U.S. patent application number 10/187783 was filed with the patent office on 2003-06-26 for nucleic acid molecules capable of distinguishing the isoforms prpc and prpsc of prion proteins and processes for their production.
Invention is credited to Famulok, Michael, Weiss, Stefan, Winnacker, Ernst-Ludwig.
Application Number | 20030119019 10/187783 |
Document ID | / |
Family ID | 8219750 |
Filed Date | 2003-06-26 |
United States Patent
Application |
20030119019 |
Kind Code |
A1 |
Winnacker, Ernst-Ludwig ; et
al. |
June 26, 2003 |
Nucleic acid molecules capable of distinguishing the isoforms PrPc
and PrPSc of prion proteins and processes for their production
Abstract
The invention describes a process for the identification and
isolation of nucleic acid molecules capable of distinguishing the
isoforms PrP.sup.c and PrP.sup.Sc of prion proteins as well as
nucleic acid molecules obtainable by this process. Furthermore,
pharmaceutical compositions and diagnostic compositions are
described which comprise nucleic acid molecules specifically
binding prion protein isoforms as well as diagnostic methods using
such molecules.
Inventors: |
Winnacker, Ernst-Ludwig;
(Munchen, DE) ; Weiss, Stefan; (Munchen, DE)
; Famulok, Michael; (Munchen, DE) |
Correspondence
Address: |
Roylance, Abrams, Berdo & Goodman, L.L.P.
Suite 600
1300 19th Street, N.W.
Washington
DC
20036
US
|
Family ID: |
8219750 |
Appl. No.: |
10/187783 |
Filed: |
July 3, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10187783 |
Jul 3, 2002 |
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09051962 |
Oct 2, 1998 |
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6426409 |
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Current U.S.
Class: |
435/6.18 ;
435/91.2; 536/23.2 |
Current CPC
Class: |
C12Q 1/6811 20130101;
C12N 2310/16 20130101; C12Q 1/6883 20130101; C07H 21/00 20130101;
C12N 15/115 20130101 |
Class at
Publication: |
435/6 ; 435/91.2;
536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12P 019/34 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 26, 1995 |
EP |
95 11 6890.5 |
Claims
1. A process for the identification and isolation of nucleic acid
molecules which are capable of distinguishing between the isoforms
PrP.sup.c and PrP.sup.Sc of prion proteins associated with
transmissible spongiform encephalopathies comprising the steps of
(i) incubating a prion protein isoform or peptide fragment or
derivative of this prion protein isoform with a pool of nucleic
acid molecules comprising different sequences; (ii) selecting and
isolating those nucleic acid molecules which are capable of binding
to said prion protein isoform or fragment or derivative thereof;
(iii) optionally, amplifying the isolated nucleic acid molecules
and repeating steps (i) and (ii); and (iv) determining the binding
specificity of the isolated nucleic acid molecules for the
PrP.sup.c and PrP.sup.Sc isoforms of prion proteins.
2. The process according to claim 1, wherein the nucleic acid
molecule is RNA.
3. The process according to claim 1 or 2, wherein the pool of
nucleic acid molecules is a randomized RNA pool.
4. The process according to claim 3, wherein the pool of nucleic
acid molecules is the RNA pool M 111.1 as described in Famulok (J.
Am. Chem. Soc. 116 (1994), 1698-1706).
5. The process according to claim 1, wherein the nucleic acid
molecule is DNA.
6. The process according to claim 5, wherein the DNA is single
stranded DNA.
7. The process according to claim 5, wherein the DNA is double
stranded DNA.
8. The process according to any one of claims 5 to 7, wherein the
pool of nucleic acid molecules is a randomized DNA pool.
9. The process according to any one of claims 1 to 8 wherein the
transmissible spongiform encephalopathy is Scrapie, bovine
spongiform encephalopathy (BSE), Creutzfeld-Jacob Disease (CJD),
Gerstmann-Stru.beta.ler-Scheinker-Syndrome (GSS), Kuru, fatal
familial insomnia (FFI) or transmissible minc encephalopathy (TME),
feline spongiform encephalopathy (FSE) or chronic wasting disease
(CWD).
10. The process according to any one of claims 1 to 9, wherein the
prion protein isoform or fragment or derivative thereof is
immobilized.
11. The process according to any one of claims 1 to 9, wherein the
prion protein isoform or fragment or derivative thereof is in
solution.
12. The process according to any one of claims 1 to 11, wherein the
prion protein isoform is the isoform PrP.sup.Sc or a fragment or
derivative of this isoform.
13. The process according to claim 12, wherein the prion protein is
the derivative PrP27-30 or a fragment thereof
14. The process according to any one of claims 1 to 11, wherein the
prion protein isoform is the isoform PrP.sup.c or a fragment or
derivative of this isoform.
15. The process according to claim 14, wherein the prion protein is
the processed form PrP.sup.c23-231.
16. The process according to any one of claims 1 to 15, wherein the
prion protein is a recombinant protein.
17. The process according to claim 16, wherein the prion protein is
part of a fusion protein.
18. The process according to claim 17, wherein the prion protein is
part of a fusion protein with oligohistidine, calmoduline binding
protein, S-peptide, FLAG, green-fluorescent protein, BTag, maltose
binding protein or glutathione-S-transferase.
19. A nucleic acid molecule obtainable by the process according to
any one of claims 1 to 18.
20. The nucleic acid molecule according to claim 19 which
specifically binds to an isoform of a prion protein as defined in
any one of claims 10 to 15 or to a fragment or derivative
thereof.
21. The nucleic acid molecule according to claim 19 or 20 which is
an RNA molecule.
22. The nucleic acid molecule according to claim 19 or 20 which is
a DNA molecule.
23. The nucleic acid molecule according to any one of claims 19 to
22 which comprises four stretches of three consecutive guanosine
residues separated by regions between four and seven nucleotides
long.
24. The nucleic acid molecule according to claim 23 which comprises
a nucleotide sequence as depicted in SEQ ID NO: 15, SEQ ID NO: 16,
or SEQ ID NO: 17.
25. The nucleic acid molecule according to any one of claims 19 to
23, which comprises a nucleotide sequence as depicted in any one of
SEQ ID NO: 1 to SEQ ID NO: 13.
26. The nucleic acid molecule according to any one of claims 19 to
22 which comprises a nucleotide sequence as depicted in SEQ ID NO:
18.
27. The nucleic acid molecule according to any one of claims 19 to
26 which is modified at one or more positions in order to increase
its stability and/or to alter its biophysical and/or biochemical
properties.
28. A pharmaceutical composition comprising a nucleic acid molecule
according to any one of claims 19 to 27 and optionally a
pharmaceutically acceptable carrier.
29. A diagnostic composition comprising a nucleic acid molecule
according to any one of claims 19 to 27.
