U.S. patent application number 09/291704 was filed with the patent office on 2002-03-14 for method for producing diverse libraries of encoded polypeptides.
Invention is credited to BARTEL, DAVID P., MERRYMAN, CHARLES EVERETT.
Application Number | 20020031762 09/291704 |
Document ID | / |
Family ID | 26767257 |
Filed Date | 2002-03-14 |
United States Patent
Application |
20020031762 |
Kind Code |
A1 |
MERRYMAN, CHARLES EVERETT ;
et al. |
March 14, 2002 |
METHOD FOR PRODUCING DIVERSE LIBRARIES OF ENCODED POLYPEPTIDES
Abstract
tRNA analogues which comprise a tRNA, such as tRNA.sup.phe; an
amino acid moiety which acts as an acceptor substrate, but not as a
donor substrate, for ribosome-directed peptidyl transfer and, thus,
is stably linked to the acceptor stem of the tRNA; and a reactive
or activatible moiety near or within the anticodon stem loop of the
tRNA that can medidate the covalent coupling of the tRNA analogue
to mRNA. Also described are polypeptide-tRNA analogue-mRNA fusions;
libraries of encoded polypeptides; methods of producing and
screening the libraries; and target members and their uses.
Inventors: |
MERRYMAN, CHARLES EVERETT;
(BEVERLY FARMS, MA) ; BARTEL, DAVID P.;
(BROOKLINE, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Family ID: |
26767257 |
Appl. No.: |
09/291704 |
Filed: |
April 14, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60082252 |
Apr 17, 1998 |
|
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|
Current U.S.
Class: |
435/6.16 ;
435/68.1; 435/69.1; 435/69.7; 530/322; 536/25.3 |
Current CPC
Class: |
C12N 15/1062 20130101;
C12N 15/11 20130101 |
Class at
Publication: |
435/6 ; 435/68.1;
435/69.1; 435/69.7; 536/25.3; 530/322 |
International
Class: |
C12Q 001/68; C07H
021/02; C07H 021/04; C12P 021/04; C07K 009/00 |
Claims
What is claimed is:
1. A tRNA analogue, comprising: (a) a tRNA; (b) an amino acid
moiety which acts as an acceptor substrate, but not as a donor
substrate, for ribosome-directed peptidyl transfer; and (c) a
reactive or activatible moiety near or within the anticodon
stem-loop that can mediate the stable coupling of the tRNA analogue
to mRNA.
2. The tRNA analogue of claim 1, wherein the tRNA analogue is a
3'-amino-3'-deoxyadenosine-substituted tRNA or
puromycin-substituted tRNA and the amino acid moiety is any amino
acid or the methoxytyrosine moiety of a puromycin-substituted
tRNA.
3. The tRNA analogue of claim 1, wherein the tRNA is yeast
tRNA.sup.phe.
4. The tRNA analogue of claim 3, wherein the 3' terminal nucleotide
of the yeast tRNA.sup.phe has been replaced by
3'-amino-3'-deoxyadenosine or puromycin.
5. A tRNA analogue which is a tRNA in which the 3' terminal
nucleotide is replaced by 3'-amino-3'-deoxyadenosine and then
linked to an amino acid moiety or replaced by puromycin and in
which the anticodon loop comprises a reactive or activatible moiety
that can mediate covalent coupling of the tRNA analogue to
mRNA.
6. The tRNA analogue of claim 5, wherein the tRNA is yeast
tRNA.sup.phe.
7. The tRNA analogue of claim 5, wherein the reactive or
activatible moiety is a modified base near or within the tRNA stem
loop.
8. The tRNA analogue of claim 7 wherein the reactive or activatible
moiety is a naturally modified guanine base at position 37.
