) the RNA from silencing transcription
of the target gene in a cell comprising the caged RNA.
[0024] Essentially all of the various optional configurations and
features noted for the embodiments above apply here as well, to the
extent they are relevant, e.g., for percent inhibition by the
caging groups, structure of the RNA, label configurations (e.g.,
use of fluorescent labels, fluorescent label/quencher, and
donor/acceptor combinations), signal output types, use of caging
groups (e.g., photolabile caging groups), appropriate uncaging
energies (light, heat, sonic, etc.), use of cellular delivery
modules (e.g., amphipathic peptides, cationic peptides, protein
transduction domains, and lipids), and the like. In another aspect,
systems and/or apparatus comprising the compositions (e.g., the
caged RNAs) noted above and, e.g., components such as detectors,
fluid handling apparatus, sources of uncaging energy, or the like,
are a feature of the invention, as are kits for making or using the
caged RNAs.
[0025] Another general class of embodiments provides methods of
selectively attenuating expression of a target gene in a cell. In
the methods, a caged RNA is introduced into the cell. The caged RNA
comprises an RNA capable of silencing transcription of the target
gene. The caged RNA also includes one or more first caging groups
associated with the RNA that inhibit (e.g., prevent) the RNA from
silencing transcription of the target gene in the cell. Silencing
of transcription of the target gene is initiated by exposing the
cell to uncaging energy, freeing the RNA from inhibition by the
caging groups.
[0026] Essentially all of the features noted for the embodiments
above apply to these methods as well, as relevant, e.g., for types
of uncaging energy, temporal and spatial control of uncaging,
introduction of the RNA into the cell through use of a cellular
delivery module, label detection, and the like.
[0027] In another general class of methods for selectively
attenuating expression of a gene in a cell, a first caged DNA and a
second caged DNA are introduced into the cell. The first caged DNA
includes a first DNA encoding an RNA sense strand and one or more
caging groups. The second caged DNA comprises a second DNA encoding
an RNA antisense strand and one or more caging groups. The presence
of the caging groups prevents transcription of the first and second
DNAs, the first and second DNAs each comprising at least a portion
of the target gene, and the sense and antisense strands being at
least partially complementary and able to form a duplex over at
least a portion of their lengths. Translational repression is
initiated by generating double-stranded RNA by exposing the cell to
uncaging energy, whereby exposure to the uncaging energy frees the
first and second DNAs from the caging groups and permits
transcription of the first and second DNAs to occur. All of the
above optional method variations apply to this method as well, to
the extent they are relevant. Further, the various composition
components noted above can be adapted for use in this method, as
appropriate; e.g., with respect to configuration of the RNA, use of
caging groups (e.g., photolabile caging groups), appropriate
uncaging energies (light, heat, sonic, etc.), use of cellular
delivery modules (e.g., amphipathic peptides, protein transduction
domains, and lipids), and the like.
[0028] Protein transduction domains can be used to introduce RNAs,
including caged RNAs, into cells. Thus, one class of embodiments
provides a composition comprising a protein transduction domain
covalently attached to an RNA. The RNA can comprise at least one
double-stranded region, the double-stranded region comprising a
sense strand and an antisense strand, the antisense strand
comprising a region which is substantially complementary to a
region of a target mRNA. Alternatively, the RNA can comprise a
single polyribonucleotide strand comprising an antisense strand,
the antisense strand comprising a region which is substantially
complementary to a region of a target mRNA.
[0029] The RNA can have any of a variety of structures, lengths,
and/or the like. Thus, in one class of embodiments, the region of
the antisense strand which is substantially complementary to the
region of the target mRNA is completely complementary to the region
of the target mRNA. In another class of embodiments, the region of
the antisense strand which is substantially complementary to the
region of the target mRNA comprises at least a first and a second
subregion, each of which is completely complementary to the target
mRNA, flanking one or more nucleotides (e.g., two, three, four, or
more nucleotides) which are not complementary to the target
mRNA.
[0030] Essentially all of the features noted for the embodiments
above apply to these methods as well, as relevant. For example, one
or more first caging groups can be associated with the RNA,
inhibiting (e.g., preventing) the RNA from repressing translation
of the target mRNA in a cell. The composition is optionally also
includes the target mRNA and/or a cell, e.g., a cell comprising the
target mRNA and/or the RNA.
[0031] The invention also provides related methods of introducing
an RNA into a cell. In the methods, a composition comprising an RNA
and a protein transduction domain covalently attached to the RNA is
provided. The RNA can comprise at least one double-stranded region,
the double-stranded region comprising a sense strand and an
antisense strand, the antisense strand comprising a region which is
substantially complementary to a region of a target mRNA.
Alternatively, the RNA can comprise a single polyribonucleotide
strand comprising an antisense strand, the antisense strand
comprising a region which is substantially complementary to a
region of a target mRNA. The composition and the cell are
contacted, whereby the protein transduction domain mediates
introduction of the RNA into the cell.
[0032] Essentially all of the features noted for the embodiments
above apply to these methods as well, as relevant. For example, the
RNA can be caged. Thus, in one class of embodiments, one or more
first caging groups are associated with the RNA, inhibiting (e.g.,
preventing) the RNA from repressing translation of the target mRNA
in the cell, and the methods include initiating translational
repression of the target mRNA by exposing the cell to uncaging
energy of a first type, whereby exposure to the uncaging energy
frees the RNA from inhibition by the first caging groups.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 Panels A-I schematically illustrate example
interfering RNA sensors. The top strand corresponds to the sense
strand and the bottom the antisense strand. Boxes A and B represent
either a fluorescent label and a quencher (or vice versa) or a
donor and acceptor (or vice versa).
[0034] FIG. 2 schematically illustrates multiplexed interfering RNA
sensors having different fluorescent label (F)/quencher (Q)
combinations.
[0035] FIG. 3 schematically illustrates multiplexed interfering RNA
sensors, one example with a donor/acceptor combination (F1/F2), one
example with a donor/acceptor combination suitable for TR-FRET
(APC/Eu), and one example with a fluorescent label/quencher (F5/Q)
combination.
[0036] FIG. 4 schematically illustrates caged siRNAs with a
photolabile linker between the two strands. The top strand
corresponds to the sense strand and the bottom the antisense
strand.
[0037] FIG. 5 schematically illustrates example caged siRNAs. The
top strand corresponds to the sense strand and the bottom the
antisense strand.
[0038] FIG. 6 schematically illustrates example caged siRNAs. The
top strand corresponds to the sense strand and the bottom the
antisense strand.
[0039] FIG. 7 schematically illustrates example siRNAs in which a
cellular delivery module is attached to the siRNA by a photolabile
linker. The top strand corresponds to the sense strand and the
bottom the antisense strand.
[0040] FIG. 8 schematically illustrates example siRNAs in which a
protein carrier is attached to the siRNA by a photolabile linker.
The top strand corresponds to the sense strand and the bottom the
antisense strand.
[0041] FIG. 9 schematically illustrates an example siRNA in which a
protein transduction domain- and endosomal release agent-coated
bead is attached to the siRNA by a photolabile linker. The top
strand corresponds to the sense strand and the bottom the antisense
strand.
[0042] FIG. 10 schematically illustrates an example siRNA in which
a lipid is attached to the siRNA by a photolabile linker. The top
strand corresponds to the sense strand and the bottom the antisense
strand.
[0043] FIG. 11 schematically illustrates caging of an siRNA in a
photolabile vesicle (solid oval) associated with a protein
transduction domain (PTD). The siRNA is released when the vesicle
dissociates (broken oval) after exposure to light.
[0044] FIG. 12 schematically illustrates caged siRNAs that can be
used, e.g., as probes for mRNA.
[0045] FIG. 13 schematically illustrates caged shRNAs that can be
used, e.g., as probes for mRNA.
[0046] FIG. 14 schematically depicts a caged siRNA linked with a
peptide transport moiety.
[0047] FIG. 15 schematically illustrates measurement of mRNA with a
FRET siRNA.
[0048] FIG. 16 schematically illustrates the use of multiple siRNAs
per target gene.
[0049] FIG. 17 depicts a flowchart illustrating an example workflow
for assays using photoactivatable mRNA sensors in a live cell assay
format.
[0050] FIG. 18 schematically depicts the detection of splice
variants with siRNA sensors that span splice junctions.
[0051] FIG. 19 presents the sequence of a portion of human GAPDH
(SEQ ID NO:1). The positions of the sense strand of three
interfering RNAs against GAPDH (RNAi 1-RNAi 3) are also
indicated.
[0052] FIG. 20 Panel A schematically illustrates an annealed GAPDH
interfering RNA sensor; Panel B schematically illustrates a
denatured GAPDH interfering RNA sensor; and Panel C shows
fluorescent emission spectra for the antisense strand (curve 1),
the sense strand (curve 2), and the annealed strands (curve 3) of a
GAPDH interfering RNA sensor.
[0053] FIG. 21 shows the GAPDH mRNA level as measured by a bDNA
assay at the indicated time points after lipofection of labeled
RNAi 1 (Panel A), as compared to a negative control (Panel B, no
lipofection reagent).
[0054] FIG. 22 compares the percentage knockout of GAPDH
expression, as measured by the bDNA assay, for labeled RNAi
1-3.
[0055] FIG. 23 shows the results of bDNA assays (RLU, luminescence)
compared to FITC signals (FLU) for cells lipofected with the RNAi 1
(Panel A), RNAi 2 (Panel B), and RNAi 3 (Panel C) sensors.
[0056] FIG. 24 shows the ratio of the bDNA assay measurement of
GAPDH mRNA levels at 20 h/4 h and the ratio of the FITC signal from
labeled RNAi's 1-3 at 20 h/4 h.
[0057] FIG. 25 Panel A presents a graph of the results of bDNA
assays (RLU, relative luminescent units, indicating the GAPDH mRNA
level) and a graph of the fluorescent signal (RFU, relative
fluorescence units) from the labeled RNAi sensor, at the indicated
time points after lipofection of the sensor. Panel B presents a
graph of the fluorescent signals against the bDNA assay
results.
[0058] FIG. 26 schematically illustrates use of an environmentally
responsive polymer as a noncovalently associated caging group.
[0059] FIG. 27 schematically illustrates use of an environmentally
responsive polymer as a covalently associated caging group for an
siRNA. The top strand of the RNA corresponds to the sense strand
and the bottom the antisense strand.
[0060] FIG. 28 Panel A schematically illustrates the caged
double-stranded siRNA RNAi 1. Panel B depicts the caged antisense
strand of caged RNAi 1 (SEQ ID NO:2).
[0061] FIG. 29 Panel A presents a graph of GAPDH expression
relative to cyclophilin expression in untransfected cells, cells
transfected with RNAi 1, cells transfected with in vitro uncaged
caged RNAi 1, cells transfected with caged RNAi 1 but not exposed
to UV light, and cells transfected with caged RNAi 1 and exposed to
UV light, as measured by a bDNA assay. Panel B presents a graph of
relative GAPDH expression levels in cells transfected with RNAi 1,
transfected with in vitro uncaged caged RNAi 1, transfected with
caged RNAi 1 but not exposed to UV light, and transfected with
caged RNAi 1 and exposed to UV light, as measured by a bDNA assay
and normalized to the expression level in cells transfected with
RNAi 1.
[0062] FIG. 30 schematically illustrates induction of expression of
an interfering RNA by uncaging of a first activation component,
tetracycline in this example.
[0063] FIG. 31 schematically illustrates induction of expression of
an interfering RNA by uncaging of a first activation component, IP3
in this example.
[0064] FIG. 32 schematically illustrates processing of an example
pre-miRNA to form an miRNA and binding of the miRNA antisense
strand to the 3'UTR of an mRNA.
[0065] FIG. 33 presents a graph of relative GAPDH expression in
cells transfected with a scrambled negative control GAPDH siRNAi,
cells transfected with caged RNAi 1 but not exposed to UV light,
cells transfected with in vitro uncaged caged RNAi 1, and cells
transfected with caged RNAi 1 and exposed to UV light.
[0066] FIG. 34 presents a graph of normalized GAPDH expression in
cells transfected with caged RNAi 1 and exposed to varying doses of
UV light.
DEFINITIONS
[0067] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which the invention pertains. The
following definitions supplement those in the art and are directed
to the current application and are not to be imputed to any related
or unrelated case, e.g., to any commonly owned patent or
application. Although any methods and materials similar or
equivalent to those described herein can be used in the practice
for testing of the present invention, the preferred materials and
methods are described herein. Accordingly, the terminology used
herein is for the purpose of describing particular embodiments
only, and is not intended to be limiting.
[0068] As used in this specification and the appended claims, the
singular forms "a," "an" and "the" include plural referents unless
the content clearly dictates otherwise. Thus, for example,
reference to "a protein" includes a plurality of proteins;
reference to "a cell" includes mixtures of cells, and the like.
[0069] An "antisense strand" is a nucleic acid strand comprising a
sequence complementary to that of a given mRNA, while a "sense
strand" is a nucleic acid strand comprising a sequence
corresponding to that of the mRNA.
[0070] "Attenuating" expression of a target gene refers to
decreasing the level of expression of the gene, e.g., as compared
to the level in the absence of a relevant interfering RNA.
[0071] A "caging group" is a moiety that can be employed to
reversibly block, inhibit, or interfere with the activity (e.g.,
the biological activity) of a molecule (e.g., a polypeptide, a
nucleic acid, a small molecule, a drug, etc.). The caging groups
can, e.g., physically trap an active molecule inside a framework
formed by the caging groups. Typically, however, one or more caging
groups are associated (covalently or noncovalently) with the
molecule but do not necessarily surround the molecule in a physical
cage. For example, a single caging group covalently attached to an
amino acid side chain required for the catalytic activity of an
enzyme can block the activity of the enzyme; the enzyme would thus
be caged even though not physically surrounded by the caging group.
Caging groups can be, e.g., relatively small moieties such as
carboxyl nitrobenzyl, 2-nitrobenzyl, nitroindoline,
hydroxyphenacyl, DMNPE, or the like, or they can be, e.g., large
bulky moieties such as a protein or a bead. Caging groups can be
removed from a molecule, or their interference with the molecule's
activity can be otherwise reversed or reduced, by exposure to an
appropriate type of uncaging energy and/or exposure to an uncaging
chemical, enzyme, or the like.
[0072] A "photoactivatable" or "photoactivated" caging group is a
caging group whose blockage, inhibition of, or interference with
the activity of a molecule with which the photoactivatable caging
group is associated can be reversed or reduced by exposure to light
of an appropriate wavelength. For example, exposure to light can
disrupt a network of caging groups physically surrounding the
molecule, reverse a noncovalent association with the molecule,
trigger a conformational change that renders the molecule active
even though still associated with the caging group, or cleave a
photolabile covalent attachment to the molecule.
[0073] A "photolabile" caging group is one whose covalent
attachment to a molecule is reversed (cleaved) by exposure to light
of an appropriate wavelength. The photolabile caging group can be,
e.g., a relatively small moiety such as carboxyl nitrobenzyl,
2-nitrobenzyl, nitroindoline, hydroxyphenacyl, DMNPE, or the like,
or it can be, e.g., a relatively bulky group (e.g. a macromolecule,
a protein) covalently attached to the molecule by a photolabile
linker (e.g., a polypeptide linker comprising a 2-nitrophenyl
glycine residue).
[0074] A "cellular delivery module" or "cellular delivery agent" is
a moiety that can mediate introduction into a cell of a molecule
with which the module is associated (covalently or
noncovalently).
[0075] The term "eukaryote" refers to organisms belonging to the
phylogenetic domain Eucarya such as animals (e.g., mammals,
insects, reptiles, birds, etc.), ciliates, plants, fungi (e.g.,
yeasts, etc.), flagellates, microsporidia, protists, etc.
Additionally, the term "prokaryote" refers to non-eukaryotic
organisms belonging to the Eubacteria (e.g., Escherichia coli,
Thermus thermophilus, etc.) and Archaea (e.g., Methanococcus
jannaschii, Methanobacterium thermoautotrophicum, Halobacterium
species, etc.) phylogenetic domains.
[0076] "Expression of a gene" or "expression of a nucleic acid"
means transcription of DNA into RNA (optionally including
modification of the RNA, e.g., splicing) and/or translation of
encoded RNA (e.g., mRNA) into a polypeptide (possibly including
subsequent modification of the polypeptide, e.g.,
post-translational modification), as indicated by the context.
[0077] The term "gene" is used broadly to refer to any nucleic acid
associated with a biological function. Genes typically include
coding sequences and/or the regulatory sequences required for
expression of such coding sequences.
[0078] A "label" is a moiety that facilitates detection of a
molecule. Common labels in the context of the present invention
include fluorescent, luminescent, and/or colorimetric labels.
Suitable labels include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescent moieties, chemiluminescent
moieties, magnetic particles, and the like. Patents teaching the
use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Many
labels are commercially available and can be used in the context of
the invention.
[0079] The term "nucleic acid" encompasses any physical string of
monomer units that can be corresponded to a string of nucleotides,
including a polymer of nucleotides (e.g., a typical DNA or RNA
polymer), PNAs, modified oligonucleotides (e.g., oligonucleotides
comprising nucleotides that are not typical to biological RNA or
DNA, such as 2'-O-methylated oligonucleotides), and/or the like. A
nucleic acid can be, e.g., single-stranded or double-stranded.
Unless otherwise indicated, a particular nucleic acid sequence of
this invention optionally comprises or encodes complementary
sequences, in addition to any sequence explicitly indicated. A
nucleic acid (e.g., a polyribonucleotide, a double-stranded RNA, or
the like) of this invention is optionally nuclease resistant.
[0080] A nucleic acid that is "nuclease resistant" or "resistant to
nuclease activity" is cleaved more slowly under typical reaction
conditions for a given nuclease (e.g., a 5' to 3' nuclease and/or
an endonuclease) than is a corresponding nucleic acid comprising
only the four conventional deoxyribonucleotides (A, T, G, and/or
C), or the four conventional ribonucleotides (U, A, G, and/or C),
and phosphodiester linkages. For example, nucleic acids that
incorporate 2'O methylated nucleotides are typically more nuclease
resistant than nucleic acids that incorporate only conventional
nucleotides. Many such modifications that impart nuclease
resistance are known and can be adapted to the present
invention.
[0081] An "oligonucleotide" or "polynucleotide" is a polymer
comprising two or more nucleotides. (For example, a
"polyribonucleotide" is a polymer comprising two or more
ribonucleotides.) The polymer can additionally comprise
non-nucleotide elements such as labels, quenchers, blocking groups,
or the like. The nucleotides of the oligonucleotide can be
deoxyribonucleotides, ribonucleotides and/or nucleotide analogs,
can be natural or non-natural, and can be unsubstituted,
unmodified, substituted or modified. The nucleotides can be linked
by phosphodiester bonds, or by phosphorothioate linkages,
methylphosphonate linkages, boranophosphate linkages, or the
like.
[0082] The "5' end" of a polynucleotide refers to the nucleotide
located at the 5' terminus of the polynucleotide. A moiety
"attached at the 5' end" of the polynucleotide can thus be attached
to any part of the 5' terminal nucleotide, e.g., the terminal 5'
phosphate or hydroxyl, the base, or the ribose. Similarly, the "3'
end" of a polynucleotide refers to the nucleotide located at the 3'
terminus of the polynucleotide. A moiety "attached at the 3' end"
of the polynucleotide can thus be attached to any part of the 3'
terminal nucleotide, e.g., the terminal 3' hydroxyl, the base, or
the ribose.
[0083] A "subcellular delivery module" or "subcellular delivery
agent" is a moiety that can mediate delivery and/or localization of
an associated molecule to a particular subcellular location (e.g.,
a subcellular compartment, a membrane, and/or neighboring a
particular macromolecule). The subcellular delivery module can be
covalently or noncovalently associated with the molecule.
Subcellular delivery modules include, e.g., peptide tags such as a
nuclear localization signal or mitochondrial matrix-targeting
signal.
[0084] A "synthetic oligonucleotide" or a "chemically synthesized
oligonucleotide" is an oligonucleotide made through in vitro
chemical synthesis, as opposed to an oligonucleotide made either in
vitro or in vivo by a template-directed, enzyme-dependent
reaction.
[0085] A "polypeptide" is a polymer comprising two or more amino
acid residues (e.g., a peptide or a protein). The polymer can
additionally comprise non-amino acid elements such as labels,
quenchers, blocking groups, or the like and can optionally comprise
modifications such as glycosylation or the like. The amino acid
residues of the polypeptide can be natural or non-natural and can
be unsubstituted, unmodified, substituted or modified.
[0086] A "protein transduction domain" is a polypeptide sequence
that can mediate introduction of a covalently associated molecule
into a cell. Protein transduction domains are typically short
peptides (e.g., often less than about 16 residues). Example protein
transduction domains have been derived from the HIV-1 protein Tat,
the herpes simplex virus protein VP22, and the Drosophila protein
antennapedia; model protein transduction domains have also been
designed.
[0087] A "quencher" is a moiety that alters a property of a label
(typically, a fluorescent label) when it is in proximity to the
label. The quencher can actually quench an emission, but it does
not have to, i.e., it can simply alter some detectable property of
the label, or, when proximal to the label, cause a different
detectable property than when not proximal to the label. A quencher
can be e.g., an acceptor fluorophore that operates via energy
transfer and re-emits the transferred energy as light; other
similar quenchers, called "dark quenchers," do not re-emit
transferred energy via fluorescence. A variety of labels and
quenchers are found in Haughland (2003) Handbook of Fluorescent
Probes and Research Products Ninth Edition, available from
Molecular Probes. A straightforward discussion of FRET can be found
in the Handbook at page 25-26 and the references cited therein.
[0088] A "target mRNA" is an mRNA that is to be detected and/or
whose expression is to be affected. A "target gene" is a gene whose
expression is to be detected (e.g., one or more corresponding mRNAs
are to be detected) and/or whose expression is to be affected. A
target gene can be, e.g., an endogenous gene or a heterologous gene
(e.g., a gene introduced into the cell through infection by a
pathogen, or a gene introduced through recombinant means). A target
gene can be, e.g., a constitutively expressed gene or an inducible
gene; similarly, a target mRNA can be, e.g., a constitutively
expressed mRNA or an inducible mRNA.
[0089] "Uncaging energy" is energy that removes one or more caging
groups from a caged molecule (or otherwise reverses the caging
groups' blockage of the molecule's activity). Uncaging energy can
be supplied, e.g., by light, sonication, a heat source, a magnetic
field, electromagnetic radiation, or the like, as appropriate for
the particular caging group(s).
[0090] The term "vector" refers to a means by which a nucleic acid
can be propagated and/or transferred between organisms, cells, or
cellular components. Vectors include plasmids, viruses,
bacteriophage, pro-viruses, phagemids, transposons, and artificial
chromosomes, and the like, that replicate autonomously or can
integrate into a chromosome of a host cell. A vector can also be a
naked RNA polynucleotide, a naked DNA polynucleotide, a
polynucleotide composed of both DNA and RNA within the same strand,
a poly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or
RNA, a liposome-conjugated DNA, or the like, that are not
autonomously replicating. Most commonly, the vectors of the present
invention are plasmids.
[0091] A variety of additional terms are defined or otherwise
characterized herein.
DETAILED DESCRIPTION
[0092] The term "RNA interference" ("RNAi," sometimes called
RNA-mediated interference, post-transcriptional gene silencing, or
quelling) refers to a phenomenon in which the presence of
double-stranded RNA in a cell results in inhibition of expression
of a gene comprising a sequence identical, or nearly identical, to
that of the double-stranded RNA. The double-stranded RNA
responsible for inducing RNAi is called an "interfering RNA."
Expression of the gene is inhibited by the mechanism of RNAi as
described below, in which the presence of the interfering RNA
results in degradation of mRNA transcribed from the gene and thus
in decreased levels of the mRNA and any encoded protein.
[0093] The mechanism of RNAi has been and is being extensively
investigated in a number of eukaryotic organisms and cell types.
See, for example, the following reviews: McManus and Sharp (2002)
"Gene silencing in mammals by small interfering RNAs" Nature
Reviews Genetics 3:737-747; Hutvagner and Zamore (2002) "RNAi:
Nature abhors a double strand" Curr Opin Genet & Dev
200:225-232; Hannon (2002) "RNA interference" Nature 418:244-251;
Agami (2002) "RNAi and related mechanisms and their potential use
for therapy" Curr Opin Chem Biol 6:829-834; Tuschl and Borkhardt
(2002) "Small interfering RNAs: A revolutionary tool for the
analysis of gene function and gene therapy" Molecular Interventions
2:158-167; Nishikura (2001) "A short primer on RNAi: RNA-directed
RNA polymerase acts as a key catalyst" Cell 107:415-418; and Zamore
(2001) "RNA interference: Listening to the sound of silence" Nature
Structural Biology 8:746-750. RNAi is also described in the patent
literature; see, e.g., CA 2359180 by Kreutzer and Limmer entitled
"Method and medicament for inhibiting the expression of a given
gene"; WO 01/68836 by Beach et al. entitled "Methods and
compositions for RNA interference"; WO 01/70949 by Graham et al.
entitled "Genetic silencing"; and WO 01/75164 by Tuschl et al.
entitled "RNA sequence-specific mediators of RNA interference."