30. A method for the in-vitro diagnosis of a transmissible
spongiform encephalopathy, wherein at least one of the nucleic acid
molecules according to any one of claims 19 to 27 is incubated with
a probe and the interaction between the nucleic acid molecules and
the PrP.sup.c or PrP.sup.Sc isoforms of a prion protein or fragment
or derivative of these isoforms is determined.
31. The method according to claim 30 wherein at least one of the
nucleic acid molecules according to any one of claims 19 to 27 is
used to quantitatively determine the amount of at least one isoform
of a prion protein or a fragment or derivative thereof in a
probe.
32. The method according to claim 31, wherein nucleic acid
molecules are used which specifically bind to the cellular isoform
PrP.sup.c or fragment or derivatives thereof in combination with
nucleic acid molecules which specifically bind to the isoform
PrP.sup.Sc or fragment or derivatives thereof and the absolute
and/or relative amount of the isoforms PrP.sup.c and PrP.sup.Sc in
the probe is determined.
33. The method according to any one of claims 30 to 32, wherein the
probe is from an organ tissue.
34. The method of claim 32 wherein the organ tissue is from brain,
tonsils, ileum, cortex, dura mater, Purkinje cells, lymphnodes,
nerve cells, spleen, muscle cells, placenta, pancreas, eyes,
backbone marrow or peyer'sche plaque.
35. The method according to any one of claims 30 to 32, wherein the
probe is from a body fluid.
36. The method according to claim 35, wherein the body fluid is
blood, cerebrospinal fluid, milk or semen.
37. A chemical compound other than a nucleic acid molecule based on
information derived from a three dimensional structure of nucleic
acid molecules according to any one of claims 19 to 27 selected
from the group consisting of inorganic or organic compounds.
38. The chemical compound according to claim 37, which is a sugar,
an amino acid, a protein or a carbohydrate.
Description
[0001] The present invention relates to a process for the
identification and isolation of nucleic acid molecules capable of
distinguishing the isoforms PrP.sup.c and PrP.sup.Sc of prion
proteins as well as to the nucleic acid molecules obtainable by
this process. Furthermore, the invention relates to pharmaceutical
and diagnostic compositions comprising said nucleic acid
molecules.
[0002] Proteinaceous infectious particles called prions are thought
to be the causative agent of transmissible spongiform
encephalopathies (TSEs) such as Scrapie of sheep, bovine spongiform
encephalopathy (BSE) of calf, transmissible minc encephalopathy
(TME) of mink as well as Kuru, Gerstmann-Strussler-Scheinker
syndrome (GSS), Creutzfeldt-Jakob-Disease (CJD) and fatal familial
insomnia (FFI) in the case of humans (Prusiner, 1982). The main
component of prions associated in amyloid-like rods (Prusiner et
al., 1983; 1984) or scrapie associated fibrils (SAF; Hope et al.,
1986) was found to be the prion protein PrP27-30 (Prusiner et al.,
1981; Prusiner et al., 1983), an N-terminal truncated, highly
protease resistant version of the prion protein PrP.sup.Sc (Oesch
et al., 1994), which is also found to a minor extend in prion
preparations (Prusiner et al. 1983). PrP27-30, which is devoid of
67 amino acids at the aminoterminal end, results from PrP.sup.Sc by
proteinase K digestions (Prusiner et al., 1984; Stahl et al., 1993)
or by lysosomal protease digestion (Caughey et al., 1991). The
distribution of PrP.sup.Sc and PrP27-30 in prion preparations
varies dependent from the absence or presence of proteases.
[0003] No specific nucleic acid could be detected so far in prion
preparations (Kellings et al., 1992) suggesting that the prion is
infectious and can replicate in the absence of any nucleic acid
(Prusiner, 1982). According to the protein-only hypothesis
(Prusiner, 1982) exogenous PrP.sup.Sc/PrP27-30 could convert the
ubiquitous cellular isoform PrP.sup.c to PrP.sup.Sc/PrP27-30. It is
assumed that chaperons may be involved in this process (Edenhofer
et al., 1996). PrP.sup.Sc/PrP27-30 could appear as a monomer
(Prusiner. 1982) or as a nucleation or crystal seed consisting of a
PrP.sup.Sc/PrP27-30 oligomer (Lansbury and Caughey. 1995).
PrP.sup.c differs from PrP27-30 only with respect to its secondary
structure: the .alpha.-helical and .beta.-sheet contents of
PrP.sup.c are 42% and 3%, respectively (Pan et al., 1993). In
contrast, the .alpha.-helical and .beta.-sheet contents of PrP27-30
were proven to be 21% and 54%, respectively (Pan et al., 1993).
These results indicate that the conversion of PrP.sup.c to
PrP.sup.Sc/PrP27-30 is most likely concomitant with extreme
alterations in the secondary structure of the prion protein.
Although a series of experiments employing knock-out mice which no
longer express PrP.sup.c suggest that cellular prion proteins could
play a crucial role in a number of cellular processes (Collinge et
al., 1994; Sakaguchi et al., 1996: Tobler et al., 1996), a precise
physiological role of PrP.sup.c remains speculative. It is,
however, proven that PrP.sup.c is necessary for the development of
transmissible spongiform encephalopathies (Bueler et al., 1993;
Brandner et al., 1996).
[0004] Translation of the mRNA from Scrapie infected Syrian golden
hamster has led to a 254 amino acid protein including a 22 amino
acid signal peptide at the NH.sub.2-terminus and a 23 amino acid
signal sequence at the carboxy terminus (Oesch et al., 1985; Basler
et al., 1986). The mature protein PrP.sup.c as well as the Scrapie
isoform PrP.sup.Sc contain amino acids 23 to 231. Only PrP.sup.Sc
can be processed to the proteinase K resistant isoform PrP27-30
(amino acids 90-231) consisting of 142 amino acids (Prusiner et al,
1984).
[0005] This property has been used to design a diagnostic assay for
diseases in connection with prion proteins in which a probe is
treated with proteinase K in order to degrade all PrP.sup.c and
then reacted with an antibody directed against prion proteins
(Groschup et al., 1994). However, this assay has the disadvantage
that sensitivity might be hampered by the fact that the proteinase
K digestion of PrP.sup.c is not complete, thereby leading to false
positive results. Furthermore, the additional step of proteinase K
digestion is time consuming. In order to be able to directly assay
for the presence or absence of PrP.sup.c and/or PrP.sup.Sc one
would need antibodies which could distinguish between these two
isoforms.
[0006] However, so far attempts to provide antibodies that can
distinguish between the cellular isoform PrP.sup.c and the isoforms
PrP.sup.Sc, as well as the truncated version PrP27-30, have failed
(Groschup et al., 1994 and ref. therein).
[0007] Thus, up to now it was not possible to distinguish the
isoforms PrP.sup.c and PrP.sup.Sc of prion proteins by
immunological or other means which would be the prerequisite for a
simple and reliable method of diagnosing a transmissible spongiform
encephalopathy.