9. A polypeptide-tRNA analogue-mRNA fusion, comprising: (a) a
polypeptide; (b) a tRNA analogue comprising: (i) a tRNA; (ii) an
amino acid moiety which can act as an acceptor substrate, but not
as a donor substrate, for ribosome-directed peptidyl transfer; and
(iii) a reactive or activatible moiety near or within the anticodon
stem-loom that can mediate the stable coupling of the tRNA analogue
to mRNA; and (c) mRNA which encodes the polypeptide of (a), wherein
the tRNA analogue is: located between the polypeptide and the mRNA;
linked to the polypeptide by a stable bond between the terminal
amino acid residue of the polypeptide and the amino acid moiety
and, linked to the mRNA by crosslinks between a reactive or
activatible moiety of the tRNA analogue and the mRNA.
10. The polypeptide-tRNA analogue-mRNA fusion of claim 9, wherein
the tRNA analogue is a 3'-amino-3'-deoxyadenosine-substituted tRNA
or puromycin-substituted tRNA and the amino acid moiety is any
amino acid or the methoxytyrosine moiety of puromycin.
11. The polypeptide-tRNA analogue-mRNA fusion of claim 9, wherein
the tRNA is yeast tRNA.sup.phe.
12. The fusion of claim 11, wherein the tRNA is yeast tRNA.sup.phe
in which the 3' terminal nucleotide has been replaced by
3'-amino-3'-deoxyadenosine or puromycin.
13. A polypeptide-tRNA analogue-mRNA fusion, comprising: (a) a
polypeptide; (b) a tRNA analogue comprising: (i) a tRNA; (ii) an
amino acid moiety which can act as an acceptor substrate, but not
as a donor substrate, for ribosome-directed peptidyl transfer; and
(iii) a reactive or activatible moiety near or within the anticodon
stem-loop that can mediate the stable coupling of the tRNA analogue
to mRNA; and (c) mRNA which encodes the polypeptide of (a), wherein
the tRNA analogue is: located between the polypeptide and the mRNA;
linked to the polypeptide by a stable bond between the terminal
amino acid residue of the polypeptide and the amino acid moiety;
and linked to the mRNA by the action of UV irradiation that
produces a crosslink between a modified base near or within the
tRNA stem loop and the mRNA.
14. The fusion of claim 13, wherein the tRNA analogue is a
3'-amino-3'-deoxyadenosine-substituted tRNA or
puromycin-substituted tRNA and the amino acid moiety is any amino
acid or the methoxytyrosine moiety of puromycin-substituted
tRNA.
15. The fusion of claim 13, wherein the tRNA is yeast tRNA.sup.phe
in which the 3' terminal nucleotide has been replaced by
3'-amino-3'-deoxyadenosine or puromycin.
16. A diverse library of encoded polypeptides, wherein the encoded
polypeptides comprise: (a) a polypeptide; (b) a tRNA analogue
comprising: (i) a tRNA; (ii) an amino acid moiety which can act as
an acceptor substrate, but not as a donor substrate, for
ribosome-directed peptidyl transfer; and (iii) a reactive or
activatible moiety near or within the anticodon stem-loop that can
mediate the stable coupling of the tRNA analogue to mRNA; and (c)
mRNA which encodes the polypeptide of (a), wherein the tRNA
analogue is: located between the polypeptide and the mRNA; linked
to the polypeptide by a stable bond between the terminal amino acid
residue of the polypeptide and the amino acid moiety; and linked to
the mRNA by crosslinks between a reactive or activatible moiety of
the tRNA analogue and the mRNA.
17. The diverse library of claim 16, wherein the tRNA analogue is a
3'-amino-3'-deoxyadenosine-substituted tRNA or
puromycin-substituted tRNA and the amino acid moiety is any amino
acid or the methoxytyrosine moiety of puromycin-substituted
tRNA.
18. The diverse library of claim 17, wherein the tRNA is yeast
tRNA.sup.phe.
19. The library of claim 18, wherein the 3' terminal nucleotide of
yeast tRNA.sup.phe has been replaced by 3'-amino-3'-deoxyadenosine
or puromycin.