[0094] In brief, double-stranded RNA introduced into a cell (e.g.,
into the cytoplasm) is processed, for example by an RNAse III-like
enzyme called Dicer, into shorter double-stranded fragments called
small interfering RNAs (siRNAs, also called short interfering
RNAs). The length and nature of the siRNAs produced is dependent on
the species of the cell, although typically siRNAs are 21-25
nucleotides long (e.g., an siRNA may have a 19 base pair duplex
portion with two nucleotide 3' overhangs at each end). Similar
siRNAs can be produced in vitro (e.g., by chemical synthesis or in
vitro transcription) and introduced into the cell to induce RNAi.
The siRNA becomes associated with an RNA-induced silencing complex
(RISC). Separation of the sense and antisense strands of the siRNA,
and interaction of the siRNA antisense strand with its target mRNA
through complementary base-pairing interactions, optionally occurs.
Finally, the mRNA is cleaved and degraded.
[0095] Expression of a target gene in a cell can thus be
specifically inhibited by introducing an appropriately chosen
double-stranded RNA into the cell. Guidelines for design of
suitable interfering RNAs are known to those of skill in the art.
For example, interfering RNAs are typically designed against exon
sequences, rather than introns or untranslated regions.
Characteristics of high efficiency interfering RNAs may vary by
cell type. For example, although siRNAs may require 3' overhangs
and 5' phosphates for most efficient induction of RNAi in
Drosophila cells, in mammalian cells blunt ended siRNAs and/or RNAs
lacking 5' phosphates can induce RNAi as effectively as siRNAs with
3' overhangs and/or 5' phosphates (see, e.g., Czauderna et al.
(2003) "Structural variations and stabilizing modifications of
synthetic siRNAs in mammalian cells" Nucl Acids Res 31:2705-2716).
As another example, since double-stranded RNAs greater than 30-80
base pairs long activate the antiviral interferon response in
mammalian cells and result in non-specific silencing, interfering
RNAs for use in mammalian cells are typically less than 30 base
pairs (for example, Caplen et al. (2001) "Specific inhibition of
gene expression by small double-stranded RNAs in invertebrate and
vertebrate systems" Proc. Natl. Acad. Sci. USA 98:9742-9747,
Elbashir et al. (2001) "Duplexes of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian cells" Nature 411:494-498 and
Elbashir et al. (2002) "Analysis of gene function in somatic
mammalian cells using small interfering RNAs" Methods 26:199-213
describe the use of 21 nucleotide siRNAs to specifically inhibit
gene expression in mammalian cell lines, and Kim et al. (2005)
"Synthetic dsRNA Dicer substrates enhance RNAi potency and
efficacy" Nature Biotechnology 23:222-226 describes use of 25-30
nucleotide duplexes). The sense and antisense strands of a siRNA
are typically, but not necessarily, completely complementary to
each other over the double-stranded region of the siRNA (excluding
any overhangs). The antisense strand is typically completely
complementary to the target mRNA over the same region, although
some nucleotide substitutions can be tolerated (e.g., a one or two
nucleotide mismatch between the antisense strand and the mRNA can
still result in RNAi, although at reduced efficiency). The ends of
the double-stranded region are typically more tolerant to
substitution than the middle; for example, as little as 15 bp (base
pairs) of complementarity between the antisense strand and the
target mRNA in the context of a 21 mer with a 19 bp double-stranded
region has been shown to result in a functional siRNA (see, e.g.,
Czauderna et al. (2003) "Structural variations and stabilizing
modifications of synthetic siRNAs in mammalian cells" Nucl Acids
Res 31:2705-2716). Any overhangs can but need not be complementary
to the target mRNA; for example, TT (two 2'-deoxythymidines)
overhangs are frequently used to reduce synthesis costs.
[0096] Although double-stranded RNAs (e.g., double-stranded siRNAs)
were initially thought to be required to initiate RNAi, several
recent reports indicate that the antisense strand of such siRNAs is
sufficient to initiate RNAi. Single-stranded antisense siRNAs can
initiate RNAi through the same pathway as double-stranded siRNAs
(as evidenced, for example, by the appearance of specific mRNA
endonucleolytic cleavage fragments). As for double-stranded
interfering RNAs, characteristics of high-efficiency
single-stranded siRNAs may vary by cell type (e.g., a 5' phosphate
may be required on the antisense strand for efficient induction of
RNAi in some cell types, while a free 5' hydroxyl is sufficient in
other cell types capable of phosphorylating the hydroxyl). See,
e.g., Martinez et al. (2002) "Single-stranded antisense siRNAs
guide target RNA cleavage in RNAi" Cell 110:563-574; Amarzguioui et
al. (2003) "Tolerance for mutations and chemical modifications in a
siRNA" Nucl. Acids Res. 31:589-595; Holen et al. (2003) "Similar
behavior of single-strand and double-strand siRNAs suggests that
they act through a common RNAi pathway" Nucl. Acids Res.
31:2401-2407; and Schwarz et al. (2002) Mol. Cell 10:537-548.
[0097] Due to currently unexplained differences in efficiency
between siRNAs corresponding to different regions of a given target
mRNA, several siRNAs are typically designed and tested against the
target mRNA to determine which siRNA is most effective. Interfering
RNAs can also be produced as small hairpin RNAs (shRNAs, also
called short hairpin RNAs), which are processed in the cell into
siRNA-like molecules that initiate RNAi (see, e.g., Siolas et al.
(2005) "Synthetic shRNAs as potent RNAi triggers" Nature
Biotechnology 23:227-231).
[0098] The present invention provides a number of novel methods,
compositions, and kits related to RNAi. For example, the invention
provides methods in which a labeled interfering RNA is used as an
in cell sensor to detect and/or quantitate a target mRNA. The
labeled RNA includes a label whose signal output changes when the
labeled RNA initiates RNA interference (e.g., if the target mRNA is
present in the cell). The labeled RNA sensor is optionally caged to
permit temporal and/or spatial control over activation of the
sensor. Related kits, systems, and compositions (e.g., comprising
labeled interfering RNAs for use as in cell sensors) are also
provided.
[0099] As another example, the invention also provides caged
interfering RNAs (e.g., photoactivatable interfering RNAs). Such a
caged RNA includes, e.g., one or more caging groups that inhibit
(e.g., prevent) the RNA from initiating RNA interference and whose
removal or change in conformation permits the RNA to initiate RNA
interference. Kits for making the caged RNA and kits and systems
comprising the caged RNA are also features of the invention, as are
methods of using a caged interfering RNA to selectively attenuate
expression of a target gene in a cell. Using a caged interfering
RNA in the methods permits the initiation of RNAi to be precisely
controlled, temporally and/or spatially.
[0100] As yet another example, the invention provides novel methods
and compositions for introducing interfering RNAs into cells. An
interfering RNA can be covalently attached to a protein
transduction domain and/or to a lipid (e.g., a myristoyl group)
that can mediate its introduction into a cell. Methods of
introducing an interfering RNA into a cell, by contacting the cell
with the protein transduction domain- and/or lipid-linked
interfering RNA, are also provided. The covalent attachment between
the protein transduction domain or lipid and the RNA is optionally
reversible (e.g., the attachment can be a photolabile linker or a
disulfide bond).
[0101] As yet another example, the invention provides additional
methods of selectively attenuating expression of a target gene in a
cell. In some embodiments, transcription of an interfering RNA is
controlled by use of a caged activation component. In other
embodiments, caged DNAs encoding an interfering RNA are introduced
into a cell and then uncaged to permit transcription of the
interfering RNA. The following sections describe the invention in
more detail.
[0102] Related compositions, methods, systems, and kits involving
RNAs capable of repressing translation of a target mRNA or
silencing transcription of a target gene are also described.
[0103] Interfering RNA Sensors
[0104] The use of double-stranded RNAs to attenuate expression of
target genes by RNAi has been well described, including, e.g., the
use of labels on siRNAs to localize the siRNAs in transfected cells
(e.g., to the cytoplasm, endosome, nucleus, or the like; see, e.g.,
Byrom et al. "Visualizing siRNA in mammalian cells: Fluorescence
analysis of the RNAi effect" Ambion TechNotes 9(3)). Such labels
are typically "nonfunctional labels"; that is, they provide a
signal output which remains constant regardless of whether the
labeled siRNA initiates RNAi (e.g., interacts with RISC and/or the
target mRNA). This invention, however, provides novel methods of
using labeled interfering RNAs as in cell sensors to detect
expression of target genes. In the methods, the labels are
"functional labels"; that is, the labels provide a signal output
that is dependent on whether the labeled interfering RNA (e.g., the
labeled siRNA) initiates RNAi of the target mRNA (e.g., interacts
with RISC and/or the target mRNA and undergoes strand separation,
leading to endonucleolytic cleavage of the target mRNA).
Compositions, systems, and kits related to the methods are also
provided. The methods, compositions, systems, and kits overcome the
above noted difficulties associated with current methods of
detecting and/or quantitating mRNA transcripts from cells, by
enabling detection and/or quantitation of mRNA in living cells and
without requiring mRNA purification, reverse transcription, or cell
lysis or fixation.
[0105] Methods
[0106] A first general class of embodiments provides methods of
detecting a target mRNA in a cell. In the methods, a labeled RNA is
provided. The labeled RNA comprises an RNA comprising at least one
double-stranded region, the double-stranded region comprising a
sense strand and an antisense strand, the antisense strand
comprising a region which is substantially complementary to a
region of the target mRNA, and at least one label. The labeled RNA
is introduced into the cell, whereby the labeled RNA initiates RNA
interference of the target mRNA. This results in an
initiation-dependent change in a signal output of the label. The
signal output, which provides an indication of the presence of the
target mRNA in the cell, is detected. The target mRNA can be, for
example, a constitutively expressed mRNA or an mRNA whose
expression is inducible.
[0107] It is worth noting that initiation of RNAi of the target
mRNA by the labeled RNA can, but need not, result in a substantial
attenuation of expression of the target mRNA. For example,
expression of the target mRNA can be unaffected, or expression of
the target mRNA can be decreased by at least about 0.001%, at least
about 0.01%, at least about 0.1%, at least about 1%, at least about
5%, at least about 10%, at least about 25%, at least about 50%, or
at least about 75% or more, or can even be reduced to an
undetectable level.
[0108] In a preferred class of embodiments, the label is a
fluorescent label, and the initiation-dependent change in the
signal output of the label is a change in fluorescent emission. The
methods can optionally be used to quantitate the amount of target
mRNA present in the cell. For example, the intensity of the
fluorescent emission can be measured. The intensity provides an
indication of the quantity of the target mRNA present in the
cell.
[0109] In one class of embodiments, the labeled RNA also includes
at least one quencher. The label and the quencher are positioned in
the RNA such that fluorescent emission by the label is quenched by
the quencher. Initiation of RNA interference by the labeled RNA
results in unquenching of the label (and thus an increase in the
fluorescent emission by the label). In this class of embodiments,
the initiation-dependent change in the signal output is thus an
increase in the fluorescent emission by the label. For example, the
label and quencher can be positioned on opposite strands, in close
enough proximity to each other that the label is quenched until the
sense and antisense strands are separated.
[0110] In a related class of embodiments, the labeled RNA comprises
two fluorescent labels, one being a donor and the other being an
acceptor. The donor and acceptor are positioned within the RNA such
that energy transfer (e.g., FRET) occurs between them (e.g.,
excitation of the donor results in fluorescence by the acceptor).
Initiation of RNA interference by the labeled RNA results in loss
of energy transfer between the donor and the acceptor. This can be
observed as an increase in fluorescence by the donor or as a
decrease in fluorescence by the acceptor. Thus, in a preferred
class of embodiments, the initiation-dependent change in the signal
output is a decrease in fluorescent emission by the acceptor
following excitation of the donor. As in the preceding embodiments,
the donor and acceptor can, for example, be positioned on opposite
strands in close enough proximity to each other that energy
transfer occurs until the sense and antisense strands are
separated.
[0111] The RNA can have any of a variety of structures, lengths,
and/or the like. Thus, in one class of embodiments, the RNA
comprises a first polyribonucleotide comprising the sense strand
and a second polyribonucleotide comprising the antisense strand.
The RNA can be, e.g., a long double-stranded RNA that is cleaved by
Dicer in the cell, or it can be, e.g., an siRNA. For example, the
first polyribonucleotide can comprise between 19 and 25
nucleotides, the second polyribonucleotide can comprise between 19
and 25 nucleotides, and the double-stranded region can comprise
between 19 and 25 base pairs. The first and second
polyribonucleotides can form a duplex over their entire length, or
they can have overhangs (e.g., 5' or 3' overhangs; e.g., 21 nt
first and second polyribonucleotides can form a 19 bp
double-stranded region with 2 nucleotide overhangs, 23 nt
polyribonucleotides can form a 21 bp double-stranded region with 2
nucleotide overhangs, and so on). For example, in some embodiments,
the first polyribonucleotide and the second polyribonucleotide each
comprise a two nucleotide TT 3' overhang (where T is
2'-deoxythymidine). The RNA is optionally nuclease resistant and
optionally comprises one or more deoxyribonucleotides one or more
PNA monomers, and/or one or more modified nucleotides (e.g.,
2'-methyl or 2'-O-allyl ribonucleotides) or internucleotide
linkages (e.g., phosphorothioate linkages).
[0112] As described in greater detail below, the RNA sensors can
be, and in several embodiments are, caged. Thus, in some
embodiments, at least one caging group is associated with the RNA.
For example, at least one caging group can be covalently attached
to a 5' hydroxyl or a 5' phosphate of the second
polyribonucleotide. Since this 5' hydroxyl or phosphate is useful
for an siRNA to initiate RNAi (Czauderna et al. (2003) "Structural
variations and stabilizing modifications of synthetic siRNAs in
mammalian cells" Nucl Acids Res 31:2705-2716), caging the 5'
hydroxyl or phosphate of the antisense strand permits the sensor to
be uncaged and activated in a controlled manner.
[0113] In one class of embodiments, in which the first and second
polyribonucleotides comprise 19-25 nt, the label is a fluorescent
label, and the RNA further comprises at least one quencher. The
label and the quencher are positioned in the RNA such that
fluorescent emission by the label is quenched by the quencher, and
initiation of RNA interference by the labeled RNA results in
unquenching of the label. The initiation-dependent change in the
signal output is thus an increase in the fluorescent emission by
the label.
[0114] The label and quencher can be attached to a nucleic acid of
the invention at essentially any suitable position(s), e.g., at the
3' end, at the 5' end, and/or within either or both the first and
second polyribonucleotides. For example, the label can be attached
to the first polyribonucleotide and the quencher attached to the
second polyribonucleotide, or the label can be attached to the
second polyribonucleotide and the quencher attached to the first
polyribonucleotide. For example, one of the label and the quencher
can be located within three nucleotides of the 3' end of the first
polyribonucleotide and the other of the label and the quencher can
be located within three nucleotides of the 3' end of the second
polyribonucleotide (see, e.g., FIG. 1 Panels A-C). As another
example, one of the label and the quencher can be located within
three nucleotides of the 3' end of the first polyribonucleotide and
the other of the label and the quencher can be located within three
nucleotides of the 5' end of the second polyribonucleotide (see,
e.g., FIG. 1 Panels D and G). As yet another example, one of the
label and the quencher can be located within three nucleotides of
the 5' end of the first polyribonucleotide and the other of the
label and the quencher can be located within three nucleotides of
the 3' end of the second polyribonucleotide (see, e.g., FIG. 1
Panels E, F and H). As yet another example, one of the label and
the quencher can be located within three nucleotides of the 5' end
of the first polyribonucleotide and the other of the label and the
quencher can be located within three nucleotides of the 5' end of
the second polyribonucleotide (see, e.g., FIG. 1 Panel I). In other
examples, the label and/or quencher can be in the middle of the
RNA; e.g., one of the label and the quencher can be located more
than three nucleotides from the 5' end and more than three
nucleotides from the 3' end of the first polyribonucleotide, and
the other of the label and the quencher can be located more than
three nucleotides from the 5' end and more than three nucleotides
from the 3' end of the second polyribonucleotide, or, one of the
label and the quencher can be located within three nucleotides of
the 5' end or the 3' end of the first or second polyribonucleotide
and the other of the label and the quencher can be located more
than three nucleotides from the 5' end and more than three
nucleotides from the 3' end of the opposite polyribonucleotide. In
one example embodiment, the label is attached at the 3' end of the
first polyribonucleotide and the quencher is attached at the 3' end
of the second polyribonucleotide. In a related example embodiment,
the quencher is attached at the 3' end of the first
polyribonucleotide and the label is attached at the 3' end of the
second polyribonucleotide. In yet another example, one of the label
and the quencher is attached at the 5' end of the first
polyribonucleotide and the other of the label and the quencher is
attached at the 3' end of the second polyribonucleotide.
[0115] Techniques for determining and verifying suitable positions
for the label and quencher are well known in the art. For example,
the label and quencher are typically positioned such that they do
not substantially reduce RNAi of the target mRNA as compared to an
otherwise identical RNA lacking the label and quencher (e.g., the
label and quencher preferably do not interfere with siRNA binding
to RISC, strand separation of the siRNA, or binding of the
antisense strand to the target mRNA). For example, if the quencher
and label are located in the middle of the double-stranded region,
the quencher can be attached to a nucleotide one or more
nucleotides removed from the complement of the nucleotide to which
the fluorescent label is attached. As another example, although
overhangs may not be necessary for siRNA function, 3' and/or 5'
overhangs of one, two, three, four, or more nucleotides can
optionally be used to position a quencher or label such that it
does not interfere with RISC binding to the sensor (see, e.g., FIG.
1 panels G and H).
[0116] In a related class of embodiments in which the first and
second polyribonucleotides comprise 19-25 nt, the labeled RNA
comprises two fluorescent labels, one of which is a donor and the
other of which is an acceptor. The donor and acceptor are
positioned within the RNA such that energy transfer occurs between
them. Initiation of RNA interference by the labeled RNA results in
loss of energy transfer between the donor and the acceptor. The
initiation-dependent change in signal output can thus be, e.g., a
decrease in fluorescent emission by the acceptor following
excitation of the donor.
[0117] The donor and acceptor can be attached at essentially any
suitable positions, e.g., at the 3' end, at the 5' end, and/or
within either or both the first and second polyribonucleotides. For
example, the donor can be attached to the first polyribonucleotide
and the acceptor to the second polyribonucleotide, or the donor can
be attached to the second polyribonucleotide and the acceptor to
the first polyribonucleotide. For example, one of the donor and the
acceptor can be located within three nucleotides of the 3' end of
the first polyribonucleotide and the other of the donor and the
acceptor can be located within three nucleotides of the 3' end of
the second polyribonucleotide (see, e.g., FIG. 1 Panels A-C). As
another example, one of the donor and the acceptor can be located
within three nucleotides of the 3' end of the first
polyribonucleotide and the other of the donor and the acceptor can
be located within three nucleotides of the 5' end of the second
polyribonucleotide (see, e.g., FIG. 1 Panels D and G). As yet
another example, one of the donor and the acceptor can be located
within three nucleotides of the 5' end of the first
polyribonucleotide and the other of the donor and the acceptor can
be located within three nucleotides of the 3' end of the second
polyribonucleotide (see, e.g., FIG. 1 Panel E, F and H). As yet
another example, one of the donor and the acceptor can be located
within three nucleotides of the 5' end of the first
polyribonucleotide and the other of the donor and the acceptor can
be located within three nucleotides of the 5' end of the second
polyribonucleotide (see, e.g., FIG. 1 Panel I). In other examples,
the donor and/or acceptor can be in the middle of the RNA; e.g.,
one of the donor and the acceptor can be located more than three
nucleotides from the 5' end and more than three nucleotides from
the 3' end of the first polyribonucleotide, and the other of the
donor and the acceptor can be located more than three nucleotides
from the 5' end and more than three nucleotides from the 3' end of
the second polyribonucleotide, or, one of the donor and the
acceptor can be located within three nucleotides of the 5' end or
the 3' end of the first or second polyribonucleotide and the other
of the donor and the acceptor can be located more than three
nucleotides from the 5' end and more than three nucleotides from
the 3' end of the opposite polyribonucleotide. In one example
embodiment, the donor is attached at the 3' end of the first
polyribonucleotide and the acceptor is attached at the 3' end of
the second polyribonucleotide. In a related example embodiment, the
acceptor is attached at the 3' end of the first polyribonucleotide
and the donor is attached at the 3' end of the second
polyribonucleotide. In yet another example, one of the donor and
the acceptor is attached at the 5' end of the first
polyribonucleotide and the other of the donor and the acceptor is
attached at the 3' end of the second polyribonucleotide. Techniques
for determining and verifying suitable positions for the donor and
acceptor are well known in the art.
[0118] Instead of comprising two polyribonucleotides, in some
embodiments, the RNA of interest comprises a self-complementary
polyribonucleotide (e.g., a shRNA). Label/quencher or
acceptor/donor combinations can be similarly positioned within the
self-complementary polyribonucleotide.
[0119] As noted, the length of the RNA can vary. For example, the
double-stranded region can comprise fewer than about 1500 base
pairs, fewer than about 1000 base pairs, fewer than about 500 base
pairs, fewer than about 250 base pairs, fewer than about 150 base
pairs, fewer than about 80 base pairs, fewer than about 50 base
pairs, fewer than about 30 base pairs, or fewer than about 25 base
pairs.
[0120] As noted, the RNA sensors can optionally be caged. Caging a
sensor, e.g., with a photolabile group, allows the initiation of
RNAi, and thus the detection of the target mRNA, to be precisely
controlled, temporally and/or spatially. This provides a number of
advantages. For example, a caged RNA sensor can be introduced into
a cell, e.g., by lipofection. The cell can be permitted to recover
from the manipulations necessary to introduce the sensor before
uncaging of the sensor permits initiation of RNAi and detection of
the target transcript. As another example, until the sensor is
uncaged, the interfering RNA exerts no effect on the cell. As yet
another example, caging the interfering RNA can protect it from
nucleases and thus extend its half-life.
[0121] Thus, in one class of embodiments, the labeled RNA further
comprises one or more first caging groups associated with the RNA.
The first caging groups inhibit the RNA from initiating RNA
interference of the target mRNA in the cell. RNA interference of
the target mRNA is initiated by exposing the cell to uncaging
energy of a first type, whereby exposure to the uncaging energy
frees the RNA from inhibition by the first caging groups.
[0122] The first caging groups can inhibit the RNA from initiating
RNA interference of the target mRNA by at least about 25%, at least
about 30%, at least about 35%, at least about 50%, at least about
75%, at least about 90%, at least about 95%, or at least about 98%,
as compared to the RNA in the absence of the first caging groups.
For example, if introduction of an siRNA into a cell decreases
expression of its target mRNA to 10% of normal (i.e., expression in
a cell not comprising the siRNA), then introduction of the
corresponding caged siRNA into a cell would decrease expression to
55% of normal if the caging groups inhibit the RNA from initiating
RNA interference by 50% (under equivalent conditions). In one class
of embodiments, the first caging groups prevent the RNA from
initiating RNA interference of the target mRNA (i.e., introduction
of the caged RNA into a cell has no effect on expression of the
target mRNA). Removal of or an induced conformational change in the
first caging groups typically permits the RNA to initiate RNA
interference of the target mRNA.
[0123] The one or more first caging groups associated with the RNA
can be covalently attached to or non-covalently associated with the
RNA. See, e.g., FIGS. 5 and 6 for a few of the possible examples of
sites of attachment of the caging groups (e.g., at one or more
bases, riboses, phosphate groups and/or terminal hydroxyls, within
and/or at the end of either or both strands of the RNA). In one
embodiment, the RNA comprises a first polyribonucleotide comprising
the sense strand and a second polyribonucleotide comprising the
antisense strand, and the first caging group is covalently attached
to the first polyribonucleotide and to the second
polyribonucleotide. For example, the first caging group can be
attached to the 5' end of the first polyribonucleotide and to the
3' end of the second polyribonucleotide, or, preferably, it can be
attached to the 3' end of the first polyribonucleotide and to the
5' end of the second polyribonucleotide (FIG. 4). The caging group
linking the two polyribonucleotides can, for example, be
photolabile.
[0124] In a preferred aspect, the one or more first caging groups
are photoactivatable (e.g., photolabile). Thus, in a preferred
class of embodiments, exposing the cell to uncaging energy of the
first type comprises exposing the cell to light of a first
wavelength (e.g., light with a wavelength between about 60 nm and
about 400 nm, between about 400 nm and about 700 nm, and/or between
about 700 nm and about 1000 nm). Other caging groups are removable
via input of different uncaging energies, e.g., the one or more
caging groups can be removable by sonication or application of
heat, or can be removed by a chemical or enzyme.
[0125] Exposing the cell to light of a first wavelength optionally
comprises exposing the cell to light such that the intensity of the
light and the duration of exposure to the light are controlled such
that a first portion (which can be a selected amount) of the caged
labeled RNA is uncaged and a second portion of the caged labeled
RNA remains caged. Put another way, the uncaging rate can be
controlled. Furthermore, the uncaging step can be repeated until
the caged RNA is depleted.
[0126] As noted, caging permits temporal control over activation of
the sensor. For example, the method can include stimulating the
cell and uncaging the sensor at a preselected time with respect to
the stimulus. For example, the method can include contacting the
cell and a test compound and exposing the cell to the uncaging
energy at a preselected time point with respect to a time at which
the cell and the test compound are contacted (e.g., to determine if
the test compound directly or indirectly affects expression of the
target mRNA). Caging also permits spatial control over activation
of the sensor. For example, the uncaging energy can be directed at
a preselected subset of a cell population comprising the cell.
