U.S. patent application number 10/173368 was filed with the patent office on 2003-09-18 for multivalent rna aptamers and their expression in multicellular organisms.
Invention is credited to Lis, John T., Shi, Hua.
Application Number | 20030175730 10/173368 |
Document ID | / |
Family ID | 26767697 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030175730 |
Kind Code |
A1 |
Shi, Hua ; et al. |
September 18, 2003 |
Multivalent RNA aptamers and their expression in multicellular
organisms
Abstract
The present invention relates to a monovalent RNA aptamer that
binds to Drosophila splicing factor B52 and a multivalent RNA
aptamer that includes at least two RNA aptamer sequences linked
together. Also disclosed are isolated or constructed DNA molecules
which encode either a monovalent RNA aptamer or a multivalent RNA
aptamer of the present invention, an engineered gene encoding a
multivalent RNA aptamer of the present invention, and host cells
and expression systems which contain either a heterologous DNA
molecule or a heterologous gene of the present invention. Further
aspects of the present invention relate to a method of expressing a
multivalent RNA aptamer in a cell, a method of increasing activity
of a splicing factor protein in a cell, and a method of inhibiting
activity of a target molecule in a cell. A transgenic non-human
organism whose somatic and germ cell lines contain an engineered
gene encoding a multivalent RNA aptamer is also disclosed.
Inventors: |
Shi, Hua; (Ithaca, NY)
; Lis, John T.; (Ithaca, NY) |
Correspondence
Address: |
Michael L. Goldman
NIXON PEABODY LLP
Clinton Square
P.O. Box 31051
Rochester
NY
14603-1051
US
|
Family ID: |
26767697 |
Appl. No.: |
10/173368 |
Filed: |
June 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10173368 |
Jun 14, 2002 |
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09296328 |
Apr 22, 1999 |
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6458559 |
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60082652 |
Apr 22, 1998 |
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Current U.S.
Class: |
435/6.18 ;
435/320.1; 435/325; 435/455; 435/6.1; 536/23.2; 800/8 |
Current CPC
Class: |
C12N 2510/00 20130101;
C12N 15/115 20130101; C12N 2310/127 20130101; A01K 2217/05
20130101; C12N 2310/111 20130101 |
Class at
Publication: |
435/6 ; 435/455;
435/320.1; 435/325; 800/8; 536/23.2 |
International
Class: |
A01K 067/033; C12Q
001/68; C07H 021/04; C12N 015/87 |
Goverment Interests
[0002] This invention was made in part with Government support
under U.S. Public Health Service Grant GM40918 and U.S.D.A. Hatch
Project Grant NY(c)-181413. The Government may have certain rights
to this invention.
Claims
What is claimed:
1. A constructed DNA molecule comprising: a plurality of monomeric
DNA sequences linked together to form a single DNA chain, each
monomeric DNA sequence encoding an independent, functional RNA
molecule.
2. The constructed DNA molecule of claim 1, wherein each of the
plurality of monomeric sequences also encodes a cis-acting
ribozyme.
3. A method of expressing a functional RNA molecule in a cell
comprising: introducing a constructed DNA molecule of claim 1 into
a cell under conditions effective to express the functional RNA
molecule.
4. An engineered gene comprising: the constructed DNA molecule of
claim 1 and a regulatory sequence coupled to the constructed DNA
molecule to control expression thereof.
5. A method of expressing a functional RNA molecule in a cell
comprising: introducing an engineered gene of claim 4 into a cell
under conditions effective to express the functional RNA
molecule.
6. A method of inhibiting activity of a target molecule in a cell
comprising: expressing a multivalent RNA aptamer in a cell, the
multivalent RNA aptamer having an affinity for a target molecule
sufficient to inhibit activity of the target molecule.
7. The method of claim 6 further comprising: introducing into the
cell, prior to said expressing, a DNA molecule encoding the
multivalent RNA aptamer.
8. The method of claim 7, wherein the DNA molecule includes a
promoter sequence which regulates transcription of the DNA
molecule.
9. The method of claim 8, wherein said expressing includes exposing
the cell to conditions effective to induce the promoter sequence to
initiate transcription of the DNA molecule.
10. The method of claim 6, wherein the multivalent RNA aptamer has
at least two RNA aptamer sequences linked together.
11. The method of claim 10, wherein the multivalent RNA aptamer has
five aptamer sequences linked together.
12. The method of claim 6, wherein the target molecule is
Drosophila splicing factor B52.
13. A method of increasing activity of a splicing factor protein
comprising: inserting a multivalent RNA aptamer, which binds to a
splicing factor protein, into an RNA transcript, which contains
exons and introns, under conditions effective to enable splicing of
the RNA transcript.
14. The method of claim 13, wherein said inserting comprises:
inserting a heterologous DNA molecule which encodes the multivalent
RNA aptamer into the genome of a host cell under conditions
effective to cause the multivalent RNA aptamer to be transcribed in
cis with the RNA transcript.
15. The method of claim 13, wherein the splicing factor protein is
Drosophila splicing factor B52.
16. A transgenic non-human organism whose somatic and germ cell
lines contain an engineered gene encoding a multivalent RNA aptamer
which inhibits activity of a target molecule to treat a condition
associated with an expression level of the target molecule.
17. The transgenic non-human organism of claim 16, wherein the
non-human organism is an insect.
18. The transgenic non-human organism of claim 17, wherein the
insect is a species of Drosophila.
19. The transgenic non-human organism of claim 18, wherein the
target molecule is Drosophila splicing factor B52.
20. The transgenic non-human organism of claim 16, wherein the
engineered gene encoding a multivalent RNA aptamer comprises: a DNA
sequence encoding the multivalent RNA aptamer and a regulatory
sequence which controls expression of the DNA sequence encoding a
multivalent RNA aptamer.
21. The transgenic non-human organism of claim 20, wherein the DNA
sequence comprises: a plurality of monomeric DNA sequences each
encoding a multivalent RNA aptamer.
22. The transgenic non-human organism of claim 21, wherein each of
the plurality of monomeric DNA sequences is substantially
identical.
23. The transgenic non-human organism of claim 21, wherein each of
the plurality of monomeric sequences also encodes a cis-acting
ribozyme.
24. The transgenic non-human organism of claim 23, wherein the
cis-acting ribozyme is a hammerhead-type ribozyme.
25. A host cell in a non-human living organism, the host cell
comprising: a DNA molecule encoding a multivalent RNA aptamer
comprising at least two RNA aptamer sequences linked together.
26. A host cell in a non-human living organism, the host cell
comprising: a constructed DNA molecule comprising a plurality of
monomeric DNA sequences linked together to form a single DNA chain,
each monomeric DNA sequence encoding a multivalent RNA aptamer
comprising at least two RNA aptamer sequences linked together, each
of the at least two RNA aptamer sequences being capable of binding
a target molecule.
27. A host cell in a non-human living organism, the host cell
comprising: a heterologous gene comprising (i) a DNA sequence
encoding a multivalent RNA aptamer comprising at least two RNA
aptamer sequences linked together and (ii) a regulatory sequence
which controls expression of the DNA sequence encoding the
multivalent RNA aptamer.
28. A host cell in a non-human living organism, the host cell
comprising: an engineered gene comprising (i) a constructed DNA
molecule comprising a plurality of monomeric DNA sequences linked
together to form a single DNA chain, each monomeric DNA sequence
encoding a multivalent RNA aptamer comprising at least two RNA
aptamer sequences linked together, each of the at least two RNA
aptamer sequences being capable of binding a target molecule and
(ii) a regulatory sequence which controls expression of each
monomeric DNA sequence encoding a multivalent RNA aptamer.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/296,328, filed Apr. 22, 1999, which claims
the benefit of U.S. Provisional Patent Application Serial No.
60/082,652, filed Apr. 22, 1998.
FIELD OF THE INVENTION
[0003] The present invention relates to monovalent and multivalent
RNA aptamers, constructed DNA molecules and engineered genes which
encode the RNA aptamers of the present invention, as well as
expression systems, host cells, and transgenic organisms which
express the RNA aptamers of the present invention.
BACKGROUND OF THE INVENTION
[0004] Cells and organisms are complex adaptive systems in which
numerous biological processes are driven by sophisticated
macromolecular machinery and regulated by elaborate signal
transduction networks, both usually composed of multiple proteins.
To better understand and control such processes, new technologies
are needed to intervene in protein functions in the real time and
space of the living cells. In many cases, such in vivo destructive
approaches are needed to expand and extend results obtained from in
vitro reconstruction studies. On the other hand, many diseases are
known to be caused by either overexpression of certain endogenous
genes (such as oncogenes in cancer) or expression of exogenous
genes (as in the case of a virus infection), and "anti-gene"
therapies are called for to avert or ameliorate the morbidity and
mortality caused by these gene products. To inactivate a specific
gene or gene product, different techniques are directed at three
distinct types of targets: DNA, RNA, and protein. For example, a
gene can be altered by homologous recombination, the expression of
the genetic code can be blocked at the RNA level by antisense
oligonucleotides or ribozymes, and the protein function can be
altered or inhibited by antibodies or drugs.
[0005] A particularly useful tool resulting from the change of the
protein coding function of genes is a conditional allele which
displays its mutant phenotype only under certain non-permissive
conditions, making it possible to obtain viable cells or organisms
when a critical protein is under investigation. More importantly,
with a conditional allele it is also possible to target and change
specific genes in specific stages of development so that the
details of a wrongly assembled protein machine can be identified.
Recently there have been many new refinements of this technique.
Notably, Struhl and colleagues developed a two-pronged approach to
create yeast strains with conditional alleles in which the addition
of copper ion leads to the simultaneous cessation of mRNA synthesis
and destruction of the target protein in the cell (Moqtaderi et
al., "TBP-Associated Factors Are Not Generally Required for
Transcriptional Activation in Yeast," Nature 383:188-191 (1996)).
However, the generation of conditional mutants in higher (i.e.,
multicellular) eukaryotes is quite difficult. In addition, it is
often impossible to assay individual domains or discrete functional
surfaces of a protein, since the function of the whole protein is
abolished.
[0006] Small molecular mass drugs and drug derivatives that
directly target proteins have been used not only clinically to
rectify disease phenotype, but also in basic research that yielded
ample information in mechanistic studies both in vitro and in vivo.
These are usually cell-permeable, low molecular weight organic
molecules identified from natural sources or designed and
synthesized in the laboratory. Usually they are specific ligands of
proteins, affecting protein functions upon binding. In many cases
they are mimetics of the natural ligands of their targets (or
receptors, as they are called in pharmacodynamics). In vivo
experiments can be conducted easily with drugs at the cellular
level since the administration may be simple diffusion governed by
Fick's law. But systemic drug delivery to the organism is usually
complicated by many pharmacokinetic factors, making it difficult to
institute dosage regimens and assess drug effects at high
temporal-resolution. The biggest limitation of using small
molecular protein ligands is their availability. It is usually not
easy to find such a ligand for a predetermined protein target,
either from natural sources or by design. Recently, a general
procedure for manipulating protein in vivo at the cellular level
was developed, in which a gain of function results from the use of
synthetic "dimerizers" derived from an immunosuppressive drug (Ho
et al., "Dimeric Ligands Define a Role for Transcriptional
Activation Domains in Reinitiation," Nature 382:822-826 (1996)).
Although this "three-part invention" (Crabtree and Schreiber,
"Three-Part Inventions: Intracellular Signaling and Induced
Proximity," TIBS 21:418-422 1996)) may overcome the difficulty to a
certain extent, a ligand-binding domain has to be appended to the
target proteins.
[0007] As specific protein binding ligands, antibodies can be
custom-made for virtually any given protein, due to the clonal
selection and maturation function of the immune system. Antibodies
raised against specific proteins have made possible many
technological advances in the field of molecular biology, including
modem immunochemistry (Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1988)). But the in vivo utility of protein reagents like
antibodies is severely limited by difficulties in their delivery
and their own immunogenicity.
[0008] RNA has distinct advantages over proteins and small organic
molecules when considering its use to inactivate protein function
in vivo. An RNA-encoding sequence can be linked to a promoter and
this artificial gene introduced into cells or organisms. Depending
on the regulatory sequence included, this provides a unique way of
constructing a time and/or tissue specific suppresser gene. Such
RNA expressing genes are usually smaller than protein-coding genes
and can be inserted easily into gene therapy vectors. Unlike a
foreign or altered protein, RNA is less likely to evoke an immune
response. Antisense molecules and ribozymes have been developed as
"code blockers" to inactivate gene function, with their promise of
rational drug design and exquisite specificity (Altman, "RNase P in
Research and Therapy," Bio/Technology 13:327-329 (1995); Matteucci
and Wagner, "In Pursuit of Antisense," Nature 384 Suppl.
(6604):20-22 (1996)). Mechanistically, both antisense
oligodeoxynucleotides ("ODNs") and bioengineered ribozymes are
expected to achieve specific binding in the first step of their
action by forming a stable duplex (or triplex in some cases of the
ODNs) with a target nucleotide sequence based on Watson-Crick or
Hoogsteen base pairing. However, this mechanism and their ability
to disrupt the function of a single gene has never been proven.
Furthermore, a wide variety of unexpected non-antisense effects
have come to light, especially with the chemically modified
compounds. Although some of these side effects may have clinical
value, the use of antisense compounds as research reagents is
severely limited (Branch, "A Good Antisense Molecule is Hard to
Find," TIBS 23:45-50 (1998)).
[0009] Recently, RNA aptamers have also been explored as research
and therapeutic reagents for their ability directly to disrupt
protein function. Selection of aptamers in vitro allows rapid
isolation of extremely rare RNAs that have high specificity and
affinity for specific proteins. Exemplary RNA aptamers are
described in U.S. Pat. No. 5,270,1.63 to Gold et al., Ellington and
Szostak, "In vitro Selection of RNA Molecules That Bind Specific
Ligands," Nature 346:818-822 (1990), and Tuerk and Gold,
"Systematic Evolution of Ligands by Exponential Enrichment: RNA
Ligands to Bacteriophage T4 DNA Polymerase," Science 249:505-510
(1990). Unlike antisense compounds, whose targets are one
dimensional lattices, RNA aptamers can bind to the three
dimensional surfaces of a protein. Moreover, RNA aptamers can
frequently discriminate finely among discrete functional sites of a
protein (Gold et al., "Diversity of Oligonucleotide Functions,"
Annu. Rev. Biochem. 64:763-797 (1995)). As research and therapeutic
reagents, aptamers not only have the combined advantages of
antibodies and small molecular mass drugs, but in vivo production
of RNA aptamers also can be controlled genetically. The controlled
expression of high affinity RNA aptamers offers a means of rapidly
inactivating specific domains of proteins and thereby assessing
their function and mechanism of action.
[0010] Although gene therapy has the potential for treating many
diseases with very low risk of adverse reactions, the efficiency of
gene transfer and expression in vivo is still disappointingly low.
Assuming that efficient gene transfer can be developed, the next
issue would be long-term, stable, or even regulated gene expression
at the appropriate level. This is perhaps the greatest shortcoming
of present vectors for gene therapy (Anderson, "Human Gene
Therapy," Nature 392 Suppl. (6679): 25-30 (1998)). Efficient and
effective intracellular expression of functional RNA molecules such
as aptamers depends on many factors, some of them giving rise to
competing and conflicting design requirements. Ideally, the RNA
should be productively transcribed, stabilized against rapid
degradation, folded correctly, and directed to the subcellular
region where its target resides. Genes expressing various inhibitor
RNAs have been generated by modifying small RNA transcription units
that normally produce tRNAs (Sullenger et al., "Overexpression of
TAR Sequences Renders Cells Resistant to Human Immunodeficiency
Virus Replication," Cell 63:601-608 (1990)), small nuclear RNAs
(Noonberg et al., "In vivo Generation of Highly Abundant
Sequence-Specific Oligonucleotides for Antisense and Triplex Gene
Regulation," Nucleic Acids Res. 22:2830-2836 (1994)), or small
viral RNAs (Lieber and Strauss, "Selection of Efficient Cleavage
Sites in Target RNAs by Using a Ribozyme Expression Library," Mol.
Cell. Biol. 15:540-551 (1995)). Although high level RNA
accumulation has been achieved in some cases, a major disadvantage
of such transcription units is the limited ability to regulate
their expression. Also, tRNA promoters have intragenic promoter
elements, resulting in RNA transcripts carrying additional tRNA
sequence which may affect the folding of the adjoining functional
RNA moiety.
[0011] The present invention is directed to overcoming these and
other deficiencies in the art.
SUMMARY OF THE INVENTION
[0012] As used herein, the term "aptamer" refers to reagents
generated in a selection from a combinatorial library (typically in
vitro) wherein a target molecule, generally although not
exclusively a protein or nucleic acid, is used to select from a
combinatorial pool of molecules, generally although not exclusively
oligonucleotides, those that are capable of binding to the target
molecule. The selected reagents can be identified as primary
aptamers. The term "aptamer" includes not only the primary aptamer
in its original form, but also secondary aptamers derived from
(i.e., created by minimizing and/or modifying) the primary aptamer.
Aptamers, therefore, must behave as ligands, binding to their
target molecule.
[0013] One aspect of the present invention relates to a monovalent
RNA aptamer that binds to Drosophila splicing factor B52.
[0014] Another aspect of the present invention relates to a
multivalent RNA aptamer that includes at least two RNA aptamer
sequences linked together.
[0015] Yet another aspect of the present invention relates to an
isolated or constructed DNA molecule encoding either a monovalent
RNA aptamer or a multivalent RNA aptamer of the present
invention.
[0016] Still another aspect of the present invention relates to an
engineered gene encoding a multivalent RNA aptamer, where the
engineered gene includes a DNA sequence encoding a multivalent RNA
aptamer and a regulatory sequence which controls expression of the
DNA sequence encoding a multivalent RNA aptamer.
[0017] Another aspect of the present invention relates to a method
of expressing a multivalent RNA aptamer in a cell which includes
introducing either a DNA molecule or an engineered gene of the
present invention into a cell under conditions effective to express
the multivalent RNA aptamer.
[0018] Yet another aspect of the present invention relates to a
method of inhibiting activity of a target molecule in a cell which
includes expressing a multivalent RNA aptamer in the cell, the
multivalent RNA aptamer having an affinity for a target molecule
sufficient to inhibit activity of the target molecule.
[0019] Another aspect of the present invention relates to a method
of increasing activity of a splicing factor protein in a cell. This
method includes inserting a multivalent RNA aptamer, which binds to
a splicing factor protein, into an RNA transcript, which contains
exons and introns, under conditions effective to enable splicing of
the RNA transcript.
[0020] A further aspect of the present invention relates to a
transgenic non-human organism whose somatic and germ cell lines
contain an engineered gene encoding a multivalent RNA aptamer which
inhibits activity of a target molecule to treat a condition
associated with an expression level of the target molecule.