20. A method of producing a diverse library of encoded
polypeptides, which comprises polypeptide-tRNA analogue-mRNA
fusions, comprising the steps of: (a) combining: (i) mRNAs which
encode polypeptides; (ii) tRNA analogues, wherein each tRNA
analogue comprises: (a) a tRNA; (b) an amino acid moiety which can
act as an acceptor substrate, but not as a donor substrate, for
ribosome-directed peptidyl transfer; and (c) a reactive or
activatible moiety near or within the anticodon stem-loop that can
mediate the stable coupling of the tRNA analogue to mRNA; and (iii)
an appropriate in vitro translation mixture, thereby producing a
combination; (b) maintaining the combination under conditions
appropriate for translation of the mRNAs to produce the encoded
polypeptides and formation of a stable amino acid-tRNA analogue
bond between the terminal amino acid residue of a polypeptide
produced and the amino acid moiety present in the tRNA, to form
polypeptide-tRNA analogue fusions, thereby producing a mixture
which contains stalled ribosomes that contain polypeptide-tRNA
analogue fusions; and (c) exposing the mixture which contains
stalled ribosomes that contain polypeptide-tRNA analogue fusions to
conditions which favor the crosslinking of the tRNA analogue and
the mRNA which encodes the polypeptide of the polypeptide-tRNA
analogue fusion, whereby polypeptide-tRNA analogue-mRNA fusions are
produced, thereby producing a diverse library of encoded
polypeptides.
21. The method of claim 20, wherein the tRNA analogue is a
3'-amino-3'-deoxyadenosine-substituted tRNA or
puromycin-substituted tRNA and the amino acid moiety is any amino
acid or the methoxytyrosine moiety of puromycin-substituted
tRNA.
22. The method of claim 20, wherein the tRNA is yeast
tRNA.sup.phe.
23. The method of claim 20, wherein the 3' terminal nucleotide of
yeast tRNA.sup.phe has been replaced with
3'-amino-3'-deoxyadenosine or puromycin and the conditions which
favor crosslinking include mild ultraviolet irradiation.
24. A method of identifying members of a diverse library of encoded
polypeptides which exhibit a desired activity, wherein members are
polypeptide-tRNA analogue-mRNA fusions, comprising the steps of:
(a) producing a diverse library of encoded polypeptides which
comprises polypeptide tRNA analogue-mRNA fusions by: (i) combining:
(1) mRNAs which encode polypeptides; (2) tRNA analogues, wherein
each tRNA analogue comprises: (a) a tRNA; (b) an amino acid moiety
which can act as an acceptor substrate, but not as a donor
substrate, for ribosome-directed peptidyl transfer; and (c) a
reactive or activatible moiety near or within the anticodon
stem-loop that can mediate the covalent coupling of the tRNA
analogue to mRNA; and (3) an appropriate in vitro translation
mixture, thereby producing a combination; (ii) maintaining the
combination under conditions appropriate for translation of the
mRNAs to produce the encoded polypeptides and formation of a stable
amino acid-tRNA analogue bond between the terminal amino acid
residue of a polypeptide produced and the amino acid moiety present
in the tRNA analogue, to form polypeptide-tRNA analogue fusions,
thereby producing a mixture which contains stalled ribosomes that
contain polypeptide-tRNA analogue fusions; and (iii) exposing the
mixture which contains stalled ribosomes that contain
polypeptide-tRNA analogue fusions to conditions which favor the
crosslinking the tRNA analogue and the mRNA which encodes the
polypeptide of the polypeptide-tRNA analogue fusion, whereby
polypeptide-tRNA analogue-mRNA fusions are produced, thereby
producing a diverse library of encoded polypeptides; (b) enriching
the diverse library of encoded polypeptides for members which
exhibit a desired activity, thereby producing an enriched diverse
library comprised of polypeptide-tRNA analogue-mRNA fusions; (c)
amplifying the enriched diverse library by: (i) reverse
transcribing the mRNA components of the fusions, thereby producing
the corresponding cDNA; (ii) amplifying and transcribing in vitro
the corresponding cDNA, thereby producing a pool of amplified,
enriched mRNA from the corresponding cDNA; (iii) combining the pool
of amplified, enriched mRNA with an appropriate in vitro
translation mixture and tRNA analogues of (a)(i)(2), thereby
producing a combination; (iv) maintaining the combination under
conditions appropriate for translation of the mRNA to produce the
encoded polypeptides and formation of a stable amino acid-tRNA
analogue bond between the terminal amino acid residue of a
polypeptide produced and the amino acid moiety present in the tRNA
analogue, to form polypeptide-tRNA analogue fusions, thereby
producing an amplified enriched mixture which contains stalled
ribosomes that contain polypeptide-tRNA analogue fusion; and (v)
exposing the amplified enriched mixture which contains stalled
ribosomes that contain polypeptide-tRNA analogue fusions to
conditions which favor crosslinking of the tRNA analogue and the
mRNA which encodes the polypeptide of the polypeptide-tRNA analogue
fusion; (d) repeating steps (b)-(c) as necessary until members
which exhibit the desired activity are present in sufficient number
to be detected; and (e) detecting members which exhibit the desired
activity, thereby identifying members which exhibit the desired
activity.