[0127] Various techniques (e.g., lipofection, microinjection, or
electroporation) can be used to introduce the labeled RNA into the
cell. In one class of embodiments, the labeled RNA also includes a
cellular delivery module, associated with the RNA, that can mediate
introduction of the labeled RNA into the cell. In this class of
embodiments, introducing the labeled RNA into the cell comprises
contacting the cell with the labeled RNA associated with the
cellular delivery module.
[0128] The cellular delivery module optionally comprises a
polypeptide, e.g., a PEP-1 peptide or an amphipathic peptide (e.g.,
an MPG or an MPG.sup..DELTA.NLS peptide; see Simeoni et al. (2003)
"Insight into the mechanism of the peptide-based gene delivery
system MPG: Implications for delivery of siRNA into mammalian
cells" Nucl Acids Res 31: 2717-2724), covalently or noncovalently
associated with the RNA. As another example, the polypeptide can be
a cationic peptide (e.g., a homopolymer of histidine, lysine, or
D-arginine) that is covalently or noncovalently (e.g., by
electrostatic interaction with the negatively charged RNA)
associated with the RNA. In one class of embodiments, the cellular
delivery module comprises a protein transduction domain, e.g.,
derived from an HIV-1 Tat protein, from a herpes simplex virus VP22
protein, or from a Drosophila antennapedia protein (e.g.,
Penetratin.TM.). In one aspect, the protein transduction domain is
a model protein transduction domain, e.g., a homopolymer of
D-arginine, e.g., 8-D-Arg. The protein transduction domain can be
covalently attached directly to the RNA, or can be indirectly
associated with the RNA (for example, the protein transduction
domain can be covalently coupled to a bead or to a carrier protein
such as BSA, which is in turn coupled to the RNA, e.g., through a
photolabile or cleavable linker; e.g., FIGS. 8-9).
[0129] The cellular delivery module can be noncovalently associated
with the RNA, or the cellular delivery module can be covalently
attached to the RNA. For example, the covalent attachment between
the cellular delivery module and the RNA is optionally reversible
by exposure to light of a preselected wavelength, and the method
includes exposing the cell to light of the preselected wavelength.
As another example, the covalent attachment is optionally a
disulfide bond or an ester linkage that is reduced or cleaved once
the sensor is inside the cell.
[0130] In certain embodiments, the cellular delivery module can
also serve as a caging group. For example, the RNA can comprise a
first polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand, and the
cellular delivery module can be covalently attached to the first
polyribonucleotide and/or to the second polyribonucleotide (e.g.,
by a photolabile linker; see, e.g., FIGS. 7-10). The cellular
delivery module can mediate introduction of the RNA into the cell,
where the presence of the cellular delivery module prevents the RNA
from initiating RNA interference until the cellular delivery module
is removed (e.g., by exposing the cell to light of an appropriate
wavelength to cleave the photolabile linker).
[0131] Optionally, the cellular delivery module covalently attached
to the RNA comprises a lipid, e.g., a fatty acid. For example, the
RNA can be covalently attached to one or more myristoyl groups,
e.g., via a photolabile linker (FIG. 10).
[0132] In one aspect, the cellular delivery module is associated
with one or more second caging groups, which inhibit (e.g.,
prevent) the cellular delivery module from mediating introduction
of the labeled RNA into the cell. In this aspect, the method
includes initiating introduction of the labeled RNA into the cell
by exposing the labeled RNA to uncaging energy of a second type
(which is typically different from the uncaging energy of the first
type if first caging groups are present on the RNA), whereby
exposure to the uncaging energy frees the cellular delivery module
from inhibition by the second caging groups.
[0133] The methods can optionally be used to monitor gene
expression, for example, induction of transcription of the target
mRNA in response to a stimulus. Thus, in one class of embodiments,
the methods include stimulating the cell, e.g., by adding a test
compound (e.g., a drug, a candidate drug, a receptor agonist or
putative agonist, or the like), by changing growth conditions, by
adding other cells, etc.
[0134] The methods can be used to examine expression of the target
mRNA, e.g., in two different cell populations, one stimulated and
one not. Similarly, expression of the target mRNA can be monitored
in a single cell (or a single cell population) before and after
stimulation of the cell. Thus, in one embodiment, the signal output
is detected at a plurality of time points with respect to a time at
which the cell is stimulated.
[0135] The methods optionally include introducing a plurality of
RNA sensors (labeled interfering RNAs) into the cell to
simultaneously monitor expression of a plurality of target mRNAs.
The labels on the different RNAs typically have detectably
different signal outputs. For example, the different RNAs can
comprise different fluorescent label/quencher combinations (see,
e.g., FIG. 2) or different donor/acceptor combinations or a
combination of FRET, fluorophore/quencher, and TR-FRET sensors can
be used (see, e.g., FIG. 3). The different sensors are optionally
caged, e.g., with photolabile caging groups removable by different
wavelengths of light, such that the different sensors can be
uncaged at different time points.
[0136] In a related class of embodiments, a reference sensor is
also introduced into the cell. Signal output from the target
sensor's label (indicating the presence of the target mRNA, e.g.,
an inducible mRNA, in the cell) can be normalized by comparison
with a signal output from the reference sensor. Such a reference
sensor can comprise a labeled interfering RNA against a
constitutively expressed or housekeeping gene (e.g., GAPDH, actin,
or the like).
[0137] In one class of embodiments, the methods are used to
determine how efficiently the RNA attenuates (or knocks-down)
expression of the target mRNA. In these embodiments, an intensity
of the signal output (e.g., intensity of a fluorescent emission) is
measured. The intensity provides an indication of the quantity
(e.g., relative or absolute quantity) of the target mRNA present in
the cell, which provides an indication of the efficiency with which
the labeled RNA reduces expression of the target mRNA.
[0138] Kits
[0139] Another aspect of the invention includes kits related to the
methods. For example, one class of embodiments provides a kit for
detecting a target mRNA in a cell. The kit includes a labeled RNA
and instructions for using the labeled RNA to detect the presence
of the target mRNA in the cell, packaged in one or more containers.
The labeled RNA comprises an RNA comprising at least one
double-stranded region, the double-stranded region comprising a
sense strand and an antisense strand, the antisense strand
comprising a region which is substantially complementary to a
region of the target mRNA. The labeled RNA also comprises at least
one label, wherein initiation of RNA interference of the target
mRNA by the labeled RNA in the cell results in an
initiation-dependent change in a signal output of the label.
[0140] The instructions can include, for example, instructions for
introducing the labeled RNA into the cell, detecting a fluorescent
signal from the RNA, interpreting the fluorescent signal (including
quantitating the mRNA based on the intensity of the fluorescent
signal), and the like.
[0141] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for label configurations (e.g., use of
fluorescent labels, fluorescent label/quencher, and donor/acceptor
combinations), signal output types, RNA configurations (e.g., one
or two polyribonucleotides, of various lengths, with or without
overhangs, etc.), use of caging groups (e.g., photolabile caging
groups), appropriate uncaging energies (light, heat, sonic, etc.),
use of cellular delivery modules (e.g., amphipathic peptides,
cationic peptides, protein transduction domains, and lipids), and
the like.
[0142] In addition, it is worth noting that the kit optionally also
includes at least one buffer and/or at least one delivery reagent.
The delivery reagent can be essentially any reagent that can
mediate introduction of the labeled RNA into the cell; for example,
the delivery reagent can comprise a polypeptide (e.g., a PEP-1
peptide, an amphipathic peptide, e.g., an MPG or MPG.sup..DELTA.NLS
peptide, or a cationic peptide, e.g., poly-His, poly-Lys, or
poly-D-Arg) or at least one lipid (e.g., a lipid optimized for
lipofection of the given labeled RNA, or a lipid comprising at
least one myristoyl group to be covalently attached to the labeled
RNA). In one class of embodiments, the labeled RNA is caged. In
this class of embodiments, the kit optionally includes a control
reagent for monitoring uncaging efficiency (e.g., a caged
fluorophore, e.g., caged FITC) and/or an uncaged version of the
labeled RNA (e.g., to be used as a control to monitor uncaging of
the caged labeled RNA, maximal knock down of the target mRNA,
and/or the like). The kit also optionally includes packaging or
instructional materials for such additional reagents.
[0143] An additional class of embodiments also provides a kit for
detecting a target mRNA in a cell. In this class of embodiments,
the kit comprises a target RNA sensor and a reference RNA sensor,
packaged in one or more containers. The target RNA sensor comprises
a first RNA comprising at least one double-stranded region, the
double-stranded region comprising a sense strand and an antisense
strand, the antisense strand comprising a region which is
substantially complementary to a region of a target mRNA, and at
least one first label, wherein initiation of RNA interference of
the target mRNA by the first RNA in the cell results in an
initiation-dependent change in a signal output of the first label.
The reference RNA sensor comprises a second RNA comprising at least
one double-stranded region, the double-stranded region comprising a
sense strand and an antisense strand, the antisense strand
comprising a region which is substantially complementary to a
region of a reference mRNA, and at least one second label, wherein
initiation of RNA interference of the reference mRNA by the second
RNA in the cell results in an initiation-dependent change in a
signal output of the second label; packaged in one or more
containers. Typically, the signal output of the first label is
detectably different from the signal output of the second
label.
[0144] The target and reference mRNAs can be essentially any mRNAs.
For example, the target mRNA can be an inducible mRNA while the
reference mRNA is a constitutively expressed or housekeeping mRNA
(e.g., GAPDH, actin, or the like).
[0145] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for label configurations (e.g., use of
fluorescent labels, fluorescent label/quencher, and donor/acceptor
combinations), signal output types, RNA configurations (e.g., one
or two polyribonucleotides, of various lengths, with or without
overhangs, etc.), use of caging groups (e.g., photolabile caging
groups), appropriate uncaging energies (light, heat, sonic, etc.),
use of cellular delivery modules (e.g., amphipathic peptides,
protein transduction domains, and lipids), and the like.
[0146] In addition, it is worth noting that the kit optionally also
includes one or more of: instructions (e.g., for using the target
and reference RNA sensors to detect the presence of the target mRNA
in the cell and/or for using the target and reference RNA sensors
to quantitate an amount of the target mRNA present in the cell), a
buffer, or a delivery reagent which can mediate introduction of the
labeled RNA into the cell (for example, a polypeptide, e.g., a
PEP-1 peptide, an amphipathic peptide, e.g., an MPG or
MPG.sup..DELTA.NLS peptide, or a cationic peptide, or at least one
lipid, e.g., a lipid optimized for lipofection of the given labeled
RNA or a lipid comprising at least one myristoyl group to be
covalently attached to the labeled RNA).
[0147] Compositions
[0148] Yet another aspect of the invention provides compositions
related to the methods (e.g., compositions produced by the methods
or facilitating use of the methods). For example, one class of
embodiments provides a composition comprising a population of
labeled RNAs (e.g., identical labeled RNAs) for detecting a target
mRNA in a cell. The target mRNA and/or the cell are also optionally
features of the composition. Each labeled RNA comprises an RNA
comprising at least one double-stranded region, the double-stranded
region comprising a sense strand and an antisense strand, the
antisense strand comprising a region which is substantially
complementary to a region of the target mRNA, and at least one
label. The label is located a preselected position in the RNA, and
initiation of RNA interference of the target mRNA by the labeled
RNA in the cell results in an initiation-dependent change in a
signal output of the label.
[0149] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for label configurations (e.g., use of
fluorescent labels, fluorescent label/quencher, and donor/acceptor
combinations), signal output types, RNA configurations (e.g., one
or two polyribonucleotides, of various lengths, with or without
overhangs, etc.), use of caging groups (e.g., photolabile caging
groups), appropriate uncaging energies (light, heat, sonic, etc.),
use of cellular delivery modules (e.g., amphipathic peptides,
protein transduction domains, and lipids), and the like. It is
worth noting that a quencher or a second label, if present in the
RNA, is optionally also located a preselected position in the RNA.
The composition comprising the population optionally also includes
the target mRNA and/or a cell, e.g., a cell comprising the
population and/or the target mRNA.
[0150] Using a population of RNA sensors in which the label is
located at a preselected position in the RNA has several advantages
over using labeled RNAs where the labels are located at random
positions in the RNA. For example, the preselected label position
can be chosen such that the initiation-dependent change in the
signal output of the label is maximized (e.g., label and quencher
or acceptor and donor positions can be selected to maximize the
change in signal output). As another example, the preselected label
position can be chosen such that the presence of the label does not
substantially interfere with initiation of RNAi by the labeled RNA,
whereas random labeling of an interfering RNA can result in labels
being attached at positions where they interfere with RISC binding
to the labeled RNA or the like.
[0151] In another class of embodiments, the invention provides a
composition comprising a target RNA sensor and a reference RNA
sensor. The target RNA sensor comprises a first RNA comprising at
least one double-stranded region, the double-stranded region
comprising a sense strand and an antisense strand, the antisense
strand comprising a region which is substantially complementary to
a region of a target mRNA, and at least one first label, wherein
initiation of RNA interference of the target mRNA by the first RNA
in the cell results in an initiation-dependent change in a signal
output of the first label. The reference RNA sensor comprises a
second RNA comprising at least one double-stranded region, the
double-stranded region comprising a sense strand and an antisense
strand, the antisense strand comprising a region which is
substantially complementary to a region of a reference mRNA, and at
least one second label, wherein initiation of RNA interference of
the reference mRNA by the second RNA in the cell results in an
initiation-dependent change in a signal output of the second label.
Typically, the signal output of the first label is detectably
different from the signal output of the second label.
[0152] The target and reference mRNAs can be essentially any mRNAs.
For example, the target mRNA can be an inducible mRNA while the
reference mRNA is a constitutively expressed or housekeeping mRNA
(e.g., GAPDH, actin, or the like).
[0153] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for label configurations (e.g., use of
fluorescent labels, fluorescent label/quencher, and donor/acceptor
combinations), signal output types, RNA configurations (e.g., one
or two polyribonucleotides, of various lengths, with or without
overhangs, etc.), use of caging groups (e.g., photolabile caging
groups), appropriate uncaging energies (light, heat, sonic, etc.),
use of cellular delivery modules (e.g., amphipathic peptides,
protein transduction domains, and lipids), and the like. It is
worth noting that the composition optionally also includes the
target mRNA, the reference mRNAs and/or a cell, e.g., a cell
comprising the target and reference sensors and/or the target
mRNA.
[0154] Systems
[0155] In another aspect, systems and/or apparatus comprising the
compositions (e.g., the labeled RNAs, cells comprising the labeled
RNAs, or the like) noted above and, e.g., components such as
detectors, fluid handling apparatus, sources of uncaging energy, or
the like, are a feature of the invention.
[0156] In general, various automated systems can be used to perform
some or all of the method steps as noted herein. In addition to
practicing some or all of the method steps herein, digital or
analog systems, e.g., comprising a digital or analog computer, can
also control a variety of other functions such as a user viewable
display (e.g., to permit viewing of method results by a user)
and/or control of output features.
[0157] For example, certain of the methods described above are
optionally implemented via a computer program or programs (e.g.,
that perform or assist in detection of target mRNA). Thus, the
present invention provides digital systems, e.g., computers,
computer readable media, and/or integrated systems comprising
instructions (e.g., embodied in appropriate software) for
performing the methods herein. For example, a digital system
comprising instructions for interpreting the change in signal
output from the label to determine whether the target mRNA is
present in the cell and/or to determine the quantity of the target
mRNA present in the cell, as described herein, is a feature of the
invention. The digital system can also include information (data)
corresponding to signal output intensities or the like. The system
can also aid a user in performing mRNA detection according to the
methods herein, or can control laboratory equipment which automates
introduction of the labeled RNAs into the cells, detection of the
signal outputs, or the like.
[0158] Standard desktop applications such as word processing
software (e.g., Microsoft Word.TM. or Corel WordPerfect.TM.) and/or
database software (e.g., spreadsheet software such as Microsoft
Excel.TM., Corel Quattro Pro.TM., or database programs such as
Microsoft Access.TM. or Paradox.TM.) can be adapted to the present
invention by inputting data which is loaded into the memory of a
digital system and performing an operation as noted herein on the
data. For example, systems can include the foregoing software
having the appropriate signal intensity (e.g., fluorescent
intensity) data, etc., e.g., used in conjunction with a user
interface (e.g., a GUI in a standard operating system such as a
Windows, Macintosh or LINUX system) to perform any analysis noted
herein, or simply to acquire data (e.g., in a spreadsheet) to be
used in the methods herein.
[0159] Systems typically include, e.g., a digital computer with
software for performing signal output interpretation and/or mRNA
quantitation, or the like, as well as data sets entered into the
software system comprising signal output intensities or the like.
The computer can be, e.g., a PC (Intel x86 or Pentium
chip-compatible DOS,.TM. OS2,.TM. WINDOWS,.TM. WINDOWS NT,.TM.
WINDOWS95,.TM. WINDOWS98,.TM. LINUX, Apple-compatible,
MACINTOSH.TM. compatible, Power PC compatible, or a UNIX compatible
(e.g., SUN.TM. work station) machine) or other commercially common
computer which is known to one of skill. Software for performing
analysis of signal output and/or mRNA quantitation can be
constructed by one of skill using a standard programming language
such as Visualbasic, Fortran, Basic, Java, or the like, according
to the methods herein.
[0160] Any system controller or computer optionally includes a
monitor which can include, e.g., a cathode ray tube ("CRT")
display, a flat panel display (e.g., active matrix liquid crystal
display, liquid crystal display), or others. Computer circuitry is
often placed in a box which includes numerous integrated circuit
chips, such as a microprocessor, memory, interface circuits, and
others. The box also optionally includes a hard disk drive, a
floppy disk drive, a high capacity removable drive such as a
writeable CD-ROM, and other common peripheral elements. Inputting
devices such as a keyboard or mouse optionally provide for input
from a user and for user selection of the wavelength of fluorescent
emission to be monitored, or the like, in the relevant computer
system.
[0161] The computer typically includes appropriate software for
receiving user instructions, either in the form of user input into
a set parameter fields, e.g., in a GUI, or in the form of
preprogrammed instructions, e.g., preprogrammed for a variety of
different specific operations. The software then converts these
instructions to appropriate language for instructing the system to
carry out any desired operation. For example, in addition to
performing signal output analysis, a digital system can control
laboratory equipment for liquid handling, signal detection, or the
like according to the relevant method herein.
[0162] The invention can also be embodied within the circuitry of
an application specific integrated circuit (ASIC) or programmable
logic device (PLD). In such a case, the invention is embodied in a
computer readable descriptor language that can be used to create an
ASIC or PLD. The invention can also be embodied within the
circuitry or logic processors of a variety of other digital
apparatus, such as PDAs, laptop computer systems, displays, image
editing equipment, etc.
[0163] Applications
[0164] The methods, compositions, systems, and kits described above
have a number of applications. For example, as noted, labeled
interfering RNA sensors can be used to detect the presence of a
target mRNA, to quantitate an amount of a target mRNA present in a
cell, and/or to detect activation of a target gene. Additional
applications include, but are not limited to, detection of specific
biological activities (e.g., GCPR activation, disease, cell
migration, cell death, and the like) via detection of activation of
biomarker target genes; detection of splice variants of mRNAs,
e.g., as biomarkers of biological activities; detection of drug
effects via detection of activation of biomarker target genes; and
performance of ADME (Absorption, Distribution, Metabolism and
Excretion) toxicity assays via detection of activation of biomarker
target genes. If desired, high throughput cell-based assays using
labeled interfering RNA sensors can be designed for the above
example applications.
[0165] Caged Interfering RNA
[0166] In one aspect of this invention, caging groups (e.g.,
photo-labile caging groups) are used to precisely control the
timing and/or location of RNA interference. For example, this
invention features photoactivatable (photo activated, PA)
interfering RNA sensors suitable for monitoring transcript
expression in cells; the sensors are designed for simple operation
and are suitable for use in a wide array of instruments (e.g.,
fluorescent instrument platforms). The caged (e.g., PA) interfering
RNAs and methods of use thereof described herein are suitable for
applications in, e.g., clinical and basic research and drug
discovery.
[0167] The advantages of using a caged (e.g., PA) reaction format
include: (1.) controlled activation of reaction components (e.g.,
controlled initiation of RNAi), (2.) improved assay precision,
achieved e.g., (a.) by reducing number of fluidic handling steps in
HTS assays--reducing the number of steps (each additional pipetting
step can introduce more error into an assay) and/or (b.) by
facilitating simultaneous activation of large numbers of assays
within millisecond, and (3.) simplified automation and design of
miniaturized platforms by reducing the number of steps required.
Finally, the caged (e.g., PA) reaction format permits specific
activation of a reaction in specific locations--e.g., a subset of
wells or locations within a microarray, microfluidic device and/or
other miniaturized formats, or even within an organism (e.g.,
activation of specific locations separated by no more than about a
micron is possible).
[0168] An additional advantage of using caged compounds in cells is
that caging a molecule frequently renders the molecule more
resistant to nucleases, proteases, lipases, and the like, thus
extending its half-life in the cell. Caging a molecule which
already possesses enhanced resistance to degradation (e.g., by a
nuclease or protease, e.g., by incorporation of unnatural amino
acids and/or nucleotides into the molecule) offers similar
advantages in terms of molecule half-life in the cell or lysate
(e.g., thus minimizing false-positive results from undesirable
cleavage of a FRET-based sensor or probe).
[0169] Yet another advantage of using caged compounds (e.g., caged
interfering RNAs) in cells is that caging a toxic molecule (e.g.,
an interfering RNA against an essential gene) frequently protects
the cell from the effect of the molecule. This permits compounds
that might otherwise be too disruptive to the cell to be utilized
in in-cell assays; the cell is not subject to the adverse effect of
the compound until the compound is uncaged during the assay.
[0170] Compositions
[0171] One general class of embodiments provides a composition
comprising a caged interfering RNA. The caged RNA includes an RNA
having at least one double-stranded region, the double-stranded
region comprising a sense strand and an antisense strand, the
antisense strand comprising a region which is substantially
complementary to a region of a target mRNA. The caged RNA also
includes one or more first caging groups associated with the RNA.
The first caging groups inhibit (e.g., prevent) the RNA from
initiating RNA interference of the target mRNA in a cell comprising
the caged RNA.
[0172] The first caging groups can inhibit the RNA from initiating
RNA interference of the target mRNA by at least about 25%, at least
about 30%, at least about 35%, at least about 50%, at least about
75%, at least about 90%, at least about 95%, or at least about 98%,
as compared to the RNA in the absence of the first caging groups.
In one class of embodiments, the first caging groups prevent the
RNA from initiating RNA interference of the target mRNA (i.e.,
introduction of the caged RNA into a cell has no effect on
expression of the target mRNA). Removal of or an induced
conformational change in the first caging groups typically permits
the RNA to initiate RNA interference of the target mRNA.
[0173] Note that the first caging groups "inhibiting the RNA from
initiating RNA interference" is not meant to imply that the first
caging groups inhibit any particular step(s) in the RNAi pathway.
For example, the first caging groups can interfere with
phosphorylation of a 5' hydroxyl of the antisense strand, RISC
binding to the interfering RNA, strand separation of the sense and
antisense strands of the interfering RNA, and/or any other step in
the RNAi pathway leading to cleavage and degradation of the target
mRNA. Initiation of RNAi by the RNA can be indicated, for example,
by a decrease in the expression levels of the target mRNA and/or
appearance of specific endonucleolytic cleavage fragments of the
target mRNA, as is known in the art.
[0174] It is also worth noting that initiation of RNAi of the
target mRNA by the RNA need not, but typically does, result in a
substantial attenuation of expression of the target mRNA. For
example, expression of the target mRNA can be unaffected, or
expression of the target mRNA can be decreased by at least about
0.001%, at least about 0.01%, at least about 0.1%, at least about
1%, at least about 5%, at least about 10%, or preferably at least
about 25%, at least about 50%, or at least about 75% or more, or
can even be reduced to an undetectable level.
[0175] The RNA can have any of a variety of structures, lengths,
and/or the like. Thus, in one class of embodiments, the RNA
comprises a first polyribonucleotide comprising the sense strand
and a second polyribonucleotide comprising the antisense strand.
The RNA can be, e.g., a long double-stranded RNA that is cleaved by
Dicer in the cell, or it can be, e.g., an siRNA. For example, the
first polyribonucleotide can comprise between 19 and 25
nucleotides, the second polyribonucleotide can comprise between 19
and 25 nucleotides, and the double-stranded region can comprise
between 19 and 25 base pairs. The first and second
polyribonucleotides can form a duplex over their entire length, or
they can have overhangs (e.g., 5' or 3' overhangs; e.g., 21 nt
first and second polyribonucleotides can form a 19 bp
double-stranded region with 2 nucleotide overhangs, 23 nt
polyribonucleotides can form a 21 bp double-stranded region with 2
nucleotide overhangs, and so on). For example, in some embodiments,
the first polyribonucleotide and the second polyribonucleotide each
comprise a two nucleotide TT 3' overhang (where T is
2'-deoxythymidine). As another example, the first and second
polyribonucleotides can each comprise between 25 and 30 nucleotides
(e.g., 27 nucleotides) and form a duplex over their entire length.