[0021] Additional aspects of the present invention include a
constructed DNA molecule that contains a plurality of monomeric
sequences each encoding a functional RNA molecule; an engineered
gene that includes a DNA sequence containing a plurality of
monomeric sequences each encoding a functional RNA molecule and a
regulatory sequence which controls expression of the DNA sequence;
and a transgenic non-human organism whose somatic and germ cell
lines contain an engineered gene encoding a functional RNA
molecule, where the functional RNA molecule inhibits the activity
of a target molecule to treat a condition associated with an
expression level of the target molecule.
[0022] Still further aspects of the invention relate to methods of
expressing a functional RNA molecule in a cell by introducing
either a constructed DNA molecule or an engineered gene, which
encode the functional RNA molecule, into a cell under conditions
effective to express the functional RNA molecule.
[0023] By coupling in vitro selection with in vivo transcriptional
regulation, a multivalent RNA aptamer can be constructed that has a
higher affinity for its target molecule (e.g., protein, nucleic
acid, etc.) than its component RNA aptamers. When its in vivo
transcription is regulated, the multivalent RNA aptamer of the
present invention can be used according to a general methodology to
inhibit in vivo functions of a specific target molecule. As shown
herein using the Drosophila splicing factor protein B52 as a model
system, a multivalent RNA aptamer of the present invention, when
expressed in cells of cell culture or in somatic and germ cells of
a transgenic organism, can act as a protein antagonist in vivo.
When the multivalent RNA aptamer is expressed in the somatic and
germ cells of a transgenic organism, activity of the target protein
is inhibited to treat a condition associated with an expression
level of the target protein. The multivalent RNA aptamers of the
present invention have the combined advantages of prior art systems
described above, but it eliminates their major shortcomings. Like
antibodies, the multivalent RNA aptamers can be made to inhibit
activity of specific target proteins. Like small organic molecules,
multivalent RNA aptamers can directly target specific domains or
discrete functional surfaces of the target protein within cells.
Like conditional alleles, administration and expression of the
multivalent RNA aptamers can be controlled genetically in whole
organisms. In addition, expression of the multivalent RNA aptamers
can be limited to specific tissues, cells, or stages of
development.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 is a schematic diagram depicting the construction and
expression of an engineered gene of the invention which encodes the
multimeric immature RNA transcript. The construction phase shows
the polymerization of hierarchically encapsulated sequence units in
four levels: the primary RNA aptamer sequence, the monomeric unit,
the transcriptional template, and the iaRNA gene. In the expression
phase, the major functional elements on each level, including the
promoter, the hammerhead ribozyme, and the hairpin structure, are
indicated symbolically.
[0025] FIGS. 2A and 2B identify individual RNA sequences selected
by B52 from a randomized pool and the predicted secondary
structures of the B52-selected sequences. Referring to FIG. 2A, the
names of the individual clones are given to the left of each
selected sequence. The variable region is represented by capital
letters and the shared sequence motif is represented in bold type
characters. Nucleotides belonging to the flanking constant region
are shown in lower case characters. Nucleotides only shared by BBS
#4, 14, 15 (SEQ. ID. No. 1) and BBS #8 (SEQ. ID. No. 2) are
underlined. The full sequence of the constant flanking region is
shown at the bottom. The predicted secondary structures shown in
FIG. 2B were generated by the computer program MulFold (Jaeger et
al., "Improved Predictions of Secondary Structures for RNA," Proc.
Natl. Acad. Sci. USA 86:7706-7710 (1989), and Zuker, "On Finding
All Suboptimal Foldings of an RNA Molecule," Science 244:48-52
(1989), which are hereby incorporated by reference). Foldings with
lowest free energy are displayed. Nucleotides forming the shared
sequence motif are represented in bold type characters. Residues
derived from the constant flanking region are represented in lower
case characters.
[0026] FIGS. 3A and 3B are images which show the affinity and
specificity of B52-binding RNA aptamers. As shown in FIG. 3A, the
binding of B52 tightly to selected sequences is depicted in a band
shift assay on a 2.5% agarose gel of 20 .mu.l binding reactions
with increasing amounts of purified B52 as indicated. G0 (SEQ. ID.
No. 24) is a control sequence randomly picked up from the original
pool. FIG. 3B shows competitive binding of selected sequences for
B52. Reactions were performed as in FIG. 3A, except that an excess
amount of cold competitor RNA (as indicated) was added prior to the
radioactive probes.
[0027] FIG. 4 lists the sequences used for mapping of the minimal
binding elements on RNA isolated from the pool. Deletion analysis
was used to define a minimal binding site and substitution
mutations were used to define key features of the binding site.
Anti-sense sequences of the consensus do not bind B52. Short RNA
transcripts were produced by in vitro transcription and their
affinity to B52 was assayed by band shift. The highest affinity
(e.g., ++++) is identical to that of BBS #8. Each + sign indicates
about a 3-fold difference in affinity as compared to BBS #8. The
predicted secondary structure of each RNA is designated under the
sequence, with paired bases denoted by matching parentheses "( )"
to either side of unpaired bases marked by dashes "-". The
stability of these structures is indicated by their folding energy
(kcal/mole). The substituted bases are signified by italics.
Although the base pairs represented by "< >" were expected
for the construct bbs-II as in its parental construct BBS-II, the
computer program predicted an alternative structure with negligible
(0.2 kcal/mole) folding energy.
[0028] FIGS. 5A and 5B identify the active domain for B52 binding
on BBS #8. FIG. 5A contains images of RNase footprinting assays.
RNase footprinting using RNase T2 (Lanes 2-4) or RNase VI (Lanes
9-11) identifies the hairpin loop of BBS #8 (SEQ. ID. No. 2) as the
B52 binding site. RNase was added to binding reactions either
lacking B52 (Lanes 2 and 9), containing 2 pmole of B52 (Lanes 3 and
10), or containing 20 pmole of B52 (Lanes 4 and 11), and the
resulting RNA products were analyzed by primer extension. The input
RNA is shown to identify nicks in the RNA as well as reverse
transcriptase stops (Lane 1). The sequencing ladder (Lanes 5-9) was
used to identify the bases of interaction. FIG. 5B contains a
corresponding diagram which illustrates the localization of the
B52-binding site on a predicted secondary structure of BBS #8 (SEQ.
ID. No. 2). Open circles indicate weak T2 protection and filled
circles and squares indicate strong T2 and V1 protection,
respectively. The single-stranded regions and the sequence in the
stem of the hairpin loop structure are indicated next to the
sequencing ladder by thin and thick lines, respectively. The
sequence of BBS #8 is indicated by the nucleotides and the constant
flanking region and vector sequences depicted by a skeletal
diagram.
[0029] FIGS. 6A and 6B contain images which indicate that the
RNA-binding site on B52 was localized to both RNA recognition
motifs. .sup.35S-Methionine labeled B52 deletion constructs were
made by in vitro translation, and their ability to bind BBS #8 RNA
was examined in two different assays. The binding reactions with
different .sup.35S-labeled proteins or combinations thereof were
followed by band shift assay on a native agarose gel, shown in FIG.
6A, or UV crosslinking and SDS-PAGE, shown in FIG. 6B. The filled
arrowhead in FIG. 6B signifies the B52/R12-BBS #8 complex, whereas
the open arrow head points to where the B52/R1-BBS #8 complex would
be expected to migrate. F=full length B52, R1=RRM 1, R2=RRM 2,
R12=RRM 1 and RRM2, and S=SR domain.
[0030] FIG. 7 is a list of the engineered genes of the present
invention. Different combinations of promoters and transcriptional
templates, which vary by length and orientation, are listed. The
number of BBS's contained in a transcriptional template is
indicated in its name as the product of the numbers within the
parentheses. For example, BBS(5.12) has 60 BBS units in twelve
pentavalent monomers. A minus sign indicates antisense, Hic stands
for "heat inducible cassette," and Mtn stands for
"metallothionein."
[0031] FIGS. 8A and 8B illustrate predicted secondary structures
for a multivalent RNA aptamer of the present invention. In
particular, FIG. 8A shows the sequence and free-energy-minimized
secondary structure of the monomeric unit of an immature
pentavalent RNA transcript (i.e., from a multimeric RNA
transcript). Parts of the selected aptamers are incorporated into
the construct in their original or modified form. The original
aptamer sequences are enclosed in the boxes. Bold type letters
indicate important functional sequences as annotated. FIG. 8B shows
the sequence and free-energy-minimized secondary structure of the
mature pentavalent RNA aptamer having an affinity for Drosophila
B52. After self-cleavage of the immature pentavalent RNA
transcript, the residual sequence of the ribozyme at both the 5'
and 3' ends of the molecule is brought together to form a virtually
closed structure. The stem formed between the 3' and 5' termini,
the S35 motif, is enclosed in a box. Both structures in FIGS. 8A
and 8B are generated by the computer program MulFold (Jaeger et
al., "Improved Predictions of Secondary Structures for RNA," Proc.
Natl. Acad. Sci. USA 86:7706-7710 (1989), and Zuker, "On Finding
All Suboptimal Foldings of an RNA Molecule," Science 244:48-52
(1989), which are hereby incorporated by reference). Foldings with
lowest free energy are displayed.
[0032] FIGS. 9A and 9B are images depicting the result of binding
assays which illustrates the avidity of the mature pentavalent RNA
aptamer for B52. FIG. 9A compares the avidity of the mature
pentavalent RNA aptamer for B52 to the affinity of a single
aptamer. A template containing a single pentavalent monomeric unit,
P1-2-3/BBS(5.1), was transcribed in both orientations to produce
the probes BBS(5+) and BBS(5-). Their avidity to B52 was compared
with the affinity of BBS #14 in a band shift assay. The adjacent
lanes in each set have a 10-fold difference in B52 concentration,
the lowest being 5 nM. The molar ratio of pentamer to monomer used
in different sets was 1:5, so that the concentration of BBS units
was identical in each reaction. The efficiency of ribozyme cleavage
was also assessed on this 25 cm native agarose gel. The uncut
transcript of BBS(5+), the Fragment A, and the Fragment C are
indicated by U, A, and C, respectively. FIG. 9B illustrates a band
shift assay with RNA transcribed from templates with different
length and orientations. The length of the templates are indicated
by the number of the monomeric units located after the decimal
point in the parentheses. The number of BBS's contained in a
template is the product of the numbers within the parentheses. The
orientation of the template is indicated by a plus or minus sign.
Fragment C is indicated. The assignments of bands representing
Fragments A and B are mainly based on their mobility and intensity,
and are not unambiguous due to the possible alternative
conformations of RNA fragments and their different movement on this
native gel.
[0033] FIGS. 10A and 10B are images which illustrate the
effectiveness of large scale production of the mature pentavalent
RNA and its binding to B52. FIG. 10A illustrates a 5%
polyacrylamide 7 M urea preparative gel, loading 20 .mu.l overnight
transcription reaction mixture in two lanes. Fragments A and B are
indicated. The small Fragment C ran off the gel. "Actin" RNA was
transcribed from a plasmid having a fragment of the mouse-actin
gene inserted in the antisense orientation under the
transcriptional control of T7 promoter, and provided as a control
template in the MAXIscript kit (Ambion). Its transcript is 334-nt
in length. The RNA was visualized by UV shadowing and the bands
representing Fragment B were excised and eluted. FIG. 10B
illustrates a gel purified mature pentavalent RNA aptamer as a
competitor in a binding reaction. BBS #8, the strongest-binding
monovalent aptamer, was used as the probe in a gel shift assay with
gel purified mature pentavalent RNA aptamer and its antisense RNA,
BBS(5-), as competitors. The same amount of purified Torulla yeast
RNA (Ambion) consisting of fragments of 300-500 bases (yRNA) was
used as a control.
[0034] FIGS. 11A and 11B are images which illustrate the ability of
the mature pentavalent RNA aptamer to modify B52 function in vitro.
FIG. 11A illustrates suppression of B52 function by mature
pentavalent RNA in trans. Labeled ftz pre-mRNA was used as the
substrate in a splicing assay, in which B52 complements the
splicing-deficient S100 extract. This activity was challenged by
adding the gel purified mature pentavalent RNA (Fragment B), its
antisense RNA (BBS(5-)), or purified yeast RNA. FIG. 11B
illustrates creation of a B52-specific splicing enhancer with BBS
in cis. ftz+BBS(5) was tested in a splicing assay containing S100
extract with or without increasing amounts of B52. The original ftz
without BBS and a ftz derivative with the antisense sequence of
BBS(5), fitz-BBS(5), were used as controls. Splicing products and
intermediates offtz are indicated schematically between the two
gels and those of ftz+BBS(5) are indicated to the right of FIG.
11B. Exons are represented by boxes, introns by lines, BBS(5) by
filled boxes.
[0035] FIGS. 12A and 12B are images which illustrate in vivo
expression of mature pentavalent RNA aptamer specific for B52. FIG.
12A illustrates transient expression of mature pentavalent RNA
aptamer in Drosophila S2 cell cultures. The transcriptional
templates BBS(5.2) and BBS(5.12) were driven by a heat shock
promoter and a metallothionein promoter, respectively. Steady-state
mature pentavalent RNA aptamer levels without induction and peak
level after induction (90.degree. heat shock or 24 hour Cu.sup.2+)
were compared. To measure the half-life of the mature pentavalent
RNA aptamer, actinomycin D was added to the media immediately after
the 90.degree. heat shock treatment, and total RNA was prepared at
the time indicated. FIG. 12B illustrates expression of the mature
pentavalent RNA aptamer in transgenic flies. HicBBS(5.12) is a
homozygous strain containing the engineered gene bearing the same
name used in the transient expression experiments. hsGAL4-UASBBSs
are heterozygous flies generated by mating different UASBBS
transgenic strains (length and orientation of the BBS constructs as
indicated) with the strain containing the transgene hsGAL4, which
is driven by a heat shock promoter. RNA samples were prepared from
third instar larvae with and without heat treatment. The mature
pentavalent RNA aptamer standards were transcribed in vitro and gel
purified. The mature pentavalent RNA aptamer in these samples was
measured by RNase protection assay with a probe covering part of
the monomeric pentavalent unit.
[0036] FIGS. 13A and 13B are images which illustrate in situ
visualization of the transgene encoding the mature pentavalent RNA
aptamer, its expression, and its binding to B52. FIG. 13A
illustrates subcellular localization of the mature pentavalent RNA
aptamer. A Texas Red labeled RNA probe was used to visualize the
mature pentavalent RNA aptamer in the nuclei of whole mount
salivary glands in late third instar larvae of the HicBBS(5.12)
transgenic line after heat treatment. The mature pentavalent RNA
aptamer is seen to fill the intranuclear space between the giant
polytene chromosomes. DNA was stained with
4',6-diamidino-2-phenylindole (DAPI). FIG. 13B illustrates the
mapping of the transgene in the HicBBS(5.12) strain to locus 12A on
the X chromosome (left panel). After heat induction, the expression
of the mature pentavalent RNA aptamer resulted in a medium sized
puff (middle panel). B52 was strongly recruited to this site (right
panel). The transgene and its expression were visualized using the
same probe as in FIG. 13A. B52 was visualized by immunofluorescence
with an anti-B52 antibody. The images were pseudo-colored in red
and merged with those of DAPI stained chromosomes (pseudo-colored
in cyan) to facilitate the localization. The transgenic insertion
site is indicated by a white arrow head in each panel. The major
heat shock loci at 87A and 87C are indicated by white dots in the
right panel.
[0037] FIG. 14 is a schematic illustration of crosses for
synthesizing triple transgenic fly lines by manipulating
chromosomes two and three. The scheme shown here illustrates the
synthesis of a UASB52 transgene on the second chromosome and a
UASBBS(5.12) transgene on the third chromosome to make a homozygous
double transgenic line UASB52-UASBBS, which is then mated with a
homozygous GAL4 line to generate the heterozygous triple transgenic
flies in which both B52 and BBS are actively transcribed. An
additional strain is used in the scheme to mark and balance these
two chromosomes. Synthesized double transgenic lines are maintained
either as homozygous or double balanced stocks.
[0038] FIGS. 15A and 15B illustrate transgenic fly lines and the
phenotypic effects of pentavalent RNA aptamer expression. FIG. 15A
is schematic depicting self-crossed, double-balanced fly strains.
FIG. 15B is a chart showing the reduced viability caused by
continuous high level expression of the mature pentavalent RNA
aptamer. The genotype of the isogenetic double transgenic flies are
shown in FIG. 15A. Each pair has a hsGAL4-UASBBS line and a
UASBBS-UASBBS line. The only difference between the two lines is
the chromosome carrying hsGAL4 in one line is replaced by a
chromosome carrying an UASBBS in the other. Selfing the double
balanced stocks of both lines, as shown in FIG. 15A, yielded four
different genotypes among the progeny with different copy number of
hsGAL 4 and UASBBS, as tabulated. The viability is calculated as
the percentage of surviving BBS-expressing progeny with regard to
its isogenetic counterparts in which BBS is dormant.
[0039] FIG. 16 is an image of a RNase protection assay which
confirms GAL4 driven expression of BBS and over expression of B52
in the heterozygous triple transgenic flies. An RNA probe derived
from the sequence coding for the RRMs was used to detect B52 mRNA,
and a probe covering part of the monomeric unit of the mature
pentavalent RNA aptamer was used to detect BBS. HsGAL4 driven
expression of B52 and/or BBS, and the response of transcription to
heat, treatment in animals produced by different crosses are shown.
Genotypes of the heterozygous flies are indicated. 12 .mu.g total
RNA was used in the assay for B52 mRNA, while 1 .mu.g total RNA was
used for detecting BBS. The samples from same flies were then
pooled and run in the same lane.
[0040] FIGS. 17A-17K illustrate the effects on phenotypes of B52
overexpression with the co-expression of mature pentavalent RNA
aptamer. FIG. 17A depicts a generic mating scheme used to prepare
transgenic flies. Active transcription of the UASB52 transgene
and/or the UASBBS(5.12) transgene is indicated by the names of gene
product in braces. The parental transgenic strains used to
synthesize the double transgenic UASB52-UASBBS strain were used in
{B52} and {BBS} controls. Despite decreased viability, the
surviving {BBS} flies were morphologically normal. FIGS. 17B-17D
are images of salivary glands dissected from third instar larvae
and FIGS. 17E-17G are images of bristles appearing on the adult
notum. FIG. 17H is a chart showing a comparison of the phenotypes
of the three classes of progeny. Different GAL4 sources were used
to drive the overexpression of B52 and the expression of BBS
(5.12). FIG. 17I depicts a generic mating scheme used to prepare
transgenic flies, which overexpress B52 and co-express the mature
pentavalent RNA aptamer. The use of the double balanced
hsGAL4-UASB52 strain produced three other genotypes among the
progeny that serve as internal controls for quantitation. Two sets
of independent transformants of each BBS construct were used in the
crosses. FIGS. 17J and 17K are graphs which illustrate the effects
of different dosages of BBS expression and a constant dosage of B52
overexpression, both driven by the hsGAL4 transgene. Twenty female
adults were scored to assess bristle development. Error bars
indicate one standard deviation on each side of the mean.