[0008] Therefore, the technical problem underlying the present
invention is to provide a process for the identification and
isolation of molecules which are capable of distinguishing between
the isoforms PrP.sup.c and PrP.sup.Sc or PrP27-30 of prion proteins
and which are useful tools for diagnosis and therapy of
transmissible spongiform encephalopathies.
[0009] The solution to said technical problem is achieved by the
provision of the embodiments characterized by the patent
claims.
[0010] Thus, the present invention relates to a process for the
identification and isolation of nucleic acid molecules which are
capable of distinguishing between the isoforms PrP.sup.c and
PrP.sup.Sc or PrP27-30 of prion proteins associated with
transmissible spongiform encephalopathies comprising the steps
of
[0011] (i) incubating a prion protein isoform or peptide fragment
or derivative of this prion protein isoform with a pool of nucleic
acid molecules comprising different sequences;
[0012] (ii) selecting and isolating those nucleic acid molecules
which are capable of binding to said prion protein isoform or
fragment or derivative thereof;
[0013] (iii) optionally, amplifying the isolated nucleic acid
molecules and repeating steps (i) and (ii); and
[0014] (iv) determining the binding specificity of the isolated
nucleic acid molecules for the PrP.sup.c and PrP.sup.Sc or PrP27-30
isoforms of prion proteins.
[0015] The process according to the invention is based on a method
called "in vitro selection". This method allows for the
identification of nucleic acid molecules (RNA modified RNA, ssDNA
or dsDNA) which bind with high affinity to a defined molecular
target from a large randomized population of nucleic acid molecules
(Tuerk and Gold, 1990, Famulok and Szostak, 1992). Using this
method it has been possible to isolate nucleic acids specifically
recognizing a variety of protein targets including HIV-1 reverse
transcriptase (Tuerk et al., 1992), HIV-1 Integrase (Allen et al.,
1995), human .alpha.-thrombin (Kubik et al., 1994) and Drosphila
sex-lethal protein (Sakashita and Sakamoto, 1994). However, up to
now it has not been possible to provide by this method nucleic acid
molecules being capable of distinguishing the two isoforms of prion
proteins, PrP.sup.c and PrP.sup.Sc. In the scope of the present
invention the term PrP.sup.c comprises the cellular isoform of the
prion protein as well as fragments and derivatives thereof
irrespective of the source organism. The term PrP.sup.Sc comprises
the isoform of the prion protein associated with various
transmissible spongiform encephalopathies. This term also comprises
fragments of this prion protein isoform such as the truncated
version of the isoform PrP.sup.Sc, the prion protein PrP27-30,
which is the main component of prions. In particular, this term
also includes PrP.sup.Sc proteins of the various Scrapie strains
including those adapted to hamster, mouse or other vertebrates.
Also included are derivatives of the prion protein isoform
PrP.sup.Sc.
[0016] The term derivatives includes chemically modified versions
of the prion protein isoforms PrP.sup.c and PrP.sup.Sc as well as
mutants of these proteins, namely proteins which differ from the
naturally occurring prion protein isoforms at one or more positions
in the amino acid sequence, as well as proteins that show deletions
or insertions in comparison to the naturally occurring prion
protein isoforms. Such mutants can be produced by recombinant DNA
technology or can be naturally occurring mutants. The term
derivatives also embraces proteins which contain modified amino
acids or which are modified by glycosylation, phosphorylation and
the like.
[0017] According to the invention it is possible to use as nucleic
acid molecules single or double stranded nucleic acid molecules,
such as RNA, modified RNA, single stranded DNA or double stranded
DNA.
[0018] A pool of nucleic acid molecules, which constitutes the
starting material from which nucleic acid molecules are selected
which specifically bind to one of the isoforms of the prion
protein, is defined as a mixture of nucleic acid molecules of
different sequences. This pool can be any mixture of nucleic acid
molecules, preferably a pool of randomized molecules. Preferably
the nucleic acid molecules of the pool are chemically synthesized
or produced by in vitro transcription.
[0019] In the case of RNA molecules the RNA pool which is screened
for molecules specifically binding to one of the isoforms of a
prion protein is preferably the RNA pool M111.1 described in
Famulok (1994). This pool consists of RNA molecules of 111
nucleotides randomized at 74 positions and results from the
transcription of corresponding DNA sequences. The pool M111.1
contains RNA molecules with approximately 1.times.10.sup.15
different sequences.
[0020] The process according to the invention can be used to
identify and isolate nucleic acid molecules which can distinguish
between the two isoforms of prion proteins, PrP.sup.c and
PrP.sup.Sc, associated with a transmissible spongiform
encephalopathy such as Scrapie of sheep, bovine spongiform
encephalopathy (BSE) of calf, transmissible mink encephalopathy
(TME) of mink, Kuru, Gerstmann-Strussler-Scheinker Syndrome (GSS),
fatal familial insomnia (FFI), Creutzfeldt-Jakob Disease (CJD) in
the case of humans, chronic wasting disease (CWD) of mule, deer and
elk or feline spongiform encephalopathy (FSE) of cats.
Transmissible spongiform encephalopathies are also known from
nyala, gemsbok, arabian oryx, greater kudu, eland, ankole,
moufflon, puma, cheetah, scimitar horned oryx, ocelot and
tiger.
[0021] The step of incubating the pool of nucleic acid molecules
with a prion protein can be carried out in different ways.
[0022] In one preferred embodiment of the invention the protein is
immobilized, for example, on a matrix such as a gel or a resin for
chromatography. The immobilization can be achieved by means known
to the person skilled in the art. For example, the protein can be
covalently linked to a matrix or can be bound to it by a specific
interaction between a group present on the matrix and a domain of
the protein specifically recognizing this group. Such a domain can
be fused to a prion protein by recombinant DNA technology as will
be discussed below.
[0023] If the prion protein is immobilized, nucleic acid molecules
which do not bind to the prion protein can be removed after
incubation by washing with an appropriate buffer Subsequently the
nucleic acid molecules binding to the prion protein can be eluted
from the immobilized protein, for example by 8M urea, and further
purified, for example, by phenol extraction and precipitation.
[0024] In another preferred embodiment the prion protein is in
solution. In this case the nucleic acid molecules binding to the
prion protein can be isolated, for example, by carrying out a gel
retardation assay and isolating the protein/nucleic acid complex.
Subsequently the nucleic acid molecules can be isolated from the
complex and further purified by known methods.
[0025] According to the invention it is possible to amplify the
nucleic acid molecules obtained by steps (i) and (ii), for example
by in vitro transcription, reverse transcription or polymerase
chain reaction or a combination of these techniques, and to repeat
steps (i) and (ii). This leads to a further selection and
amplification of nucleic acid molecules which bind specifically to
the used prion protein.