The RNA is optionally nuclease resistant and optionally comprises
one or more deoxyribonucleotides, one or more PNA monomers, and/or
one or more modified nucleotides (e.g., 2'-methyl or 2'-O-allyl
ribonucleotides) or internucleotide linkages (e.g.,
phosphorothioate linkages). In one embodiment, at least one of the
one or more first caging groups is covalently attached to a 5'
hydroxyl or a 5' phosphate of the second polyribonucleotide. Since
this 5' hydroxyl or phosphate is useful for an siRNA to initiate
RNAi, caging the 5' hydroxyl or phosphate of the antisense strand
permits the RNA to be uncaged and activated in a controlled
manner.
[0176] As noted, in certain embodiments, the RNA comprises a first
polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand. In other
embodiments, the RNA comprises a self-complementary
polyribonucleotide (e.g., a hairpin, a shRNA). In either case, the
double-stranded region optionally comprises fewer than about 25
base pairs, fewer than about 30 base pairs, fewer than about 50
base pairs, fewer than about 80 base pairs, fewer than about 150
base pairs, fewer than about 250 base pairs, fewer than about 500
base pairs, fewer than about 1000 base pairs, or fewer than about
1500 base pairs. Although a double-stranded region comprising about
19-25 base pairs is typically sufficient to initiate RNAi, longer
regions may be convenient or desirable in certain applications
(e.g., double-stranded RNAs longer than 25 bp can stimulate the
mammalian immune system, which can be advantageous in certain
applications).
[0177] The one or more first caging groups associated with the RNA
can be covalently attached to or non-covalently associated with the
RNA (e.g., at one or more bases, riboses, phosphate groups and/or
terminal hydroxyls, within and/or at the end of either or both
strands of the RNA). In one embodiment, the RNA comprises a first
polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand, and the first
caging group is covalently attached to the first polyribonucleotide
and to the second polyribonucleotide. For example, the first caging
group can be attached to the 5' end of the first polyribonucleotide
and to the 3' end of the second polyribonucleotide, or, preferably,
it can be attached to the 3' end of the first polyribonucleotide
and to the 5' end of the second polyribonucleotide (FIG. 4). The
caging group linking the two polyribonucleotides can, for example,
be photolabile.
[0178] In a preferred aspect, the one or more first caging groups
are photoactivatable (e.g., photolabile). For example, the caging
groups can be removed by exposure to light with a wavelength
between about 60 nm and about 400 nm, between about 400 nm and
about 700 nm, and/or between about 700 nm and about 1000 nm. Other
caging groups are removable via input of different uncaging
energies; e.g., the one or more caging groups can be removable by
sonication or application of heat, or can be removed by a chemical
or enzyme.
[0179] In one class of embodiments, the one or more first caging
groups each comprises a first binding moiety. The composition also
includes a second binding moiety that can bind at least one first
binding moiety. For example, the first binding moiety on the caging
groups can comprise biotin (see, e.g., FIG. 28), and the second
binding moiety can comprise avidin or streptavidin. Streptavidin,
for example, can thus be bound to the first caging group,
increasing its bulkiness and its effectiveness at inhibiting the
caged RNA from participating in RNAi. In some embodiments, the
caged RNA comprises two or more first caging groups each comprising
the first binding moiety, and the second binding moiety can bind
two or more first binding moieties simultaneously. For example, the
caged RNA can comprise at least two biotinylated caging groups
(e.g., one at the 5' end of the sense strand and one at the 5' end
of the antisense strand); binding of streptavidin to multiple
biotin moieties on multiple caged RNA molecules links the caged
RNAs into a large network. Cleavage of the photolabile group
attaching the biotin to the RNA results in dissociation of the
network. The uncaged RNA can then participate in RNAi.
[0180] The RNA optionally also includes at least one label, wherein
initiation of RNA interference of the target mRNA by the labeled
RNA in the cell results in an initiation-dependent change in a
signal output of the label. In a preferred class of embodiments,
the label is a fluorescent label, and the initiation-dependent
change in the signal output of the label is a change in fluorescent
emission.
[0181] In one class of embodiments, the labeled RNA also includes
at least one quencher. The label and the quencher are positioned in
the RNA such that fluorescent emission by the label is quenched by
the quencher. Initiation of RNA interference by the labeled RNA
results in unquenching of the label (and thus an increase in the
fluorescent emission by the label). In this class of embodiments,
the initiation-dependent change in the signal output is thus an
increase in the fluorescent emission by the label. For example, the
label and quencher can be positioned on opposite strands, in close
enough proximity to each other that the label is quenched until the
sense and antisense strands are separated.
[0182] In one class of embodiments in which the RNA comprises a
fluorescent label and a quencher, the RNA comprises a first
polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand. The first
polyribonucleotide comprises between 19 and 25 nucleotides, the
second polyribonucleotide comprises between 19 and 25 nucleotides,
and the double-stranded region comprises between 19 and 25 base
pairs. The label and quencher can be attached at essentially any
suitable positions, e.g., at the 3' end, at the 5' end, and/or
within either or both the first and second polyribonucleotides,
e.g., as described for the embodiments above. As noted previously,
techniques for determining and verifying suitable positions for the
label and quencher are well known in the art.
[0183] In a related class of embodiments, the labeled RNA comprises
two fluorescent labels, one of which is a donor and the other of
which is an acceptor. The donor and acceptor are positioned within
the RNA such that energy transfer (e.g., FRET) occurs between them
(e.g., excitation of the donor results in fluorescence by the
acceptor). Initiation of RNA interference by the labeled RNA
results in loss of energy transfer between the donor and the
acceptor. This can be observed as an increase in fluorescence by
the donor or as a decrease in fluorescence by the acceptor. Thus,
in a preferred class of embodiments, the initiation-dependent
change in the signal output is a decrease in fluorescent emission
by the acceptor following excitation of the donor. For example, the
donor and acceptor can be positioned on opposite strands, in close
enough proximity to each other that energy transfer occurs until
the sense and antisense strands are separated.
[0184] In one class of embodiments in which the RNA comprises a
donor and an acceptor, the RNA comprises a first polyribonucleotide
comprising the sense strand and a second polyribonucleotide
comprising the antisense strand. The first polyribonucleotide
comprises between 19 and 25 nucleotides, the second
polyribonucleotide comprises between 19 and 25 nucleotides, and the
double-stranded region comprises between 19 and 25 base pairs. The
donor and acceptor can be attached at essentially any suitable
positions, e.g., at the 3' end, at the 5' end, and/or within either
or both the first and second polyribonucleotides, e.g., as
described for the embodiments above. Techniques for determining and
verifying suitable positions for the donor and acceptor are well
known in the art.
[0185] In another class of embodiments, the sense strand comprises
a first label and the antisense strand a second label. The two
labels are different, non-interacting fluorophores with distinct
emission spectra (e.g., red and green, such that the
double-stranded RNA is yellow while the single strands are red and
green).
[0186] The composition optionally also includes the target mRNA
and/or a cell, e.g., a cell comprising the caged RNA and/or the
target mRNA. Various techniques (e.g., lipofection, microinjection,
or electroporation) can be used to introduce the caged RNA into the
cell. In one class of embodiments, the caged RNA also includes a
cellular delivery module, associated with the RNA, that can mediate
introduction of the caged RNA into the cell. All of the various
optional configurations and features noted for the embodiments
above apply here as well, to the extent they are relevant, e.g.,
for types of cellular delivery modules (e.g., polypeptides,
amphipathic peptides, protein transduction domains, and lipids),
use of one or more second caging groups, and the like.
[0187] Optionally, in the embodiments herein, the caged RNA is
bound to a matrix (e.g., electrostatically, covalently, directly or
via a linker). In one aspect, the matrix is a surface and the RNA
is bound to the surface at a predetermined location within an array
comprising other RNAs. In other embodiments, the matrix comprises a
bead (e.g., color-coded or otherwise addressable).
[0188] Kits for making the caged RNA (e.g., comprising an RNA, one
or more first caging groups, and instructions for assembling the
RNA and the first caging groups to form the caged RNA, packaged in
one or more containers, and/or one or more first caging groups and
instructions for assembling the first caging groups and an RNA
supplied by a user of the kit to form the caged RNA, packaged in
one or more containers) are also a feature of the invention.
Similarly, the invention provides kits for making caged and labeled
RNA, e.g., a kit comprising one or more first caging groups, at
least one label, and instructions for assembling the first caging
groups, at least one label, and an RNA supplied by a user of the
kit to form the caged RNA, packaged in one or more containers.
[0189] Kits comprising the caged RNA are another feature of the
invention. For example, one class of embodiments provides a kit
comprising the caged RNA and one or more of: instructions for using
the caged RNA (e.g., to attenuate and/or to detect expression of
the target mRNA in a cell), a delivery reagent that can mediate
introduction of the caged RNA into a cell, or a buffer, packaged in
one or more containers.
[0190] Caging the interfering RNA allows, e.g., precise control
over the timing of gene silencing by controlling initiation of RNA
interference (also called RNAi or RNA-mediated interference). Use
of RNAi for inhibiting gene expression in a number of cell types
(including, e.g., mammalian cells) and organisms is well described
in the literature, as are methods for determining appropriate
interfering RNA(s) to target a desired gene and for generating such
interfering RNAs. For example, RNA interference is described e.g.,
in U.S. patent application publications 20020173478, 20020162126,
and 20020182223 and in Hannon G. J. "RNA interference" Nature. Jul.
11, 2002;418 (6894):244-51; Ueda R. "RNAi: a new technology in the
post-genomic sequencing era" J Neurogenet. 2001;15(3-4):193-204;
Ullu et al "RNA interference: advances and questions" Philos Trans
R Soc Lond B Biol Sci. Jan. 29, 2002;357(1417):65-70; and Schmid et
al "Combinatorial RNAi: a method for evaluating the functions of
gene families in Drosophila" Trends Neurosci. Feb. 25, 2002
(2):71-4. A kit for producing interfering RNAs is commercially
available, e.g., from Ambion, Inc. (www.ambion.com, the
Silencer.TM. siRNA construction kit); kits for randomly labeling
such RNAs are available from the same source.
[0191] As noted, single-stranded siRNAs can also initiate RNAi.
Thus, another, related general class of embodiments provides a
caged interfering RNA. The caged RNA includes an RNA comprising a
single polyribonucleotide strand comprising an antisense strand,
the antisense strand comprising a region which is substantially
complementary to a region of a target mRNA. The caged RNA also
includes one or more first caging groups associated with the RNA.
The first caging groups inhibit (e.g., prevent) the RNA from
initiating RNA interference of the target mRNA in a cell comprising
the caged RNA. The RNA is typically not self-complementary.
[0192] The single-stranded RNA can have any of a variety of
lengths. For example, the polyribonucleotide strand can comprise
between 10 and 100 nucleotides, between 10 and 80 nucleotides,
between 10 and 50 nucleotides, preferably between 10 and 30
nucleotides, or more preferably between 15 and 30 nucleotides or
between 19 and 25 nucleotides.
[0193] The RNA optionally comprises at least one label. In one
class of embodiments, initiation of RNA interference of the target
mRNA by the labeled RNA in the cell results in an
initiation-dependent change in a signal output of the label. For
example, the single-stranded RNA can have a fluorescent label at or
near one end of the polyribonucleotide and a quencher at or near
the other end, or it can have a donor at or near one end of the
polyribonucleotide and an acceptor at or near the other end.
Alternatively, signal output of the label can be unaffected by
participation of the RNA in the RNAi pathway.
[0194] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for label configurations (e.g., use of
fluorescent labels, fluorescent label/quencher, and donor/acceptor
combinations), signal output types, use of caging groups (e.g.,
photolabile caging groups), appropriate uncaging energies (light,
heat, sonic, etc.), use of cellular delivery modules (e.g.,
amphipathic peptides, cationic peptides, protein transduction
domains, and lipids), and the like.
[0195] Methods
[0196] In one class of methods of the invention, methods of
selectively attenuating expression of a target gene in a cell are
provided. In the methods, a caged RNA is introduced into the cell.
The caged RNA can include an RNA comprising at least one
double-stranded region, the double-stranded region comprising a
sense strand and an antisense strand, the antisense strand
comprising a region which is substantially complementary to a
region of a target mRNA corresponding to the target gene.
Alternatively, the caged RNA can include an RNA comprising a single
polyribonucleotide strand comprising an antisense strand, the
antisense strand comprising a region which is substantially
complementary to a region of a target mRNA corresponding to the
target gene. The caged RNA comprises one or more caging groups
associated with the RNA, the caging groups inhibiting (e.g.,
preventing) the RNA from initiating RNA interference of the target
mRNA in the cell. RNA interference is initiated by exposing the
cell to uncaging energy (e.g., light of a predetermined
wavelength), freeing the RNA from inhibition by the caging
groups.
[0197] Exposing the cell to uncaging energy optionally includes
exposing the cell to light of a first wavelength. This exposure can
be addressable; e.g., the caged RNA can be exposed to light of the
first wavelength by exposing one or more preselected areas (e.g.,
wells of a microtiter plate or portions thereof, or the like) to
the light. As another example, the uncaging energy can be directed
at a preselected subset of a cell population comprising the
cell.
[0198] Exposing the cell to light of the first wavelength
optionally comprises exposing the cell to light such that the
intensity of the light and the duration of exposure to the light
are controlled such that a first portion (which can be a selected
amount) of the caged labeled RNA is uncaged and a second portion of
the caged labeled RNA remains caged. Put another way, the uncaging
rate can be controlled. Furthermore, the uncaging step can be
repeated until the caged RNA is depleted.
[0199] As noted, caging the RNA permits temporal control over
initiation of RNA interference. For example, the method can include
contacting the cell and a test compound and exposing the cell to
the uncaging energy at a preselected time point with respect to a
time at which the cell and the test compound are contacted.
[0200] All of the above optional method variations apply to this
method as well. Further, the various composition components noted
(particularly the caged RNA embodiments) above can be adapted for
use in this method, as appropriate. For example, in one class of
embodiments, the caged RNA further comprises a cellular delivery
module that can mediate introduction of the caged RNA into the
cell, the cellular delivery module being associated with the RNA.
In this class of embodiments, the caged RNA is introduced into the
cell by contacting the cell with the caged RNA associated with the
cellular delivery module. As another example, the cellular delivery
module can be covalently attached to the RNA via a photolabile
linker, which can be cleaved by exposure to light of an appropriate
wavelength once the RNA is inside the cell.
[0201] As another example, in one class of embodiments, the RNA
comprises at least one label (e.g., one with an
initiation-dependent signal output), and the methods include
detecting a signal from the label.
[0202] The methods optionally include introducing a plurality of
caged RNAs into the cell. The plurality of caged RNAs can then be
uncaged simultaneously or at different times. For example, a first
caged RNA can be uncaged, e.g., by exposure to light of a first
wavelength, and permitted to initiate RNAi of a first target mRNA.
A second caged RNA can be uncaged, e.g., by exposure to light of a
second, different wavelength, at a later time.
[0203] In another aspect, systems and/or apparatus comprising the
compositions (e.g., the caged RNAs) noted above and, e.g.,
components such as detectors, fluid handling apparatus, sources of
uncaging energy, or the like, are a feature of the invention.
[0204] Other Caged RNAs
[0205] The presence of RNA, particularly double-stranded RNA, in a
cell can result in inhibition of expression of a gene comprising a
sequence identical or nearly identical to that of the RNA through
mechanisms other than RNAi. For example, double-stranded RNAs that
are partially complementary to a target mRNA can repress
translation of the mRNA without affecting its stability. As another
example, double-stranded RNAs can induce histone methylation and
heterochromatin formation, leading to transcriptional silencing of
a gene comprising a sequence identical or nearly identical to that
of the RNA (see, e.g., Schramke and Allshire (2003) "Hairpin RNAs
and retrotransposon LTRs effect RNAi and chromatin-based gene
silencing" Science 301:1069-1074; Kawasaki and Taira (2004)
"Induction of DNA methylation and gene silencing by short
interfering RNAs in human cells" Nature 431:211-217; and Morris et
al. (2004) "Small interfering RNA-induced transcriptional gene
silencing in human cells" Science 305:1289-1292).
[0206] Short RNAs called microRNAs (miRNAs) have been identified in
a variety of species. Typically, these endogenous RNAs are each
transcribed as a long RNA and then processed to a pre-miRNA of
approximately 60-75 nucleotides that forms an imperfect hairpin
(stem-loop) structure. The pre-miRNA is typically then cleaved,
e.g., by Dicer, to form the mature miRNA. Mature miRNAs are
typically approximately 21-25 nucleotides in length, but can vary,
e.g., from about 14 to about 25 or more nucleotides. Some, though
not all, miRNAs have been shown to inhibit translation of mRNAs
bearing partially complementary sequences. Such miRNAs contain one
or more internal mismatches to the corresponding mRNA that are
predicted to result in a bulge in the center of the duplex formed
by the binding of the miRNA antisense strand to the mRNA (e.g.,
FIG. 32). The miRNA typically forms approximately 14-17
Watson-Crick base pairs with the mRNA; additional wobble base pairs
can also be formed. In addition, short synthetic double-stranded
RNAs (e.g., similar to siRNAs) containing central mismatches to the
corresponding mRNA have been shown to repress translation (but not
initiate degradation) of the mRNA. See, for example, Zeng et al.
(2003) "MicroRNAs and small interfering RNAs can inhibit mRNA
expression by similar mechanisms" Proc. Natl. Acad. Sci. USA
100:9779-9784; Doench et al. (2003) "siRNAs can function as miRNAs"
Genes & Dev. 17:438-442; Bartel and Bartel (2003) "MicroRNAs:
At the root of plant development?" Plant Physiology 132:709-717;
Schwarz and Zamore (2002) "Why do miRNAs live in the miRNP?" Genes
& Dev. 16:1025-1031; Tang et al. (2003) "A biochemical
framework for RNA silencing in plants" Genes & Dev. 17:49-63;
Meister et al. (2004) "Sequence-specific inhibition of microRNA-
and siRNA-induced RNA silencing" RNA 10:544-550; Nelson et al.
(2003) "The microRNA world: Small is mighty" Trends Biochem. Sci.
28:534-540; Scacheri et al. (2004) "Short interfering RNAs can
induce unexpected and divergent changes in the levels of untargeted
proteins in mammalian cells" Proc. Natl. Acad. Sci. USA
101:1892-1897; Sempere et al. (2004) "Expression profiling of
mammalian microRNAs uncovers a subset of brain-expressed microRNAs
with possible roles in murine and human neuronal differentiation"
Genome Biology 5:R13; Dykxhoorn et al. (2003) "Killing the
messenger: Short RNAs that silence gene expression" Nature Reviews
Molec. and Cell Biol. 4:457-467; McManus (2003) "MicroRNAs and
cancer" Semin Cancer Biol. 13:253-288; and Stark et al. (2003)
"Identification of Drosophila microRNA targets" PLoS Biol.
1:E60.
[0207] The cellular machinery involved in translational repression
of mRNAs by partially complementary RNAs (e.g., certain miRNAs)
appears to partially overlap that involved in RNAi, although, as
noted, translation of the mRNAs, not their stability, is affected
and the mRNAs are typically not degraded.
[0208] The location and/or size of the bulge(s) formed when the
antisense strand of the RNA binds the mRNA can affect the ability
of the RNA to repress translation of the mRNA. Similarly, location
and/or size of any bulges within the RNA itself can also affect
efficiency of translational repression. See, e.g., the references
above. Typically, translational repression is most effective when
the antisense strand of the RNA is complementary to the 3'
untranslated region (3' UTR) of the mRNA. Multiple repeats, e.g.,
tandem repeats, of the sequence complementary to the antisense
strand of the RNA can also provide more effective translational
repression; for example, some mRNAs that are translationally
repressed by endogenous miRNAs contain 7-8 repeats of the miRNA
binding sequence at their 3' UTRs. It is worth noting that
translational repression appears to be more dependent on
concentration of the RNA than RNA interference does; translational
repression is thought to involve binding of a single mRNA by each
repressing RNA, while RNAi is thought to involve cleavage of
multiple copies of the mRNA by a single siRNA-RISC complex.
[0209] Guidance for design of a suitable RNA to repress translation
of a given target mRNA can be found in the literature (e.g., the
references above and Doench and Sharp (2004) "Specificity of
microRNA target selection in translational repression" Genes &
Dev. 18:504-511; Rehmsmeier et al. (2004) "Fast and effective
prediction of microRNA/target duplexes" RNA 10:1507-1517; Robins et
al. (2005) "Incorporating structure to predict microRNA targets"
Proc Natl Acad Sci 102:4006-4009; and Mattick and Makunin (2005)
"Small regulatory RNAs in mammals" Hum. Mol. Genet. 14:R121-R132,
among many others) and herein. However, due to differences in
efficiency of translational repression between RNAs of different
structure (e.g., bulge size, sequence, and/or location) and RNAs
corresponding to different regions of the target mRNA, several RNAs
are optionally designed and tested against the target mRNA to
determine which is most effective at repressing translation of the
target mRNA (preferably, without inducing endonucleolytic cleavage
and degradation of the target mRNA).
[0210] The present invention provides a number of novel methods,
compositions, systems, and kits related to translational repression
and chromatin silencing by RNAs. In one aspect, caging groups
(e.g., photolabile caging groups) are used to precisely control the
timing and/or location of translational repression or chromatin
silencing.
[0211] Compositions, Systems, and Kits
[0212] One general class of embodiments provides a caged RNA. The
caged RNA includes an RNA capable of repressing translation of a
target mRNA. The caged RNA also includes one or more first caging
groups associated with the RNA (e.g., two or more, three or more,
four or more, or the like, first caging groups). The first caging
groups inhibit (e.g., prevent) the RNA from repressing translation
of the target mRNA in a cell comprising the caged RNA.
[0213] The first caging groups can inhibit the RNA from repressing
translation of the target mRNA by at least about 25%, at least
about 30%, at least about 35%, at least about 50%, at least about
75%, at least about 90%, at least about 95%, or at least about 98%,
as compared to the RNA in the absence of the first caging groups.
In one class of embodiments, the first caging groups prevent the
RNA from repressing translation of the target mRNA (i.e.,
introduction of the caged RNA into a cell has no effect on
translation of the target mRNA). Removal of or an induced
conformational change in the first caging groups typically permits
the RNA to repress translation of the target MRNA.
[0214] Note that the first caging groups "inhibiting the RNA from
repressing translation of the target mRNA" is not meant to imply
that the first caging groups inhibit any particular step(s) in the
translational repression pathway. For example, the first caging
groups can interfere with cleavage of a long RNA hairpin by Dicer,
RISC binding to the RNA, strand separation of the sense and
antisense strands of the RNA, antisense strand binding to the
target mRNA, and/or any other step in a pathway leading to
translational repression of the target mRNA. Translational
repression can be detected, for example, by a decrease in
expression level of a protein translated from the target mRNA.
Methods for detecting protein expression levels are well known in
the art, and include Western analysis, immunoprecipitation, and
specific protein activity assays, among many others.
[0215] In a preferred class of embodiments, the RNA does not
initiate degradation of the target mRNA in a cell comprising the
RNA. Thus, for example, the RNA preferably does not initiate
endonucleolytic cleavage and/or RNAi of the target mRNA.
Alternatively, the RNA can initiate degradation of the target mRNA
as well as repress its translation; e.g., expression of the target
mRNA can be decreased by at least about 0.1%, 1%, 5%, 10%, or even
more, while translation of any remaining mRNA is repressed.
[0216] The RNA can have any of a variety of structures, lengths,
and/or the like. For example, the RNA can be single-stranded, or,
preferably, double-stranded. The RNA typically comprises at least
an antisense strand (e.g., only the antisense strand if the RNA is
single-stranded, or the antisense strand and a complementary or
partially complementary sense strand if the RNA is
double-stranded). As noted, efficient translational repression
generally requires at least one mismatch between the antisense
strand and the target mRNA. Thus, in one class of embodiments, the
antisense strand of the RNA comprises a first region which is
complementary to a second region of the target mRNA. The first
region is interrupted by one or more nucleotides which are not
complementary to the second region; for example, one or more
nucleotides which form a bulge when the antisense strand binds the
target mRNA. The first region is optionally interrupted by two,
three, four, or more (typically at most ten) nucleotides which are
not complementary to the second region. These nucleotides are
typically, but not necessarily, consecutive (e.g., a duplex formed
between the antisense strand and the target mRNA can have one, two,
or more bulges, loops, or the like). As another example, the first
region can be shorter than the second region, lacking one or more
nucleotides corresponding to, e.g., the middle of the second
region, such that a bulge forms in the mRNA strand of an antisense
strand-target mRNA duplex. Similarly, the second region can be
shorter than the first region.
[0217] The second region, the region of the mRNA to which the
antisense strand binds, can be located essentially anywhere within
the mRNA, e.g., the 5' UTR, an exon, an exon, an exon-intron
boundary, or the like. In one class of embodiments, the second
region is within the 3' UTR of the target mRNA. The target
optionally includes a plurality of repeats of the second region,
e.g., tandem repeats, and/or a region complementary to a different
RNA capable of repressing translation of the target.