DETAILED DESCRIPTION OF THE INVENTION
[0041] One aspect of the present invention relates to a novel
monovalent RNA aptamer that binds to a splicing factor ("SR")
protein. SR proteins are structurally and functionally related and
evolutionarily conserved. The SR family contains at least six
members, which are conserved from Drosophila melanogaster to humans
(Zahler et al., "SR Proteins: A Conserved Family of Pre-mRNA
Splicing Factors," Genes Dev. 6:837-847 (1992), which is hereby
incorporated by reference). The splicing factor protein is
preferably the Drosophila splicing factor B52, more preferably B52
from Drosophila melanogaster. B52 from Drosophila melanogaster is
homologous to the human splicing factor SRp55 (Champlin et al.,
"Characterization of a Drosophila Protein Associated with
Boundaries of Transcriptionally-Active Chromatin," Genes Dev.
5:1611-1621 (1991), which is hereby incorporated by reference).
[0042] The Drosophila SR protein family, which includes B52, is a
group of nuclear proteins that are both essential splicing factors
and specific splicing regulators (Fu, "The Superfamily of
Arginine/Serine-Rich Splicing Factors," RNA 1:663-680 (1995);
Manley and Tacke, "SR Proteins and Splicing Control," Genes Dev.
10(3):1569-1579 (1996), which are hereby incorporated herein by
reference). Like most other SR proteins, B52 contains two RNA
recognition motifs ("RRMs") in the N-terminus and a C-terminal
domain rich in serine-arginine dipeptide repeats (Champlin et al.,
"Characterization of a Drosophila Protein Associated With
Boundaries of Transcriptionally Active Chromatin," Genes Dev.
5:1611-1621 (1991), which is hereby incorporated by reference). In
vivo, B52 is an abundant protein and plays a critical role in
Drosophila development. B52 deletion mutants are homozygous lethal
at the second-instar larval stage (Ring and Lis, "The SR Protein
B52/SRp55 is Essential for Drosophila Development," Mol. Cell.
Biol. 14:7499-7506 (1994), which is hereby incorporated by
reference), and overexpression of B52 protein causes severe
developmental defects (Kraus and Lis, "The Concentration of B52, an
Essential Splicing Factor and Regulator of Splice Site Choice, is
Critical for Drosophila Development," Mol. Cell. Biol. 14:5360-5370
(1994), which is hereby incorporated by reference), evincing that
B52 is an essential protein which must be expressed at the
appropriate level.
[0043] Suitable monovalent RNA aptamers which bind Drosophila
splicing factor B52 are listed below and in FIG. 2A. A first
monovalent RNA aptamer, designated BBS #4,14,15, has a nucleotide
sequence corresponding to SEQ. ID. No. 1 as follows:
[0044] gggagaauuc aacugccauc uaggcagggu aacgaucaac cuggcgacag
cugcccugcc 60
[0045] guccaaguac uacaagcuuc uggacucggu 90
[0046] A second monovalent RNA aptamer, designated BBS #8, has a
nucleotide sequence corresponding to SEQ. ID. No. 2 as follows:
[0047] gggagaauuc aacugccauc uaggcugguc aaccaggcga ccgccacccg
cgcgcgcaau 60
[0048] accuaguacu acaagcuucu ggacucggu 89
[0049] A third monovalent RNA aptamer, designated BBS #11, has a
nucleotide sequence corresponding to SEQ. ID. No. 3 as follows:
[0050] gggagaauuc aacugccauc uaggcugcuc acgaguccau gaccaguacg
aucaaccagg 60
[0051] cgacaguacu acaagcuucu ggacucggu 89
[0052] A fourth monovalent RNA aptamer, designated BBS#23, has a
nucleotide sequence corresponding to SEQ. ID. No. 4 as follows:
[0053] gggagaauuc aacugccauc uaggcccaac ugcuaagaag cauccuguac
gaucaacccg 60
[0054] gcgacaguac uacaagcuuc uggacucggu 90
[0055] The monovalent RNA aptamers of the present invention, which
are specific for B52, were identified from a large pool of RNA
molecules. Identifying the primary aptamers basically involved
selecting RNA aptamers that bind full-length B52 with high affinity
(K.sub.d=20-50 nM) and specificity from a large pool of RNAs
containing a random region of about 40 bases (Shi et al., "A
Specific RNA Hairpin Loop Structure Binds the RNA Recognition
Motifs of the Drosophila SR Protein B52," Mol. Cell. Biol.
17:1649-1657 (1997); Shi, "Perturbing Protein Function with RNA
Aptamers," Thesis, Cornell University, University Microfilms, Inc.
(1997), which are hereby incorporated by reference). Both RRMs of
the protein are required for the interaction with the RNA aptamer.
The B52 binding sites ("BBS") on members of this non-clonally
derived family of RNA aptamers not only have a well "conserved"
consensus sequence, but also have a virtually identical hairpin
loop structure as predicted by the MulFold computer program using
free energy minimization (Jaeger et al., "Improved Predictions of
Secondary Structures for RNA," Proc. Natl. Acad. Sci. USA
86:7706-7710 (1989), and Zuker, "On Finding All Suboptimal Foldings
of an RNA Molecule," Science 244:48-52 (1989), which are hereby
incorporated by reference) and confirmed by structure-specific
enzymatic probing using RNase, see Example 3 infra. The conserved
sequence of the above-identified monovalent RNA aptamers of the
present invention has a nucleotide sequence of SEQ. ID. No. 5 as
follows:
[0056] gnucaaccng gcgacng 17
[0057] Of this sequence identified as SEQ. ID. No. 5, nucleotides
5-12 form the functional loop structure of the predicted hairpin
loop secondary structure of each monovalent RNA aptamer.
[0058] To identify primary aptamers of any particular target
protein, an established in vitro selection and amplification
scheme, SELEX, can be used. The SELEX scheme is described in detail
in U.S. Pat. No. 5,270,163 to Gold et al.; Ellington and Szostak,
"In vitro Selection of RNA Molecules That Bind Specific Ligands,"
Nature 346:818-822 (1990); and Tuerk and Gold, "Systematic
Evolution of Ligands by Exponential Enrichment: RNA Ligands to
Bacteriophage T4 DNA Polymerase," Science 249:505-510 (1990), which
are hereby incorporated by reference. An established
template-primer system (Bartel et al., "HIV-1 Rev Regulation
Involves Recognition of Non-Watson-Crick Base Pairs in Viral RNA,"
Cell 67:529-536 (1991), which is hereby incorporated by reference)
can be adapted to produce RNA molecules having a stretch of about
38-40 random bases sandwiched between 5' and 3' constant regions.
The 5' DNA template, which contains a T7 promoter used to drive
transcription of the variable RNA, has a nucleotide sequence
corresponding to SEQ. ID. No. 6 as follows:
[0059] gtaatacgac tcactatagg gagaattcaa ctgccatcta ggc 43
[0060] The 3' DNA template has a nucleotide sequence corresponding
to SEQ. ID. No. 7 as follows:
[0061] agtactacaa gcttctggac tcggt 25
[0062] Commercial oligonucleotide synthesis generally yields more
than 500 picomoles of the template at the 200 nmol synthesis scale.
The synthetic oligonucleotide templates can be amplified by
polymerase chain reaction ("PCR") and then transcribed to generate
the original RNA pool. Assuming that ten percent of the RNA
molecules are free of chemical lesions that prevent second-strand
synthesis and transcription, this pool would contain more than
3.times.10.sup.13 different sequences. Because filter binding is
applicable for most protein targets, it can be used as the
partitioning device, although other suitable schemes can be used.
The selected primary RNA aptamers can be cloned into any
conventional subcloning vector and sequenced using any variation of
the dideoxy method. Next, the secondary structure of each primary
RNA aptamer can be predicted by computer programs such as MulFold
(Jaeger et al., "Improved Predictions of Secondary Structures for
RNA," Proc. Natl. Acad. Sci. USA 86:7706-7710 (1989), and Zuker,
"On Finding All Suboptimal Foldings of an RNA Molecule," Science
244:48-52 (1989), which are hereby incorporated by reference).
Secondary structures of the four monovalent RNA aptamers of the
present invention are shown in FIG. 2B. Mutational studies can be
conducted by preparing substitutions or deletions to map both
binding sites on the RNA aptamer and its target molecule, as
described in Example 2 infra.
[0063] Other known RNA aptamers include, without limitation, RNA
ligands of T4 DNA polymerase, RNA ligands of HIV reverse
transcriptase, RNA ligands of bacteriophage R17 coat protein, RNA
ligands for nerve growth factor, RNA ligands of HSV-1 DNA
polymerase, RNA ligands of Escherichia coli ribosomal protein S1,
and RNA ligands of HIV-1 Rev protein (U.S. Pat. No. 5,270,163 to
Gold et al., which is hereby incorporated by reference); RNA
ligands of Bacillus subtillus ribonuclease P (U.S. Pat. No.
5,792,613 to Schmidt et al., which is hereby incorporated by
reference); RNA ligands of ATP and RNA ligands of biotin (U.S. Pat.
No. 5,688,670 to Szostak et al., which is hereby incorporated by
reference); RNA ligands of prion protein (Weiss et al., "RNA
Aptamers Specifically Interact with the Prion Protein PrP," J.
Virol. 71(11):8790-8797 (1997), which is hereby incorporated by
reference); RNA ligands of hepatitis C virus protein NS3 (Kumar et
al., "Isolation of RNA Aptamers Specific to the NS3 Protein of
Hepatitis C Virus from a Pool of Completely Random RNA," Virol.
237(2):270-282 (1997); Urvil et al., "Selection of RNA Aptamers
that Bind Specifically to the NS3 Protein of Hepatitis C Virus,"
Eur. J. Biochem. 248(1):130-138 (1997); Fukuda et al., "Specific
RNA Aptamers to NS3 Protease Domain of Hepatitis C Virus," Nucleic
Acids Symp. Ser. 37:237-238 (1997), which are hereby incorporated
by reference); RNA ligands of chloramphenicol (Burke et al., "RNA
Aptamers to the Peptidyl Transferase Inhibitor Chloramphenicol,"
Chem. Biol. 4(11):833-843 (1997), which is hereby incorporated by
reference); RNA ligands of the adenosine moiety of S-adenosyl
methionine (Burke and Gold, "RNA Aptamers to the Adenosine Moiety
of S-Adenosyl Methionine: Structural Inferences from Variations on
a Theme and the Reproducibility of SELEX," Nucleic Acids Res.
25(10):2020-2024 (1997), which is hereby incorporated by
reference); RNA ligands of protein kinase C (Conrad et al.,
"Isozyme-Specific Inhibition of Protein Kinase C by RNA Aptamers,"
J. Biol. Chem. 269(51):32051-32054 (1994); Conrad and Ellington,
"Detecting Immobilized Protein Kinase C Isozymes with RNA
Aptamers," Anal. Biochem. 242(2):261-265 (1996), which are hereby
incorporated by reference); RNA ligands of subtilisin (Takeno et
al., "RNA Aptamers of a Protease Subtilisin," Nucleic Acids Symp.
Ser. 37:249-250 (1997), which is hereby incorporated by reference);
RNA ligands of yeast RNA polymerase II (Thomas et al., "Selective
Targeting and Inhibition of Yeast RNA Polymerase II by RNA
Aptamers," J. Biol. Chem. 272(44):27980-27986 (1997), which is
hereby incorporated by reference); RNA ligands of human activated
protein C (Gal et al., "Selection of a RNA Aptamer that Binds to
Human Activated Protein C and Inhibits its Protein Function," Eur.
J. Biochem. 252(3):553-562 (1998), which is hereby incorporated by
reference); and RNA ligands of cyanocobalamin (Lorsch and Szostak,
"In vitro Selection of RNA Aptamers Specific for Cyanocobalamin,"
Biochem. 33(4):973-982 (1994), which is hereby incorporated by
reference). Additional RNA aptamers are continually being
identified and isolated by those of ordinary skill in the art.
[0064] Another aspect of the present invention relates to a
multivalent RNA aptamer that contains at least two RNA aptamer
sequences linked together.
[0065] The multivalent RNA aptamer of the present invention is
prepared from known RNA aptamers or those identified using, for
example, the SELEX procedure described above. Once the sequence and
structure information of the individual RNA aptamers has been
identified, a multivalent RNA aptamer of the present invention can
be designed.
[0066] Multivalent RNA aptamers of the present invention should
enhance the stability of the target molecule-RNA interaction,
because it is equivalent to an increased local concentration of
aptamers for the target molecule that binds to RNA, thus providing
a decreased overall off rate. Many RNA aptamers have distinct
secondary structures such as hairpin loops (see FIG. 2B), and
correct folding of each individual aptamer in an array is critical.
To avoid unwanted pairing of sequences in the stem of the
individual RNA aptamers (i.e., to achieve an overall multivalent
RNA aptamer structure that is both kinetically favored and
thermodynamically stable), the stem of some individual RNA aptamers
can be reinforced and/or elongated with different sequences to
reduce the general sequence similarity among them. Energy-minimized
secondary structures can be generated using any conventional
program, such as the Mulfold program (Jaeger et al., "Improved
Predictions of Secondary Structures for RNA," Proc. Natl. Acad.
Sci. USA 86:7706-7710 (1989), and Zuker, "On Finding All Suboptimal
Foldings of an RNA Molecule," Science 244:48-52 (1989), which are
hereby incorporated by reference). The secondary structures for
both monomeric unit of the immature RNA transcript and the mature
multivalent aptamer can be generated and the folding pattern of
individual moieties can be compared to the previously established
folding pattern of the monovalent RNA aptamer(s). The sequence of
these monomeric units can then be adjusted iteratively until each
individual aptamer (i.e., in the immature RNA transcript and the
mature multivalent RNA aptamer) is folded correctly.
[0067] Each of the at least two RNA aptamer sequences preferably
has a hairpin loop structure, with a neck portion of various
lengths that is characterized by a high degree of base-pairing and
a loop portion that is characterized by non-paired bases of a
target-binding sequence.
[0068] In addition to the target-binding region of the individual
RNA aptamers, which together form the major functional sequence of
the mature multivalent RNA aptamer, different regulatory sequences
or structural elements can be incorporated into the mature
multivalent RNA aptamer as ancillary sequences. A preferred
ancillary sequence is an exonuclease-blocking sequence linked to
one of the at least two RNA aptamer sequences.
[0069] In particular, a stable tetra-loop near the 3' end of the
mature, multivalent RNA aptamer can be engineered. Because of its
highly stacked and relatively inaccessible structure, the UUCG
tetra-loop (Cheong et al., "Solution Structure of an Unusually
Stable RNA Hairpin, 5'GGAC(UUCG)GUCC," Nature 346:680-682 (1990),
which is hereby incorporated by reference) is included to stabilize
the mature multivalent RNA aptamer against degradation by 3'
exonucleases and to serve as a nucleation site for folding (Varani,
"Exceptionally Stable Nucleic Acid Hairpins," Annu. Rev. Biophys.
Biomol. Struct. 24:379-404 (1995), which is hereby incorporated by
reference).
[0070] In addition, the mature multivalent RNA aptamer can contain
an "S35 motif" which yields a virtually closed structure resistant
to nucleolytic degradation. The S35 motif, constructed by creating
complementary 5' and 3' ends, has been shown to cause an over
100-fold increase in accumulation of a tRNA-ribozyme chimerical
transcript in stably transduced cell lines (Thompson et al.,
"Improved Accumulation and Activity of Ribozymes Expressed from a
tRNA-based RNA Polymerase III Promoter," Nucleic Acids Res.
23:2259-2268 (1995), which is hereby incorporated by
reference).
[0071] By way of example, a preferred mature multivalent RNA
aptamer of the present invention is a pentavalent RNA aptamer that
includes five tandemly arranged RNA aptamer sequences which bind to
the Drosophila splicing factor B52, a UUCG tetraloop, and an S35
motif (FIG. 8B). By combining the various monovalent RNA aptamers
of the present invention to create a pentavalent RNA aptamer, it
was possible to create an aptamer having higher avidity for B52.
The five tandemly arranged RNA aptamer sequences correspond to BBS
#11 (SEQ. ID. No. 3), BBS #23 (SEQ. ID. No. 4), two copies of BBS
#8 (SEQ. ID. No. 2), and BBS #4, 14, 15 (SEQ. ID. No. 1). Each of
the five tandemly arranged RNA aptamer sequences has a hairpin loop
structure that has a neck portion of various lengths and a loop
portion. Specifically, each of the loop portions contains
nucleotides 5-12 of SEQ. ID. No. 5 as non-paired bases. The
nucleotide sequence for the mature multivalent RNA aptamer
corresponds to SEQ. ID. No. 8 as follows:
1 gcggccgccu ccgcggccgc cugaugaguc cgugaggacg aaacaugcau gucgagagua
60 cgaucaacca ggcgacagua cucucgacga ucaaccaggc gacaguggcu
ggucaaccag 120 gcgaccgcca cugcagggua acggucaacc aggcgaccgu
uacccggacg gucaaccagg 180 cgaccguuga cuucggucag ucgagaugca uguc
214
[0072] Once the structure and sequence of the multivalent RNA
aptamer has been established, a gene capable of encoding such an
RNA aptamer can be prepared. Therefore, another aspect of the
present invention relates to a DNA molecule and, more particularly,
a gene which encodes the RNA aptamers of the present invention.
[0073] According to one embodiment, the DNA molecule encodes a
monovalent RNA aptamer of the present invention.
[0074] One such DNA molecule encodes the monovalent RNA aptamer BBS
#4,14,15 and has a nucleotide sequence corresponding to SEQ. ID.
No. 9 as follows:
2 gtaatacgac tcactatagg gagaattcaa ctgccatcta ggcagggtaa cgatcaacct
60 ggcgacagct gccctgccgt ccaagtacta caagcttctg gactcggt 108
[0075] Another such DNA molecule encodes the monovalent RNA aptamer
BBS#8 and has a nucleotide sequence corresponding to SEQ. ID. No.
10 as follows:
3 gtaatacgac tcactatagg gagaattcaa ctgccatcta ggctggtcaa ccaggcgacc
60 gccacccgcg cgcgcaatac ctagtactac aagcttctgg actcggt 107
[0076] Another such DNA molecule encodes the monovalent RNA aptamer
BBS#11 and has a nucleotide sequence corresponding to SEQ. ID. No.