25. The method of claim 24, wherein the polypeptide-tRNA
analogue-mRNA fusion is a 3'-amino-3'-deoxyadenosine-substituted
tRNA or puromycin-substituted tRNA and the amino acid moiety is any
amino acid or the methoxytyrosine moiety of puromycin.
26. The method of claim 25, wherein in the polypeptide-tRNA
analogue-mRNA fusion, the tRNA is yeast tRNA.sup.phe.
27. The method of claim 26, wherein in the yeast tRNA.sup.phe, the
3' terminal nucleotide has been replaced by
3'-amino-3'-deoxyadenosine or puromycin.
28. A member of a diverse library of encoded polypeptides which
exhibits a desired activity, identified by the method of claim
24.
29. A polypeptide fragment of a member of a diverse library of
encoded polypeptides, wherein the member exhibits a desired
activity and is identified by the method of claim 24.
30. A tRNA analogue-mRNA fragment of a member of a diverse library
of encoded polypeptides, wherein the member exhibits a desired
activity and is identified by the method of claim 24.
Description
RELATED APPLICATION
[0001] This application claims the benefit of U.S. provisional
application 60/082,252, entitled "Method for producing Diverse
Libraries of Encoded Peptides," by Charles E. Merryman and David P.
Bartel, filed Apr. 17, 1998. The entire teachings of the referenced
provisional application are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Large combinatorial libraries of biopolymers are starting
points for isolating new enzymes, binding motifs and other useful
molecules. For example, current technologies can generate
populations of nucleic acids with complexities on the order of
10.sup.15 molecules and then isolate and identify a single molecule
with a desired activity. Random polypeptide populations have
greater chemical diversity than do polynucleotides, making them an
attractive alternative to nucleic acids. Current systems are
limited in their ability to easily generate large complex libraries
of polypeptides that are in a form that allows the isolation and
identification of rare molecules with a desired activity.
SUMMARY OF THE INVENTION
[0003] The present invention relates to tRNA analogues;
polypeptide-tRNA-analogue-mRNA fusions; diverse libraries of
encoded polypeptides; and a method of producing the diverse
libraries. tRNA analogues of the present invention comprise a tRNA
(such as a yeast tRNA); an amino acid moiety that acts as an
acceptor substrate, but not as a donor substrate, for
ribosome-directed peptidyl transfer; and a reactive or activatible
moiety near or within the anticodon stemp loop of the tRNA that can
mediate the stable coupling of the tRNA analogue to mRNA. An amino
acid moiety is stably linked to the tRNA if the linkage between the
two or the chemical environment allows the amino acid moiety to act
as an acceptor substrate but not as a donor substrate for
ribosome-directed peptidyl transfer. In a specific embodiment, the
tRNA analogue is a 3'-amino-3'-deoxyadenosine-substituted tRNA in
which the 3'-terminal A of yeast tRNA.sup.phe is replaced or
substituted by 3'-amino-3'-deoxyadenosine and then the substituted
tRNA is charged with phenylalaine. This tRNA analogue is termed
PHE-N-tRNA. In another specific embodiment, the tRNA analogue is a
puromycin-substituted tRNA in which the 3'-terminal A of yeast
tRNA.sup.phe is replaced or substituted by puromycin, with the
result that the amino acid moiety is the methoxytyrosine moiety of
puromycin. In both embodiments, the reactive or activatible moiety
near or within the anticodon stem-loop is the modified Y base of
yeast tRNA.sup.phe.