[0218] The RNA optionally comprises at least one double-stranded
region that includes the antisense strand and a sense strand. The
sense strand can be completely complementary to the antisense
strand over the double-stranded region. Alternatively, in some
embodiments, the sense strand is not completely complementary to
the antisense strand over the double-stranded region. For example,
the double-stranded region can include one or more mismatches,
bulges, loops, and/or the like. The mismatched nucleotides can be
the same or different nucleotides than those mismatched to the
target mRNA.
[0219] In one class of embodiments, the RNA comprises a first
polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand. For example,
the first polyribonucleotide can comprise between 14 and 29
nucleotides (e.g., between 17 and 29 or between 18 and 25
nucleotides), the second polyribonucleotide can comprise between 14
and 29 nucleotides (e.g., between 17 and 29 or between 18 and 25
nucleotides), and the double-stranded region can comprise between
14 and 29 base pairs (e.g., between 17 and 29 or between 18 and 25
base pairs). The first and second polyribonucleotides can form a
duplex over their entire length, or they can have overhangs. For
example, in some embodiments, the first polyribonucleotide and the
second polyribonucleotide each comprise a two nucleotide TT 3'
overhang (where T is 2'-deoxythymidine). In one embodiment, at
least one of the one or more first caging groups is covalently
attached to a 5' hydroxyl or a 5' phosphate of the second
polyribonucleotide.
[0220] As noted, in certain embodiments, the RNA comprises a first
polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand. In other
embodiments, the RNA comprises a self-complementary
polyribonucleotide (e.g., a hairpin, a perfect or imperfect
hairpin). The stem and/or loop of the hairpin can be any of a
variety of different lengths. For example, the hairpin can
correspond to a pre-miRNA that is processed in a cell to produce an
RNA that represses translation of the target mRNA.
[0221] The RNA is optionally nuclease resistant and optionally
comprises one or more deoxyribonucleotides, one or more PNA
monomers, and/or one or more modified nucleotides (e.g., 2'-methyl
or 2'-O-allyl ribonucleotides) or internucleotide linkages (e.g.,
phosphorothioate linkages).
[0222] The one or more first caging groups associated with the RNA
can be covalently attached to or non-covalently associated with the
RNA (e.g., at one or more bases, riboses, phosphate groups and/or
terminal hydroxyls, within and/or at the end of either or both
strands of the RNA). In one embodiment, the RNA comprises a first
polyribonucleotide comprising the sense strand and a second
polyribonucleotide comprising the antisense strand, and the first
caging group is covalently attached to the first polyribonucleotide
and to the second polyribonucleotide. For example, the first caging
group can be attached to the 5' end of the first polyribonucleotide
and to the 3' end of the second polyribonucleotide, or, preferably,
it can be attached to the 3' end of the first polyribonucleotide
and to the 5' end of the second polyribonucleotide. The caging
group linking the two polyribonucleotides can, for example, be
photolabile.
[0223] In a preferred aspect, the one or more first caging groups
are photoactivatable (e.g., photolabile). For example, the caging
groups can be removed by exposure to light with a wavelength
between about 60 nm and about 400 nm, between about 400 nm and
about 700 nm, and/or between about 700 nm and about 1000 nm. Other
caging groups are removable via input of different uncaging
energies; e.g., the one or more caging groups can be removable by
sonication or application of heat, or can be removed by a chemical
or enzyme.
[0224] In one class of embodiments, the one or more first caging
groups each comprises a first binding moiety. The composition also
includes a second binding moiety that can bind at least one first
binding moiety. For example, the first binding moiety on the caging
groups can comprise biotin, and the second binding moiety can
comprise avidin or streptavidin, as described in the RNAi
embodiments above. In some embodiments, the caged RNA comprises two
or more first caging groups each comprising the first binding
moiety, and the second binding moiety can bind two or more first
binding moieties simultaneously. For example, the caged RNA can
comprise at least two biotinylated caging groups (e.g., one at the
5' end of the sense strand and one at the 5' end of the antisense
strand); binding of streptavidin to multiple biotin moieties on
multiple caged RNA molecules links the caged RNAs into a large
network. Cleavage of the photolabile group attaching the biotin to
the RNA results in dissociation of the network. The uncaged RNA can
then participate in translational repression.
[0225] In some embodiments, the RNA also includes at least one
label, e.g., a fluorescent label. Optionally, binding and/or
repression of translation of the target mRNA by the RNA results in
a binding and/or repression-dependent change in a signal output of
the label. The labeled RNA optionally also includes at least one
quencher. For example, the label and quencher can be positioned on
opposite strands of the RNA, in close enough proximity to each
other that the label is quenched until the sense and antisense
strands are separated. In a related class of embodiments, the
labeled RNA comprises two fluorescent labels, one of which is a
donor and the other of which is an acceptor. The donor and acceptor
are positioned within the RNA such that energy transfer (e.g.,
FRET) occurs between them (e.g., excitation of the donor results in
fluorescence by the acceptor). For example, the donor and acceptor
can be positioned on opposite strands, in close enough proximity to
each other that energy transfer occurs until the sense and
antisense strands are separated. In embodiments in which the RNA
includes a sense strand and the antisense strand, the sense strand
can comprise a first label and the antisense strand a second label.
The two labels can be different, non-interacting fluorophores with
distinct emission spectra (e.g., red and green, such that the
double-stranded RNA is yellow while the single strands are red and
green). As noted previously, techniques for determining and
verifying suitable positions for the label(s) or label and quencher
are well known in the art.
[0226] The composition optionally also includes the target mRNA
and/or a cell, e.g., a cell comprising the caged RNA and/or the
target mRNA. Various techniques (e.g., lipofection, microinjection,
or electroporation) can be used to introduce the caged RNA into the
cell. In one class of embodiments, the caged RNA also includes a
cellular delivery module, associated with the RNA, that can mediate
introduction of the caged RNA into the cell. All of the various
optional configurations and features noted for the embodiments
above apply here as well, to the extent they are relevant, e.g.,
for types of cellular delivery modules (e.g., polypeptides,
amphipathic peptides, protein transduction domains, and lipids),
use of the first caging group as a cellular delivery module, use of
one or more second caging groups, and the like.
[0227] Optionally, in the embodiments herein, the caged RNA is
bound to a matrix (e.g., electrostatically, covalently, directly or
via a linker). In one aspect, the matrix is a surface and the RNA
is bound to the surface at a predetermined location within an array
comprising other RNAs. In other embodiments, the matrix comprises a
bead (e.g., color-coded or otherwise addressable).
[0228] Kits for making the caged RNA are also a feature of the
invention. Thus, one class of embodiments provides a kit including
an RNA, one or more first caging groups, and instructions for
assembling the RNA and the first caging groups to form the caged
RNA, packaged in one or more containers. Another class of
embodiments provides a kit comprising one or more first caging
groups and instructions for assembling the first caging groups and
an RNA supplied by a user of the kit to form the caged RNA,
packaged in one or more containers.
[0229] Kits comprising the caged RNA are another feature of the
invention. For example, one class of embodiments provides a kit
comprising the caged RNA and one or more of: instructions for using
the caged RNA (e.g., to attenuate expression of the target mRNA in
a cell), a delivery reagent that can mediate introduction of the
caged RNA into a cell, or a buffer, packaged in one or more
containers.
[0230] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for label configurations (e.g., use of
fluorescent labels, fluorescent label/quencher, and donor/acceptor
combinations), signal output types, appropriate uncaging energies
(light, heat, sonic, etc.), and the like.
[0231] In another aspect, systems and/or apparatus comprising the
compositions (e.g., the caged RNAs) noted above and, e.g.,
components such as detectors, fluid handling apparatus, sources of
uncaging energy, or the like, are a feature of the invention.
[0232] Another aspect of the invention deals with RNAs capable of
inducing histone methylation and chromatin silencing. Such RNAs can
be designed and tested by techniques known in the art, e.g., for
assaying heterochromatin formation, mRNA expression levels, and the
like.
[0233] Thus, one general class of embodiments provides a caged RNA
that includes an RNA capable of silencing transcription of a target
gene and one or more first caging groups associated with the RNA.
The first caging groups inhibit (e.g., prevent) the RNA from
silencing transcription of the target gene in a cell comprising the
caged RNA. Transcriptional silencing can reduce the amount of
target mRNA present in a cell; for example, expression of the
target mRNA can be decreased by at least about 5%, at least about
10%, at least about 25%, at least about 50%, or at least about 75%
or more, or can even be reduced to an undetectable level.
[0234] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for percent inhibition by the caging
groups, structure of the RNA, label configurations (e.g., use of
fluorescent labels, fluorescent label/quencher, and donor/acceptor
combinations), signal output types, use of caging groups (e.g.,
photolabile caging groups), appropriate uncaging energies (light,
heat, sonic, etc.), use of cellular delivery modules (e.g.,
amphipathic peptides, cationic peptides, protein transduction
domains, and lipids), and the like.
[0235] In another aspect, systems and/or apparatus comprising the
compositions (e.g., the caged RNAs) noted above and, e.g.,
components such as detectors, fluid handling apparatus, sources of
uncaging energy, or the like, are a feature of the invention, as
are kits for making or using the caged RNAs.
[0236] Methods
[0237] In one class of methods of the invention, methods of
selectively attenuating expression of a target gene in a cell are
provided. In the methods, a caged RNA is introduced into the cell.
The caged RNA includes an RNA capable of repressing translation of
a target mRNA transcribed from the target gene. The caged RNA also
comprises one or more caging groups associated with the RNA, the
caging groups inhibiting (e.g., preventing) the RNA from repressing
translation of the target mRNA in the cell. Repression of
translation is initiated by exposing the cell to uncaging energy
(e.g., light of a predetermined wavelength), freeing the RNA from
inhibition by the caging groups. In a preferred class of
embodiments, the amount of the target mRNA present in the cell is
not affected by the presence of the RNA in the cell; i.e., uncaging
the RNA does not initiate RNAi.
[0238] Exposing the cell to uncaging energy optionally includes
exposing the cell to light of a first wavelength. This exposure can
be addressable; e.g., the caged RNA can be exposed to light of the
first wavelength by exposing one or more preselected areas (e.g.,
wells of a microtiter plate or portions thereof, or the like) to
the light. As another example, the uncaging energy can be directed
at a preselected subset of a cell population comprising the
cell.
[0239] Exposing the cell to light of the first wavelength
optionally comprises exposing the cell to light such that the
intensity of the light and the duration of exposure to the light
are controlled such that a first portion (which can be a selected
amount) of the caged RNA is uncaged and a second portion of the
caged RNA remains caged. Put another way, the uncaging rate can be
controlled. Furthermore, the uncaging step can be repeated until
the caged RNA is depleted.
[0240] As noted, caging the RNA permits temporal control over
initiation of translational repression. For example, the method can
include contacting the cell and a test compound and exposing the
cell to the uncaging energy at a preselected time point with
respect to a time at which the cell and the test compound are
contacted.
[0241] All of the above optional method variations apply to this
method as well. Further, the various composition components noted
(particularly the caged RNA embodiments) above can be adapted for
use in this method, as appropriate. For example, in one class of
embodiments, the caged RNA further comprises a cellular delivery
module that can mediate introduction of the caged RNA into the
cell, the cellular delivery module being associated with the RNA.
In this class of embodiments, the caged RNA is introduced into the
cell by contacting the cell with the caged RNA associated with the
cellular delivery module. As another example, the cellular delivery
module can be covalently attached to the RNA via a photolabile
linker, which can be cleaved by exposure to light of an appropriate
wavelength once the RNA is inside the cell.
[0242] As another example, in one class of embodiments, the RNA
comprises at least one label (e.g., one with a binding and/or
repression-dependent signal output), and the methods include
detecting a signal from the label.
[0243] The methods optionally include introducing a plurality of
caged RNAs into the cell. The plurality of caged RNAs can then be
uncaged simultaneously or at different times. For example, a first
caged RNA can be uncaged, e.g., by exposure to light of a first
wavelength, and permitted to repress translation of a first target
mRNA. A second caged RNA can be uncaged, e.g., by exposure to light
of a second, different wavelength, at a later time.
[0244] Another class of methods of the invention also provides
methods of selectively attenuating expression of a target gene in a
cell. In the methods, a caged RNA is introduced into the cell. The
caged RNA comprises an RNA capable of silencing transcription of
the target gene. The caged RNA also includes one or more first
caging groups associated with the RNA that inhibit (e.g., prevent)
the RNA from silencing transcription of the target gene in the
cell. Silencing of transcription of the target gene is initiated by
exposing the cell to uncaging energy, freeing the RNA from
inhibition by the caging groups.
[0245] All of the above optional method variations apply to this
method as well, e.g., for types of uncaging energy, temporal and
spatial control of uncaging, introduction of the RNA into the cell
through use of a cellular delivery module, label detection, and the
like.
[0246] Interfering RNAs with Protein Transduction Domains
[0247] As noted, interfering RNAs and other RNAs can be introduced
into cells using protein transduction domains. Thus, one class of
embodiments provides a composition comprising an RNA and a protein
transduction domain covalently attached to the RNA. The RNA can
comprise at least one double-stranded region, the double-stranded
region comprising a sense strand and an antisense strand, the
antisense strand comprising a region which is substantially
complementary to a region of a target mRNA. Alternatively, the RNA
can comprise a single polyribonucleotide strand comprising an
antisense strand, the antisense strand comprising a region which is
substantially complementary to a region of a target mRNA
corresponding to the target gene. The composition optionally also
includes the target mRNA and/or a cell, e.g., a cell comprising the
RNA and/or the target mRNA.
[0248] The RNA can be, for example, an interfering RNA, an RNA
capable of repressing translation of the target mRNA, or an RNA
capable of silencing transcription of a gene from which the target
mRNA is transcribed. Thus, in some embodiments, the region of the
antisense strand that is substantially complementary to a region of
the target mRNA is completely complementary to the region of the
target mRNA. In other embodiments, the region of complementarity is
interrupted. For example, the region of the antisense strand that
is substantially complementary to the region of the target mRNA can
comprise at least a first and a second subregion, each of which is
completely complementary to the target mRNA, flanking one or more
nucleotides (e.g., two, three, four, or more nucleotides) which are
not complementary to the target mRNA.
[0249] The protein transduction domain can be essentially any
protein transduction domain that can mediate introduction of the
RNA into the cell. In one class of embodiments, the protein
transduction domain is derived from an HIV-1 Tat protein, from a
herpes simplex virus VP22 protein, or from a Drosophila
antennapedia protein (e.g., Penetratin.TM.). In other embodiments,
the protein transduction domain is a model protein transduction
domain, e.g., a homopolymer of lysine, histidine, or D-arginine,
e.g., 8-D-Arg.
[0250] The covalent attachment between the protein transduction
domain and the RNA is optionally reversible by exposure to light of
a preselected wavelength. Similarly, the protein transduction
domain can be attached to the RNA through a disulfide bond or an
ester linkage that can be reduced or cleaved once the RNA is inside
the cell.
[0251] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for RNA configurations (e.g., one or two
polyribonucleotides, of various lengths, with or without overhangs,
etc.), use of caging groups (e.g., photolabile caging groups),
appropriate uncaging energies (light, heat, sonic, etc.), label
configurations (e.g., use of fluorescent labels, fluorescent
label/quencher, and donor/acceptor combinations), signal output
types, binding to a matrix, and the like.
[0252] Kits for making the protein transduction domain-linked RNAs
are also a feature of the invention. For example, one embodiment
provides a kit comprising an RNA, a protein transduction domain,
and instructions for assembling the RNA and the protein
transduction domain to form the composition, packaged in one or
more containers. A related embodiment provides a kit comprising a
protein transduction domain and instructions for assembling the
protein transduction domain and an RNA supplied by a user of the
kit to form the composition, packaged in one or more
containers.
[0253] The invention also provides related methods of introducing
an RNA into a cell. In the methods, a composition comprising an RNA
and a protein transduction domain covalently attached to the RNA is
provided. The RNA can comprise at least one double-stranded region,
the double-stranded region comprising a sense strand and an
antisense strand, the antisense strand comprising a region which is
substantially complementary to a region of a target mRNA.
Alternatively, the RNA can comprise a single polyribonucleotide
strand comprising an antisense strand, the antisense strand
comprising a region which is substantially complementary to a
region of a target mRNA corresponding to the target gene. The
composition and the cell are contacted, whereby the protein
transduction domain mediates introduction of the RNA into the
cell.
[0254] In some embodiments, the composition comprises one or more
first caging groups associated with the RNA, which inhibit the RNA
from initiating RNA interference of the target mRNA in the cell.
The method includes initiating RNA interference of the target mRNA
by exposing the cell to uncaging energy of a first type, freeing
the RNA from inhibition by the first caging groups. In related
embodiments, the composition includes one or more first caging
groups associated with the RNA, which inhibit the RNA from
repressing translation of the target mRNA in the cell. The method
then includes initiating translational repression of the target
mRNA by exposing the cell to uncaging energy of a first type,
freeing the RNA from inhibition by the first caging groups.
Similarly, the composition optionally includes one or more first
caging groups associated with the RNA, which inhibit the RNA from
silencing transcription of a gene corresponding to the target mRNA
in the cell. The method then includes initiating transcriptional
silencing of the target gene by exposing the cell to uncaging
energy of a first type, freeing the RNA from inhibition by the
first caging groups.
[0255] All of the above optional method variations apply to this
method as well. Further, the various composition components noted
(particularly the protein transduction domain-linked RNA
embodiments) above can be adapted for use in this method, as
appropriate.
[0256] In another aspect, systems and/or apparatus comprising the
compositions noted above and, e.g., components such as detectors,
fluid handling apparatus, sources of uncaging energy, or the like,
are a feature of the invention.
[0257] Interfering RNAs with Lipids
[0258] Interfering RNAs and other RNAs can also be introduced into
cells by covalently or non-covalently associated lipids. Thus, one
class of embodiments provides a composition comprising an RNA and a
lipid covalently or non-covalently attached to the RNA. The RNA can
comprise at least one double-stranded region, the double-stranded
region comprising a sense strand and an antisense strand, the
antisense strand comprising a region which is substantially
complementary to a region of a target mRNA; alternatively, the RNA
can comprise a single polyribonucleotide strand comprising an
antisense strand, the antisense strand comprising a region which is
substantially complementary to a region of a target mRNA. The lipid
can be, e.g., a fatty acid. In one example class of embodiments,
the lipid comprises (or, e.g., consists of) a myristoyl group.
[0259] All of the various optional configurations and features
noted for the embodiments above apply here as well, to the extent
they are relevant, e.g., for label configurations (e.g., use of
fluorescent labels, fluorescent label/quencher, and donor/acceptor
combinations), signal output types, RNA configurations (e.g., one
or two polyribonucleotides, of various lengths, with or without
overhangs, etc.), use of caging groups (e.g., photolabile caging
groups), appropriate uncaging energies (light, heat, sonic, etc.),
use of cellular delivery modules (e.g., amphipathic peptides,
protein transduction domains, and lipids), and the like. It is
worth noting that the composition optionally also includes the
target mRNA and/or a cell, e.g., a cell comprising the target mRNA
and/or the RNA. It is also worth noting that the RNA can be, for
example, an interfering RNA, an RNA capable of repressing
translation of the target mRNA, or an RNA capable of silencing
transcription of a gene from which the target mRNA is transcribed.
Thus, for example, the region of the antisense strand that is
substantially complementary to a region of the target mRNA can be
completely complementary to the region of the target mRNA, or the
region of complementarity can be interrupted.
[0260] Kits for making the lipid-linked RNAs are also a feature of
the invention. For example, one embodiment provides a kit
comprising an RNA, a lipid, and instructions for assembling the RNA
and the lipid to form the composition, packaged in one or more
containers. A related embodiment provides a kit comprising a lipid
and instructions for assembling the lipid and an RNA supplied by a
user of the kit to form the composition, packaged in one or more
containers.
[0261] The invention also provides related methods of introducing
an RNA into a cell. In the methods, a composition comprising an RNA
and a lipid covalently attached to the RNA is provided. The RNA can
comprise at least one double-stranded region, the double-stranded
region comprising a sense strand and an antisense strand, the
antisense strand comprising a region which is substantially
complementary to a region of a target mRNA; alternatively, the RNA
can comprise a single polyribonucleotide strand comprising an
antisense strand, the antisense strand comprising a region which is
substantially complementary to a region of a target mRNA. The
composition and the cell are contacted, whereby the lipid mediates
introduction of the RNA into the cell.
[0262] All of the above optional method variations apply to this
method as well. Further, the various composition components noted
(particularly the lipid-linked RNA embodiments) above can be
adapted for use in this method, as appropriate.
[0263] In some embodiments, the composition comprises one or more
first caging groups associated with the RNA, which inhibit the RNA
from initiating RNA interference of the target mRNA in the cell.
The method includes initiating RNA interference of the target mRNA
by exposing the cell to uncaging energy of a first type, freeing
the RNA from inhibition by the first caging groups. In related
embodiments, the composition includes one or more first caging
groups associated with the RNA, which inhibit the RNA from
repressing translation of the target mRNA in the cell. The method
then includes initiating translational repression of the target
mRNA by exposing the cell to uncaging energy of a first type,
freeing the RNA from inhibition by the first caging groups.
Similarly, the composition optionally includes one or more first
caging groups associated with the RNA, which inhibit the RNA from
silencing transcription of a gene corresponding to the target mRNA
in the cell. The method then includes initiating transcriptional
silencing of the target gene by exposing the cell to uncaging
energy of a first type, freeing the RNA from inhibition by the
first caging groups.
[0264] In another aspect, systems and/or apparatus comprising the
compositions noted above and, e.g., components such as detectors,
fluid handling apparatus, sources of uncaging energy, or the like,
are a feature of the invention.
[0265] Induction of RNA Expression
[0266] In one aspect, the invention includes methods of selectively
attenuating expression of a target mRNA in a cell. In the methods,
one or more vectors that comprise or encode an RNA are introduced
into the cell. The RNA comprises at least one double-stranded
region, the double-stranded region comprising a sense strand and an
antisense strand, the antisense strand comprising a region which is
substantially complementary to a region of the target mRNA. A caged
first activation component is also introduced into the cell. The
caged first activation component includes one or more caging groups
associated with a first activation component. The first activation
component directly or indirectly increases expression of the RNA
from the one or more vectors, and the one or more caging groups
inhibit (e.g., prevent) the first activation component from
increasing expression of the RNA. The cell is exposed to uncaging
energy (e.g., light of a first wavelength), whereby exposure to the
uncaging energy frees the first activation component from
inhibition by the caging groups. This results in increased
expression of the RNA, which can then initiate RNA interference of
the target mRNA, repress translation of the target mRNA, or silence
transcription of a gene from which the target mRNA is transcribed,
for example.
[0267] In one class of embodiments, the first activation component
directly increases expression of the RNA from the one or more
vectors. For example, the first activation component can be a
transcription factor (i.e., a transcriptional activator) or an RNA
polymerase, e.g., T7 polymerase.
[0268] In another class of embodiments, the first activation
component indirectly increases expression of the RNA by binding to
a second activation component, whereby the bound second activation
component directly increases expression of the RNA. An example
embodiment is schematically illustrated in FIG. 30, which depicts
tetracycline caged with a photolabile caging group (the caged first
activation component). Exposure to light frees the tetracycline
from the caging group. In this example, the tetracycline binds a
tetracycline-controlled transactivator (tTA, the second activation
component), which stimulates transcription of the interfering RNA
from a promoter comprising tet operator sequences.
[0269] In yet another class of embodiments, the first activation
component indirectly increases expression of the RNA by indirectly
activating a third activation component, whereby the activated
third activation component directly increases expression of the
RNA. An example embodiment is schematically illustrated in FIG. 31,
which depicts IP3 (inositol 1,4,5-triphosphate) caged with a
photolabile caging group (the caged first activation component).
Exposure to light frees the IP3 from the caging group, leading to a
rise in intracellular Ca.sup.2+ concentration. The increased
Ca.sup.2+ concentration stimulates calcineurin to dephosphorylate
the NF-AT (nuclear factor of activated T cells) transcription
factor complex, which then migrates into the nucleus and activates
expression of the interfering RNA from a promoter comprising
NF-AT-response elements. Caged Ca.sup.2+, for example, can also be
used as a first activation component in this system.
[0270] Other examples of suitable first activation components
include, but are not limited to, cAMP, non-mammalian steroid
hormones and small molecules that bind immunophilins. See, e.g.,
Gossen and Bujard (1992) "Tight control of gene expression in
mammalian cells by tetracycline-responsive promoters" Proc. Natl.
Acad. Sci. USA 89:5547-5551; Saez et al. (1997) "Inducible gene
expression in mammalian cells and transgenic mice" Curr. Opin.
Biotechnol. 8:608-616; Li et al. (1998) "Cell-permeant caged InsP3
ester shows that Ca2+ spike frequency can optimize gene expression"
Nature 392:936-541; and Lin et al. (2002) "Spatially discrete,
light-driven protein expression" Chem. Biol. 9:1347-1353.
[0271] Methods of expressing interfering RNAs of various lengths
and structures from vectors are well known in the art. See, e.g.,
Patterson and Hannon (2002) "Stable suppression of gene expression
by RNAi in mammalian cells" Proc. Natl. Acad. Sci. USA 99:1443-1448
and Garbarek and Glover (2003) "RNA interference by production of
short hairpin dsRNA in ES cells, their differentiated derivatives,
and somatic cell lines" BioTechniques 34:734-744. Methods of
expressing other RNAs are similarly known.