11 as follows:
4 gtaatacgac tcactatagg gagaattcaa ctgccatcta ggctgctcac gagtccatga
60 ccagtacgat caaccaggcg acagtactac aagcttctgg actcggt 107
[0077] Still another such DNA molecule encodes the monovalent RNA
aptamer BBS#23 and has a nucleotide sequence corresponding to SEQ.
ID. No. 12 as follows:
5 gtaatacgac tcactatagg gagaattcaa ctgccatcta ggcccaactg ctaagaagca
60 tcctgtacga tcaacccggc gacagtacta caagcttctg gactcggt 108
[0078] According to another embodiment, the DNA molecule encodes a
multivalent RNA aptamer of the present invention. For DNA molecules
encoding a multivalent RNA aptamer, it is preferable for the DNA
molecule to contain a plurality of monomeric DNA sequences ligated
"head-to-tail", each of which encodes a multivalent RNA aptamer.
This is particularly useful for augmenting the number of
multivalent RNA aptamers produced during each transcriptional
event. By plurality, it is intended that the number of monomeric
DNA sequences be at least two, preferably at least four, more
preferably at least eight, and most preferably at least twelve.
Such tandemly arrayed sequences are known to be relatively stable
in bacteria (Lindquist, "Varying Patterns of Protein Synthesis in
Drosophila During Heat Shock: Implications for Regulation," Dev.
Biol. 77:463-479 (1980), which is hereby incorporated herein by
reference) and can persist for many generations in transgenic fly
lines (Xiao and Lis, "A Consensus Sequence Polymer Inhibits in vivo
Expression of Heat Shock Genes," Mol. Cell. Biol. 6:3200-3206
(1986); Shopland and Lis, "HSF Recruitment and Loss at Most
Drosophila Heat Shock Loci is Coordinated and Depends on Proximal
Promoter Sequences," Chromosoma 105:158-171 (1996), which are
hereby incorporated by reference). This strategy should be
applicable to other organisms. For example, long direct repeating
sequences have been used in yeast (Robinett et al., "In vivo
Localization of DNA Sequences and Visualization of Large-scale
Chromatin Organization Using lac Operator/Repressor Recognition,"
J. Cell. Biol. 135:1685-700 (1996), which is hereby incorporated by
reference). It should be apparent to those of ordinary skill in the
art, however, that the number of monomeric DNA sequences can vary
for each application of the DNA molecule.
[0079] Depending upon the desired application and intended use for
the DNA molecule, it is possible to produce homopolymers containing
a plurality of substantially identical monomeric DNA sequences or
copolymers containing a plurality of substantially different
monomeric DNA sequences. The mature multivalent RNA aptamers
produced from such a homopolymer are a single type, each capable of
inhibiting the activity of the same target. In contrast, the mature
multivalent RNA aptamers produced from such a copolymer are
different types, each capable of inhibiting the activity of a
distinct target or, alternatively, binding to discrete surfaces of
the same target. Thus, the plurality of monomeric DNA sequences can
be substantially identical (i.e., producing substantially the same
multivalent RNA aptamer) or they can be substantially different
(i.e., producing substantially different multivalent RNA aptamers).
When the plurality of monomeric DNA sequences are substantially
different, the resulting RNA multivalent aptamers can be directed
to the same or to different target molecules.
[0080] When the DNA molecule encodes a plurality of monomeric DNA
sequences, it is important that the resulting RNA transcript be
cleaved into the individual multivalent RNA aptamers. To this end,
it is particularly desirable for each of the plurality of monomeric
DNA sequences to also encode a cis-acting ribozyme that can cleave
the immature RNA transcript of the DNA molecule to yield multiple
copies of the mature multivalent RNA aptamers. Although any
ribozyme sequence can be utilized, a hammerhead ribozyme sequence
(Haseloff and Gerlach, "Simple RNA Enzymes with New and High
Specific Endoribonucleases Activities," Nature 334:585-591 (1988),
which is hereby incorporated by reference) is preferred because of
its simplified and efficient structure. The sequence encoding the
hammerhead ribozyme is incorporated into each of the plurality of
monomeric DNA sequences, resulting in the hammerhead ribozyme being
located at the 3' end of each monomeric unit of the immature RNA
transcript. The immature RNA transcript is self-cleaved by the
cis-acting ribozyme to yield the mature multivalent RNA
aptamer.
[0081] When the DNA molecule is a polymer encoding an immature RNA
transcript containing more than one multivalent RNA aptamer, this
self-cleavage produces three different kind of RNA fragments of the
immature RNA transcript, as shown in FIG. 1. Fragment A contains
the target-binding region, the tetra-loop, and a portion of the
ribozyme sequence, while fragment C is the terminal fragment of the
immature RNA transcript and has no aptamer sequence. Fragment B is
the mature multivalent RNA aptamer molecule of the present
invention, which is expected to fold into a stable structure when
an eight base pair stem forms following the cleavage (i.e., by the
ribozyme) and holds both 5' and 3' ends together in the S35 motif
as described above. The structure of a mature pentavalent RNA
aptamer specific for Drosophila B52 (SEQ. ID. No. 8) is shown in
FIG. 8B. The molar fraction of Fragment B in the cleavage product
increases in proportion to the number of monomeric DNA sequences
contained in the template.
[0082] One such DNA molecule of the present invention is a monomer
which encodes an immature pentavalent RNA aptamer and has a
nucleotide sequence corresponding to SEQ. ID. No. 13 as
follows:
6 gtcgagagta cgatcaacca ggcgacagta ctctcgacga tcaaccaggc gacagtggct
60 ggtcaaccag gcgaccgcca ctgcagggta acggtcaacc aggcgaccgt
tacccggacg 120 gtcaaccagg cgaccgttga cttcggtcag tcgagatgca
tgtcgcggcc gcctccgcgg 180 ccgcctgatg agtccgtgag gacgaaacat gcat
214
[0083] The nucleotide sequence for the monomeric immature RNA
transcript encoded by this DNA molecule corresponds to SEQ. ID. No.
14 as follows:
7 gucgagagua cgaucaacca ggcgacagua cucucgacga ucaaccaggc gacaguggcu
60 ggucaaccag gcgaccgcca cugcagggua acggucaacc aggcgaccgu
uacccggacg 120 gucaaccagg cgaccguuga cuucggucag ucgagaugca
ugucgcggcc gccuccgcgg 180 ccgccugaug aguccgugag gacgaaacau gcau
214
[0084] The structure of this immature RNA transcript is shown in
FIG. 8A.
[0085] Once the DNA molecule of the present invention has been
constructed, it can be incorporated in cells using conventional
recombinant DNA technology. Generally, this involves inserting the
DNA molecule into an expression system to which the DNA molecule is
heterologous (i.e., not normally present). The heterologous DNA
molecule is inserted into the expression system or vector in proper
sense orientation. The vector contains the necessary elements for
the transcription of the RNA molecule of the present invention.
[0086] U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby
incorporated by reference, describes the production of expression
systems in the form of recombinant plasmids using restriction
enzyme cleavage and ligation with DNA ligase. These recombinant
plasmids are then introduced by means of transformation and
transfection, and replicated in cultures including prokaryotic
organisms and eukaryotic cells grown in tissue culture.
[0087] Recombinant or engineered genes may also be introduced into
viruses, such as vaccinia virus. Recombinant viruses can be
generated by transfection of plasmids into cells infected with
virus.
[0088] Suitable vectors include, but are not limited to, the
following viral vectors such as lambda vector system gt11, gt
WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325,
pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37,
pKC101, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene
Cloning Systems" Catalog (1993) from Stratagene, La Jolla, Calif.,
which is hereby incorporated by reference), pQE, pIH821, pGEX, pET
series (see Studier et. al., "Use of T7 RNA Polymerase to Direct
Expression of Cloned Genes," Gene Expression Technology, vol. 185
(1990), which is hereby incorporated by reference), and any
derivatives thereof. Suitable vectors are continually being
developed and identified. Recombinant molecules can be introduced
into cells via transformation, transduction, conjugation,
mobilization, or electroporation. The DNA sequences are cloned into
the vector using standard cloning procedures in the art, as
described by Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y. (1982),
which is hereby incorporated by reference.
[0089] A variety of host-vector systems may be utilized to express
the monovalent RNA aptamer-encoding sequence(s) or the multivalent
RNA aptamer-encoding sequence(s). Primarily, the vector system must
be compatible with the host cell used. Host-vector systems include
but are not limited to the following: bacteria transformed with
bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such
as yeast containing yeast vectors; mammalian cell systems infected
with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell
systems infected with virus (e.g., baculovirus); and plant cells
infected by bacteria or transformed via particle bombardment (i.e.,
biolistics). The expression elements of these vectors vary in their
strength and specificities. Depending upon the host-vector system
utilized, any one of a number of suitable transcription elements
can be used.
[0090] Transcription of the DNA molecule of the present invention
is dependent upon the presence of a promoter which is a DNA
sequence that directs the binding of RNA polymerase and thereby
promotes RNA synthesis. The DNA sequences of eukaryotic promoters
differ from those of procaryotic promoters. Furthermore, eukaryotic
promoters and accompanying genetic signals may not be recognized in
or may not function in a prokaryotic system and, further,
prokaryotic promoters are not recognized and do not function in
eukaryotic cells.
[0091] Promoters vary in their "strength" (i.e., their ability to
promote transcription). For the purposes of expressing the
constructed DNA molecule or engineered gene, it is desirable to use
strong promoters in order to obtain a high level of transcription
and, hence, expression of the gene. Depending upon the host cell
system utilized, any one of a number of suitable promoters may be
used. For instance, when cloning in E. coli, its bacteriophages, or
plasmids, promoters such as the T7 phage promoter, lac promoter,
trp promoter, recA promoter, ribosomal RNA promoter, the P.sub.R
and P.sub.L promoters of coliphage lambda and others, including but
not limited, to lacUV5, ompF, bla, lpp, and the like, may be used
to direct high levels of transcription of adjacent DNA segments.
Additionally, a hybrid trp-lacUV5 (tac) promoter or other E. coli
promoters produced by recombinant DNA or other synthetic DNA
techniques may be used to provide for transcription of the inserted
gene.
[0092] Bacterial host cell strains and expression vectors may be
chosen which inhibit the action of the promoter unless specifically
induced. In certain operons, the addition of specific inducers is
necessary for efficient transcription of the inserted DNA. For
example, the lac operon is induced by the addition of lactose or
IPTG (isopropylthio-beta-D-galac- toside). A variety of other
operons, such as trp, pro, etc., are under different controls.
[0093] Specific initiation signals are also required for efficient
gene transcription in procaryotic cells. These transcription
initiation signals may vary in "strength" as measured by the
quantity of gene specific messenger RNA and protein synthesized,
respectively. The DNA expression vector, which contains a promoter,
may also contain any one of various "strong" transcription
initiation signals.
[0094] Once the constructed DNA molecules encoding the monovalent
RNA aptamers or multivalent RNA aptamers, as described above, have
been cloned into an expression system, they are ready to be
incorporated into a host cell. Such incorporation can be carried
out by the various forms of transformation noted above, depending
upon the vector/host cell system. Suitable host cells include, but
are not limited to, bacteria, yeast, mammalian cells, insect cells,
plant cells, and the like. The host cell is preferably present
either in a cell culture or in a non-human living organism.
[0095] Plant tissue suitable for transformation include leaf
tissue, root tissue, meristems, zygotic and somatic embryos, and
anthers. It is particularly preferred to utilize embryos obtained
from anther cultures.
[0096] The expression system of the present invention can be used
to transform virtually any plant tissue under suitable conditions.
Tissue cells transformed in accordance with the present invention
can be grown in vitro in a suitable medium to control expression of
a target molecule (e.g., a protein or nucleic acid) using an RNA
aptamer of the present invention, preferably a multivalent RNA
aptamer of the present invention. Transformed cells can be
regenerated into whole plants such that the expressed RNA aptamer
regulates the function or activity of the target protein in the
intact transgenic plants.
[0097] In producing transgenic plants, the DNA construct in a
vector described above can be microinjected directly into plant
cells by use of micropipettes to transfer mechanically the
recombinant DNA (Crossway, Mol. Gen. Genetics, 202:179-85 (1985),
which is hereby incorporated by reference). The genetic material
may also be transferred into the plant cell using polyethylene
glycol (Krens, et al., Nature, 296:72-74 (1982), which is hereby
incorporated by reference).
[0098] One technique of transforming plants with the DNA molecules
in accordance with the present invention is by contacting the
tissue of such plants with an inoculum of a bacteria transformed
with a vector comprising a DNA molecule or an engineered gene in
accordance with the present invention. Generally, this procedure
involves inoculating the plant tissue with a suspension of bacteria
and incubating the tissue for 48 to 72 hours on regeneration medium
without antibiotics at 25-28.degree. C.
[0099] Bacteria from the genus Agrobacterium can be utilized to
transform plant cells. Suitable species of such bacterium include
Agrobacterium tumefaciens and Agrobacterium rhizogenes.
Agrobacterium tumefaciens (e.g., strains C58, LBA4404, or EHA105)
is particularly useful due to its well-known ability to transform
plants.
[0100] Heterologous genetic sequences can be introduced into
appropriate plant cells, by means of the Ti plasmid of A.
tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri
plasmid is transmitted to plant cells on infection by Agrobacterium
and is stably integrated into the plant genome (Schell, Science,
237:1176-83 (1987), which is hereby incorporated by reference).
[0101] After transformation, the transformed plant cells must be
regenerated.
[0102] Plant regeneration from cultured protoplasts is described in
Evans et al., Handbook of Plant Cell Cultures, Vol. 1, MacMillan
Publishing Co., New York (1983) and Vasil (ed.), Cell Culture and
Somatic Cell Genetics of Plants, Acad. Press, Orlando, Vol. I
(1984) and Vol. III (1986), which are hereby incorporated by
reference.
[0103] It is known that practically all plants can be regenerated
from cultured cells or tissues.
[0104] Thus, another aspect of the present invention relates to an
engineered gene which includes the DNA sequence encoding a
multivalent RNA aptamer, as described above, and a regulatory
sequence which controls expression of the DNA sequence encoding the
multivalent RNA aptamer.
[0105] As described above, one type of regulatory sequence is a
promoter located upstream or 5' to the DNA sequence encoding the
multivalent RNA aptamer. Depending upon the desired activity, it is
possible to select the promoter for not only in vitro production of
the multivalent RNA aptamers of the present invention, but also in
vivo production in cultured cells or whole organisms, as described
above. As shown in FIGS. 1 and 7, the in vivo production can be
regulated genetically. Thus, a preferable type of promoter is an
inducible promoter which induces transcription of the DNA sequence
in response to specific conditions, thereby enabling expression of
the multivalent RNA aptamer according to desired therapeutic needs
(i.e., expression within specific tissues, or at specific temporal
and/or developmental stages).
[0106] Preferred promoters for use in the engineered gene of the
present invention include a T7 promoter, a hsp70 promoter, a Mtn
promoter, a UAShs promoter, and functional fragments thereof. The
T7 promoter is a well-defined, short DNA sequence that can be
recognized and utilized by T7 RNA polymerase of the bactieriophage
T7. The T7 RNA polymerase can be purified in large scale and is
commercially available. The transcription reaction with T7 promoter
can be conducted in vitro to produce a large amount of the RNA
aptamers of the present invention (Milligan et al.,
"Oligoribonucleotide Synthesis Using T7 RNA Polymerase and
Synthetic DNA Templates," Nucleic Acids Res. 15(21):8783-8798
(1987), which is hereby incorporated by reference. The heat shock
promoters are heat inducible promoters driven by the RNA polymerase
II in eukaryotes. The frequency with which RNA polymerase II
transcribes the major heat shock genes can be increased rapidly in
minutes over 100-fold upon heat shock. The heat shock promoter used
in the present invention is preferably a Drosophila hsp70 promoter,
more preferably a portion of the Drosophila hsp70 promoter which is
fully functional with regard to heat inducibility and designated
heat inducible cassette, or Hic (Kraus et al., "Sex-specific
Control of Drosophila melanogaster Yolk Protein 1 Gene Expression
is Limited to Transcription," Mol. Cell. Biol. 8:4756-4764 (1988),
which is hereby incorporated by reference). Another inducible
promoter driven by RNA polymerase II used in the preferred
embodiment of the present invention is a metallothionein promoter,
which is inducible to the similar degree as the heat shock promoter
in a time course of hours (Stuart et al., "A 12-base-pair Motif
that is Repeated Several Times in Metallothionein Gene Promoters
Confers Metal Regulation to a Heterologus Gene," Proc. Natl. Acad.
Sci. USA 81:7318-7322 (1984), which is hereby incorporated by
reference). An additional promoter used in the present invention is
a constructed hybrid promoter in which the yeast upstream
activation sequence for the GAL1 genes was fused to the core
Drosophila hsp70 promoter (Brand and Perrimon, "Targeted Gene
Expression as a Means of Altering Cell Fates and Generating
Dominant Phenotypes," Development 118:401-415 (1993), which is
hereby incorporated by reference). This promoter is no longer
activated by heat shock. Rather, it is activated by the yeast GAL4
protein, a transcription activator that is normally not present in
Drosophila. The yeast GAL4 gene has been introduced into
Drosophila, and is itself under a variety of transcriptional
control in different fly lines.
[0107] For example, in vitro production of a pentavalent RNA
aptamer from a DNA molecule of the present invention was driven by
a T7 promoter. The accurate trimming of the immature RNA transcript
at both 5' and 3' ends by the ribozyme allowed the use of a
circular template for higher transcriptional efficiency than the
conventional run-off transcription (Taira et al., "Construction of
a Novel RNA-Transcript Trimming Plasmid Which Can Be Used Both in
vitro in Place of Run Off and (G) Free Transcriptions and in vivo
as Multi-Sequence Transcription Vectors," Nucleic Acids Res.
19:5125-5130 (1991), which is hereby incorporated by
reference).
[0108] In contrast, in vivo production of the mature pentavalent
RNA aptamer was achieved using several different promoters driven
by the RNA polymerase II. These promoters are very strong, yet
tightly regulated. For example, a BBS dodeca-pentamer transcribed
from the promoter of the induced heat shock genes would yield about
1000 B52 binding sites in a minute, which would build up to an
intranuclear concentration of more than a hundred nanomolar in
about 10 minutes, assuming the half-life of the RNA is
significantly longer than this time scale. (The diameter of a
Drosophila nucleus is 2 .mu.m, which results in a nuclear volume of
3.4.times.10.sup.-12. When fully induced, an hsp70 promoter fires
productively once every 4 seconds, which follows from the density
of RNA polymerase II being one per 80 bp and the elongation rate
being 1.2 kb/min.)