[0026] If several cycles of steps (i) to (iii) of the process are
performed, it is possible to use in one or more cycles an
immobilized protein and in one or more cycles a protein in
solution. A cycle in which a protein in solution is used permits
the elimination of nucleic acid molecules binding to the matrix on
which the immobilized protein is fixed.
[0027] The prion protein used in the process can be any of the
known prion protein isoforms or a fragment or derivative of such a
protein.
[0028] In a preferred embodiment the prion protein is the isoform
PrP.sup.Sc present in the prion. In a specifically preferred
embodiment the N-terminally truncated version of PrP.sup.Sc,
PrP27-30, is used. In this context PrP.sup.Sc and PrP27-30 refer to
any of these isoforms which can be found in an organism affected
with a transmissible spongiform encephalopathy.
[0029] In a further preferred embodiment the prion protein used in
the process is the cellular isoform PrP.sup.c, most preferably the
processed form PrP.sup.c23-231 which comprises amino acids 23 to
231 of PrP.sup.c.
[0030] In another preferred embodiment the prion protein used in
the process is a recombinant protein. This means that the protein
is produced by recombinant DNA technology, namely by expression
from a cloned DNA sequence.
[0031] More preferably, the prion protein is part of a fusion
protein. Such a fusion protein can comprise beside the prion
protein a protein or protein domain which confers to the fusion
protein a specific binding capacity. For example, such a domain may
be an oligohistidine (Le-Grice et al., 1990), Calmoduline binding
peptide (CBP) (Carr et al., 1991), S-peptide (ribonuclease A) (Kim
and Raines, 1993), FLAG (Kawase et al., 1995), green-fluorescent
protein (GFP) (Hampton et al., 1996), BTag (Wang et al., 1996), or
maltose-binding protein (MBP) (Aitken et al., 1994, Richards and
Wyckoff, 1971). Proteins comprising such a domain can be
immobilized for example, on IMAC-Ni.sup.2+, Calmodulin, S-protein
104 aa (Kim and Raines, 1993), anti-FLAG-antibodies,
anti-GFP-antibodies, BTag-antibodies or maltose. Elution can then
be achieved by a method well-known in the art. In a preferred
embodiment the prion protein is fused to glutathione-S-transferase.
Such a fusion protein possesses a high affinity for glutathione and
can thus be immobilized on a matrix comprising glutathione, such as
glutathione-sepharose.
[0032] In the last step of the process according to the invention
the isolated nucleic acid molecules are tested for their binding to
the different isoforms, PrP.sup.c and PrP.sup.sc of a prion
protein. Those nucleic acid molecules are selected which
specifically bind to only one of the isoforms.
[0033] Thus, the process according to the invention allows the
identification and isolation of nucleic acid molecules which
specifically bind to one of the isoforms of a prion protein or a
fragment or derivative thereof and thereby allow the distinguishing
of the different isoforms. These nucleic acid molecules therefore
show an unexpected high specificity, which is even higher than the
specificity of poly- or monoclonal antibodies which cannot
distinguish between the isoforms of prion proteins.
[0034] The process of the invention has been successfully carried
out to isolate RNA molecules which can distinguish between the
isoforms PrP.sup.c23-231 and PrP27-30 from Syrian Golden Hamster.
In this case the isoforms were recombinant proteins fused to
glutathione-s-transferase (GST::PrP.sup.c23-231 and
GST::rPrP27-30). The recombinant rPrP27-30 protein is identical in
sequence to the natural PrP27-30 protein but reveals in contrast to
the natural isoform proteinase K sensitivity.
[0035] Furthermore, the present invention relates to nucleic acid
molecules obtainable by a process according to the invention,
namely to RNA, single stranded DNA or double stranded DNA molecules
which bind to one of the isoforms of a prion protein. These include
nucleic acid molecules which specifically bind to the cellular
isoform PrP.sup.c, namely to the processed form PrP.sup.c23-231, or
specifically to the isoform PrP.sup.Sc, namely to the truncated
version PrP27-30, or specifically to derivatives of these
proteins.
[0036] In a preferred embodiment the nucleic acid molecules of the
invention comprise four stretches of three consecutive guanosine
residues separated by single stranded regions between four and
seven nucleotides long. More preferably, the nucleic acid molecules
comprise a nucleotide sequence as depicted in SEQ ID NO: 15, SEQ ID
NO: 16 or SEQ ID NO: 17.
[0037] In another preferred embodiment, the region comprising the
four guanosine stretches is flanked by two variable regions of
predominantly Watson-Crick covariation. In particular, the nucleic
acid molecules preferably comprise a nucleotide sequence as
depicted in any one of SEQ ID NO: 1 to 13 and more preferably a
nucleotide sequence as depicted in SEQ ID NO: 18.
[0038] In a preferred embodiment the nucleic acid molecules
according to the invention are further modified at one or more
positions in order to increase their stability and/or to alter
their biochemical and/or biophysical properties.
[0039] The present invention also relates to pharmaceutical
compositions comprising nucleic acid molecules according to the
invention. Such compositions can optionally comprise
pharmaceutically acceptable carriers.
[0040] These compositions may be useful for the therapy of
transmissible spongiform encephalopathies such as those listed
above. It may be possible, for example, to suppress the conversion
of the isoform PrP.sup.c into the prion associated isoform
PrP.sup.Sc by applying nucleic acid molecules which specifically
bind to PrP.sup.c.
[0041] Furthermore, the present invention relates to diagnostic
compositions comprising nucleic acid molecules according to the
invention. Such compositions may contain additives commonly used
for diagnostic purposes. The nucleic acid molecules and the
diagnostic compositions according to the invention can be used in
methods for the diagnosis of transmissible spongiform
encephalopathies. Such a method comprises, for example, the
incubation of a probe taken from a body with at least one kind of
nucleic acid molecules according to the invention and the
subsequent determination of the interaction of the nucleic acid
molecules with the isoforms PrP.sup.c and PrP.sup.Sc of a prion
protein.
[0042] Since during the occurrence of a transmissible spongiform
encephalopathy the amount of the isoform PrP.sup.Sc increases and
the total amount of the cellular isoform PrP.sup.c decreases, it is
in principle possible to use for diagnosis nucleic acid molecules
which bind to one or the other of the two isoforms.
[0043] On the one hand, it is possible to use at least one kind of
nucleic acid molecule according to the invention in order to
quantitatively determine the amount of at least one isoform of a
prion protein in a probe.
[0044] On the other hand, it is possible to use nucleic acid
molecules which specifically bind the PrP.sup.c isoform in
combination with nucleic acid molecules which specifically bind the
PrP.sup.Sc isoform in order to determine the absolute and/or
relative amount of the isoforms in a probe.