[0272] The invention also provides compositions related to the
methods. Thus, one general class of embodiments provides a
composition comprising one or more vectors and a caged first
activation component. The one or more vectors comprise or encode an
RNA comprising at least one double-stranded region, the
double-stranded region comprising a sense strand and an antisense
strand, the antisense strand comprising a region which is
substantially complementary to a region of a target mRNA. The caged
first activation component comprises one or more caging groups
associated with a first activation component, which first
activation component directly or indirectly increases expression of
the RNA from the one or more vectors in a cell comprising the one
or more vectors and the first activation component, and which one
or more caging groups inhibit the first activation component from
increasing expression of the RNA in the cell. The composition
optionally includes the target mRNA and/or a cell, e.g., a cell
comprising the one or more vectors and the caged first activation
component and/or the target mRNA.
[0273] The composition optionally also includes a second activation
component, which second activation component directly increases
expression of the RNA when bound by the first activation component
(e.g., tetracycline). In a related class of embodiments, the
composition optionally also includes a third activation component,
which third activation component directly increases expression of
the RNA when indirectly activated by the first activation
component. For example, the first activation component can comprise
IP3 or Ca.sup.2+ and the third activation component can comprise an
NF-AT transcription factor complex. Other examples of suitable
first activation components include, but are not limited to, cAMP,
non-mammalian steroid hormones and small molecules that bind
immunophilins.
[0274] The length and/or structure of the RNA can vary. For
example, the RNA can comprise a first polyribonucleotide comprising
the sense strand and a second polyribonucleotide comprising the
antisense strand. The double-stranded region formed by annealing of
the sense and antisense strands can, e.g., comprise more than about
1500 base pairs, comprise fewer than about 1500 base pairs, fewer
than about 1000 base pairs, fewer than about 500 base pairs, fewer
than about 250 base pairs, fewer than about 150 base pairs, fewer
than about 80 base pairs, fewer than about 50 base pairs, fewer
than about 30 base pairs, or even fewer than about 25 base pairs.
Instead of comprising a two-stranded interfering RNA (e.g., a
siRNA), the RNA comprises a self-complementary polyribonucleotide
(e.g., an shRNA). As noted for the embodiments above, the RNA can
be, for example, an interfering RNA, an RNA capable of repressing
translation of the target mRNA, or an RNA capable of silencing
transcription of a gene from which the target mRNA is transcribed,
for example.
[0275] Kits form another feature of the invention. Thus, one class
of embodiments provides a kit comprising one or more vectors and a
caged first activation component, packaged in one or more
containers. The kit can also include a vector that comprises or
encodes a second or a third activation component, and/or
instructions for using the kit, e.g., instructions for using the
kit to attenuate expression of a target mRNA.
[0276] All of the various optional configurations and features
noted for the embodiments above apply to the methods and
compositions here as well, to the extent they are relevant, e.g.,
RNA configurations (e.g., one or two polyribonucleotides, of
various lengths, with or without overhangs, etc.), use of caging
groups (e.g., photolabile caging groups), appropriate uncaging
energies (light, heat, sonic, etc.), use of cellular delivery
modules (e.g., amphipathic peptides, protein transduction domains,
and lipids), and the like.
[0277] Caged DNAs Encoding Interfering RNAs and Other RNAs
[0278] The invention also includes other methods of selectively
attenuating expression of a target gene in a cell. In one general
class of methods, a first caged DNA and a second caged DNA are
introduced into the cell. The first caged DNA includes a first DNA
encoding an RNA sense strand and one or more caging groups. The
second caged DNA comprises a second DNA encoding an RNA antisense
strand and one or more caging groups. The presence of the caging
groups prevents transcription of the first and second DNAs, the
first and second DNAs each comprising at least a portion of the
target gene, and the sense and antisense strands being at least
partially complementary and able to form a duplex over at least a
portion of their lengths. RNA interference is initiated by
generating double-stranded RNA by exposing the cell to uncaging
energy, whereby exposure to the uncaging energy frees the first and
second DNAs from the caging groups and permits transcription of the
first and second DNAs to occur.
[0279] The resulting double-stranded RNA can comprise two distinct
polyribonucleotides (i.e., the sense strand can comprise a first
polyribonucleotide while the antisense strand comprises a second
polyribonucleotide), or the resulting double-stranded RNA can
comprise a single, self-complementary polyribonucleotide that
includes the sense and antisense strands (e.g., an shRNA).
[0280] All of the above optional method variations apply to this
method as well, to the extent they are relevant. Further, the
various composition components noted above can be adapted for use
in this method, as appropriate; e.g., use of caging groups (e.g.,
photolabile caging groups), appropriate uncaging energies (light,
heat, sonic, etc.), use of cellular delivery modules (e.g.,
amphipathic peptides, protein transduction domains, and lipids),
and the like. It is worth noting that when the resulting
double-stranded RNA comprises a single, self-complementary
polyribonucleotide, the first and second DNAs are covalently joined
in proximity to each other as a single transcription unit, e.g., on
a plasmid. When the resulting double-stranded RNA comprises two
distinct polyribonucleotides, the first and second DNAs can be on
separate plasmids or can optionally be included on a single plasmid
(see, e.g., U.S. patent application publication 20020182223). The
DNAs are optionally nuclease resistant.
[0281] In another general class of methods, a first caged DNA and a
second caged DNA are introduced into the cell. The first caged DNA
includes a first DNA encoding an RNA sense strand and one or more
caging groups. The second caged DNA comprises a second DNA encoding
an RNA antisense strand and one or more caging groups. The presence
of the caging groups prevents transcription of the first and second
DNAs, the first and second DNAs each comprising at least a portion
of the target gene, and the sense and antisense strands being at
least partially complementary and able to form a duplex over at
least a portion of their lengths. Translational repression is
initiated by generating double-stranded RNA by exposing the cell to
uncaging energy, whereby exposure to the uncaging energy frees the
first and second DNAs from the caging groups and permits
transcription of the first and second DNAs to occur.
[0282] The resulting double-stranded RNA can comprise two distinct
polyribonucleotides (i.e., the sense strand can comprise a first
polyribonucleotide while the antisense strand comprises a second
polyribonucleotide), or the resulting double-stranded RNA can
comprise a single, self-complementary polyribonucleotide that
includes the sense and antisense strands.
[0283] All of the above optional method variations apply to this
method as well, to the extent they are relevant. Further, the
various composition components noted above can be adapted for use
in this method, as appropriate; e.g., use of caging groups (e.g.,
photolabile caging groups), appropriate uncaging energies (light,
heat, sonic, etc.), use of cellular delivery modules (e.g.,
amphipathic peptides, protein transduction domains, and lipids),
and the like. It is worth noting that when the resulting
double-stranded RNA comprises a single, self-complementary
polyribonucleotide, the first and second DNAs are covalently joined
in proximity to each other as a single transcription unit, e.g., on
a plasmid. When the resulting double-stranded RNA comprises two
distinct polyribonucleotides, the first and second DNAs can be on
separate plasmids or can optionally be included on a single
plasmid, as described above. The DNAs are optionally nuclease
resistant.
[0284] RNAs capable of silencing transcription of a target gene can
be similarly expressed.
[0285] Caging Groups
[0286] A large number of caging groups, and a number of reactive
compounds that can be used to covalently attach caging groups to
other molecules, are well known in the art. Examples of photolabile
caging groups include, but are not limited to: 2-nitrobenzyl;
1-(4,5-dimethoxy-2-nitrophenyl)eth- yl (DMNPE); brominated
7-hydroxycoumarin-4-ylmethyls (e.g.,
6-Bromo-7-hydroxycoumarin-4-ylmethyl (Bhc)); nitroindolines;
N-acyl-7-nitroindolines; phenacyls; hydroxyphenacyl; benzoin
esters; dimethoxybenzoin; meta-phenols; 4,5-dimethoxy-2-nitrobenzyl
(DMNB); alpha-carboxy-2-nitrobenzyl (CNB); 1-(2-nitrophenyl)ethyl
(NPE); 5-carboxymethoxy-2-nitrobenzyl (CMNB);
(5-carboxymethoxy-2-nitrobenzyl)ox- y)carbonyl;
(4,5-dimethoxy-2-nitrobenzyl)oxy) carbonyl; desoxybenzoinyl; and
the like. See e.g., WO 2004/046339, U.S. Pat. No. 5,635,608 to
Haugland and Gee (Jun. 3, 1997) entitled ".alpha.-carboxy caged
compounds"; Neuro 19, 465 (1997); J Physiol 508.3, 801 (1998); Proc
Natl Acad Sci USA September 1998; 85(17):6571-5; J Biol Chem Feb.
14, 1997; 272(7):4172-8; Neuron 20,619-624, 1998; Nature Genetics,
vol. 28:2001:317-325; Nature, vol. 392,1998:936-941; Pan, P., and
Bayley, H. "Caged cysteine and thiophosphoryl peptides" FEBS
Letters 405:81-85 (1997); Pettit et al. (1997) "Chemical two-photon
uncaging: a novel approach to mapping glutamate receptors" Neuron
19:465-471; Furuta et al. (1999) "Brominated
7-hydroxycoumarin-4-ylmethyls: novel photolabile protecting groups
with biologically useful cross-sections for two photon photolysis"
Proc. Natl. Acad. Sci. 96(4):1193-1200; Zou et al. "Catalytic
subunit of protein kinase A caged at the activating
phosphothreonine" J. Amer. Chem. Soc. (2002) 124: 8220-8229; Zou et
al. "Caged Thiophosphotyrosine Peptides" Angew. Chem. Int. Ed.
(2001) 40: 3049-3051; Conrad II et al. "p-Hydroxyphenacyl
Phototriggers: The Reactive Excited State of Phosphate
Photorelease" J. Am. Chem. Soc. (2000) 122:9346-9347; Conrad II et
al. "New Phototriggers 10: Extending the .pi.,.pi.* Absorption to
Release Peptides in Biological Media" Org. Lett. (2000)
2:1545-1547; Givens et al. "A New Phototriggers 9:
p-Hydroxyphenacyl as a C-Terminus Photoremovable Protecting Group
for Oligopeptides" J. Am. Chem. Soc. (2000) 122:2687-2697; Bishop
et al. "40-Aminomethyl-2,20-bipyr- idyl-4-carboxylic Acid (Abc) and
Related Derivatives: Novel Bipyridine Amino Acids for the
Solid-Phase Incorporation of a Metal Coordination Site Within a
Peptide Backbone" Tetrahedron (2000)56:4629-4638; Ching et al
"Polymers As Surface-Based Tethers with Photolytic triggers
Enabling Laser-Induced Release/Desorption of Covalently Bound
Molecules" Bioconjugate Chemistry (1996) 7:525-8; U.S. Pat. No.
5,888,829 to Gee and Millard (Mar. 30, 1999) entitled "Photolabile
caged ionophores and method of using in a membrane separation
process"; U.S. Pat. No. 6,043,065 to Kao et al. (Mar. 28, 2000)
entitled "Photosensitive organic compounds that release
2,5,-di(tert-butyl) hydroquinone upon illumination"; U.S. Pat. No.
5,430,175 to Hess et al. (Jul. 4, 1995) entitled "Caged carboxyl
compounds and use thereof"; U.S. Pat. No. 5,872,243; PNAS (1980)
77:7237-41; BioProbes Handbook, 2002 from Molecular Probes, Inc.;
and Handbook of Fluorescent Probes and Research Products, Ninth
Edition or Web Edition, from Molecular Probes, Inc, as well as the
references below. Many compounds, kits, etc. for use in caging
various molecules are commercially available, e.g., from Molecular
Probes, Inc. (www.molecularprobes.com).
[0287] Environmentally responsive polymers suitable for use as
caging groups have also been described. Such polymers undergo
conformational changes induced by light, an electric or magnetic
field, a change in pH and/or ionic strength, temperature, or
addition of an antigen or saccharide, or other environmental
variables. For example, Shimoboji et al. (2002) "Photoresponsive
polymer-enzyme switches" Proc. Natl. Acad. Sci. USA
99:16,592-16,596 describes polymers that undergo reversible
conformational changes in response to light; such polymers can,
e.g., be used as photoactivatable caging groups. See also Ding et
al. (2001) "Size-dependent control of the binding of biotinylated
proteins to streptavidin using a polymer shield" Nature 411:59-62;
Miyata et al. (1999) "A reversibly antigen-responsive hydrogel"
Nature 399:766-769; Murthy et al. (2003) "Bioinspired pH-responsive
polymers for the intracellular delivery of biomolecular drugs"
Bioconjugate Chem. 14:412-419; and Galaev and Mattiasson (1999)
"`Smart` polymers and what they could do in biotechnology and
medicine" Trends Biotech. 17:335-340. FIGS. 26 and 27 schematically
illustrate use of environmentally responsive polymers as caging
groups. FIG. 26 illustrates noncovalent association of a polymer
with a component to be caged (e.g., an siRNA). In its folded
conformation, the polymer physically surrounds and traps the
component (Panel B). The caged RNA is optionally introduced into a
cell. A conformational change in the polymer induced by light, pH,
temperature, or the like results in release of the RNA from the
unfolded conformation of the polymer (Panel D). FIG. 27 illustrates
covalent association of a polymer with an example double-stranded
siRNA. In its folded conformation, the polymer prevents the siRNA
from initiating RNAi (e.g., by preventing the siRNA from
interacting with a kinase, RISC, or other components of the RNAi
cellular machinery) (Panel A). A conformational change in the
polymer induced by light, pH, temperature, or the like permits the
siRNA to initiate RNAi (Panel B).
[0288] Caged polypeptides (including, e.g., polypeptide cellular
delivery modules, e.g., protein transduction domains) can be
produced, for example, by reacting a polypeptide with a caging
compound or by incorporating a caged amino acid during synthesis of
a polypeptide. See, e.g., U.S. Pat. No. 5,998,580 to Fay et al.
(Dec. 7, 1999) entitled "Photosensitive caged macromolecules";
Kossel et al. (2001) PNAS 98:14702-14707; Trends Plant Sci (1999)
4:330-334; PNAS (1998) 95:1568-1573; J Am Chem Soc (2002)
124:8220-8229; Pharmacology & Therapeutics (2001) 91:85-92; and
Angew Chem Int Ed Engl (2001) 40:3049-3051. A photolabile
polypeptide linker (e.g., for connecting a protein transduction
domain and an RNA, or the like) can, for example, comprise a
photolabile amino acid such as that described in U.S. Pat. No.
5,998,580 (supra).
[0289] Caged nucleic acids (e.g., DNA, RNA or PNA, e.g.,
interfering RNAs) can be produced by reacting the nucleic acids
with caging compounds or by incorporating a caged nucleotide during
synthesis of a nucleic acid. For example, U.S. Pat. No. 6,242,258
to Haselton and Alexander (Jun. 5, 2001) entitled "Methods for the
selective regulation of DNA and RNA transcription and translation
by photoactivation" and U.S. Pat. No. 6,410,327 to Haselton, III,
et al. entitled "Methods for the selective regulation of DNA and
RNA transcription and translation by photoactivation" describe
DMNPE caging of DNA by postsynthetic reactions; Ando et al. (2001)
"Photo-mediated gene activation using caged RNA/DNA in zebrafish
embryos" Nature Genetics 28: 317-325 describes Bhc caging of RNA
and DNA by postsynthetic reactions; and Chaulk and MacMillan (1998)
"Caged RNA: Photo-control of a ribozyme reaction" Nucl Acids Res.
26:3173-3178 describes 2-nitrobenzyl caging of RNA by incorporation
of a caged phosphoramidite during RNA synthesis. A caged RNA or an
RNA that is to be caged optionally includes one or more
deoxyribonucleotides and/or nonnatural or modified nucleotides,
e.g., that are less reactive than standard ribonucleotides, to
facilitate attachment of the caging group(s), e.g., to a 5'
hydroxyl.
[0290] Caging groups can be attached at random and/or predetermined
sites within a molecule. Useful site(s) of attachment of and/or
conditions for attaching caging groups to a given molecule can be
determined by techniques known in the art. For example, a molecule
with a known activity (e.g., an interfering RNA or a protein
transduction domain) can be reacted with a caging compound. The
resulting caged molecule can then be tested to determine if its
activity (e.g., ability to initiate RNAi or to mediate introduction
of an associated molecule into a cell) is sufficiently abrogated.
As another example, amino acid residues central to the activity of
a polypeptide (e.g., residues located at a binding interface of a
protein transduction domain, or the like) can be identified by
routine techniques such as scanning mutagenesis, sequence
comparisons and site-directed mutagenesis, or the like. Such
residues can then be caged, and the activity of the caged
polypeptide (e.g., its ability to mediate introduction of an
associated molecule into a cell) can be assayed to determine the
efficacy of caging. Similarly, an RNA can be caged at positions
and/or groups identified as being required for activity (e.g., the
5' phosphate or 5' hydroxyl of the antisense strand of an siRNA can
be caged).
[0291] An alternative method for caging a molecule (e.g., an siRNA)
is to enclose the molecule in a photolabile vesicle (e.g., a
photolabile lipid vesicle), optionally including a protein
transduction domain or the like (FIG. 11). Similarly, the molecule
can be loaded into the pores of a porous bead which is then encased
in a photolabile gel.
[0292] Appropriate methods for uncaging caged molecules are also
known in the art. For example, appropriate wavelengths of light for
removing many photolabile groups have been described; e.g., 300-360
nm for 2-nitrobenzyl, 350 nm for benzoin esters, and 740 nm for
brominated 7-hydroxycoumarin-4-ylmethyls (two-photon) (see, e.g.,
references herein). Conditions for uncaging any caged molecule
(e.g., the optimal wavelength for removing a photolabile caging
group) can be determined according to methods well known in the
art. Instrumentation and devices for delivering uncaging energy are
likewise known (e.g., sonicators, heat sources, light sources,
other sources of electromagnetic radiation, and the like). For
example, well known and useful light sources include e.g., a lamp,
a laser (e.g., a laser optically coupled to a fiber-optic delivery
system) or a light-emitting compound.
[0293] In vivo and in vitro Cellular Delivery
[0294] Molecules (e.g., double-stranded RNAs, including caged
and/or labeled RNAs) can be introduced into cells by traditional
methods such as lipofection, electroporation, microinjection,
optofection, laser transfection, calcium phosphate precipitation,
and/or particle bombardment. Double-stranded RNA can also be
introduced into cells by pinocytosis or by using streptolysin-O
(SLO). See, e.g., WO 03/040375 by Wolff entitled "Compositions and
processes using siRNA, amphipathic compounds and polycations."
Reagents for delivery of double-stranded RNAs are commercially
available, e.g., TransIT-TKO.TM. (Mirus Corporation,
www.genetransfer.com). If the molecule is caged, such delivery can
be accomplished without uncaging and thereby activating the
molecule; for example, a photoactivatable interfering RNA is not
active during the delivery process until exposed to light of
appropriate wavelength. However, these methods require manipulation
of the cells, e.g., adding and removing transfection materials,
pre-treating cells, and special apparatus and equipment, etc. In
addition, some cells (particularly primary cells) are difficult to
transfect by methods such as lipofection.
[0295] While the methods above are suitable for introducing
molecules (e.g., interfering RNAs and caged DNAs) into cells, this
invention features a simpler and more effective method of
introducing molecules into the cell. That is, the molecule is
optionally associated (covalently or non-covalently) with a
cellular delivery module that can mediate its introduction into the
cell. The cellular delivery module is typically, but need not be, a
polypeptide, for example, a PEP-1 peptide, an amphipathic peptide,
e.g., an MPG peptide (Simeoni et al. (2003) "Insight into the
mechanism of the peptide-based gene delivery system MPG:
Implications for delivery of siRNA into mammalian cells" Nucl Acids
Res 31: 2717-2724), a cationic peptide (e.g., a homopolymer of
lysine, histidine, or D-arginine), or a protein transduction domain
(a polypeptide that can mediate introduction of a covalently
associated molecule into a cell). See, e.g., Lane (2001)
Bioconjugate Chem., 12:825-841; Bonetta (2002) The Scientist 16:38;
and Curr Opin Mol Ther (2000) 2:162-7. For example, an interfering
RNA (including a caged and/or labeled interfering RNA) can be
covalently associated with a protein transduction domain (e.g., an
HIV TAT sequence, which most cells naturally uptake, or a short
D-arginine homopolymer, e.g., 8-D-Arg, eight contiguous D-arginine
residues). The protein transduction domain-coupled RNA can simply
be, e.g., added to cell culture or injected into an animal for
delivery. (Note that TAT and D-arginine homopolymers, for example,
can alternatively be noncovalently associated with the interfering
RNA and still mediate its introduction into the cell.)
[0296] A number of polypeptides capable of mediating introduction
of associated molecules into a cell are known in the art and can be
adapted to the present invention; see, e.g., the references above
and Langel (2002) Cell Penetrating Peptides CRC Press, Pharmacology
& Toxicology Series.
[0297] As noted, an RNA, or a caged DNA, can also be introduced
into cells by covalently or noncovalently attached lipids, e.g., by
a covalently attached myristoyl group. In any of the cellular
delivery modules herein, lipids used for lipofection are optionally
excluded from cellular delivery modules in some embodiments.
[0298] In summary, an RNA or a caged DNA can be introduced into a
cell by any of several methods, including without limitation,
lipofection, electroporation, microinjection, and association with
a cellular delivery module (including covalent association with a
protein transduction domain). RNA and caged DNA can optionally be
introduced into specific tissues and/or cell types (e.g., explanted
or in an organism), for example, by laser transfection, gold
particle bombardment, microinjection, coupling to viral proteins,
or covalent association with a protein transduction domain, among
other techniques. See, e.g., Robbins et al. (2002) "Peptide
delivery to tissues via reversibly linked protein transduction
sequences" Biotechniques 33:190-192 and Rehman et al. (2003)
"Protection of islets by in situ peptide-mediated transduction of
the I-kappa B kinase inhibitor Nemo-binding domain peptide" J Biol
Chem 278:9862-9868.
[0299] The cell into which an RNA or a caged DNA of this invention
is introduced is typically a eukaryotic cell (e.g., a yeast, a
vertebrate cell, a mammalian cell, a rodent cell, a primate cell, a
human cell, a plant cell, an insect cell, or essentially any other
type of eukaryotic cell). The cell can be, e.g., in culture or in a
tissue, fluid, etc. and/or from or in an organism.
[0300] The cellular delivery modules optionally can be caged.
Covalently associated cellular delivery modules (e.g., protein
transduction domains) can optionally be released from the
associated molecule (e.g., by placement of a photolabile linkage, a
disulfide or ester linkage that is reduced or cleaved in the cell,
or the like, between the cellular delivery module and the
molecule). For example, 8-D-Arg can be covalently linked through a
disulfide linker to an interfering RNA. The 8-D-Arg module mediates
entry of the RNA into a cell, where the linker is reduced in the
reducing environment of the cytoplasm, freeing the interfering RNA
from the 8-D-Arg module.
[0301] The amount of a nucleic acid delivered to a cell can
optionally be controlled by controlling the number of cellular
delivery modules associated with the nucleic acid (covalently or
noncovalently). For example, increasing the ratio of 8-D-Arg to
interfering RNA can increase the percentage of interfering RNA that
enters the cell.
[0302] The RNAs and caged DNAs of this invention optionally also
comprise a subcellular delivery module (e.g., a peptide, nucleic
acid, and/or carbohydrate tag) or other means of achieving a
desired subcellular localization. For example, an interfering RNA
is typically most effective at initiating RNAi when it is localized
to the cytoplasm. Thus, if a method that results in localization of
the interfering RNA to the endosome is used to introduce the RNA
into the cell (e.g., lipofection, certain protein transduction
domains, and the like), performance of the interfering RNA can be
improved by including an endosomal release agent on the RNA (e.g.,
HA-2, PEI, or a dendrimer). See, e.g., Journal of Controlled
Release (1999) 61:137-143; J Biol Chem 277:27135-43; Proc Natl Acad
Sci 89:7934-38; and Bioconjugate Chem (2002) 13:996-1001. Examples
of subcellular delivery modules include nuclear localization
signals, chloroplast stromal targeting sequences, and many others
(see, e.g., Molecular Biology of the Cell (3rd ed.) Alberts et al.,
Garland Publishing, 1994; and Molecular Cell Biology (4th ed.)
Lodish et al., W H Freeman & Co, 1999). Similarly, localization
can be to a target protein; that is, the subcellular delivery
module can comprise a binding domain that binds the target
protein.
[0303] Labels
[0304] The compositions of this invention optionally include one or
more labels; e.g., optically detectable labels, such as fluorescent
or luminescent labels, and/or non-optically detectable labels, such
as magnetic labels. A number of fluorescent labels are well known
in the art, including but not limited to, quantum dots, hydrophobic
fluorophores (e.g., coumarin, rhodamine and fluorescein), and green
fluorescent protein (GFP) and variants thereof (e.g., cyan
fluorescent protein and yellow fluorescent protein). See e.g.,
Haughland (2002) Handbook of Fluorescent Probes and Research
Products, Ninth Edition or the current Web Edition, both available
from Molecular Probes, Inc. Likewise, a variety of donor/acceptor
and fluorophore/quencher combinations, using e.g., fluorescence
resonance energy transfer (FRET)-based quenching, non-FRET based
quenching, or wavelength-shifting harvester molecules, are known.