[0109] In addition, the mature multivalent RNA aptamers can be
directed to specific subcellular compartments to ensure that they
will encounter the intended target and be concentrated in the
organelle where the target resides. To direct in vivo produced RNA
to specific subcellular locations, several approaches can be used.
RNA will stay in the nuclei if it does not have an exporting signal
such as a polyadenyl tail. To export RNA from the nucleus, a
specific RNA sequence or structure, such as the Constitutive
Transport Element of the type D retrovirus (Bray et al., "A Small
Element from the Mason-Pfizer Monkey Virus Genome Makes Human
Immunodeficiency Virus Type 1 Expression and Replication
Rev-independent," Proc. Natl. Acad. Sci. USA 91:1256-1260 (1994);
Ernst et al., "A Structured Retroviral RNA Element that Mediates
Nucleocytoplasmic Export of Intron-containing RNA," Mol. Cell.
Biol. 17:135-144(1997), which are hereby incorporated by reference)
can be appended to the RNA constructs as ancillary elements. To
direct RNA aptamers to other subcellular locations, specific
proteins may be attached to the RNA aptamer to carry the RNA to its
destiny. A second level of spatial control is achieved by
tissue-specific promoters, which have to be driven by the RNA
polymerase II. The many types of cells in animals and plants are
created largely through mechanisms that cause different genes to be
transcribed in different cells, and many specialized animal cells
can maintain their unique character when grown in culture. The
tissue-specific promoters involved in such special gene switching
mechanisms, which are driven by RNA polymerase II, can be used to
drive the transcription templates that code for the RNA aptamers of
the present invention, providing a means to restrict the expression
of the aptamers in particular tissues.
[0110] Additional aspects of in vitro and in vivo production of the
mature pentavalent RNA aptamer of the present invention are
described in Shi, "Perturbing Protein Function with RNA Aptamers,"
Thesis, Cornell University, University Microfilms, Inc. (1997), and
Shi et al., "Artificial Genes Expressing RNA Aptamers as Specific
Protein Inhibitors in vivo," Nucleic Acids Symp. Ser. 36:194-196
(1997), which are hereby incorporated by reference.
[0111] Another aspect of the present invention relates to a
transgenic non-human organism whose somatic and germ cell lines
contain an engineered gene encoding a multivalent RNA aptamer which
inhibits activity of a target molecule to treat a condition
associated with an expression level of the target molecule. The
engineered gene is a gene of the present invention. The target
molecule can be any target used in the selection process,
preferably a protein or nucleic acid.
[0112] The transgenic non-human organism is preferably a
multicellular organism, such as a plant, an animal, or an insect.
The plant can be a monocot or a dicot. The animal can be a mammal,
an amphibian, a fish, a reptile, or a bird. Preferred transgenic
mammals of the present invention include sheep, goats, cows, dogs,
cats, all primates, such as monkeys and chimpanzees, and all
rodents, such as rats and mice. Preferred insects include all
species of Drosophila, particularly Drosophila melanogaster.
[0113] According to one embodiment of the present invention, the
transgenic organism is a transgenic insect, namely Drosophila
melanogaster, whose somatic and germ cell lines contain an
engineered gene encoding a multivalent RNA aptamer which inhibits
activity of Drosophila splicing factor B52 to treat various
conditions associated with over-expression of Drosophila splicing
factor B52.
[0114] Related aspects of the present invention involve methods of
expressing a multivalent RNA aptamer in a cell which include
introducing either a DNA molecule of the present invention or an
engineered gene of the present invention into a cell under
conditions effective to express the multivalent RNA aptamer. As
described above, the conditions under which expression will occur
are dependent upon the particular promoter or other regulatory
sequences employed.
[0115] Another aspect of the present invention relates to a method
of inhibiting the activity of a target molecule in a cell which
includes expressing a multivalent RNA aptamer in a cell, where the
multivalent RNA aptamer has an affinity for the target molecule
sufficient to inhibit activity of the target molecule. The target
molecule can be any target used in the selection process,
preferably a protein or nucleic acid. This method also includes
introducing into the cell, prior to the step of expressing, a DNA
molecule encoding the multivalent RNA aptamer. As described above,
expression of the DNA molecule can be under the control of any one
of a variety of regulatory sequences such as promoters, preferably
inducible promoters. The cell can be in an in vitro environment, in
an in vivo cell culture, or in vivo within an organism.
[0116] Another aspect of the present invention relates to a method
of increasing activity of a splicing factor protein in a cell. This
method includes inserting a multivalent RNA aptamer, which binds to
a splicing factor protein, into an RNA transcript, which contains
exons and introns, under conditions effective to enable splicing of
the RNA transcript. The splicing factor protein is preferably
Drosophila splicing factor B52 or a homologous splicing factor. To
be effective, the RNA aptamer must be transcribed in cis with the
RNA transcript containing the exons and introns. This can be
accomplished by inserting a heterologous DNA molecule of the
present invention into the genome of a host cell using the
techniques described above.
[0117] Thus, in trans, the pentavalent RNA aptamer specific for
Drosophila splicing factor B52 can suppress the splicing of a
pre-mRNA substrate. In cis, the RNA aptamer sequence can enhance
B52-dependent pre-mRNA splicing. When the pentavalent RNA aptamer
was expressed in vivo, it was efficiently synthesized, it was
stable, and it accumulated to high level within the nuclei where
its target resides. Moreover, Drosophila splicing factor B52 was
demonstrated to be recruited to the chromosome site of production
for the pentavalent RNA aptamer, providing direct evidence of their
interaction in vivo. The efficacy of the pentavalent RNA aptamer as
a Drosophila splicing factor B52 antagonist at the organismal level
in Drosophila was demonstrated by its capability of averting all
phenotypes caused by B52 overexpression.
[0118] Although the use of a plurality of monomeric DNA sequences
and a cis-acting ribozyme has been described above in connection
with the multivalent RNA aptamer, it should be apparent to one of
ordinary skill in the art that this approach is applicable to the
expression, in a cell, of any functional RNA molecule, e.g.,
monovalent or multivalent RNA aptamers, ribozymes, and antisense
RNA. Thus, other aspects of the invention include: (1) a
constructed DNA molecule that contains a plurality of monomeric
sequences each encoding a functional RNA molecule; (2) an
engineered gene that includes a DNA sequence that contains a
plurality of monomeric sequences each encoding a functional RNA
molecule and a regulatory sequence which controls expression of the
DNA sequence; (3) methods of expressing a functional RNA molecule
in a cell by introducing such a constructed DNA molecule or
engineered gene into a cell under conditions effective to express
the functional RNA molecule; and (4) a transgenic non-human
organism whose somatic and germ cell lines contain an engineered
gene that contains a plurality of monomeric sequences each encoding
a functional RNA molecule, where the functional RNA molecule
encoded by the engineered gene inhibits the activity of a target
molecule to treat a condition associated with an expression level
of the target molecule.
[0119] These aspects of the present invention are further
illustrated by the examples below.
EXAMPLES
[0120] The following examples are provided to illustrate
embodiments of the present invention but they are by no means
intended to limit its scope.
[0121] The materials and methods described below are applicable for
all of the following examples.
[0122] Protein Expression and Purification
[0123] Target protein from different organisms can be prepared
either directly from tissue samples or through recombinant DNA
methodology using knowledge in the art. The target in the
Drospohila melanogaster model system, the full length B52 protein,
was expressed in Sf9 cells using the baculovirus expression system.
The B52 cDNA used in these Examples was described previously (Kraus
and Lis, "The Concentration of B52, an Essential Splicing Factor
and Regulator of Splice Site Choice, is Critical for Drosophila
Development," Mol. Cell. Biol. 14:5360-5370 (1994), which is hereby
incorporated by reference) and was cloned into the vector pJVP10Z
(Ueda et al., "Human Monocyte Chemoattractant Protein-1 Expressed
in a Baculovirus System," Gene 140:267-272 (1994), which is hereby
incorporated by reference). The transfection, purification, and
culturing of the recombinant baculovirus was performed as described
previously (Summers and Smith, A Manual of Methods for Baculovirus
Vectors and Insect Cell Culture Procedures, College Station, Tex.,
Texas Agricultural Experiment Station (Bulletin No. 1555) (1987);
Groebe et al., "Cationic Lipid-Mediated Co-transfection of Insect
Cells," Nucleic Acids Res. 18:4033 (1990), which are hereby
incorporated by reference). The over-expressed B52 was purified
using the standard SR protein purification procedure (Zahler et
al., "SR Proteins: A Conserved Family of Pre-mRNA Splicing
Factors," Genes Dev. 6:837-847 (1992), which is hereby incorporated
by reference). B52 deletion constructs were generated by PCR
amplification of the corresponding regions of the B52 cDNA. They
were then cloned into the vector pGEM.RTM.-3Z (Promega Corporation,
Madison, Wis.). Truncated versions of B52 were made by in vitro
translation using the TNT.RTM. Coupled Reticulocyte Lysate System
(Promega Corporation, Madison, Wis.) with L-(.sup.35S) Methionine
(in vivo cell labeling grade, Amersham Life Science Inc.,
Cleveland, Ohio) according to the manufacturer's instructions. The
quality of translation products was checked on SDS-PAGE prior to
use.
[0124] Oligonucleotides
[0125] The template-primer system consists of three
oligonucleotides identical or similar to those used by Bartel et
al. ("HIV-1 Rev Regulation Involves Recognition of Non-Watson-Crick
Base Pairs in Viral RNA," Cell 67:529-536 (1991), which is hereby
incorporated by reference). The synthesized template, designated
Temp, has a nucleotide sequence corresponding to SEQ. ID. No. 15 as
follows:
[0126] accgagtcca gaagcttgta gtactnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn
[0127] nnnnngccta gatggcagtt gaattctccc tatagtgagt cgtattac 108
[0128] where the internal 40 bases is a random sequence. A first
primer, designated T7Univ, has a nucleotide sequence corresponding
to SEQ. ID. No. 16 as follows:
[0129] gtaatacgac tcactatagg gagaattcaa ctgccatcta 40
[0130] A second primer, designated RevUniv, has a nucleotide
sequence corresponding to SEQ. ID. No. 17 as follows:
[0131] accgagtcca gaagcttgta gt 22
[0132] These oligonucleotides were synthesized at 0.2 .mu.M
quantity by Integrated DNA Technologies, Inc. (Coralville,
Iowa).
[0133] The templates of the deletion, mutation and antisense
constructs of BBS were made as oligonucleotides appended with the
T7 promoter sequence. They were synthesized as Gibco BRL Custom
Primers.
[0134] The monomeric template of the immature pentavalent RNA
aptamer was made by ligation of three fragments, each of which was
synthesized as a pair of oligonucleotides as Gibco BRL custom
primers. Their sequences are as follows:
8 P1+ accgctcgag agtacgatca accaggcgac agtactctcg acgatcaacc
aggcgacagt 60 (SEQ. ID. No.18) P1- aaactgcagt ggcggtcgcc tggttgacca
gccactgtcg cctggttgat cgtcgagagt 60 (SEQ. ID. No19) P2+ aaactgcagg
gtaacggtca accaggcgac cgttacccgg acggtcaacc aggcg 55 (SEQ. ID.
No.20) P2- acgcgtcgac tgaccgaagt caacggtcgc ctggttgacc gtccgggtaa
cggtc 55 (SEQ. ID. No.21) P3+ accgctcgag atgcatgtcg cggccgcctc
cgcggccgcc tgatgagtcc 50 (SEQ. ID. No.22) P3- acgcgtcgac atgcatgttt
cgtcctcacg gactcatcag gcggccgcgg 50 (SEQ. ID. No.23)
[0135] In vitro Selection
[0136] Pool construction: Gel-purified synthetic oligo
deoxynucleotides Temp (SEQ. ID. No. 15), T7Univ (SEQ. ID. No. 16),
and RevUniv (SEQ. ID. No. 17) were used as template and primers for
a 5-ml PCR reaction carried out in 10 Eppendorf tubes. The
temperature cycling was performed by manual transfer among three
water baths and stopped after four cycles when the product was
still increasing nearly two-fold per cycle. The amplified DNA
template was phenol-extracted and ethanol precipitated.
[0137] Cycles of selection: RNA for each round was produced using
the T7-MEGAshortscript.TM. in vitro transcription kit for large
scale synthesis of short transcript RNAs (Ambion Inc., Austin,
Tex.) according to the manufacturer's instructions. Gel purified
RNA was quantified by spectrophotometry and diluted into 1.times.
binding buffer (50 mM Tris/Cl, pH 7.6, 200 mM KoAc, 5 mM
MgCl.sub.2, 2.5 mM dithiothreitol). The pool was heated to
70.degree. C. for 3 minutes and then cooled to ambient temperature
over 5 minutes before adding the protein. Prior to every other
cycle, the folded RNA pool was also passed through a nitrocellulose
filter (HAWP 02500 available from Millipore Inc., New Bedford,
Miss.). The binding reaction was performed in 100 .mu.l volume,
with 30 .mu.M RNA and 30 nM B52 protein. In the first three cycles,
40 u of rRNasin (Promega Corporation, Madison, Wis.) was also
included. The reaction was incubated for 80 minutes, with
temperature cycling between 25.degree. C. (or ambient temperature)
and 37.degree. C., such that the reaction was at 25.degree. C. for
three-quarters of the time (Bartel and Szostak, "Isolation of New
Ribozymes From a Large Pool of Random Sequences," Science
261:1411-1418 (1993), which is hereby incorporated by reference).
Protein-RNA complexes were isolated by filtration and extracted as
previously described (Tuerk and Gold, "Systematic Evolution of
Ligands by Exponential Enrichment: RNA Ligands to Bacteriophage T4
DNA Polymerase," Science 249:505-510 (1990), which is hereby
incorporated by reference). The selected RNAs were amplified using
the SUPERSCRIPT.TM. Pre-amplification System for First Strand cDNA
Synthesis and Taq DNA Polymerase (Life Technologies Corporation,
Gaithersburg, Md.) according to the manufacturer's
instructions.
[0138] Cloning and Sequencing
[0139] The pool DNAs were cloned in the vector pGEM.RTM.-3Z,
between the EcoRI and HindIII sites of the poly-linker region.
Individual clones were sequenced using the Taq cycle sequencing
method with DyeDeoxy.TM. Terminators on a Applied Biosystems 737A
automated DNA Sequencer by the DNA Services facility at Cornell
Biotechnology Programs.
[0140] RNA-Protein Binding Assay
[0141] The RNA probes were uniformly labeled with [a-.sup.32P] UTP
(Amersham Life Science Inc., Cleveland, Ohio) using the
T7-MAXIscript.TM. in vitro transcription kit (Ambion, Inc., Austin,
Tex.) according to the manufacturer's instructions. When the cloned
sequences from the selected pools were to be used, the plasmids
were linearized by digestion with ScaI to serve as templates. The
templates of the deletion, mutation, and antisense constructs of
BBS were made as oligonucleotides appended with the T7 promoter
sequence. They were synthesized as GibcoRBL Custom Primers. The
template plasmids of the immature pentavalent RNA aptamer were
linearized by digestion with EcoRI. The RNAs used as competitors
were prepared using the T7-MAGAshortscrip.TM. in vitro
transcription kit (Ambion, Inc., Austin, Tex.) according to the
manufacturer's instructions. Prior to use in a binding assay, the
majority of transcripts of each RNA preparation were shown to be of
the expected size by electrophoresis on an 8% polyacrylamide, 7 M
urea gel.
[0142] All binding assays were performed in 20 .mu.l volume in
1.times. binding buffer described above. A typical binding assay
using labeled RNA contains about 20 finole of .sup.32P-labeled RNA
probe and differing amounts (1-10 pmole) of B52 protein. A typical
binding assay using the truncated versions of B52 contains 1 .mu.l
of the .sup.35S-labeled translation reaction mixture with 2 pmoles
of unlabeled RNA transcript (the final concentration of RNAs in
this reaction was 100 nM and concentration of the labeled B52
proteins was much lower). The reactions were allowed to equilibrate
for 15-20 minutes at ambient temperature before being subjected to
filter binding, gel shift, or UV crosslinking.
[0143] Gel shifts were performed at 4.degree. C. The binding
reaction mixtures were set at 4.degree. C. for 5-10 minutes before
being loaded onto a 2.5% agarose gel in 1/4 TBE buffer. The avidity
of the pentavalent RNA aptamer to B52 was examined on longer
agarose gels (25 cm) to improve the resolution of RNA bands. The
affinity of the RNA for the in vitro translated polypeptides was
estimated by comparing the intensity of the bands representing the
complex on the agarose gel and that of the corresponding
polypeptides run on SDS-PAGE. To crosslink proteins to RNA, the
binding reaction mixtures were irradiated for 20 minutes from 5 cm
directly above by inverting a short-wavelength UV
trans-illuminator. The products of crosslinking were analyzed using
standard SDS-PAGE.
[0144] Footprinting
[0145] Footprinting procedures were modified from those previously
reported (Christiansen et al., "Analysis of rRNA Structure:
Experimental and Theoretical Considerations" in Ribosomes and
Protein Synthesis: A Practical Approach, Spedding (ed.), IRL Press,
New York (1990), which is hereby incorporated by reference). The
RNA substrate (100 ng) was incubated with or without B52 under
binding conditions. RNase T2 (1 u) or RNase V1 (0.35 u) was added
and incubated at room temperature for 1 minute. The digestion was
stopped by a phenol and chloroform extraction followed by ethanol
precipitation. The precipitated RNAs were re-suspended and analyzed
by primer extension with .sup.32P labeled pUC/M13 reverse
sequencing primer and Superscript II Reverse Transcriptase (Life
Technologies) and then separated on either a 6% (RNase T2
digestion) or 8% (RNase V1 digestion) sequencing gel.
[0146] Splicing Assay
[0147] The inhibitory RNA was prepared in vitro with the
T7-MAGAshort script.TM. in vitro transcription kit (Ambion, Inc.,
Austin, Tex.) and purified on a 5% polyacrylamide gel with 7M urea.
The ftz pre-mRNA was produced by run-off transcription from XhoI
linearized plasmid pGEM2 V61 S/B (Rio, "Accurate and Efficient
pre-mRNA Splicing in Drosophila Cell-free Extracts," Proc. Natl.
Acad. Sci. USA 89:2904-2908 (1998), which is hereby incorporated by
reference). The pentavalent monomeric unit of BBS, P1-2-3/BBS(5),
was cloned into the same XhoI site of the pGEM V61 S/B in both
directions, resulting in the plasmids V61+1.times.5 (ftz+BBS(5))
and V61-1.times.5 (ftz-BBS(5)). When used as templates,
V61+1.times.5 was linearized by NotI, and V61-1.times.5 by ScaI.