[0045] In a preferred embodiment the probe may be obtained from
various organs, perferably from tissue, for example, from brain,
tonsils, ileum, cortex, dura mater, Purkinje cells, lymphnodes,
nerve cells, spleen, muscle cells, placenta, pancreas, eyes,
backbone marrow or peyer'sche plaques, for example in the form of
thin sections. Alternatively the probe may be obtained from a body
fluid, preferably from blood cerebrospinal fluid, milk or
semen.
[0046] In the case that brain is used as a probe, diagnosis is in
most cases performed post mortem. Exceptionally, brain biopsies can
be performed on the alive organism. The brain can originate from
any organism that might be afflicted with a transmissible
spongiform encephalopathy, such as sheep, calf, mice, cats,
hamster, mule, deer, elk or humans or from other organisms which
may be afflicted by a TSE as mentioned above. The brain should
originate from organisms which are PrP.sup.0/0 (knock-out),
PrP.sup.Sc (infected) and PrP.sup.c (wild-type) or of unknown
PrP-status.
[0047] In the case that blood, milk, cerebrospinal fluid, semen or
tissue from other organs as mentioned above is used as a probe,
diagnosis is possible for living individuals.
[0048] Furthermore, the nucleic acid molecules according to the
invention can be used to identify three dimensional structures
which are necessary for the specific binding of a prion protein
isoform. With the help of this information other chemical compounds
can be isolated or synthesized which can specifically bind prion
protein isoforms. Thus, the present invention also relates to
chemical compounds other than nucleic acid molecules which are
based on the information derived from a three dimensional structure
of a nucleic acid molecule according to the invention, selected
from the group consisting of inorganic or organic compounds,
preferably sugars, amino acids, proteins or carbohydrates.
[0049] FIG. 1A: Illustrates schematically the method for in vitro
selection of RNA molecules specifically binding to the immobilized
fusion protein GST::rPrP.sup.c (GST::rPrP23-231)
(GST=glutathione-S-transferase; PCR=Polymerase chain reaction).
[0050] In the following, (r)PrP.sup.c stands for rPrP23-231.
Furthermore, rPrP27-30 stands for rPrP90-231 (Syrian Golden
Hamster).
[0051] FIG. 1B: Illustrates schematically a further step in the in
vitro selection of RNA molecules specifically binding to
GSTL:rPrP.sup.c using GST::rPrP.sup.c in solution and a gel
retardation assay
[0052] FIG. 2: Schematically illustrates the construction by in
vitro transcription of the randomized RNA pool M111.1 (Famulok,
1994) (Ntes=nucleotides).
[0053] FIG. 3: Shows the percentage of RNA binding to immobilized
GST::rPrP.sup.c23-231 after each cycle of the process described in
Example 1.
[0054] Radioactivity associated with GST::rPrP.sup.c beads after
removal of the supernatant was set to 100%. Radioactivity retained
after 4 washing steps represents the percentage of RNA binding.
[0055] FIG. 4A: Shows the binding of selected RNAs and unselected
RNAs to GST, GST::rPrP.sup.c and GST::rPrP27-30. 5' labeled RNA was
incubated in the presence of the proteins, filtered over BA85
nitrocellulose on a millipore slot blot apparatus. Retained
radioactivity was quantified by Cerenkov counting.
[0056] FIG. 4B: Shows that the in vitro selected RNA molecules of
Example 1 distinguish between PrP.sup.c and rPrP27-30 from the
Syrian Golden Hamster. Gel a: 5' labeled RNA molecules after 9
cycles were incubated in the presence of GST, GST::PrP.sup.c and
GST::rPrP27-30, and analyzed on 0.7% non-denaturing agarose gels.
Gels were fixed by 5% TCA, dried and subjected to autoradiography.
The GST::rPrP.sup.c/RNA complex from the 9th cycle was excised from
the gel, the RNA extracted, reverse transcribed, PCR amplified and
in vitro transcribed (see FIG. 1B). This procedure was repeated
twice for cycle 10 and 11. Gel b: The 5' labeled RNA--after 11
cycles--was again incubated in the presence of GST, GST::rPrP.sup.c
and GST::rPrP27-30 and analyzed as described above.
[0057] FIG. 5: Sequences of selected RNA aptamers directed against
rPrP.sup.c23-231 fused to GST from hamster by in vitro selection.
The aptamers belong to several groups of molecules. RNA aptamers of
group (A) (motif I) and (B) (motif II) can harbor G-quartet motifs
and distinguish between rPrP23-231 (rPrP.sup.c) and rPrP90-231
(rPrP27-30). RNA aptamers of group (C) (motif III) could also have
G-quartet motifs but interact with rPrP23-231 (rPrP.sup.c) and
rPrP90-231 (rPrP27-30). (D) Aptamers with unique G-quartets (5 out
of 6 aptamers shown). Aptamers of group (E) lack any G-quartet
motif and bind to GST (one out of 6 aptamers shown).
[0058] FIG. 6: RNA aptamers motif I and II distinguish the
recombinant prion protein isoforms rPrP23-231 (rPrP.sup.c) and
rPrP90-231 (rPrP27-30) from hamster and calf. (A) 4 pMols of
labeled RNA Ap1 (motif I; lanes 1-3) were incubated in the presence
of 40 pMols each of recombinant GST::rPrP23-231 (rPrP.sup.c) (lane
2) and GST::rPrP90-231 (rPrP27-30) from Syrian golden hamster (lane
3). (B) 4 pMols of labeled RNA Ap1 (motif I; lanes 1-3) were
incubated in the presence of 40 pMols each of recombinant
GST::bov-rPrP25-242 (rPrP.sup.c) (lane 2) and GST::bov-rPrP93-242
(rPrP27-30+1 octarepeat) from calf (lane 3).(C) 4 pMol of labeled
RNA motif II (lanes 1-3) were incubated in the presence of
GST::rPrP23-231 (rPrP.sup.c) (lane 2) and GST::rPrP90-231
(rPrP27-30) (lane 3) from hamster. Reaction assays were analyzed on
0.7% agarose gels. (D) 4 pMols of labeled RNA Ap2 (motif II; lanes
1-3) were incubated in the presence of 40 pMols each of recombinant
GST::bov-rPrP25-242 (rPrP.sup.c) (lane 2) and GST::bov-rPrP93-242
(rPrP27-30+1 octarepeat) from calf (lane 3). The additional bovine
octarepeat extends from aa 93 to 101.