Example combinations include cyan fluorescent protein and yellow
fluorescent protein, terbium chelate and TRITC (tetrarhodamine
isothiocyanate), lanthanide (e.g., europium or terbium) chelates
and allophycocyanin (APC) or Cy5, europium cryptate and
Allophycocyanin, fluorescein and tetramethylrhodamine, IAEDANS and
fluorescein, EDANS and DABCYL, fluorescein and DABCYL, fluorescein
and fluorescein, BODIPY FL and BODIPY FL, and fluorescein and QSY 7
dye. Nonfluorescent acceptors such as DABCYL and QSY 7 and QSY 33
dyes have the particular advantage of eliminating background
fluorescence resulting from direct (i.e., nonsensitized) acceptor
excitation. See, e.g., U.S. Pat. Nos. 5,668,648, 5,707,804,
5,728,528, 5,853,992, and 5,869,255 to Mathies et al. for a
description of FRET dyes.
[0305] For use of quantum dots as labels for biomolecules, see,
e.g., Dubertret et al. (2002) Science 298:1759; Nature
Biotechnology (2003) 21:41-46; and Nature Biotechnology (2003)
21:47-51. In the context of the present invention, such quantum
dots can be used to label any nucleic acid of interest, e.g., an
interfering RNA, e.g., a caged interfering RNA.
[0306] Other optically detectable labels can also be used in the
invention. For example, gold beads can be used as labels and can be
detected using a white light source via resonance light scattering.
See, e.g., http://www.geniconsciences.com. Suitable non-optically
detectable labels are also known in the art. For example, magnetic
labels can be used in the invention (e.g., 3 nm superparamagnetic
colloidal iron oxide as a label and NMR detection; see e.g., Nature
Biotechnology (2002) 20:816-820).
[0307] Labels can be introduced to nucleic acids during synthesis
or by postsynthetic reactions by techniques established in the art.
For example, a fluorescently labeled nucleotide can be incorporated
into an RNA or DNA during enzymatic or chemical synthesis of the
nucleic acid, e.g., at a preselected or random nucleotide position.
Alternatively, fluorescent labels can be added to RNAs or DNAs by
postsynthetic reactions, at either random or preselected positions
(e.g., an oligonucleotide can be chemically synthesized with a
terminal amine or free thiol at a preselected position, and a
fluorophore can be coupled to the oligonucleotide via reaction with
the amine or thiol). Reagents for fluorescent labeling of nucleic
acids are commercially available; for example, a variety of kits
for fluorescently labeling nucleic acids are available from
Molecular Probes, Inc. (www.probes.com), and a kit for randomly
labeling double-stranded RNA is available from Ambion, Inc.
(www.ambion.com, the Silencer.TM. siRNA labeling kit). Quenchers
can be introduced by analogous techniques.
[0308] Attachment of labels to oligos during automated synthesis
and by post-synthetic reactions has been described. See, e.g.,
Tyagi and Kramer (1996) "Molecular beacons: probes that fluoresce
upon hybridization" Nature Biotechnology 14:303-308; U.S. Pat. No.
6,037,130 to Tyagi et al. (Mar. 14, 2000), entitled
"Wavelength-shifting probes and primers and their use in assays and
kits"; and U.S. Pat. No. 5,925,517 (Jul. 20, 1999) to Tyagi et al.
entitled "Detectably labeled dual conformation oligonucleotide
probes, assays and kits." Additional details on synthesis of
functionalized oligos can be found in Nelson, et al. (1989)
"Bifunctional Oligonucleotide Probes Synthesized Using A Novel CPG
Support Are Able To Detect Single Base Pair Mutations" Nucleic
Acids Research 17:7187-7194.
[0309] Labels and/or quenchers can be introduced to the
oligonucleotides, for example, by using a controlled-pore glass
column to introduce, e.g., the quencher (e.g., a
4-dimethylaminoazobenzene-4'-sulfonyl moiety (DABSYL). For example,
the quencher can be added at the 3' end of oligonucleotides during
automated synthesis; a succinimidyl ester of
4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL) can be used when
the site of attachment is a primary amino group; and
4-dimethylaminophenylazo- phenyl-4'-maleimide (DABMI) can be used
when the site of attachment is a sulfhydryl group. Similarly,
fluorescein can be introduced into oligos, either using a
fluorescein phosphoramidite that replaces a nucleoside with
fluorescein, or by using a fluorescein dT phosphoramidite that
introduces a fluorescein moiety at a thymidine ring via a spacer.
To link a fluorescein moiety to a terminal location,
iodoacetoamidofluorescein can be coupled to a sulfhydryl group.
Tetrachlorofluorescein (TET) can be introduced during automated
synthesis using a 5'-tetrachloro-fluorescein phosphoramidite. Other
reactive fluorophore derivatives and their respective sites of
attachment include the succinimidyl ester of 5-carboxyrhodamine-6G
(RHD) coupled to an amino group; an iodoacetamide of
tetramethylrhodamine coupled to a sulfhydryl group; an
isothiocyanate of tetramethylrhodamine coupled to an amino group;
or a sulfonylchloride of Texas red coupled to a sulfhydryl group.
Labeled oligonucleotides can be purified, if desired, e.g., by high
pressure liquid chromatography or other methods.
[0310] Similarly, signals from the labels (e.g., absorption by
and/or fluorescent emission from a fluorescent label) can be
detected by essentially any method known in the art. For example,
multicolor detection, detection of FRET (including, e.g.,
time-resolved or TR-FRET, e.g., between lanthanide chelate donors
and fluorescent dye acceptors; see, e.g., Journal of Biomolecular
Screening (2002) 7:3-10), and the like, are well known in the art.
In brief, FRET (Fluorescence Resonance Energy Transfer) is a
non-radiative energy transfer phenomenon in which two fluorophores
with overlapping emission and excitation spectra, when in
sufficiently close proximity, experience energy transfer by a
resonance dipole induced dipole interaction. The phenomenon is
commonly used to study the binding of analytes such as nucleic
acids, proteins and the like. FRET is a distance dependent excited
state interaction in which emission of one fluorophore is coupled
to the excitation of another which is in proximity (close enough
for an observable change in emissions to occur). Some excited
fluorophores interact to form excimers, which are excited state
dimers that exhibit altered emission spectra (e.g., phospholipid
analogs with pyrene sn-2 acyl chains); see, e.g., Haughland (2003)
Handbook of Fluorescent Probes and Research Products Ninth Edition,
available from Molecular Probes. A straightforward discussion of
FRET can be found in the Handbook and the references cited
therein.
[0311] As another example, fluorescence polarization can be used.
Briefly, in the performance of such fluorescent binding assays, a
typically small, fluorescently labeled molecule, e.g., a ligand,
antigen, etc., having a relatively fast rotational correlation
time, is used to bind to a much larger molecule, e.g., a receptor
protein, antibody etc., which has a much slower rotational
correlation time. The binding of the small labeled molecule to the
larger molecule significantly increases the rotational correlation
time (decreases the amount of rotation) of the labeled species,
namely the labeled complex over that of the free unbound labeled
molecule. This has a corresponding effect on the level of
polarization that is detectable. Specifically, the labeled complex
presents much higher fluorescence polarization than the unbound,
labeled molecule.
[0312] Generally, fluorescence polarization level is calculated
using the following formula:
P=[I.sub.1-I.sub.2]/[I.sub.1+I.sub.2]
[0313] where I.sub.1 is the fluorescence detected in the plane
parallel to the excitation light, and I.sub.2 is the fluorescence
detected in the plane perpendicular to the excitation light.
References which discuss fluorescence polarization and/or its use
in molecular biology include Perrin (1926) "Polarization de la
lumiere de fluorescence. Vie moyenne de molecules dans l'etat
excite" J Phys Radium 7:390; Weber (1953) "Rotational Brownian
motion and polarization of the fluorescence of solutions" Adv
Protein Chem 8:415; Weber (1956) J Opt Soc Am 46:962; Dandliker and
Feigen (1961) "Quantification of the antigen-antibody reaction by
the polarization of fluorescence" Biochem Biophys Res Commun 5:299;
Dandliker and de Saussure (1970) "Fluorescence polarization in
immunochemistry" Immunochemistry 7:799; Dandliker et al. (1973)
"Fluorescence polarization immunoassay. Theory and experimental
method" Immunochemistry 10:219; Levison et al. (1976) "Fluorescence
polarization measurement of the hormone-binding site interaction"
Endocrinology 99:1129; Jiskoot et al. (1991) "Preparation and
application of a fluorescein-labeled peptide for determining the
affinity constant of a monoclonal antibody-hapten complex by
fluorescence polarization" Anal Biochem 196:421; Wei and Herron
(1993) "Use of synthetic peptides as tracer antigens in
fluorescence polarization immunoassays of high molecular weight
analytes" Anal Chem 65:3372; Devlin et al. (1993) "Homogeneous
detection of nucleic acids by transient-state polarized
fluorescence" Clin Chem 39:1939; Murakami et al. (1991)
Fluorescent-labeled oligonucleotide probes detection of hybrid
formation in solution by fluorescence polarization spectroscopy"
Nuc. Acids Res 19:4097; Checovich et al. (1995) "Fluorescence
polarization-a new tool for cell and molecular biology" Nature
375:354-256; Kumke et al. (1995) "Hybridization of
fluorescein-labeled DNA oligomers detected by fluorescence
anisotropy with protein binding enhancement" Anal Chem 67:21,
3945-3951; and Walker et al. (1996) "Strand displacement
amplification (SDA) and transient-state fluorescence polarization
detection of mycobacterium tuberculosis DNA" Clinical Chemistry
42:1, 9-13.
[0314] Arrays
[0315] In certain embodiments, the RNA is arranged in an array. In
an array on a matrix (e.g., a surface), each nucleic acid is bound
(e.g., electrostatically or covalently bound, directly or via a
linker) to the matrix at a unique location. Methods of making,
using, and analyzing such arrays (e.g., microarrays) are well known
in the art, including methods of using arrays by overlaying the
arrays with cells into which the components of the array can be
introduced. See e.g., U.S. Pat. No. 6,197,599; Ziauddin and
Sabatini "Microarrays of cells expressing defined cDNAs" Nature May
3, 2001;411(6833):107-10; and Falsey et al. Bioconjug. Chem. (2001)
12:346-53.
[0316] Molecular Biological Techniques
[0317] In practicing the present invention, many conventional
techniques in molecular biology, microbiology, and recombinant DNA
technology are optionally used (e.g., for making and/or
manipulating nucleic acids, polypeptides, and/or cells of the
invention). These techniques are well known, and detailed protocols
for numerous such procedures (including, e.g., in vitro
amplification of nucleic acids, cloning, mutagenesis,
transformation, cellular transduction with nucleic acids, protein
expression, and/or the like) are described in, for example, Berger
and Kimmel, Guide to Molecular Cloning Techniques, Methods in
Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
(Berger); Sambrook et al., Molecular Cloning--A Laboratory Manual
(3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York, 2002 ("Sambrook") and Current Protocols in
Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, a
joint venture between Greene Publishing Associates, Inc. and John
Wiley & Sons, Inc., (supplemented through 2004) ("Ausubel")).
Other useful references, e.g. for cell isolation and culture (e.g.,
for subsequent nucleic acid or protein isolation) include Freshney
(1994) Culture of Animal Cells, a Manual of Basic Technique, third
edition, Wiley-Liss, New York and the references cited therein;
Payne et al (1992) Plant Cell and Tissue Culture in Liquid Systems
John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips
(Eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental
Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New
York) and Atlas and Parks (Eds.) The Handbook of Microbiological
Media (1993) CRC Press, Boca Raton, Fla.
[0318] Oligonucleotide Synthesis
[0319] In general, synthetic methods for making oligonucleotides
and PNAs (including labeled oligos and PNAs) are well known. For
example, oligonucleotides can be synthesized chemically according
to the solid phase phosphoramidite triester method described by
Beaucage and Caruthers (1981), Tetrahedron Letts.,
22(20):1859-1862, e.g., using a commercially available automated
synthesizer, e.g., as described in Needham-VanDevanter et al.
(1984) Nucleic Acids Res., 12:6159-6168. Synthesis of PNAs and
modified oligonucleotides (e.g., oligonucleotides comprising
2'-O-methyl nucleotides and/or phosphorothioate, methylphosphonate,
or boranophosphate linkages) are described in e.g.,
Oligonucleotides and Analogs (1991), IRL Press, New York; Shaw et
al. (1993), Methods Mol. Biol. 20:225-243; Nielsen et al. (1991),
Science 254:1497-1500; and Shaw et al. (2000) Methods Enzymol.
313:226-257.
[0320] Oligonucleotides, including modified oligonucleotides (e.g.,
oligonucleotides comprising fluorophores and quenchers, unnatural
nucleotides, 2'-O-methyl nucleotides, and/or phosphorothioate,
methylphosphonate, or boranophosphate linkages) can also be ordered
from a variety of commercial sources known to persons of skill.
There are many commercial providers of oligo synthesis services,
and thus, this is a broadly accessible technology. Any nucleic acid
can be custom ordered from any of a variety of commercial sources,
such as The Midland Certified Reagent Company (www.mcrc.com), The
Great American Gene Company (www.genco.com), ExpressGen Inc.
(www.expressgen.com), QIAGEN (http://oligos.qiagen.com), Dharmacon
(www.dharmacon.com), and many others.
[0321] A variety of nuclease-resistant nucleic acids can optionally
be created, e.g., comprising modified nucleotides and/or modified
internucleotide linkages such as those currently used in the
synthesis of antisense oligonucleotides. For example, a nuclease
resistant oligonucleotide can comprise one or more 2'-O-methyl
nucleotides. For example, an oligonucleotide comprising standard
deoxyribonucleotides can also comprise one or more 2'-O-methyl
nucleotides (e.g., at its 5' end), or an oligonucleotide can
consist entirely of 2'-O-methyl nucleotides. As another example, a
nuclease resistant oligonucleotide can comprise one or more
phosphorothioate linkages (oligonucleotides comprising such
linkages are sometimes called "S-oligos"). An oligonucleotide can
comprise, e.g., only phosphorothioate linkages or a mixture of
phosphodiester and phosphorothioate linkages. In other embodiments,
the oligonucleotide comprises one or more methylphosphonate
linkages, one or more boranophosphate linkages, or the like.
Combinations of typical nuclease resistance modification strategies
can also be employed; for example, a nuclease resistant
oligonucleotide can comprise both 2'-O-methyl nucleotides and
phosphorothioate linkages.
[0322] As noted, a nucleic acid can be produced by chemical
synthesis or can be custom ordered. In addition, nucleic acids can
be produced by enzymatic synthesis (in vitro or in vivo). For
example, interfering RNAs can be produced by in vitro transcription
using techniques well known in the art. Kits for in vitro
transcription are commercially available; for example, the
Silencer.TM. siRNA construction kit from Ambion, Inc.
(www.ambion.com).
[0323] Polypeptide Production
[0324] Polypeptides (e.g., polypeptide cellular delivery modules,
e.g., protein transduction domains) can optionally be produced by
expression in a host cell transformed with a vector comprising a
nucleic acid encoding the desired polypeptide(s). Expressed
polypeptides can be recovered and purified from recombinant cell
cultures by any of a number of methods well known in the art,
including ammonium sulfate or ethanol precipitation, acid
extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography (e.g., using any of the
tagging systems noted herein), hydroxylapatite chromatography, and
lectin chromatography, for example. Protein refolding steps can be
used, as desired, in completing configuration of the mature
protein. Finally, high performance liquid chromatography (HPLC) can
be employed in the final purification steps. See, e.g., the
references noted above and Deutscher, Methods in Enzymology Vol.
182: Guide to Protein Purification, Academic Press, Inc. N.Y.
(1990); Sandana (1997) Bioseparation of Proteins, Academic Press,
Inc.; Bollag et al. (1996) Protein Methods, 2.sup.nd Edition
Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana
Press, NJ; Harris and Angal (1990) Protein Purification
Applications: A Practical Approach IRL Press at Oxford, Oxford,
U.K.; Scopes (1993) Protein Purification: Principles and Practice
3.sup.rd Edition Springer Verlag, NY; Janson and Ryden (1998)
Protein Purification: Principles, High Resolution Methods and
Applications, Second Edition Wiley-VCH, NY; and Walker (1998)
Protein Protocols on CD-ROM Humana Press, NJ.
[0325] Alternatively, cell-free transcription/translation systems
can be employed to produce polypeptides encoded by nucleic acids. A
number of suitable in vitro transcription and translation systems
are commercially available. A general guide to in vitro
transcription and translation protocols is found in Tymms (1995) In
vitro Transcription and Translation Protocols: Methods in Molecular
Biology Volume 37, Garland Publishing, NY.
[0326] In addition, polypeptides (including, e.g., polypeptides
comprising fluorophores and quenchers and/or unnatural amino acids)
can be produced manually or by using an automated system, by direct
peptide synthesis using solid-phase techniques (see, e.g., Stewart
et al. (1969) Solid-Phase Peptide Synthesis, WH Freeman Co, San
Francisco; Merrifield J (1963) J. Am. Chem. Soc. 85:2149-2154).
Exemplary automated systems include the Applied Biosystems 431A
Peptide Synthesizer (Perkin Elmer, Foster City, Calif.). In
addition, there are many commercial providers of peptide synthesis
services. If desired, subsequences can be chemically synthesized
separately, and combined using chemical methods to provide
full-length polypeptides.
EXAMPLES
[0327] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
Accordingly, the following examples are offered to illustrate, but
not to limit, the claimed invention.
[0328] PA Sensors: Constructs and Methods for Measuring RNA
Transcripts in Living Cells
[0329] In one aspect, the present invention provides sensors for
detecting and measuring mRNA in living cells (also known as PAC
probes for mRNA) and methods of controlling activation of such mRNA
sensors in living cells. In one class of embodiments, the sensor is
attached to one or more photo-labile groups that protect the sensor
from extra-cellular and intra-cellular degradation and, at the same
time, inactivate the sensor. Upon exposure to light of a specific
wavelength, the photolabile groups detach from the sensor and the
sensor becomes active. The mRNA sensors include one or more labels
(e.g., a combination of acceptor and donor fluorophores that
interact via FRET or ET or a fluorophore/quencher combination) on
RNAs that can initiate RNAi (e.g., siRNA, shRNA; FIGS. 12-14, in
which A and B represent either a fluorescent label and a quencher
(or vice versa) or a donor and acceptor (or vice versa)). The
signal from the sensor is used to detect and measure mRNA in living
cells. Splice variants of mRNAs, for example, can also be analyzed
using interfering RNA approaches.
[0330] Traditional or novel delivery methods can be used to
introduce a sufficient quantity of mRNA sensors into cells. A high
throughput uncaging device, such as those described in U.S. Ser.
No. 60/427,664 filed Nov. 18, 2002, 60/436,855 filed Dec. 26, 2002,
60/439,917 filed Jan. 13, 2003, 60/451,177 filed February 27, and
60/456,870 filed Mar. 21, 2003, can be used to activate
photoactivatable sensors, e.g., in cells grown in a microtiter
plate. This invention also features methods of detecting and
measuring mRNA with such sensors in living cells.
[0331] The ability to monitor immediate changes in mRNA levels in
living cells facilitates the development of a broad range of
cell-based assays for basic research, pharmaceutical industries,
clinical and agricultural diagnostics. For example, a specific GPCR
or kinase cell-based assay can be developed for screening lead
compounds using one or more PAC probes for monitoring mRNAs
downstream of the GPCR or kinase. Actual transcript or surrogate
transcript (marker, mRNA of a gene further downstream in a pathway)
response to modulation of specific pathways by the compounds can be
monitored in living cells.
[0332] An example PAC probe for an mRNA comprises a labeled
interfering RNA (e.g., an siRNA or a shRNA; see, e.g., Watanabe
(Jan. 13, 2003) Scientist 17(1):36; D Engelke (2002) Nature Biotech
29: 505; Trends in Biotech 20:49 (2002)); one or more caging
groups, e.g., photolabile caging groups (see, e.g., F R Haselton
JBC 274:20895 and H Okamoto (2001) Nature Genetics 28:317); and
optionally a cellular and/or subcellular delivery module, e.g., a
peptide delivery module such as TAT or Antp (see, e.g., Lane (2001)
Bioconjugate Chemistry 12:825).
[0333] Modified nucleotides can optionally be incorporated into
interfering RNAs to reduce degradation in cells. For example, a
phosphate backbone analog (e.g., phosphorothioate and/or a modified
nucleotide (for example, a 2'-O-methyl nucleotide, e.g.,
2'-O-methylinosine) can be used to protect the RNA from nuclease
digestion. Caging groups can also protect against nuclease
digestion.
[0334] FIG. 12 describes a small interfering RNA (siRNA, also known
as short interfering RNA) structure used for detecting mRNA in
living cells. The siRNA can be, for example, a 21-25 mer
double-stranded RNA; other lengths and/or optional overhangs (e.g.,
two nucleotide 3' overhangs) can also be used. A reporter
combination (e.g., a fluorophore/quencher pair or acceptor/donor
FRET pair) is linked at the 5' and 3' ends of one strand or at the
ends of opposite strands. The reporter molecules can also be within
the siRNA, either on the same strand or on opposite strands of the
double stranded siRNA. The reporters can be, e.g., a combination of
FRET dyes such as coumarin and FITC or a combination such as
europium and APC that permits application of time-resolved
fluorescence (TRF) techniques.
[0335] An interfering RNA can be caged, e.g., with photo-labile
groups, at the phosphates, riboses and/or bases to protect it and
to inactivate its function. It can optionally be linked to a
delivery module, e.g., a peptide delivery module (for example,
8-D-Arg, Antp, Pep-1, or the like), e.g., with a disulfide linker
as illustrated in FIG. 14. Other established delivery approaches
can also be used, e.g., lipofection.
[0336] FIG. 13 shows another type of interfering RNA for measuring
mRNA, e.g., a short hairpin RNA (shRNA, also called small hairpin
RNA; e.g., Nature Genetics 33:396). For example, a shRNA can have
about 60-70 nt that form a hairpin, e.g., with a 25-30 mer
double-stranded region and an 8 mer single-stranded loop. A
reporter combination (e.g., a donor and an acceptor fluorophore
that interact via FRET, or a fluorophore/dark quencher) can be
attached for signaling the presence of a specific RNA transcript.
As in the previous example, the shRNA can be caged, e.g., with
photolabile caging groups.
[0337] FIG. 15 shows the detection of mRNA using an interfering RNA
PAC probe. The siRNA is incorporated into the RISC complex, and the
antisense strand guides cleavage of the target mRNA (promoting its
degradation). Strand separation of the interfering RNA probe leads
to the separation of the reporter molecules on the RNA, resulting
in a detectable signal or change in signal (as indicated by the
starburst symbol). Multiple mRNA transcripts can be analyzed using
interfering RNAs with different reporter molecules (e.g.,
fluorophores that emit at different wavelengths).
[0338] FIG. 16 shows the detection of a single target using
multiple (e.g., two or more) interfering RNA sensors. The different
interfering RNAs typically emit distinguishable signals before
and/or after initiation of RNAi. Detection specificity is improved
using this design, because an actual signal or signal change
(indicating degradation of the specific target mRNA) is recorded
only when signals from both interfering RNAs are observed at about
the same time.
[0339] Applications
[0340] FIG. 17 shows an example workflow for mRNA measurement using
the sensors of this invention, where the effect of a compound
(drug, agonist, antagonist, etc. affecting or potentially affecting
an upstream signaling molecule) on mRNA level is monitored. There
are minimal fluidic handling steps and reagents required. A
photolabile PAC probe can be uncaged by exposing to a light source
(e.g., in an uncaging device such as those described in U.S. Ser.
No. 60/427,664 filed Nov. 18, 2002, 60/436,855 filed Dec. 26, 2002,
60/439,917 filed Jan. 13, 2003, 60/451,177 filed February 27, and
60/456,870 filed Mar. 21, 2003).
[0341] A PAC probe for mRNA can be used to measure amount of mRNA
transcript and location of mRNA processing in living cells. When
performing quantitative analysis, an interfering RNA sensor for a
house-keeping RNA can optionally be used to normalize for variable
target(s). Deviation between different cells can be corrected if
one or more dual-labeled FRET interfering RNAs, for example, are
used instead of a dark quencher/fluorophore probe format. With a
dual-labeled FRET probe (i.e., a probe with a donor fluorophore and
an acceptor fluorophore, where the donor and acceptor are capable
of exhibiting FRET), at least two different signals can be
obtained, i.e., the FRET signal (emission by the acceptor following
stimulation of the donor) and the acceptor signal (emission by the
acceptor following stimulation of the acceptor) using different
excitation wavelengths, e.g., produced by different lasers, to
stimulate the donor and acceptor. The ratio of these two signals
can be taken, e.g., to normalize for transfection efficiency of the
probe.