Nuclear and cytoplasmic (S100) extracts were made from Kc cells
(Dignam et al., "Accurate Transcription Initiation by RNA
Polymerase II in a Soluble Extract from Isolated Mammalian Nuclei,"
Nucleic Acids Res. 11: 1475-1489 (1983), which is hereby
incorporated by reference). In vitro splicing reactions were
assembled essentially as described in Rio, "Accurate and Efficient
Pre-mRNA Splicing in Drosophila Cell-free Extracts," Proc. Natl.
Acad. Sci. USA 89:2904-2908 (1988), which is hereby incorporated by
reference, and carried out at 20.degree. C. for 90 minutes. The
resulting RNAs were separated on a 6% polyacrylamide gel containing
7M urea.
[0148] RNase Protection Assay
[0149] Total RNA from transfected cells was prepared with Trizol
reagent (Life Technologies Corporation, Gaithersburg, Md.). Total
RNA from flies was prepared using the RNAqueous Total RNA Isolation
Kit (Ambion Inc., Austin, Tex.) from late third instar larvae.
RNase protection assay was performed using the HybSpeed RPA
protocol (Ambion Inc., Austin, Tex.). To determine the abundance of
the pentavalent RNA aptamer, the internally labeled antisense
transcript of part of the monomeric pentavalent RNA aptamer
sequence was used as probe. 4 .mu.g of the RNA samples from
transfected cells or 1-2 .mu.g of RNA samples from larvae, both
DNase treated, were used in each assay.
[0150] Cell Transfection
[0151] To construct the HicBBS series and the MtnBBS series of
plasmid, the transcriptional templates of the immature pentavalent
RNA aptamer were lifted from the pSP73 vectors as XhoI-SalI
fragments and cloned into the SalI site of Hic-L vector (Kraus et
al., "Sex-Specific Control of Drosophila melanogaster Yolk Protein
1 Gene Expression is Limited to Transcription," Mol. Cell. Biol.
8:4756-4764 (1988), which is hereby incorporated by reference) or
the XhoI site of pMtnEX vector. Both orientations of the insert
were recovered in some cases. 2.5 .mu.g plasmid DNA was used to
transform each 6 mm plate of S2 cells (initially 5.times.10.sup.6)
with Lipofectin (Life Technologies Corporation, Gaithersburg, Md.)
according to the manufacturer's instruction. The genes were induced
24 hours after transfection by either heat shock at 36.5.degree. C.
for 90 minutes or adding CuSO.sub.4 to final concentration of 0.5
mM for 24 hours. The half life of the mature pentavalent RNA
aptamer was measured by treating the cells with actinomycin (Life
Technologies, 35 .mu.l, 1 mg/ml) immediately after heat shock, and
harvesting cells at 0, 2, 4, and 8 hours thereafter.
[0152] Generation of Transgenic Fly Lines
[0153] The gene encoding the mature pentavalent RNA aptamer was
moved from the HicBBS(5.12) plasmid as a BamHI-EcoRV fragment to
the BamHI-HpaI site of the pW8 vector (Klemenz et al., "The White
Gene as a Marker in a New P-Element Vector for Gene Transfer in
Drosophila," Nucleic Acid Res. 15:3947-3959 (1987), which is hereby
incorporated by reference) to generate the pW8-HicBBS (5.12)
plasmid. To generate the pUASBBS series of plasmids, the different
length transcriptional templates (i.e., dimers, tetramers,
octamers, or dodecamers) of the pentavalent RNA aptamer, as
XhoI-SalI fragments, were cloned into the XhoI site of the pUAST
vector (Brand and Perrimon, "Targeted Gene Expression as a Means of
Altering Cell Fates and Generating Dominant Phenotypes,"
Development 118:401-415 (1993), which is hereby incorporated by
reference). Drosophila germ line transformation was performed
according to a previously developed protocol (Park and Lim, "A
Microinjection Technique for Ethanol-Treated Eggs and a Mating
Scheme for Detection of Germ Line Transformants," Dro. Inf. Serv.
76:197-199 (1995), which is hereby incorporated by reference) with
minor modifications. Briefly, the embryos from w, .DELTA.2-3 (99B)
were collected from orange juice collection plates and immersed in
95% ethanol for 3.5 minutes before being arranged on an orange agar
coated coverslip. The DNA preparation was injected at concentration
of 500 ng/.mu.l. The coverslip was put on a grape plate and kept at
room temperature. The flies recovered were mated individually with
z.sup.1w.sup.11e4 to establish two or three broods and all progeny
were examined for colored eyes. Independent transformants were back
crossed to z.sup.1w.sup.11e4 several times to generate stable lines
before homozygosing. At least two independent transformants were
isolated for each construct.
[0154] To express the UASBBS genes, a number of GAL4 lines were
mated with different UASBBS lines. To generate the triple
transgenic flies containing the B52, BBS, and GAL4 transgenes, one
representative transgene from each suite of GAL4, UASB52, and
UASBBS transgenes was first selected to synthesize three double
transgenic fly lines, UASB52-UASBBS, hsGAL4-UASB52, and
hsGAL4-UASBBS. The double transgenic fly lines were synthesized by
manipulating the second and the third chromosome with an additional
multiple balancer line, CUX, whose genotype is In(2LR)O, Cy; TM2,
Ubx.sup.130/T(2;3)ap.sup.Xa. Each of the double transgenic lines
was then mated with a series of corresponding single transgenic
lines to produce heterozygous triple transgenic flies. A scheme is
shown in FIG. 14 that illustrates the synthesis of an UASB52
transgene on the second chromosome and an UASBBS(5.12) transgene on
the third chromosome to make a homozygous double transgenic line
UASB52-UASBBS, which is then mated with a homozygous GAL4 line to
generate the heterozygous triple transgenic flies in which both B52
and BBS are actively transcribed. All transgene-bearing chromosomes
were kept homozygous, except those in the hsGAL4-UASB52, which were
balanced. All fly lines were maintained at 24.degree. C. For heat
shock treatment, flies in glass vials were kept in a 36.5.degree.
C. incubator for the indicated time. In most cases, reciprocal
crosses of each genotype was set up by mating 5 females with 3
males in a glass vial. Larval phenotype were examined at the 6th
day after mating; surviving adults were examined and counted at
16th day.
[0155] In situ Hybridization and Immunofluorescence
[0156] The RNA probe was internally labeled with ChromaTide Texas
Red-5-UTP (Molecular Probes, Inc., Eugene, Oreg.) by in vitro
transcription with T7 RNA polymerase. Hybridization of the probe to
whole, formaldehyde-fixed salivary gland tissue was performed at
60.degree. C. overnight in solution containing 50% formamide,
5.times.SSC, 100 .mu.g/ml yeast RNA, 50 .mu.g/ml heparin, and 0.1%
Tween-20. The glands were subsequently washed at 60.degree. C. for
3-4 hours in eight changes of solution in which the hybridization
buffer is gradually displaced by the PBT buffer (Drosophila PBS
plus 0.1% Tween-20). Polytene chromosome spreads were prepared from
salivary glands of late third instar larvae according to (Champlin
et al., "Distribution of B52 Within a Chromosomal Locus Depends on
the Level of Transcription," Molec. Biol. Cell. 5:71-79 (1991),
which is hereby incorporated by reference) with minor
modification.
[0157] The anti-B52 antibody was described in (Kraus and Lis, "The
Concentration of B52, an Essential Splicing Factor and Regulator of
Splice Site Choice, is Critical for Drosophila Development," Mol.
Cell. Biol. 14:5360-5370 (1994), which is hereby incorporated by
reference). Immunofluorescence was performed as described in
(Champlin et al., "Distribution of B52 Within a Chromosomal Locus
Depends on the Level of Transcription," Molec. Biol. Cell. 5:71-79
(1991), which is hereby incorporated by reference).
Example 1
Identification of B52 Aptamers
[0158] The B52 target protein used in the selection was
overexpressed from a baculovirus construct in insect cells. The B52
produced by these insect cells appears to be full length and
properly modified, as it has the same electrophoretic mobility as
the Drosophila protein. Immunoblot analysis of this same
preparation using a B52 specific antibody displays the identical
mobility of B52 when produced in baculovirus or assayed in
Drosophila nuclear extract. In addition, baculovirus produced B52
is fully active in an in vitro splicing assay. Truncated versions
of B52 were not used as target because the involvement of the SR
domain in possible sequence-specific binding could not be excluded
a priori.
[0159] The pool of random RNA was carried through nine rounds of
selection and amplification. RNA-protein complexes were selected by
binding to nitrocellulose filters. A significant increase in
affinity by the pool for B52 was observed as the selection
progressed. Fractions of two final pools were cloned and sequenced.
Some cloned sequences were found in duplicate or triplicate,
indicating that the complexity of these selected pools was quite
low.
[0160] The B52 aptamer family in the selected pools consists of
four different sequences, one of which occurred in three separate
isolates (FIG. 2). Since all six members of this family showed
specific binding to B52, they are designated as a B52-Binding
Sequence or Site ("BBS").
[0161] As previously described, BBS #4,14,15 has a nucleotide
sequence corresponding to SEQ. ID. No. 1, BBS #8 has a nucleotide
sequence corresponding to SEQ. ID. No. 2, BBS #11 has a nucleotide
sequence corresponding to SEQ. ID. No. 3, and BBS #23 has a
nucleotide sequence corresponding to SEQ. ID. No. 4.
[0162] These sequences have a conserved region (SEQ. ID. No. 5)
which contains two absolutely conserved hexamers separated by a
variable nucleotide. There is also a conserved G in the flanking
region of either side one base from the end of the double-hexamer.
This sequence motif is found in different sequence contexts and in
different positions of the randomized region. In two of these
sequences, BBS #8 (SEQ. ID. No. 2) and BBS #4/14/15 (SEQ. ID. No.
1), sequence similarity extends further downstream, covering a
region of additional 16 nucleotides in length.
[0163] The computer program MulFold (Jaeger et al., "Improved
Predictions of Secondary Structures for RNA," Proc. Natl. Acad.
Sci. USA 86:7706-7710 (1989); Zuker, "On Finding All Suboptimal
Foldings of an RNA Molecule," Science 244:48-52 (1989), which are
hereby incorporated by reference) was utilized to examine the free
energy minimized secondary structures of the BBS's with their
flanking constant region (i.e., the RNA molecules as they were
selected). The conserved region of all four different sequences was
predicted to fold into a common secondary structure element core
(FIG. 2B): the double-hexamer forms the loop portion and part of
the stem portion of a hairpin loop structure, and its flanking
regions form the extended stem which may contain a bulge or
internal loops. In different structures, the hairpin loop
encompasses from 23 to 38 nucleotides with 6 to 12 Watson-Crick
base pairs and some G-U pairs. The loops and the top of the stems
are identical in all four structures, with the loop portion
containing nucleotides 5-12 of SEQ. ID. No. 5. Since the random
sequence was only 40 nucleotides in length, it is not surprising
that this structural element includes sequences from the primer
annealing region in some cases. Some bases in the constant region
on either side of the random stretch even become part of the
conserved sequence of different BBS's. These structures suggest
that the common structural element may be selected for binding to
B52.
[0164] When individual RNA's transcribed from cloned sequences were
tested by filter binding assay the BBS RNA's were all able to bind
B52 to generate signals that were 20 times the background produced
by RNA's from the unselected pools. A band shift assay was also
developed as an independent way to assess the affinity and
specificity of binding of the selected sequences to B52, and these
results are shown in FIGS. 3A and 3B. After cloning the selected
sequences, RNA transcripts without most of the 3' constant region
were made by cutting the template with the restriction endonuclease
Scal. For BBS #8 (SEQ. ID. No. 2) and BBS #14 (SEQ. ID. No. 1) this
trimming did not interfere with stem formation. The band shift
assay was first performed with these sequences. Two sequences
randomly picked from the original unselected pool were used as
controls. Increasing amounts of B52 were added to .sup.32P-labeled
RNA probes, which contain the 5' constant region, in the presence
of a large excess of tRNA. Stable complexes were formed with BBS
sequences but not with the control sequence from the unselected
pool. Since only one shifted complex is observed, and B52 is a
monomer, it could be assumed the interaction between B52 and RNA
has a 1:1 stoichiometry. Because the concentration of the RNA
probes in these reactions is negligible as compared to that of B52,
the apparent dissociation constant of the complex was estimated to
be roughly equal to the protein concentration at which 50% RNA is
bound. For example, lane 6 of FIG. 3A, where 50% of RNA is bound,
indicates that the K.sub.d of the B52-BBS #14 (SEQ. ID. No. 1)
complex is about 50 nM. BBS #8 (SEQ. ID. No. 2), also on this gel,
displayed an even higher affinity to B52, with a K.sub.d of
approximately 20 nM.
[0165] The specificity of the B52-RNA interaction was examined by
subjecting it to the challenge of different specific competitors.
As shown in FIG. 3B, a constant amount of B52 (5 pmole) was
incubated with increasing amounts of three different unlabeled RNA
competitors before the addition of either the BBS #8 (SEQ. ID. No.
2) or BBS #14 (SEQ. ID. No. 1) probes. It is obvious that both BBS
#8 (SEQ. ID. No. 2) and BBS #14 (SEQ. ID. No. 1) can compete with
themselves (Lanes 1-3 & 13-15), and with each other (Lanes 4-6
& 10-12), but the random control sequence, designated G0, does
not compete with these interactions (Lanes 7-9 & 16-18). G0 has
a nucleotide sequence corresponding to SEQ. ID. No. 24 as
follows:
[0166] gagacccacc gacacctcgg ccggcggggc ttttagcgag 40
[0167] To further test the specificity of the BBS, additional
binding assays were performed. RBP1, another Drosophila SR protein,
and HSF, the Drosophila heat shock factor did not show any
measurable affinity for the BBS RNA's. Also, since B52 has been
shown previously to crosslink with DNA in vivo (Champlin et al.,
"Distribution of B52 Within a Chromosomal Locus Depends on the
Level of Transcription," Molec. Biol. Cell. 5:71-79 (1991), which
is hereby incorporated by reference), single-stranded DNA
containing the BBS motif was also tested for its ability to bind
B52. No B52-DNA complex was detected in the band shifting assay
under similar conditions used for BBS RNA transcripts. These
results indicate that B52 possesses distinct RNA binding
specificity for the BBS RNA.
Example 2
Defining the Minimum RNA Sequence Requirements for Binding to
B52
[0168] To determine the sequence/structure requirements for binding
by B52, deletion and substitution mutations, and antisense
constructs of BBS were designed in light of the shared sequence
motif and predicted secondary structures (FIG. 4). These short RNA
transcripts were produced by in vitro transcription of synthetic
templates, and their affinity to B52 was assayed by band shift.
[0169] The approximate 5' and 3' termini of the aptamer binding
site were determined by deletion analysis. Based on the sequence of
BBS #8 (SEQ. ID. No. 2), an RNA construct, BBS-I/Long, was made to
contain the region shared by all BBS RNAs plus the region only
shared with #14, but missing both 5' and 3' constant regions. The
nucleotide sequence of BBS-I/Long (SEQ. ID. No. 25) is as
follows:
[0170] ggcuggucaa ccaggcgacc gccacccgcg cgc 33
[0171] It was predicted to retain the BBS #8 hairpin loop
structure, which is the shortest and most stable hairpin among the
four BBS's, plus a 3' unfolded tail. This construct demonstrated
full binding activity.
[0172] Next, the 3' tail was deleted to make the construct BBS-I,
which contained only the hairpin loop structure; it was also fully
active. The nucleotide sequence of BBS-I (SEQ. ID. No. 26) is as
follows:
[0173] ggcuggucaa ccaggcgacc gcc 23
[0174] A third deletion construct, designated BBS-I/NoBulge, was
prepared by deleting the bulged U in the stem. Its binding activity
was not compromised either. The nucleotide sequence of
BBS-I/NoBulge (SEQ. ID. No. 27) is as follows:
[0175] gqcggucaac caggcgaccg cc 22
[0176] Another construct, designated BBS-II, which is part of the
BBS #11 sequence (SEQ. ID. No. 3) and has the conserved hairpin
loop with an internal loop in the stem, binds B52 as well as BBS-I.
The nucleotide sequence of BBS-II (SEQ. ID. No. 28) is as
follows:
[0177] ggquacgauc aaccaggcga caguaccc 28
[0178] However, when several nucleotides on each side of BBS-II
were deleted to decrease the stability of the stem, the affinity to
B52 decreased almost by a factor of three. This construct,
designated bbs-II, was comparable to BBS-I in length, containing
the sequence shared by all members of BBS family, and was predicted
to have no stable secondary structure in solution. However, the
possibility that non-Watson-Crick base pairs, base-ribose, and/or
base-phosphate interactions may occur in the internal loop region
to produce a compact and stable structure, or that binding to B52
may stabilize the remaining weak pairs to give a structure like the
one in the full sequences cannot be ruled out. The nucleotide
sequence of bbs-II (SEQ. ID. No. 29) is as follows:
[0179] ggacgaucaa ccaggcgaca gu 22
[0180] The last deletion construct, designated AltStem, contained
only the double-hexamer region plus one of the two conserved G's at
the 5' end. The resulting predicted structure, a smaller and
slightly different hairpin, still retained some low affinity for
B52, but much less than that of bbs-II. The sequence of AltStem
(SEQ. ID. No. 30) is as follows:
[0181] ggucaaccag gcgac 15
[0182] The above data suggest that both primary sequence and
secondary structure of the RNA's contribute to their affinity for
B52. Therefore, substitution variants were prepared to separate the
effect of sequence and structure. Based on the sequence of BBS-II
(SEQ. ID. No. 28), the variant FlipBBS-II was prepared by
interchanging part of the sequences in each half of the stem. The
nucleotide sequence of FlipBBS-II (SEQ. ID. No. 31) is as
follows:
[0183] ggcaugaauc aaccaggcga cgcaugcc 28
[0184] Based on the sequence of BBS-I (SEQ. ID. No. 26), the
variant TransBBS-I was prepared by making two G-C to A-U
transitions in the stem. Both these constructs, in which the
conserved flanking G's were replaced, exhibited poor binding even
though their predicted secondary structures were unaffected by the
changes. The nucleotide sequence of TransBBS-I (SEQ. ID. No. 32) is
as follows:
[0185] ggaugucaac caggcgacau cc 22
[0186] Then based on bbs-II, the unstable internal loop region was
converted into a stem by changing sequences on either side of the
loop, resulting in the variants bbs-II/5'Stem and bbs-II/3'Stem,
respectively. The nucleotide sequence of bbs-II/5'Stem (SEQ. ID.