[0059] FIG. 7: Mapping of the RNA aptamer--PrP interaction site of
hamster and calf. (A) 4 pMols of labeled RNA aptamer motif I (lanes
1-9) and (B) 4 pmols of labelled RNA aptamer motif II (lanes 1-9)
were incubated in the presence of 40 pMol each of GST::rPrP23-231
(rPrP.sup.C) (lanes 8), GST::rPrP90-231 (rPrP27-30) (lanes 9) from
hamster and 20 pMol each of GST::P.sub.23-52 (lanes 2),
GST::P.sub.55-93 (lanes 3). GST::P.sub.90-109 (lanes 4)
GST::P.sub.129-175 (lanes 5), GST::P.sub.218-231 (lanes 6) and
GST::P.sub.180-210 (lanes 7). Reaction assays were analyzed on 0.7%
agarose gels. (Top) Schematic presentation of the hamster PrP
region. Hatched box, PrP region interacting with the aptamers. Void
boxes, PrP region not interacting with the aptamers. (C) 4 pMols of
labelled RNA aptamer motif II (lanes 1-4) were incubated in the
presence of 40 pMol each of bovine GST::bovP.sub.25-92 (lane 2),
GST::bovP.sub.93-120 (lane 3) and 20 pMol each of bovine
GST::bov-rPrP93-242 (rPrP27-30+1 octarepeat; lane 1) and hamster
GST::P.sub.23-89 (lane 4).
[0060] The Examples illustrate the invention.
EXAMPLE 1
[0061] In vitro Selection of RNA Molecules Specifically Binding
GST::rPrP.sup.c23-231
[0062] An in vitro selection procedure (schematically outlined in
FIGS. 1A and B) was carried out using recombinant PrP23-231
(rPrP.sup.c) from the Syrian Golden Hamster fused to GST (Weiss et
al., 1995) and RNA pool M 111.1 (Famulok, 1994)
[0063] Cycles 1-9: 5'[.gamma.-.sup.32P]-ATP labeled (1.Cycle) or
[.alpha.-.sup.32P]-UTP labeled (Cycles 2-10) RNA M111.1 (Famulok,
1994) (6,8 nMol (first), 1.82 nMol (2nd), 914 pMol (3rd), 665 pMol
(4th), 2.07 nMol (5th), 831 pMol (6th), 2.7 nMol (7th), 1.94 nMol
(8th and 9th cycle) was incubated in the presence of immobilized
GST (185 pMol) synthesized in the Baculovirus system (Weiss et al.,
1995) in binding buffer comprised of 8 mM Na.sub.2HPO.sub.4, 0.87
mM KH.sub.2PO.sub.4, 136 mM NaCl, 112.6 mM KCl, 2 mM DTT and 2 mM
MgCl.sub.2(FIG. 1A). Incubation was done at 37.degree. C. in an
overhead incubator.
[0064] Cycle 1-7: After 60 min. beads were collected for 10 min. at
700 g.
[0065] Cycle 8-9: Incubation with immobilized GST was done for 30
min. as described above. Subsequently the beads were removed by
centrifugation and the supernatant incubated with freshly
immobilized GST for another 30 minutes.
[0066] Cycle 1-9: The supernatant from the preselection(s) was
incubated with immobilized GST::rPrP.sup.c23-231 (53 pMol)
synthesized in the Baculovirus system (Weiss et al., 1995) as
described above. After 60 minutes the beads were washed four times
with binding buffer and the RNA eluted in the presence of 8 M urea
in 100 mM sodium citrate pH 8.0 and 3 mM EDTA. The RNA was phenol
(pH 5.0)/chloroform extracted and EtOH precipitated in the presence
of 2 M NH.sub.4-acetate (FIG. 1A).
[0067] Cycle 9 to 11: Selected RNA (40 pMol in the 9th; 4 pMol each
in the 10th and 11th cycle) was 5' labeled and incubated with
soluble GST::rPrP.sup.c23-231 (Weiss et al., 1995; 140 pMol in the
9th cycle, 40 pMol in the 10th and 11th cycle) for 60 min. at
37.degree. C. in binding buffer and analyzed by an gel retardation
assay on an 0.7% native agarose gel (FIG. 1B) as described (Weiss
et al., 1992). Following electrophoresis the
RNA/GST::PrP.sup.c23-231 complex was excised and extracted by
employing an Qiaex extraction kit (Qiagen).
[0068] Cycle 1-11: 50% each of the extracted RNA was subjected to a
reverse transcription reaction according to the Superscript reverse
transcriptase kit (Gibco, BRL). 50% of the resulting cDNA was
amplified by PCR according to Saiki et al., 1988 using the primers
shown in FIG. 2B. 50% of the amplified cDNA was in vitro
transcribed as described (Weiss et al., 1992).
[0069] Nitrocellulose binding assay: 4 pMol 5' labeled (Sambrook et
al., 1989) RNA was incubated in the presence of 0 to 500 nM of
protein in the presence of binding buffer for 60 min. at 37.degree.
C. The incubation mixture was filtered over a BA85 nitrocellulose
membrane in a Millipore slot blot apparatus, the filter washed with
4 ml of incubation buffer, excised and measured by Cerenkov
Counting.
[0070] Gel retardation assay: RNA and protein were incubated as
described above and the reaction mixture loaded on a 0.7% native
agarose gel as described (Weiss et al., 1992). Following
electrophoresis, the gel was fixed by 5% TCA, dried and subjected
to autoradiography.
[0071] RNA Pool M111.1: The RNA pool was prepared by in vitro
transcription from a DNA pool (138 bases) as described (Famulok,
1994). In brief, M111.1 reveals a randomized sequence of 74, a base
permutation of 474=3.56.times.10.sup.44 molecules, a molecular
weight of 36630 Dalton. Synthesis yielded 175 .mu.g (4.76 nMol)
RNA; that is 6.times.10.sup.23
(Avogadro).times.4.76.times.10.sup.-9=2.86.times.10.sup- .15
molecules. 36% of the synthesized ssDNA pool are extendible by PCR
which resulted in a pool with approximately 1.03.times.10.sup.15
different sequences and a complexity of 1.03.times.10.sup.15, which
is equivalent to one pool copy (i.e. each individual RNA molecule
is represented one fold in the pool). In particular, the technical
features of RNA pool M111.1 are the following:
[0072] Nucleotide Sequence of Fixed Region 1
[0073] 5' CCGAATTCTAATACGACTCACTATAGGAGCTCAGC CTTCACTGC (SEQ ID NO:
19)
[0074] Nucleotide Sequence of Fixed Region 2
[0075] 5' GTGGATCCGACCGTGGTGCC (SEQ ID NO: 20)
[0076] randomized sequence=74 nucleotides
[0077] base permutation 4.sup.74=3.56.times.10.sup.44;
[0078] MW=36630; 1 nM=36.63 .mu.g;
[0079] 175 .mu.g (4.76 nMol) were synthesized; that is
6.times.10.sup.23.times.4.76.times.10.sup.-9=2.86.times.10.sup.15
molecules
[0080] 36% extendable
[0081] pool complexity=1.03.times.10.sup.15 molecules
[0082] =1 pool copy i.e. each individual RNA molecule is
represented one fold in the pool
[0083] A schematic view of RNA pool M111.1 is shown in FIG. 2. The
nucleotide sequence 5'-CCGAATTCTAATACGACTCACTATA (nucleotides 1 to
25 of SEQ. ID NO: 19) of the fixed region 1 only belongs to the DNA
pool since it is not transcribed.