[0342] As noted, interfering RNA PAC probes can be used to analyze
splice variants (including, e.g., in living cells). Examples of
genes with a variety of splice variants are beta-actin and cyclic
nucleotide phosphodiesterases (Current Opinion in Cell Biology
(2000) 12:174-179), among many others. To analyze alternatively
spliced mRNAs, for example, a siRNA probe can be designed to
recognize the splice junction. One or more such siRNAs can be used
to detect various isoforms. For example, FIG. 18 illustrates how
multiple siRNAs can be used to determine splice variants. A nuclear
RNA containing three exons and two introns is transcribed from
chromosomal DNA. The nuclear RNA is spliced to form the mRNA, which
in this example includes all three exons and no introns. A siRNA is
designed to be at the splice junction. The isoform with the correct
splice variant is digested. Similarly, siRNA can be made to bind to
the exon regions and not between the splice junctions, or a siRNA
can be designed against an intron. Splice variants containing the
intron are digested and result in a signal from the siRNA
probe.
[0343] Cell Based Assay using Labeled Interfering RNA as in vivo
mRNA Sensor
[0344] The following sets forth a series of experiments that
demonstrate design and use of interfering RNA sensors to detect
GAPDH mRNA. GAPDH is constitutively expressed.
[0345] Three different interfering RNAs were designed against GAPDH
(FIG. 19; SEQ ID NO:1): RNAi 1, corresponding to nt 690-708 (each
strand has 19 GAPDH bases plus a TT 3' overhang), RNAi 2,
corresponding to nt 915-936 (each strand has 21 GAPDH bases,
forming a 19 bp double-stranded region and two nucleotide
overhangs), and RNAi 3, corresponding to nt 601-621 (each strand
has 21 GAPDH bases, forming a 19 bp double-stranded region and two
nucleotide overhangs). Each RNA was labeled with 6-FAM on the 3'
end of the antisense strand, and a Dabcyl quencher was attached to
the 3' end of the sense strand. The FAM label and Dabcyl quencher
were incorporated during oligonucleotide synthesis. FIG. 20
illustrates one of the three GAPDH RNAi sensors. When the sense and
antisense strands are annealed, the FAM label is quenched (Panel
A); when the strands are denatured, the label is not quenched and
fluoresces (Panel B). Panel C shows fluorescent emission spectra
for the antisense strand (curve 1), the sense strand (curve 2), and
the annealed strands (curve 3), illustrating that the FAM label is
quenched in the annealed sensor.
[0346] To verify that the labeled RNAs were able to attenuate
expression of GAPDH, labeled RNAi 1-3 were lipofected into HeLa
cells (1000 cells) at a concentration of 0.5 .mu.g/.mu.l for 4
hours. Cells were maintained at two temperatures (37.degree. C. and
45.degree. C.) and lysed at different time points after lipofection
(4 h, 10 h, 20 h, 34 h, and 44 h). GAPDH mRNA was measured using a
branched DNA (bDNA) assay (see, e.g., Journal of Clinical Virology
(2002) 25:205-216; QuantiGene bDNA assay kits are commercially
available from Genospectra, Inc., www.genospectra.com).
[0347] FIG. 21 shows the GAPDH mRNA level as measured by the bDNA
assay at the indicated time points after lipofection of labeled
RNAi 1 (Panel A), as compared to a negative control (Panel B, no
lipofection reagent). FIG. 22 compares the percentage knockout of
GAPDH expression, as measured by the bDNA assay, for labeled RNAi
1-3. RNAi 1 was the most potent silencer of the three interfering
RNAs tested, knocking out GAPDH expression in HeLa cells by as much
as 90%.
[0348] To test the labeled RNAi's as in vivo mRNA sensors, the
three GAPDH RNAis (labeled with 6-FAM and Dabcyl) were lipofected
into HeLa cells (1000 cells) at a concentration of 0.5 .mu.g/.mu.l
for 4 h at 37.degree. C. The cells were incubated with fresh medium
at 37.degree. C. Cells were fixed 4 h and 20 h after lipofection
and scanned on a Packard scanner for FITC signal, and bDNA assays
were performed at the same time points. FIG. 23 shows the results
of the bDNA assays (RLU, luminescence) compared to the FITC signals
(FLU) for cells lipofected with the RNAi 1 (Panel A), RNAi 2 (Panel
B), and RNAi 3 (Panel C) sensors. We observed opposing trends over
time between the signals for the labeled RNAi sensors (increased
FITC signal, reflecting degradation of GAPDH mRNA) and the bDNA
data (reduced GAPDH mRNA level in the presence of interfering
RNA).
[0349] FIG. 24 shows the ratio of the bDNA assay measurement of
GAPDH mRNA levels at 20 h/4 h and the ratio of the FITC signal from
labeled RNAi's 1-3 at 20 h/4 h, and demonstrates that RNAi 1 is the
most effective in silencing GAPDH and the most prominent in
generating FRET signal.
[0350] Labeled RNAi 1 was further tested as an in vivo mRNA sensor.
2000 HeLa cells were plated in each well in eight well chambers
with complete DMEM medium overnight at 37.degree. C. Medium was
changed to OptiMEM, and the cells were lipofected with GAPDH RNAi 1
(2 .mu.g, 4 .mu.g) for 4 h in reduced serum medium at 37.degree. C.
At three different time points (0 h, 4 h, and 10 h after
lipofection), duplicate slides were plated. One slide was used for
a bDNA assay, the other for scanning the FAM signal. For the bDNA
assay, cells were lysed with bDNA lysis buffer at 0 h, 4 h, and 10
h time points, and lysate from approximately 300 cells was assayed
for GAPDH mRNA using the bDNA assay. (Note that "0 h" is the time
point after the lipofection process, which takes about 4 hours.) At
each time point, the duplicate slide was fixed and scanned on a
Packard scanner in the FAM channel at 90% power and 70% PMT
gain.
[0351] Fluorescent signal from the RNAi 1 sensor increased over
time (from 0 to 4 h and from 4 to 10 h) at both amounts of sensor
tested (data not shown). Fluorescent signal from the sensor also
increased with increasing amount of sensor; 4 .mu.g of RNAi 1
produced a more intense signal than 2 .mu.g at each time point. At
both amounts of RNAi 1 tested (2 .mu.g and 4 .mu.g), the level of
GAPDH mRNA as measured by the bDNA assay typically decreased over
time.
[0352] An additional test of labeled RNAi 1 as an in vivo mRNA
sensor was performed. 2000 HeLa cells were plated in each well in
eight well glass slides with complete DMEM medium overnight at
37.degree. C. Medium was changed to OptiMEM, and the cells were
lipofected with 4 .mu.g of GAPDH RNAi 1 for 4 h in reduced serum
medium at 37.degree. C. At three different time points (0 h, 4 h,
and 10 h after lipofection), duplicate slides were plated. One
slide was used for a bDNA assay, the other for scanning the FAM
signal. For the bDNA assay, cells were lysed with bDNA lysis buffer
at 0 h, 4 h, and 10 h time points, and lysate from approximately
350 cells was assayed for GAPDH mRNA using the bDNA assay. (Note
that "0 h" is the time point after the lipofection process, which
takes about 4 hours.) At each time point, the duplicate slide was
fixed and scanned on a Packard microarray scanner in the FAM
channel at 90% power and 60% PMT gain; for each well, fluorescent
signal from the entire well was analyzed. The experiment was
performed in duplicate on three independent days, and the resulting
data were averaged to obtain the results plotted in FIG. 25.
[0353] FIG. 25 Panel A shows the results of the bDNA assay
(diamonds, RLU, representing the GAPDH mRNA level in the cells at
the indicated time points) and the fluorescent signal for the
labeled RNAi 1 mRNA PAC probe (circles, RFU), at each time point.
FIG. 25 Panel B plots the fluorescent signals from the labeled RNAi
1 sensor against the results of the bDNA assay. We note an inverse
linear relationship between the signal from the interfering RNA
sensor and the amount of GAPDH mRNA remaining in the cells.
[0354] It is worth noting that in the examples above, the level of
fluorescent signal from the siRNA sensor is correlated to the
cumulative destruction of GAPDH mRNA in the cells. As more GAPDH
mRNA gets degraded, the signal from the sensor increases. (Clearly,
in these examples, the increase in sensor signal level from 0 h to
4 h to 10 h (e.g., for the RNAi 1 sensor in FIG. 25) does not mean
that the GAPDH mRNA level is increasing.) Therefore, for
transcripts already abundant in cells (e.g., constitutively
expressed genes, such as GAPDH), an siRNA sensor can provide an
indication of the knock-down efficiency of the siRNA. The methods
can similarly be applied to determine the knock-down efficiency of
an siRNA against an inducible target mRNA.
[0355] In summary, we conclude from the above experiments that the
magnitude of the FRET signal for the FAM label on the RNA sensor
correlates to the level of GAPDH expression knockdown as measured
by the bDNA assay and inversely correlates with the level of GAPDH
remaining in the cell, confirming that labeled GAPDH interfering
RNA functions as an inhibitor sensor.
[0356] Discussion
[0357] It will be evident to one of skill that the methods of
detecting target mRNA in a cell using a labeled interfering RNA
sensor described herein have a number of applications, and that the
signal output detected from the sensor can provide different types
of information under different circumstances. The signal output is
typically proportional to the amount of target mRNA degraded;
depending on the circumstances, the signal output can be, e.g.,
proportional to the amount of target mRNA initially present or
induced in the cell and/or inversely proportional to the amount of
mRNA remaining in the cell, as illustrated in the following
examples.
[0358] For example, the methods can be used to determine how
effective any given siRNA is at knocking down (or knocking out)
expression of its target mRNA, e.g., in real time in living cells.
The siRNA can be labeled to produce an siRNA sensor, which can be
used in the methods described herein. For example, in the
experiments described above, the signal output from RNAi 1 is
stronger than that from RNAi 2, indicating that RNAi 1 leads to the
degradation of more GAPDH mRNA then RNAi 2, and thus indicating
that RNAi 1 is better at knocking down GAPDH expression then is
RNAi 2 (see, e.g., FIGS. 22-24).
[0359] As another example, the methods can be used for real-time,
dynamic monitoring of target mRNA levels. The experiment summarized
in FIG. 25, for example, illustrates an inverse linear relationship
between target mRNA levels and signal output from the sensor. In
this example, a stronger fluorescent signal from the sensor
indicates more of the constitutively expressed GAPDH transcript has
been degraded and thus that less of the transcript is currently
present in the cell.
[0360] The methods can be used to monitor both constitutively
expressed and/or inducible target mRNA levels. Thus, in yet another
example, the methods can be used to detect expression of an
inducible gene, e.g., in real time in living cells. For example, an
siRNA sensor for an inducible target gene (e.g., IL-8) can be
introduced into cells, expression of the target gene can be
induced, and the level of signal from the siRNA sensor (e.g., the
slope, intercept(s), and/or maximum value(s) from a plot of signal
strength versus time) can be used as an indication of the onset of
target gene expression and/or the degree of induction of the target
gene. In this example, the level of fluorescent signal from the
siRNA sensor is correlated to the degree of induction. As more
target gene transcript becomes available for RNA interference, the
sensor signal increases. Again, the level of sensor signal reflects
the amount of transcript being destroyed and not the final level of
the inducible transcript. Since the amount of transcript destroyed
is proportional to the degree of induction of the inducible gene,
the level of sensor signal is proportional to the degree of
induction: a stronger signal indicates stronger induction (more
transcripts destroyed).
[0361] As yet another example, a caged siRNA sensor can be used in
the methods to detect the mRNA level of a target gene, e.g., in
real time in living cells. The caged siRNA sensor is put into the
cells and is then uncaged (e.g., at a preselected time). The level
of signal from the sensor can be used as a measurement of the
transcript level immediately prior to uncaging. Preferably, in
these example embodiments, the concentration of the caged siRNA
sensor is higher than the concentration of the target mRNA. The
slope, intercept(s), and/or maximum value(s) from a plot of signal
strength versus time after uncaging, for example, can be used to
reflect the target mRNA level at the time of uncaging. Again in
this example, a stronger signal from the sensor indicates
degradation of more target mRNA and thus a higher concentration of
the target mRNA in the cell at the time of uncaging.
[0362] In vivo Photoactivation of Photolabile Caged siRNA
[0363] The following sets forth a series of experiments that
demonstrate use of a photolabile caged siRNA to control initiation
of RNAi of the GAPDH mRNA.
[0364] In vivo Photoactivation
[0365] The 5' phosphate of the antisense strand of RNAi 1 was caged
(FIG. 28). The caged antisense oligo (5'
PhotoCageAGUAGAGGCAGGGAUGAUGdTdT 3', SEQ ID NO:2) was synthesized
by Trilink Biotechnologies, Inc. (www.trilinkbiotech.com), as
follows. The commercially available caged phosphoramidite
[1-N-(4,4'-Dimethoxytrityl)-5-(6-biotinamidocaproamidomet-
hyl)-1-(2-nitrophenyl)-ethyl]-2-cyanoethyl-(N,N-diisopropyl)-phosphoramidi-
te (PC Biotin Phosphoramidite, from Glen Research Corp.,
www.glenres.com) was coupled to the 5' terminus of a 21-mer
oligoribonucleotide using standard phosphoramidite chemistry.
Following the coupling step, oxidation, and cleavage from the
resin, the caged oligoribonucleotide was purified using RNase-free
HPLC purification and verified using gel electrophoresis analysis
and mass spectrometry. An oligoribonucleotide corresponding to the
sense strand was also synthesized (5' CAUCAUCCCUGCCUCUACUdTdT 3',
SEQ ID NO:3), and equimolar amounts of the sense and caged
antisense strands were annealed to form the caged RNAi 1. An RNAi 1
siRNA which did not contain the caging group was also
synthesized.
[0366] HeLa cells were lipofected with 100 nM RNAi 1, caged RNAi 1,
or caged RNAi 1 that had been uncaged in vitro, using
Lipofectamine.TM. 2000 (Invitrogen, www.invitrogen.com) according
to the manufacturer's instructions. In brief, 5000 HeLa cells were
plated evenly into each well of 96 well Coming Costar black clear
bottom plates in 200 .mu.L of Dulbecco modified Eagle medium
(DMEM). The cells were incubated at 37.degree. C. for 16-24 h, and
then visually examined to ensure that each well was 70-90%
confluent and that the culture was evenly distributed in each well.
For each well, 0.25 .mu.g of the appropriate siRNA was diluted in
25 .mu.L OptiMEM and incubated at room temperature for 4 min; 0.5
.mu.L of Lipofectamine 2000 was also diluted in 25 .mu.L OptiMEM
and incubated at room temperature for 4 min. The siRNA and the
lipofection reagent were then combined, mixed gently, and incubated
at room temperature for 20 min, the volume was adjusted to 175
.mu.L with OptiMEM, medium was aspirated from the well containing
the HeLa cells, and the siRNA-lipofection reagent complex was added
to the cells. Plates were then incubated for 4 h at 37.degree. C.
with gentle shaking, then the medium was replaced with 200 .mu.L
fresh complete DMEM.
[0367] To cleave the caging group from the caged siRNA, following
the 4 h lipofection with caged RNAi 1, cells were exposed from the
bottom of the well to 1 J/cm.sup.2 365 nm UV light. Uncaging light
was produced by a BlueWave.TM. UV Spot Light System fitted with a
Lightguide mount assembly, Cool Blue.TM. filter, and Lightguide rod
lens assembly (Dymax Corp., www.dymax.com, part numbers 38600,
38670, and 38699). Cells were incubated at 37.degree. C. and lysed
at different time points after uncaging: 0 h (immediately after
uncaging), 6 h and 10 h after uncaging.
[0368] As controls, cells lipofected with unmodified RNAi 1, cells
lipofected with caged RNAi 1 but not exposed to uncaging light, and
cells lipofected with caged RNAi 1 that had previously been uncaged
in vitro by exposure to 12 J/cm.sup.2 of UV light were also
maintained at 37.degree. C. and lysed at 0 h (immediately following
lipofection, corresponding to the 0 h time point for the in vivo
uncaged caged siRNA above), 6 h, and 10 h.
[0369] GAPDH mRNA was measured with a branched DNA assay using a
Quantigene Explore bDNA assay kit (Genospectra, Inc.) according to
the instructions supplied with the kit. To normalize for cell
number, GAPDH expression was normalized to cyclophilin expression
(also measured with a bDNA assay).
[0370] FIG. 29 Panel A shows GAPDH expression normalized to
cyclophilin expression in untransfected cells and cells transfected
with: RNAi 1 (unmodified), in vitro uncaged caged RNAi 1, caged
RNAi 1 (transected cells were not exposed to light), and in vivo
uncaged caged RNAi 1, as measured by the bDNA assay at the
indicated time points after uncaging (or just lipofection). FIG. 29
Panel B shows the relative GAPDH mRNA level as measured by the bDNA
assay at the indicated time points after uncaging (or just
lipofection) of: RNAi 1 (unmodified), in vitro uncaged caged RNAi
1, caged RNAi 1 (transected cells were not exposed to light), and
in vivo uncaged caged RNAi 1. Expression is normalized to that of
cells transfected with the unmodified RNAi 1. Comparing relative
GAPDH expression in cells transfected with unmodified RNAi 1 and
caged RNAi 1 indicates that the caging group inhibits initiation of
RNAi by the caged siRNA; levels of GAPDH mRNA are higher for cells
transfected with the caged siRNA but not exposed to light than for
cells transfected with unmodified RNAi 1 at all three time points.
Removal of the caging group restores the ability of the siRNA to
participate in RNAi, since relative GAPDH levels in cells
transfected with caged RNAi 1 and then exposed to UV light are
close to the levels in cells transfected with RNAi 1 at 6 and 10 h
after uncaging.
[0371] Enhanced Delivery and in vivo Photoactivation
[0372] RNAi 1 caged at the 5' phosphate of the antisense strand
(FIG. 28) was produced as described above. HeLa cells were
lipofected with 3 nM caged RNAi 1, caged RNAi 1 that had been
uncaged in vitro, or a scrambled GAPDH negative control siRNA
(Ambion catalog no. 4605, www.ambion.com), using Lipofectamine.TM.
2000 (Invitrogen, www.invitrogen.com). In brief, 5000 HeLa cells
were plated overnight in each well of 96 well black clear bottom
plates. Cells were transfected using Lipofectamine.TM. 2000, 3 nM
of the relevant siRNA, and 27 nM supercoiled pcDNA.TM.3.1 plasmid
(Invitrogen). Cells were exposed to the lipofection complex for 4 h
in minimal media.
[0373] Mixing the siRNA with plasmid to form the lipofection
complex permits use of lower concentrations of the siRNA than does
forming the lipofection complex with siRNA in the absence of
plasmid. Use of such lower concentrations of caged siRNA can be
advantageous, since any uncaged siRNA contaminating the caged siRNA
is thus also introduced into the cells at a lower concentration,
for example. As another example, using lower concentrations of the
siRNA can decrease the risk of off-target effects (in which the
siRNA affects expression of an mRNA that is not the desired
target).
[0374] To cleave the caging group from the caged siRNA, following
the 4 h lipofection with caged RNAi 1, cells were exposed from the
bottom of the well to 1.4 J/cm.sup.2 365 nm UV light. Cells were
incubated at 37.degree. C. and lysed at different time points after
uncaging: 0 h (immediately after uncaging), 20 h and 44 h after
uncaging.
[0375] As controls, cells lipofected with the scrambled negative
control siRNA, cells lipofected with caged RNAi 1 but not exposed
to uncaging light, and cells lipofected with caged RNAi 1 that had
previously been uncaged in vitro by exposure to UV light were also
maintained at 37.degree. C. and lysed at 0 h (immediately following
lipofection, corresponding to the 0 h time point for the in vivo
uncaged caged siRNA above), 20 h, and 44 h.
[0376] GAPDH mRNA was measured with a branched DNA assay using a
Quantigene Explore bDNA assay kit (Genospectra, Inc.) according to
the instructions supplied with the kit and normalized to
cyclophilin expression.
[0377] FIG. 33 shows GAPDH expression normalized to cyclophilin
expression in cells transfected with: the scrambled GAPDH negative
control siRNA, caged RNAi 1 (transected cells were not exposed to
light), in vitro uncaged caged RNAi 1, and in vivo uncaged caged
RNAi 1, as measured by the bDNA assay at the indicated time points
after uncaging (or just lipofection). Normalized expression is
shown relative to GAPDH expression in cells transfected with the
scrambled GAPDH negative control siRNA. FIG. 33 demonstrates that
the photoactivated RNAi 1 remains active in the cells for at least
44 h, while the unexposed caged RNAi 1 remains inactive for at
least 44 h. It also shows that transfection with 3 nM of the caged
RNAi in the presence of plasmid is sufficient to reduce GAPDH
expression following photoactivation.
[0378] In vivo Photoactivation at Different Light Dosages
[0379] RNAi 1 caged at the 5' phosphate of the antisense strand was
produced as described above. HeLa cells were lipofected with 3 nM
caged RNAi 1. In brief, 5000 HeLa cells were plated overnight in
each well of 96 well black clear bottom plates. Cells were
transfected using Lipofectamine 2000, 3 nM caged RNAi 1, and 27 nM
pcDNA.TM. 3.1 plasmid (Invitrogen). Cells were exposed to the
lipofection complex for 4 h in minimal media.
[0380] Following the 4 h lipofection with caged RNAi 1, cells were
exposed from the bottom of the well to varying energy densities of
365 nm UV light to cleave the caging group from the caged siRNA.
Cells were incubated at 37.degree. C. and lysed at 0 h (immediately
after uncaging) and 20 h after uncaging. GAPDH mRNA was measured
with a branched DNA assay using a Quantigene Explore bDNA assay kit
(Genospectra, Inc.) according to the instructions supplied with the
kit and normalized to cyclophilin expression.
[0381] FIG. 34 shows normalized GAPDH expression in cells
transfected with caged RNAi 1 as measured by the bDNA assay at 0 h
and 20 h after uncaging in vivo with varying doses of light (0.02
J/cm.sup.2, 0.1 J/cm.sup.2, 0.5 J/cm.sup.2, and 1.4 J/cm.sup.2).
GAPDH expression in cells transfected with caged RNAi 1 but not
exposed to uncaging light (0.0 J/cm.sup.2) is also shown. FIG. 34
demonstrates that suppression of GAPDH expression increases with
higher doses of light, indicating that increasing light dosage
photoactivates more of the caged siRNA. The caged siRNA can thus be
partially or completely photoactivated in vivo, as desired, by
controlling the energy density of the uncaging light to which the
cells are exposed (e.g., by controlling the intensity of the
uncaging light and/or the duration of exposure).
[0382] While the foregoing invention has been described in some
detail for purposes of clarity and understanding, it will be clear
to one skilled in the art from a reading of this disclosure that
various changes in form and detail can be made without departing
from the true scope of the invention. For example, all the
techniques and apparatus described above can be used in various
combinations.
[0383] All publications, patents, patent applications, and/or other
documents cited in this application are incorporated by reference
in their entirety for all purposes to the same extent as if each
individual publication, patent, patent application, and/or other
document were individually indicated to be incorporated by
reference for all purposes.
Sequence CWU 1
1
3 1 1273 DNA Homo sapiens 1 ctctctgctc ctcctgttcg acagtcagcc
gcatcttctt ttgcgtcgcc agccgagcca 60 catcgctcag acaccatggg
gaaggtgaag gtcggagtca acggatttgg tcgtattggg 120 cgcctggcac
cagggctgct tttaactctg gtaaagtgga tattgttgcc atcaatgacc 180
ccttcattga cctcaactac atggtttaca tgttccaata tgattccacc catggcaaat
240 tccatggcac cgtcaggctg agaacgggaa gcttgtcatc aatggaaatc
ccatcaccat 300 cttccaggag cgagatccct ccaaaatcaa gtggggcgat
gctggcgctg agtacgtcgt 360 ggagtccact ggcgtcttca caccatggag
aaggctgggg ctcatttgca ggggggagcc 420 aaaagggtca tcatctctgc
cccctctgct gatgccccca tgttcgtcat gggtgtgaac 480 catgagaagt
atgacaacag cctcaagaca tcagcaatgc ctcctgcacc accaactgct 540
tagcacccct ggccaaggtc atccatgaca actttggtat cgtggaagga ctcatgacca
600 cagtccatgc catcactgcc acccaaagac tgtggatggc ccctccggga
aactgtggcg 660 tgatggccgc ggggctctcc agaacatcat ccctgcctct
actggcgctg ccaaggctgt 720 gggcaaggtc atccctgagc tgacgggaag
ctcactggca tggccttccg tgtccccact 780 gccaacgtgt cagtggtgga
cctgacctgc cgtctagaaa aacctgccaa atatgatgac 840 atcaagaagg
tggtgaagca ggcgtcggag gccccctcaa gggcatcctg ggctacactg 900
agcaccaggt ggtctcctct gacttcaaca gcgacaccca ctcctccacc tttgacgctg
960 gggctggcat tgccctcaac gaccacttgt caagctcatt tcctggtatg
acaacgaatt 1020 tggctacagc aacagggtgg tggacctcat ggcccacatg
gcctccaagg agtaagaccc 1080 ctggaccacc agccccagca agagcacaag
aggagagaga gaccctcact gctggggagt 1140 ccctgccaca ctcagtcccc
caccacactg aatctcccct cctcacagtt gccatgtaga 1200 ccccttgaag
aggggagggg cctagggagc cgcaccttgt atgtaccatc aataaagtac 1260
cctgtgctca acc 1273 2 21 DNA Artificial synthetic siRNA 2
aguagaggca gggaugaugt t 21 3 21 DNA Artificial synthetic siRNA 3
caucaucccu gccucuacut t 21
* * * * *
References