No. 33) is as follows:
[0187] ggacugucaa ccaggcgaca gu 22
[0188] The nucleotide sequence of bbs-II/3'Stem (SEQ. ID. No. 34)
is as follows:
[0189] ggacggucaa ccaggcgacc gu 22
[0190] Although both have a similarly stable hairpin loop
structure, bbs-II/5'Stem, which has only one conserved flanking G,
was a weak binder, while bbs-II/3'Stem, which has both conserved
flanking G's, was a strong one. In making bbs-II/3'Stem, a sequence
identical to that of BBS-I was generated in between the two
end-most conserved G's.
[0191] As a negative control, antisense RNA of BBS-I and bbs-II
were made. The nucleotide sequence of Antibbs-I (SEQ. ID. No. 35)
is as follows:
[0192] ggcggucgcc ugguugacca gcc 23
[0193] The nucleotide sequence of AntiBBS-II (SEQ. ID. No. 36) is
as follows:
[0194] ggacugucgc cugguugauc gu 22
[0195] Both antisense constructs did not bind B52, although
AntiBBS-I (SEQ. ID. No. 35) has a hairpin loop structure similar to
BBS-I.
[0196] In addition, the importance of the single-strandedness of
the core BBS consensus was assessed by linking it to its antisense
sequence via a UUCG tetra-loop. The nucleotide sequence of UUCG/BBS
(SEQ. ID. No. 37) is as follows:
[0197] ggucgccugg uugaucuucg gaucaaccag gcgaca 36
[0198] The BBS consensus sequence trapped in an RNA duplex lost its
binding activity.
[0199] These experiments demonstrated that the sequence between and
including the two end-most conserved G's (i.e., SEQ. ID. No. 5) is
required for efficient recognition by B52. While most of the
conserved sequence resides in the loop, these variants showed that
particular sequences of the stem, particularly the conserved
flanking G's, are also critical for this interaction. The flanking
region on both sides of this conserved sequence segment can
contribute to binding. While not wishing to be bound by a
particular theory, it is believed that the flanking regions help to
form a more stable stem, probably by pre-paying some entropic cost
of the interaction.
[0200] On the basis of the binding results, it was determined that
a strong B52-binding site on RNA contains at least 17 nucleotides,
namely SEQ. ID. No. 5, and generally about 20 nucleotides, which is
the length of most hairpin loops tested.
Example 3
Probing the Secondary Structure and Critical Regions in the
B52-Binding Site of RNA
[0201] To delineate at higher resolution the B52 binding region on
the selected RNA, RNase footprinting was performed with the cloned
BBS #8 (SEQ. ID. No. 2). Two RNases with different specificities
were used to determine the region of binding in both single and
double stranded areas of RNA. These experiments have the added
benefit of confirming the predicted hairpin loop secondary
structure of the selected RNA.
[0202] RNase T2 is a single-strand specific endoribonuclease with
low specificity (a preference for adenines), while RNase V1 cleaves
RNA predominantly at double-stranded regions with no apparent
sequence specificity. Both free RNA and RNA-B52 mixtures were
treated with RNase T2 and V1, and the resulting RNA products were
detected by primer extension. This allowed an accurate
identification of the protected nucleotides, since sequencing
reactions using the same primer could be run alongside of the
footprinting reactions. A comparison of the primer extension
products from free RNA with those from RNA-B52 mixtures identified
residues that are in contact with the protein. As the B52
concentration is increased, the intensity of several bands
decreased (FIG. 5A, Lanes 2-3 and 9-11). Comparing the position of
these bands directly to the sequencing ladder served to identify
bases that are protected by the binding of B52. These B52-protected
bases reside in the predicted hairpin loop region (FIG. 5B). In
addition, the cleavage of the RNA substrate by these RNases in the
absence of B52 confirms the predicted hairpin loop secondary
structure of the selected RNA. The RNase protection results provide
physical evidence that the conserved hairpin loop of BBS RNA is the
target of B52 interaction.
Example 4
Locating the RNA-binding Site of B52 to Both RRMs
[0203] BBS RNAs were selected for their ability to bind full length
B52 protein. It is possible that B52 binds RNA through either one
or the other RRMs, the SR domain, or a combination of these
domains. To identify the specific RNA-binding site on B52, a set of
.sup.35S-methionine labeled B52 deletion constructs were prepared
by in vitro translation and then tested for their ability to bind
the selected BBS #8 RNA in two different assays (FIG. 6A). These
polypeptides also serve as a second source of target proteins in
binding assays to confirm the specificity of binding to the
selected BBS RNA.
[0204] The in vitro translated polypeptides were examined on
SDS-PAGE to verify that they were of the predicted size before
being used in the binding assays. The integrity of two
polypeptides, R1 and R12, is shown in FIG. 6B. Other polypeptides
were made in an identical way and had similar quality. FIG. 6A
shows a band shift assay with individual .sup.35S-labeled
polypeptides or these in combination. The binding reaction was set
up as previously described, but with a constant amount of BBS #8
RNA transcript in molar excess relative to protein. In the presence
of BBS #8 RNA, .sup.35S-labeled full length B52, F, showed a
discrete band (Lane 2, FIG. 6A), which has the same mobility as
that of a B52-BBS #8 complex generated with .sup.32P labeled RNA.
The polypeptide containing both RRMs, R12, gave rise to a band
running faster than the full length B52-BBS #8 complex (Lane 4,
FIG. 6A). The SR domain (S, Lane 6, FIG. 6A), the RRM I (R1, Lane
10, FIG. 6A), and a polypeptide containing both the RRM II and the
SR domain (R2S, Lane 12, FIG. 6A) did not produce any discrete band
on this native gel. When the polypeptides containing the two RRMs
(R12) and the SR domain (S) were put together in the presence of
the RNA, a band (Lane 8, FIG. 6A) was observed with similar
mobility as that seen with R12 in Lane 4. No shifted band was
produced with the other combination, polypeptides R1 plus R2S.
These data indicate that the SR domain of B52 does not participate
in any interaction with an RNA aptamer, and both RRMs are required
for the binding. In addition, the affinity of the RNA for both in
vitro synthesized polypeptides F and R12 is comparable to that
measured previously using baculovirus produced B52 protein in
excess over RNA.
[0205] To confirm the requirements of both RRMs for binding, these
binding reactions were repeated and the reaction mixtures were
subjected to UV irradiation. The UV-crosslinked species were then
analyzed on SDS-PAGE. As shown in FIG. 6B, R12 can be crosslinked
to the BBS #8 RNA, forming a complex with its apparent molecular
weight being the sum of the protein and RNA components (Lane 6). In
contrast, R1 was not crosslinked to RNA (Lane 3, FIG. 6B). In these
reactions, equal molar amounts of protein were used. Besides, both
RRMs must be in cis to bind RNA, since R2S cannot complement R1 to
restore the binding even when the RNA is at much a higher
concentration.
Example 5
Construction of the DNA Molecule Encoding the Pentavalent
RNA.sup.B52 Aptamer Specific for Drosophila and its Expression
System
[0206] The monomeric template of the pentavalent RNA aptamer was
made as three pairs of synthetic oligonucleotides. Each pair was
annealed and digested with proper restriction endonucleases to
generate compatible sticky ends (PstI, XhoI or SalI). The three
fragments were ligated to form the monomer template
P1-2-3/BBS(5.1), which was cloned in between the XhoI and SalI
sites of the pSP73 vector (Promega Corporation, Madison, Wis.).
After its sequence was confirmed, the monomer unit was prepared in
large scale as the XhoI-SalI fragment, and ligated into polymers in
the presence of both restriction enzymes in standard ligation
buffer at DNA concentrations of 100-200 ng/.mu.l (Xiao and Lis, "A
Consensus Sequence Polymer Inhibits in vivo Expression of Heat
Shock Genes," Mol. Cell. Biol. 6:3200-3206 (1986), which is hereby
incorporated by reference) to produce a head-to-tail array of the
XhoI-SalI fragments that are resistant to both restriction enzymes.
Polymers of different length (e.g., dimers, tetramers, octamers,
and dodecamers generated by one or more rounds of ligation) were
then cloned back into XhoI-SalI digested pSP73 and transformed into
the STBL2.TM. competent cells (Life Technologies, Gaithersburg,
Md.). Both orientations of the insert were recovered in some cases.
Next, the dodecamer and some shorter polymers were moved to sites
downstream of different promoters in different plasmids. For
example, they are cloned into the XhoI site of the pUAST vector
(Brand and Perrimon, "Targeted Gene Expression as a Means of
Altering Cell Fates and Generating Dominant Phenotypes,"
Development 118:401-415 (1993), which is herbey incorporated by
reference) and used in the Drosophila germ line transformation. A
list of engineered BBS genes is listed in FIG. 7.
Example 6
In vitro Large Scale Production of Pentavalent RNA Aptamer and its
Binding to Drosophila B52
[0207] The pentavalent RNA aptamer can be transcribed and
self-cleaved in vitro in large scale. The pentavalent RNA aptamer
transcribed from two different templates was compared after being
run on a polyacrylamide preparative gel and visualized by UV
shadowing as shown in FIG. 10A. A 1:1 ratio of Fragment A to
Fragment B was observed for the pentavalent RNA aptamer transcribed
from the BBS (5.2) templates (Lanes 1 and 2, FIG. 10A), and a
roughly 1:10 ratio was observed for the pentavalent RNA aptamer
transcribed from the BBS (5.12) templates (Lanes 3 and 4, FIG.
10A). No other bands representing higher molecular weight
transcripts were visible in these lanes. The self-cleavage reaction
was virtually completed during the overnight transcription. The
activity of the gel purified Fragment B--about 100 .mu.g was
recovered from gel purification--was tested in a competition
binding assay (FIG. 10B). BBS #8, the strongest-binding monovalent
RNA aptamer, was used as the probe in a gel shift assay with gel
purified pentavalent RNA aptamer and its antisense RNA, BBS(5-), as
competitors. The same amount of purified Torulla yeast RNA (Ambion)
consisting of fragments of 300-500 bases (yRNA) was used as a
control. The pentavalent RNA aptamer competed more efficiently than
monovalent aptamers for binding to B52.
Example 7
Increased Avidity of the Pentavalent RNA Aptamer for B52
[0208] As shown in FIG. 9A, the ribozyme cleavage of a single unit
of the immature RNA transcript BBS(5.1) yielded equal molar amount
of Fragment A and C. Fragment C, which contains no binding site,
served as an internal loading control in a band shift assay to
assess the avidity of the pentavalent construct. During the
transcription reaction, the extent of self-cleavage reached about
90% (FIG. 9A, Lane 1). All BBS sequences used in constructing the
pentavalent RNA aptamer had been previously tested individually for
their ability to bind B52, exhibiting an average dissociation
constant of 50 nM (ranging from 20 to 80 nM). The multivalent RNA
aptamers bound B52 10-fold more avidly than the individual
monovalent RNA aptamers, as shown here compared to the
strong-binding primary aptamer BBS #14 (Lanes 2-4 vs. Lanes 6-8,
FIG. 9A). Dimeric, tetrameric, octameric, and dodecameric
pentavalent RNA aptamer templates yielded RNA Fragments A, B, and
C, with the molar fraction of Fragment B (the functional
pentavalent RNA aptamer) increasing in proportion to the length of
the template. Indeed, all four constructs showed almost identical
pattern of bands on a native gel (FIG. 9B, Lanes 4, 7, 10, and 13),
with a notable increase in the ratio of Fragment B to C as the
length of the template increase. The pentavalent RNA aptamers
produced by each construct showed identical affinities for B52. The
similarity of the pattern of bands produced by transcriptional
templates of different length also indicated that each ribozyme
folded correctly and acted independently.
Example 8
Inhibiting B52 Function with the Pentavalent RNA Aptamer
[0209] The pentavalent RNA aptamer was produced in vitro in large
scale and tested for its ability to alter B52 function. Recombinant
B52 complements a Drosophila S100 splicing deficient extract,
allowing the accurate splicing of a ftz pre-mRNA derivative. Gel
purified pentavalent RNA aptamer (Fragment B) inhibited the
generation of spliced product as well as several splicing
intermediates (FIG. 11A, Lanes 3-5), while the same amount of the
antisense BBS (5.1) RNA or yeast genomic RNA of comparable size
caused no change in splicing activity in this assay (FIG. 11A,
Lanes 6-8 and 9-11). The lack of accumulation of splicing
intermediates in the BBS-inhibited splicing reactions indicates
that B52, like some other SR proteins, acts at an early step in
splicing (Fu, "The Superfamily of Arginine/Serine-Rich Splicing
Factors," RNA 1:663-680 (1995); Manley and Tacke, "SR Proteins and
Splicing Control," Genes Dev. 10(3):1569-1579 (1996), both of which
are hereby incorporated by reference). To confirm the specificity
of this inhibition, the splicing activity suppressed by the
pentavalent RNA aptamer was restored by adding additional amounts
of B52 to the suppressed assay mixture. These results support the
theory that the inhibition is through the pentavalent RNA aptamer
binding to the RRMs of B52, which prevents interaction of B52 with
the ftz pre-mRNA.
Example 9
Increasing B52 Activity with the Pentavalent RNA Aptamer
[0210] While B52 can be neutralized or sequestered by the addition
of pentavalent RNA aptamer in trans in a fashion similar to
antibody depletion of a protein as shown in Example 8, appending
the pentavalent RNA aptamer sequence directly to pre-mRNA has the
opposite effect. Selected aptamer RNAs that bind to other SR
proteins have been shown to function as synthetic splicing
enhancers when multiple copies are inserted in pre-mRNA substrates
(Tacke and Manley, "The Human Splicing Factors ASF/SF2 and SC35
Possess Distinct, Functionally Significant RNA Binding
Specificities," EMBO J. 14:3540-3551 (1995); Tacke et al.,
"Sequence-Specific RNA Binding by an SR Protein Requires RS Domain
Phosphorylation: Creation of an SRp40-Specific Splicing Enhancer,"
Proc. Natl. Acad. Sci. USA 94:1148-1153 (1997), both of which are
hereby incorporated by reference). A splicing substrate containing
the BBS pentamer affixed to the 3' end of the ftz pre-mRNA was
constructed and used in the splicing assay described in Example 8.
An enhancement in splicing activity was observed (FIG. 11B).
Notably, the S100 preparation has trace amount of B52, not
sufficient to prompt splicing of the original ftz substrate, but
enough to activate the splicing of the substrate bearing BBSs in
the 3' exon (FIG. 11B, Lane 5 vs. 1). The strong affinity of B52 to
the pentavalent RNA aptamer presumably allows B52 in low
concentration to bind this BBS-modified pre-mRNA and activate
splicing.
Example 10
Accumulation and Half-Life of the Pentavalent RNA Aptamer in
Cells
[0211] To evaluate the expression of the pentavalent RNA aptamer in
vivo, BBS-expressing genes were introduced into cultured Drosophila
S2 cells to assess the abundance and half-life of the pentavalent
RNA aptamer. Transient expression of the pentavalent RNA aptamer
was measured by quantitative RNase protection assay with in vitro
transcribed pentavalent RNA aptamer as standards. Two strongly
inducible promoters, a metallothionein promoter and a heat shock
promoter, were used to drive BBS transcriptional templates of
different length. While a metallothionein promoter (in the MtnBBS
constructs) can be induced by Cu.sup.2+ in a few hours, a heat
shock promoter (in the HicBBS constructs) becomes fully active
within minutes. The rapid induction of the heat shock promoter also
allows a more precise measurement of the half-life of the
pentavalent RNA aptamer. As shown in FIG. 12A, both promoters
resulted in similar levels of RNA accumulation when an identical
template was transcribed. After measuring the transfection
efficiency and by comparing the pentavalent RNA aptamer in the
total RNA sample to in vitro transcribed pentavalent RNA aptamer
standards, it was estimated that pentavalent RNA aptamer
transcribed from a dodecameric template can accumulate to a level
equivalent to 0.1% of total RNA or 10% of total mRNA. This abundant
accumulation of pentavalent RNA aptamer was also confirmed using
another Drosophila cell line, Kc.
[0212] To measure the half-life of the pentavalent RNA aptamer, the
cells were treated with actinomycin D (Lindquist, "Varying Patterns
of Protein Synthesis in Drosophila During Heat Shock: Implications
for Regulation," Dev. Biol. 77:463-479 (1980), which is hereby
incorporated by reference) to stop all transcription immediately
after heat shock, and the level of the pentavalent RNA aptamer in
cells harvested at 0, 2, 4, 8 hours thereafter was assayed. As
shown in FIG. 12A, the half-life of the pentavalent RNA aptamer was
about four hours (Lanes 2-5 and 7-10).
Example 11
Temporally and Spatially Regulated Expression of the Pentavalent
RNA Aptamer in Drosophila
[0213] To express the pentavalent RNA aptamer in flies, two systems
were compared. First, a heat shock promoter was used to directly
control the expression of the pentavalent RNA aptamer in the HicBBS
strains. Because of the precise temporal control over gene
expression, the HicBBS(5.12) strain (FIG. 12B, Lanes 9 and 10) was
used in cytological experiments. Since B52 is a nuclear protein,
the pentavalent RNA aptamer was designed to be retained inside the
nuclei. When the BBS transcriptional template is cloned into a
standard in vivo expression vector having a downstream
poly-adenylation signal, the ribozyme-cleaved pentavalent RNA
aptamer does not bear a poly-A tail and, therefore, should remain
nuclear. The exclusiveness of nuclear retention of the accumulated
pentavalent RNA aptamer was demonstrated by in situ hybridization
with whole mount salivary gland tissue as shown in FIG. 13A. The
co-compartmentalization of pentavalent RNA aptamer with its target
not only facilitated their encounter with each other, but also
achieved a considerable subcellular concentration of the
pentavalent RNA aptamer. In Drosophila, the polytene chromosomes
provided an ideal venue to visualize the in vivo interaction
between B52 and BBS, since the distribution of B52 protein on the
polytene chromosomes had been well-characterized (Champlin et al.,
"Characterization of a Drosophila Protein Associated With
Boundaries of Transcriptionally Active Chromatin," Genes Dev.
5:1611-1621 (1991); Champlin and Lis, "Distribution of B52 Within a
Chromosomal Locus Depends on the Level of Transcription," Molec.
Biol. Cell. 5:71-79 (1994), which are hereby incorporated by
reference). The locus of transgene insertion was mapped (FIG. 13B,
left panel) by polytene in situ hybridization. With a similar
technique, expression of the pentavalent RNA aptamer was visualized
as a medium-sized puff (FIG. 13B, middle panel). Immunofluorescence
with an anti-B52 antibody showed massive recruitment of B52 upon
heat shock to the insertion site of the HicBBS(5.12) transgene
(FIG. 13B, right panel). This co-localization of B52 and its
pentavalent RNA aptamer indicate an interaction between them in
vivo. Notably, this B52 recruitment to the site of nascent
pentavalent RNA aptamer synthesis far exceeded that at the native
heat shock loci, which are normally the strongest sites labeled
during heat shock. Also, at the transgenic insertion site, B52
covers the entire puff where RNA is made, in contrast to the puff
bracketing pattern seen at the native heat shock loci.