[0004] Also the subject of this invention are polypeptide-tRNA
analogue-mRNA fusions, in which: the polypeptide can be a peptide
or polypeptide of any size; the tRNA analogue is as described
herein; and the mRNA is mRNA which encodes the polypeptide
component of the fusion. In a fusion, the tRNA analogue component
is: (a) located between the polypeptide and the mRNA which encodes
the polypeptide; (b) linked to the polypeptide by a stable bond
between the terminal amino acid residue of the polypeptide and the
amino acid moiety of the tRNA analogue; and (c) linked to the mRNA
by crosslinks between a reactive or activatible moiety of the tRNA
analogue and the mRNA. In one embodiment, the polypeptide-tRNA
analogue-mRNA fusion includes a PHE-N-tRNA (and, thus, the amino
acid moiety which acts as an acceptor substrate but cannot act as a
donor substrate is phenylalanine) and the tRNA is yeast
tRNA.sup.phe. In another embodiment, the polypeptide-tRNA
analogue-mRNA fusion includes a puromycin-substituted tRNA (and,
thus, the amino acid moiety which acts as an acceptor substrate but
cannot act as a donor substrate is the methoxytyrosine moiety of
puromycin) and the tRNA is yeast tRNA.sup.phe.
[0005] Diverse libraries or collections of encoded polypeptides are
also the subject of this invention. A diverse library or collection
comprises the encoded polypeptides, which are the polypeptide-tRNA
analogue-mRNA fusions described herein. Methods of producing such
libraries are also the subject of this invention.
[0006] The invention further relates to a method of screening a
diverse encoded polypeptide library to identify target members
(library members with desired biological or biochemical properties
or activities, such as binding to a particular ligand or enzymatic
activity). In one embodiment of screening a diverse encoded
polypeptide library to identify a target member or members, the
diverse library is initially enriched in molecules with desired
properties. This is done, for example, to identify a binding
partner or ligand of interest using known enrichment methods, such
as affinity enrichment using an immobilized ligand or binding
partner or to identify a library member with enzymatic activity by
assessing affinity of a library member to a product of a reaction
in which the enzyme has modified itself or a substrate to which the
library member is attached. Library members identified in this way
are target members. In a further step in the method, a library
which has been enriched in target members (an enriched fusion or
encoded diverse polypeptide library) is amplified and subjected to
additional enrichment. For example, the enriched library is reverse
transcribed, thereby producing cDNAs of the mRNA components; the
cDNAs are, optionally, amplified. The initially produced cDNAs or
the resulting PCR products are subjected to in vitro transcription,
thereby producing an amplified pool of mRNAs that encode members of
the enriched fusion library. The amplified pool of mRNAs is
subjected to in vitro translation in the presence of the tRNA
analogue of the present invention, producing an amplified versionof
the enriched encoded polypeptide library. Library members (fusions)
amplified in this manner are, optionally, subjected to further
enrichment and amplification, as necessary, until target members
are enriched to a level where they are present in sufficient
numbers to be detected. They are detected using any known method,
such as binding to a ligand of interest or catalyzing a reaction of
interest. mRNAs of target members are cloned and then individual
fusions made from cloned mRNAs are screened for the desired
properties (e.g., by ligand binding or catalyzing a reaction of
interest). Library members identified in this way are target
members, which are also a subject of this invention.
[0007] Target members are polypeptide-tRNA analogue-mRNA fusions.