[0084] After 9 rounds of selection 7.2% of the selected RNA bound
to GST::rPrP.sup.c23-231 immobilized on glutathione-sepharose 4B
(Table 1 and FIG. 3).
[0085] The percentage of RNA-binding was determined as follows:
Radioactivity associated with GST::rPrP.sup.c beads after removal
of supernatant was set to 100%. Radioactivity retained after four
washing steps represents the percentage of the RNA binding.
1TABLE 1 RNA protein ratios and % binding of RNA to protein
dependent on the selection cycle RNA: % binding* RNA:GST
GST::rPrP.sup.c (RNA to Cycle (molar ratio) (molar ratio)
GST::rPrP.sup.c) 1 36:1 83:1 0 2 10:1 22:1 0 3 5:1 11:1 0 4 3.5:1
8:1 1 5 10:1 24:1 1.25 6 4.5:1 10:1 2.5 7 14:1 33:1 2.75 8 10:1/7:1
18:1 7.2 9 10:1/7:1 18:1 7.2 *The percentage of RNA binding was
measured as described in the legend to FIG. 3.
[0086] A binding assay employing soluble GST, GST::PrP.sup.c23-231
and GST::rPrP27-30 revealed that 2% of the enriched RNA from cycle
9 bound to GST::PrP.sup.c23-231 whereas only 1.1% bound to
GST::rPrP27-30 and GST (FIG. 4A). This result indicates that
.about.5% of the RNA bound to the matrix, i.e.
glutathione-sepharose 4B. After 6 rounds of selection only 1% of
the RNA bound to immobilized GST::PrP.sup.c23-231 and 0.7% to GST
(FIG. 4A).
[0087] A gel retardation assay with RNA isolated after 9 cycles of
selection confirms that about 2% of the RNA bound to
GST::PrP.sup.c23-231 at a molar ratio of 10:1 (protein:RNA) (FIG.
4B, panel a, lane 6), whereas no binding occurs under identical
conditions in the case of GST::rPrP27-30 (lane 9) and GST (lane
3).
[0088] To enrich RNA specifically binding to GST::PrP.sup.c23-231
and to remove RNA molecules binding to the matrix, we excised the
RNA/GST::PrP.sup.c23-231 complex. extracted and amplified the RNA
(FIG. 1B) and subjected it to two further gel retardation assays.
As demonstrated in FIG. 4B after a total of 11 cycles (panel b) we
isolated an RNA which bind specifically to GST::PrP.sup.c23-231 at
a 10 fold molar excess of protein over RNA (FIG. 4B, panel b, lane
6). No binding occurs in the presence of GST::rPrP27-30 (lane 9)
and GST (lane 3). A more detailed binding analysis revealed that
binding of RNA to GST::PrP.sup.c23-231 occurs at a molar ratio
(RNA:protein) between 1:1 and 5:1. These findings demonstrate the
selection of an RNA which can distinguish between
GST::PrP.sup.c23-231 and GST::rPrP27-30.
[0089] These results demonstrate that it is possible to isolate an
RNA aptamer (aptus=to fit) by in vitro selection which bind
specifically to the cellular prion protein isoform PrP.sup.c23-231
fused to GST. This RNA does not bind to the recombinant prion
protein rPrP27-30 fused to GST and not to GST. Therefore a RNA was
selected which can distinguish between PrP.sup.c23-231 and
rPrP27-30. Recombinant PrP27-30 share the same amino acid sequence
compared to natural PrP27-30 present in Scrapie prion preparations
(Prusiner et al., 1984) but reveals in contrast to the natural
isoform proteinase K sensitivity.
[0090] The RNA aptamer able to distinguish between PrP.sup.c23-231
and rPrP27-30 overcomes the problem that it is not possible to
produce poly- and monoclonal PrP antibodies which recognize
specifically only one PrP isoform (Groschup et al., 1984 and ref.
therein) and provides a suitable tool for a reliable diagnostic of
transmissible spongiform encephalopathy.
EXAMPLE 2
[0091] Determination of Sequences of the Identified RNA
Molecules
[0092] In order to determine the sequences of RNA molecules
identified after 11 cycles of amplification and selection, these
RNA molecules were reversed transcribed into cDNA and amplified by
PCR (Sambrook et al., 1989). The obtained cDNA was restricted with
EcoRI and BamHI, subcloned into pGEM-3-Zf(-) and the sequence of 20
different cDNA clones pGEM-Ap 1 to 20 determined according to
Sanger et al (1977). Sequences of 14 RNA molecules identified are
depicted in FIG. 5 (SEQ ID NO: 1 to 14) The obtained monoclonal
RNAs revealed sequences which may contain G-quartet motifs. Three
classes of G-quartet motifs (Table II; FIGS. 5A, B, C) could be
identified with more than one monoclonal RNA. 30% of the sequenced
DNA molecules encode for unique RNA molecules which may also
contain G-quartets (FIG. 5D), 30% of the selected RNA aptamers did
not contain any G-quartet motif (FIG. 5E).
2TABLE II Distribution of selected RNA aptamers. G-quartet no motif
G-quartet Motif I Motif II Motif III (unique) motif % of clones 10
15 15 30 30 sequenced
[0093] A detailed analysis of the 20 sequenced clones revealed that
70% of the clones contained four sets of three highly conserved
consecutive guanosine residues, separated by single stranded
regions between four and seven nucleotides long. These guanosine
rich consensus motifs are flanked by two variable regions of
predominantly Watson-Crick variation (see FIG. 6). The primary
sequence of the molecules comprising the four sets of guanosine
stretches strongly suggests that their secondary structure contains
a three layered G-tetrad motif (see FIG. 6). In 40% of the selected
RNAs three classes of aptamer motifs were identified based on their
relationship within the three single stranded loop regions (see
FIG. 6). While individual members of each identified class were
identical in the putative G-tetrad and loop regions, they showed
significant covariation in the Watson-Crick helix.
[0094] Such G-tetrad motifs had already been identified in several
other in-vitro selected nucleic acid molecules (see e.g. Bock et
al., 1992; Wang et al., 1993; Macaya et al., 1993; Lauhon and
Szostak, 1995; Huizenga and Szostak, 1995; Harada and Frankel,
1995) and appear to represent an important feature in nucleic acid
molecules which bind to a ligand with high specificity.