[0214] To further enhance the accumulation of pentavalent RNA
aptamer and to achieve spatial control of expression in different
tissues, BBS transgenes activated by the yeast transcription factor
GAL4 (Brand and Perrimon, "Targeted Gene Expression as a Means of
Altering Cell Fates and Generating Dominant Phenotypes,"
Development 118:401-415 (1993), which is hereby incorporated by
reference) were constructed. When GAL4 expression was controlled by
a heat shock promoter (in the hsGAL4 construct), an additional step
of amplification in the pentavalent RNA aptamer expression was
achieved, as shown in FIG. 12B. When identical templates (in this
case dodecameric ones) were used, indirect heat shock induction via
the GAL4-UAS system resulted in a several fold increase in
pentavalent RNA aptamer accumulation (FIG. 12B, Lanes 8 vs. 10).
Even without heat shock, the basal level transcription from the
heat shock promoter provided sufficient GAL4 to sustain a
steady-state pentavalent RNA aptamer level in the heterozygous
hsGAL4-UASBBS(5.12) flies comparable to that in the homozygous
HicBBS(5.12) flies immediately following heat treatment (FIG. 12B,
Lanes 7 vs. 10).
Example 12
Efficacy of the Pentavalent RNA Aptamer as B52 Antagonist at the
Organismic Level
[0215] Previous genetic studies had shown that the level of B52 is
critical to Drosophila development. While a B52 deletion resulted
in lethality (Ring and Lis, "The SR Protein B52/SRp55 is Essential
for Drosophila Development," Mol. Cell. Biol. 14:7499-7506 (1994),
which is hereby incorporated by reference), overproduction of B52
also caused lethality or morphological defects (Kraus and Lis, "The
Concentration of B52, an Essential Splicing Factor and Regulator of
Splice Site Choice, is Critical for Drosophila Development," Mol.
Cell. Biol. 14:5360-5370 (1994), which is hereby incorporated by
reference). To appraise the in vivo efficacy of the pentavalent RNA
aptamer as an inhibitor of B52, the phenotype caused by high level
expression of the pentavalent RNA aptamer was examined. It was
first noticed that the homozygous double transgenic line
hsGAL4-UASBBS(5.12) produces many fewer progeny than wild-type
strains, while the homozygous hsGAL4 line is as viable as
wild-type. To confirm that the reduced viability is caused by BBS
expression and to estimate the maximum tolerated dose, a genetic
test was designed in which flies carrying different copy numbers of
either transgene can be identified and counted (FIG. 14 & 15).
Two pairs of isogenetic double transgenic flies were synthesized
such that each pair has an hsGAL4-UASBBS line and an UASBBS-UASBBS
line. The only difference between the two lines is the chromosome
carrying hsGAL4 in one line is replaced by a chromosome carrying an
UASBBS in the other. By self-crossing the double balanced stocks of
both lines, the effect of the active transgenes in one line with
that of the dormant transgenes in the other could be compared. FIG.
15B shows the results of the two experiments. Reduced viability was
observed when more than one copy of UASBBS(5.12) was present. The
overall morphology of the surviving animals appeared normal.
[0216] A more rigorous verification of the pentavalent RNA
aptamer's mechanism of action and an assessment of its efficacy was
conducted in flies that over produce B52, which had been sensitized
to the change of B52 level. This was performed under the assumption
that co-expression of the pentavalent RNA aptamer may suppress the
phenotypes caused by B52 overexpression if it indeed acts as a B52
antagonist. To test this theory, transgenic fly lines were produced
containing genes that can over-express both B52 (UASB52) and
pentavalent RNA aptamer (UASBBS). Both genes have a promoter that
is strongly activated by the GAL4 activator of yeast that has been
also introduced to transgenic fly lines (FIG. 14). The GAL4 gene
itself can be controlled by a heat shock promoter or various
developmental enhancers. The expression of both B52 and BBS in the
heterozygous triple transgenic flies were examined by RNase
protection assay (FIG. 16). The expression of the GAL4 gene was
also confirmed by mating the GAL4 lines with a UASLacZ strain
followed by a .beta.-gal assay on whole mount larvae tissues.
[0217] Crosses that place the GAL4 gene and the UASB52 gene in the
same strain can produce five different phenotypes depending on the
pattern and level of GAL4 expression (Kraus and Lis, "The
Concentration of B52, an Essential Splicing Factor and Regulator of
Splice Site Choice, is Critical for Drosophila Development," Mol.
Cell. Biol. 14:5360-5370 (1994), which is hereby incorporated by
reference, and FIG. 17H). Remarkably, the introduction of a UASBBS
gene into a strain that has this B52 over production rescued all of
these phenotypes. One dramatic phenotype of B52 over-expression is
the absence of larval salivary glands. This salivary gland
development is largely restored by the co-expression of pentavalent
RNA aptamer (compare FIGS. 17B-17D). In addition, bristles of the
adult notum are missing in a line that over-expresses an UASB52
gene. Here too, the bristles are largely restored to their normal
number by co-expression of the pentavalent RNA aptamer (compare
FIGS. 17E-17G). Abnormal phenotypes of wings and abdominal
sternites, as well as the lethality caused by B52 over-expression
were also all suppressed in the presence of the pentavalent RNA
aptamer (FIG. 17H). Detailed description of the phenotypes is
provided below. Also examined was the effect of pentavalent RNA
aptamer dosage in rescuing lethality and bristle development
quantitatively using the cross scheme of FIG. 171, where the dose
of pentavalent RNA aptamer was varied by expressing pentavalent RNA
aptamer genes containing different numbers (i.e., dimers,
tetramers, octamers, or dodecamers) of pentavalent RNA aptamer
units. The degree of rescue of both B52 over-expression phenotypes
was proportional to pentavalent RNA aptamer dose. FIGS. 17J and 17K
depict the dosage-dependent effect of the pentavalent RNA aptamer
on bristle development and viability, respectively. These results
demonstrate that pentavalent RNA aptamer reverses all of the
phenotypes caused by B52 over-expression and strongly supports the
hypothesis that the pentavalent RNA aptamer can inhibit B52
function in vivo. Four different kinds of morphological changes and
lethality were observed with different level and temporal/spatial
pattern of B52 overexpression. Larval phenotypes were examined on
the 6th day after mating; surviving adults on the 16th day were
examined and counted (male and female separately). The following is
a qualitative and, where possible, quantitative description of the
phenotypes caused by B52 overexpression and its suppression by
co-expressing the pentavalent RNA aptamer. When both the additional
B52 gene and the BBS (iaRNA) gene are active the fly is designated
as {B52+BBS}. The patterns of GAL4 expression was described in
Kraus and Lis ("The Concentration of B52, an Essential Splicing
Factor and Regulator of Splice Site Choice, is Critical for
Drosophila Development," Mol. Cell. Biol. 14:5360-5370 (1994),
which is hereby incorporated by reference).
[0218] Salivary Glands
[0219] GAL4 source: hsGAL4, dppGAL4, G-17.
[0220] Phenotype: Glands are extremely small or absent
altogether.
[0221] Suppression: Glands bigger than 1/2 of its normal size were
counted as averted phenotype. Size variation and morphological
features were recorded. When dppGAL4 was used as the GAL4 source, a
100% suppression was observed in {B52+BBS}. However, the glands in
the third instar larvae appeared to be less translucent, less
smooth, and the size of both individual cells and the whole glands
appeared to be more variable.
[0222] Bristles
[0223] GAL4 source: hsGAL4
[0224] Phenotype: Long bristles or macrochaetes on the thorax, head
and legs are shorter, thinner, or absent altogether.
[0225] Suppression: Seven pairs of bristles were chosen to score:
the posterior super-alar, the dorso-centrals, the post-alars, and
the scutellars. Each full length bristle was counted as 1. The wild
type flies have the score 14. The sum of length of this set of
bristles in the transgenic flies were estimated and given a score
less or equal to 14. 20 animals were scored for each cross. The
suppression of this phenotype showed a dosage response to BBS.
[0226] Abdominal Sternites
[0227] GAL4 source: G-17, and I-65
[0228] Phenotype: Chaetes, especially on 3s, 4s, and 5s, are
missing or misplaced. The abnormal sternite usually still have more
than 10 chaetes. The defects are usually manifest in 40% adults of
both sex.
[0229] Suppression: Any missing or misplacement was counted as a
defect. When G-17 was used as the GAL4 source, almost all defects
were obviated in {B52+BBS}. When I-65 was used, about 40% viable
adults of {B52+BBS} showed this defect.
[0230] Wings
[0231] GAL4 source: dppGAL4, and hsGAL4.
[0232] Phenotype: The most severe phenotype is seen with dppGal4
(100%), where the wings are folded as they are within the pupal
case. The less severe phenotype is seen with hsGal4 (30-70%), where
the wings may be curled or wrinkled at the ends.
[0233] Suppression: The severity of the phenotype was ordered as
follows: folded>curled>wrinkled>easily ripped>normal.
Folded, curled, and wrinkled were counted. When dppGAL4 was used as
the GAL4 source, about 20% unfolded in {B52+BBS}. When hsGAL4 was
used, 5% remained curled in {B52+BBS}.
[0234] Lethality
[0235] GAL4 source: hsGAL4, dppGAL4, A-25, I-65, G-17.
[0236] Phenotype: Lethality can occur at different stages of
development. A-25: 100% third instar lethal. I-65: 100% first
instar lethal.
[0237] Suppression: Reduced viability was revealed by comparing the
number of adult living progeny from the experimental cross to that
of control crosses. When total lethality occurs, the developmental
stage was recorded. Survival beyond this stage was counted as
suppression. Surviving adults in each cross were counted.
Morphological features of the surviving animal were recorded. In
all cases viability was restored to different degrees. When A-25
was used, the surviving adults of {B52+BBS} had wing defects. When
I-65 was used, the survived adults {B52+BBS} had abdominal sternite
defects.
[0238] Although the invention has been described in detail for the
purpose of illustration, it is understood that such detail is
solely for that purpose, and variations can be made therein by
those skilled in the art without departing from the spirit and
scope of the invention which is defined by the following claims.
Sequence CWU 1
1
37 1 90 RNA Artificial Sequence Description of Artificial Sequence
monovalent RNA aptamer for Drosophila splicing factor B52 1
gggagaauuc aacugccauc uaggcagggu aacgaucaac cuggcgacag cugcccugcc
60 guccaaguac uacaagcuuc uggacucggu 90 2 89 RNA Artificial Sequence
Description of Artificial Sequence monovalent RNA aptamer for
Drosophila splicing factor B52 2 gggagaauuc aacugccauc uaggcugguc
aaccaggcga ccgccacccg cgcgcgcaau 60 accuaguacu acaagcuucu ggacucggu
89 3 89 RNA Artificial Sequence Description of Artificial Sequence
monovalent RNA aptamer for Drosophila splicing factor B52 3
gggagaauuc aacugccauc uaggcugcuc acgaguccau gaccaguacg aucaaccagg
60 cgacaguacu acaagcuucu ggacucggu 89 4 90 RNA Artificial Sequence
Description of Artificial Sequence monovalent RNA aptamer for
Drosophila splicing factor B52 4 gggagaauuc aacugccauc uaggcccaac
ugcuaagaag cauccuguac gaucaacccg 60 gcgacaguac uacaagcuuc
uggacucggu 90 5 17 RNA Artificial Sequence Description of
Artificial Sequence binding sequence of RNA aptamers for Drosophila
B52 splicing factor 5 gnucaaccng gcgacng 17 6 43 DNA Artificial
Sequence Description of Artificial Sequence 5' DNA template 6
gtaatacgac tcactatagg gagaattcaa ctgccatcta ggc 43 7 25 DNA
Artificial Sequence Description of Artificial Sequence 3' DNA
template 7 agtactacaa gcttctggac tcggt 25 8 214 RNA Artificial
Sequence Description of Artificial Sequence pentavalent RNA aptamer
for Drosophila splicing factor B52 8 gcggccgccu ccgcggccgc
cugaugaguc cgugaggacg aaacaugcau gucgagagua 60 cgaucaacca
ggcgacagua cucucgacga ucaaccaggc gacaguggcu ggucaaccag 120
gcgaccgcca cugcagggua acggucaacc aggcgaccgu uacccggacg gucaaccagg
180 cgaccguuga cuucggucag ucgagaugca uguc 214 9 108 DNA Artificial
Sequence Description of Artificial Sequence DNA encoding monovalent
RNA aptamer 9 gtaatacgac tcactatagg gagaattcaa ctgccatcta
ggcagggtaa cgatcaacct 60 ggcgacagct gccctgccgt ccaagtacta
caagcttctg gactcggt 108 10 107 DNA Artificial Sequence Description
of Artificial Sequence DNA encoding monovalent RNA aptamer 10
gtaatacgac tcactatagg gagaattcaa ctgccatcta ggctggtcaa ccaggcgacc
60 gccacccgcg cgcgcaatac ctagtactac aagcttctgg actcggt 107 11 107
DNA Artificial Sequence Description of Artificial Sequence DNA
encoding monovalent RNA aptamer 11 gtaatacgac tcactatagg gagaattcaa
ctgccatcta ggctgctcac gagtccatga 60 ccagtacgat caaccaggcg
acagtactac aagcttctgg actcggt 107 12 108 DNA Artificial Sequence
Description of Artificial Sequence DNA encoding monovalent RNA
aptamer 12 gtaatacgac tcactatagg gagaattcaa ctgccatcta ggcccaactg
ctaagaagca 60 tcctgtacga tcaacccggc gacagtacta caagcttctg gactcggt
108 13 214 DNA Artificial Sequence Description of Artificial
Sequence DNA encoding immature RNA transcript 13 gtcgagagta
cgatcaacca ggcgacagta ctctcgacga tcaaccaggc gacagtggct 60
ggtcaaccag gcgaccgcca ctgcagggta acggtcaacc aggcgaccgt tacccggacg
120 gtcaaccagg cgaccgttga cttcggtcag tcgagatgca tgtcgcggcc
gcctccgcgg 180 ccgcctgatg agtccgtgag gacgaaacat gcat 214 14 214 RNA
Artificial Sequence Description of Artificial Sequence Immature RNA
transcript 14 gucgagagua cgaucaacca ggcgacagua cucucgacga
ucaaccaggc gacaguggcu 60 ggucaaccag gcgaccgcca cugcagggua
acggucaacc aggcgaccgu uacccggacg 120 gucaaccagg cgaccguuga
cuucggucag ucgagaugca ugucgcggcc gccuccgcgg 180 ccgccugaug
aguccgugag gacgaaacau gcau 214 15 108 DNA Artificial Sequence
Description of Artificial Sequence DNA template 15 accgagtcca
gaagcttgta gtactnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60
nnnnngccta gatggcagtt gaattctccc tatagtgagt cgtattac 108 16 40 DNA
Artificial Sequence Description of Artificial Sequence DNA primer
16 gtaatacgac tcactatagg gagaattcaa ctgccatcta 40 17 22 DNA
Artificial Sequence Description of Artificial Sequence DNA primer
17 accgagtcca gaagcttgta gt 22 18 60 DNA Artificial Sequence
Description of Artificial Sequence DNA primer 18 accgctcgag
agtacgatca accaggcgac agtactctcg acgatcaacc aggcgacagt 60 19 60 DNA
Artificial Sequence Description of Artificial Sequence DNA primer
19 aaactgcagt ggcggtcgcc tggttgacca gccactgtcg cctggttgat
cgtcgagagt 60 20 55 DNA Artificial Sequence Description of
Artificial Sequence DNA primer 20 aaactgcagg gtaacggtca accaggcgac
cgttacccgg acggtcaacc aggcg 55 21 55 DNA Artificial Sequence
Description of Artificial Sequence DNA primer 21 acgcgtcgac
tgaccgaagt caacggtcgc ctggttgacc gtccgggtaa cggtc 55 22 50 DNA
Artificial Sequence Description of Artificial Sequence DNA primer
22 accgctcgag atgcatgtcg cggccgcctc cgcggccgcc tgatgagtcc 50 23 50
DNA Artificial Sequence Description of Artificial Sequence DNA
primer 23 acgcgtcgac atgcatgttt cgtcctcacg gactcatcag gcggccgcgg 50
24 40 DNA Artificial Sequence Description of Artificial Sequence
Random sequence for competitive binding 24 gagacccacc gacacctcgg
ccggcggggc ttttagcgag 40 25 33 RNA Artificial Sequence Description
of Artificial Sequence Deletion variant of monovalent RNA aptamer
25 ggcuggucaa ccaggcgacc gccacccgcg cgc 33 26 23 RNA Artificial
Sequence Description of Artificial Sequence Deletion variant of
monovalent RNA aptamer 26 ggcuggucaa ccaggcgacc gcc 23 27 22 RNA
Artificial Sequence Description of Artificial Sequence Deletion
variant of monovalent RNA aptamer 27 ggcggucaac caggcgaccg cc 22 28
28 RNA Artificial Sequence Description of Artificial Sequence
Deletion variant of monovalent RNA aptamer 28 ggguacgauc aaccaggcga
caguaccc 28 29 22 RNA Artificial Sequence Description of Artificial
Sequence Deletion variant of monovalent RNA aptamer 29 ggacgaucaa
ccaggcgaca gu 22 30 15 RNA Artificial Sequence Description of
Artificial Sequence Deletion variant of monovalent RNA aptamer 30
ggucaaccag gcgac 15 31 28 RNA Artificial Sequence Description of
Artificial Sequence Substitution variant of monovalent RNA aptamer
31 ggcaugaauc aaccaggcga cgcaugcc 28 32 22 RNA Artificial Sequence
Description of Artificial Sequence Substitution variant of
monovalent RNA aptamer 32 ggaugucaac caggcgacau cc 22 33 22 RNA
Artificial Sequence Description of Artificial Sequence Substitution
variant of monovalent RNA aptamer 33 ggacugucaa ccaggcgaca gu 22 34
22 RNA Artificial Sequence Description of Artificial Sequence
Substitution variant of monovalent RNA aptamer 34 ggacggucaa
ccaggcgacc gu 22 35 23 RNA Artificial Sequence Description of
Artificial Sequence Antisense sequence of monovalent RNA aptamer 35
ggcggucgcc ugguugacca gcc 23 36 22 RNA Artificial Sequence
Description of Artificial Sequence Antisense sequence of monovalent
RNA aptamer 36 ggacugucgc cugguugauc gu 22 37 36 RNA Artificial
Sequence Description of Artificial Sequence RNA duplex containing
binding sequence of RNA aptamer for Drosophila B52 splicing factor
and antisense sequence 37 ggucgccugg uugaucuucg gaucaaccag gcgaca
36
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