Furthermore, G-quartets have been suggested for telomeric DNA
sequences in species such as Tetrahymena (Sundquist and Klug, 1989;
Williamson et al., 1989; for review: Williamson, 1993). Guanine
rich sequences which could form G-quartets were found in
immunoglobulin switch regions, gene promotors and in chromosomal
telomers which are thought to bring the four homologous chromatids
together during meiosis and prevent the DNA from degradation (Sen
and Gilbert, 1988). G-quartets have also been discussed to play a
role in the dimerization process of retroviral genomic RNA (Weiss
et al., 1993), a prerequisite for the generation of infectious
virions. G-tetrads are held together by Hoogsteen base pairing
through hydrogen bonds between nitrogens or oxygens and hydrogens
(Sen and Gilbert, 1988). The RNA aptamers selected against the
prion protein could contain three G-quartets stacked upon each
other (FIG. 6) to form two eight-coordinate chelation cages.
Alkali-metal ions such as potassium located within the axial
channel are able to complex four oxygens of the upper and four
oxygens of the lower G-quartet. Because of the very compact
structure G-tetrads are very stable and unusual RNAse
resistant.
EXAMPLE 3
[0095] Monoclonal RNA Aptamers Harboring G-Quartet Motif I and II
Bind Specifically to rPrP23-231 (rPrP.sup.c) from Hamster and
rPrP26-242 (rPrP.sup.c) from Calf
[0096] Monoclonal RNA aptamers representing motif I (Ap1; FIGS. 6A,
B) and II (Ap2; FIGS. 6C, D) interact specifically with rPrP23-231
(rPrP.sup.c) from Syrian golden hamster (FIGS. 6A, C; lanes 2) and
rPrP25-242 (rPrP.sup.c) from cattle (FIGS. 6B, D; lanes 2) both
fused to GST. Prion proteins from hamster and cattle reveal a
sequence homology of 88%. Bovine PrP was synthesized in insect
cells infected with a recombinant baculovirus containing bovine
prn-p cDNA (Yoshimoto et al., 1992). Binding of the RNA aptamers to
bovine PrP was investigated to prove whether the aptamers are
suitable for the development of a BSE diagonostic tool. Both
aptamers do not bind to the recombinant GST fused prion proteins
rPrP90-231 (rPrP27-30) from hamster (FIGS. 6, A, C; lanes 3) and
rPrP93-242 (rPrP27-30+1 octarepeat, aa 93-101) from cattle (FIGS.
6, B, D; lanes 3) demonstrating that the molecules distinguish
between rPrP.sup.c and rPrP27-30 from both species. RNA aptamers of
group III interact with rPrP.sup.C and show weak interaction with
rPrP27-30. Some of the selected RNA aptamers lacking any G-quartet
motif interact with GST (data not shown).
[0097] Furthermore, an aptamer was constructed consisting of 60
nucleotides on the basis of an aptamer comprising motif I as shown
in FIG. 5, which, however, lacked the primer binding sites as well
as 14 nucleotides of the randomized region. This 60-mer exactly
corresponds to a part of one of the aptamers displaying the motif I
as depicted in FIGS. 6A/B and displayed the same binding
characteristics as the full-length aptamer. The sequence of this
60mer is depicted in SEQ ID NO: 18. This molecule was isolated by
the following procedure: By using two appropriate primers, DNA
containing the 60 nucleotides encoding the RNA motif I (FIG. 5A,
#II; SEQ ID NO: 18) flanked by a T7 promoter was amplified by PCR
(Sambrook et al. 1989) under the following conditions 1 min
94.degree. C. 1 min 52.degree. C. and 2 min 72.degree. C. for 25
cycles The amplified cDNA product of 80 nucleotides was subjected
to an in vitro transcription reaction (Sambrook et al., 1989) with
T7 RNA polymerase leading to the RNA aptamer consisting of 60
nucleotides (motif I, FIGS. 6A, B). The RNA was gel purified before
the use in gel retardation assays.
[0098] This 60mer was also used for the determination of the
equilibrium binding constants. For this purpose 4 pMol of 5'
.gamma.-.sup.32P-ATP labelled and .alpha.-.sup.32P-UTP labelled RNA
aptamer motif I (SEQ ID NO: 18) was incubated in the presence of 0,
4, 20, 28, 40, 60, 80, 108 pMol of GST::rPrP23-231 for 60 min at
37.degree. C. under assay conditions as described above.
RNA/protein complexes have been analyzed by an gel retardation
assay (Weiss et al., 1992). Gel was fixed by 5% TCA, dried and
subjected to autoradiography for 12 hours. Intensities of the
signals have been determined by phosphoimaging (ImageQuaNT.TM.,
Strom 860, Molecular Dynamics).
[0099] For the calculation of the equilibrium binding constant a
bimolecular reaction between the RNA and the protein was assumed.
The concentration [c] of the RNA/protein complex at equilibrium can
be determined from the amount of radioactivity in the shifted
position and the known specific activity of the RNA. For the
calculation of the equilibrium binding constant (K.sub.D) the
following formula was used (Meisteremst et al., 1988;
Schellenberger et al., 1989): 1 KD = [ R o [ PR ] eq - 1 ] .times.
[ P ] eq
[0100] R.sub.O=R.sub.eq+[RP].sub.eq;
[0101] P.sub.eq=P.sub.O-[RP].sub.eq;
[0102] R=RNA aptamer motif I (60mer)
[0103] P=GST::rPrP23-231 (GST::rPrP.sup.c),
[0104] The following equilibrium binding constant (K.sub.D) for the
complex of the RNA aptamer (60mer) and GST::rPrP23-231
(GST::PrP.sup.c) was preliminary calculated as
K.sub.D=8.times.10.sup.-7 M. Applying other models not basing on
bimolecular binding reactions K.sub.D values <8.times.10.sup.-7
M are expected.
EXAMPLE 4
[0105] Mapping of the Hamster and Bovine PrP/Aptamer Binding
Site
[0106] To map the interaction site of Syrian golden hamster
PrP.sup.c to RNA aptamers 1 and 2, we employed a series of
recombinant prion peptides (Weiss et al., 1995; FIGS. 7, A, B).
Only peptide P.sub.23-52 interact with RNA aptamers Ap1 and 2
(FIGS. 7, A, B), demonstrating that the amino terminal residues
aa23 to aa52 of the Syrian golden hamster prion protein are
sufficient for the recognition by both aptamers. Binding of the
aptamers to rPrP27-30 failed because this molecule lacks the amino
terminal 67 amino acid residues. Prion peptides P.sub.25-92 and
P.sub.93-120 (FIG. 7C, lane 3) from bovine PrP have been
synthesized to map the interaction site of bovine PrP.sup.c to RNA
aptamer motif II. Only P.sub.25-92 (FIG. 7C, lane 2) did bind to
RNA aptamer motif II (Ap2) demonstrating that it is the amino
terminus of the bovine prion protein which is recognized by the
aptamer. Hamster peptide P.sub.23-89 (FIG. 7C, lane 4) did also
interact with aptamer Ap2 confirming the interaction of the amino
terminus of the hamster prion protein with aptamer motif II.
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Sequence CWU 1
1
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