The translation products of the enriched mRNA (the polypeptide
component of a target member) which display properties of interest
and modified or engineered derivatives of the translation products
which display properties of interest are target polypeptide
fragments. These fragments are also a subject of this invention. As
used herein, the term target polypeptide fragments includes
fragments released or separated from target members in which they
occur and fragments produced or synthesized by another method
(e.g., chemical synthesis, mRNA translation in the absence of the
tRNA analogue or a recombinant DNA method in which DNA encoding a
desired target polypeptide fragment is expressed). Target
polypeptide fragments include, but are not limited to, protein
catalysts, single-chain monoclonal antibodies, binding pair members
(ligand or binding partner), receptors or their ligands, and
enzymes and their substrates.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1a and 1b are schematic representations of two
embodiments of tRNA analogues of the present invention, in which
the tRNA portion is stably linked by an amide bond (very thick
line) to the amino acid moiety of the tRNA analogue and the
reactive or activatible moiety is a modified nucleotide (Y base)
which can be used to couple the tRNA analogue to mRNA when the base
is activated by UV irradiation.
[0009] FIG. 2 is a schematic representation of one embodiment of
the present invention, in which steps which occur during in vitro
translation are presented and the tRNA analogue is a yeast
tRNA.sup.phe analogue in which the last nucleotide of the acceptor
stem has been replaced by 3-amino-3'-deoxyadenosine.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present invention relates to a modified tRNA or
functional analogue of tRNA (both referred to herein as a tRNA
analogue) that comprises 1) a tRNA or tRNA-like molecule; 2) an
amino acid moiety, which is an amino acid, a modified amino acid,
or other amino-acid like molecule that can act as an acceptor
substrate for ribosome directed peptidyl transfer, but cannot act
as a donor substrate for ribosome-directed peptidyl transfer; thus,
it is stably linked to the acceptor stem of the tRNA or tRNA-like
molecule and 3) a reactive or activatible moiety, which can be any
addition, deletion, substitution, modification or alteration of the
tRNA (such as addition, deletion, substitution, modification or
alteration of one or more bases or nucleotides near or within the
anticodon stem-loop of the tRNA), that can mediate the covalent
coupling of the tRNA analogue to messenger RNA (mRNA). For example,
the activatible moiety can be the naturally occurring modification
of guanine (the Y base) that is found in the anticodon loop at
position 37 of yeast tRNA.sup.phe. Such a tRNA analogue is a
bifunctional molecule whose amino-acid moiety can accept, but not
donate, a polypeptide that is being synthesized (under the
direction of the ribosome) and which can specifically link with the
mRNA encoding the polypeptide being synthesized. The tRNA analogue
can be, for example, a 3'-amino-3'-deoxyadenosine-substituted tRNA
or puromycin-substituted tRNA and the amino acid moiety is any
amino acid or the methoxytyrosine moiety of a puromycin-substituted
tRNA. The tRNA analogue can be, for example, a tRNA in which the
3'terminal nucleotide is replaced by 3'-amino-3'-deoxyadenosine and
then linked to an amino acid moiety or replaced by puromycin and in
which the anticodon loop comprises a reactive or activatible moiety
that can mediate covalent coupling of the tRNA analogue to the
mRNA. The tRNA analogue can be used to produce diverse encoded
polypeptide libraries, which are also the subject of this
invention.
[0011] The tRNA analogues can comprise any tRNA or tRNA-like
molecule (e.g., bacterial, yeast, mammalian, in vitro transcribed,
synthesized, tmRNA, etc.) which: 1) is modified such that the
resulting modified tRNA can form a stable link with a polypeptide
being expressed and 2) can make a specific link with the mRNA which
encodes the polypeptide being expressed (the encoding mRNA). In a
specific embodiment, the 3' end of the tRNA is modified such that
3'-amino-3'-deoxyadenosine replaces the A base at the 3' end and
then the substituted tRNA is charged with phenylalanine. In another
specific embodiment, the 3' end of the tRNA is modified such that
puromycin replaces the A base at the 3' end. The tRNA analogue can
contain any modification which can form the desired stable
peptide-tRNA link. As a result of the formation of a stable
peptide-tRNA link, protein synthesis is stalled, presumably after
translocation of the peptide-tRNA analogue complex to the P site.
The specific link between the tRNA analogue and the mRNA which
encodes the polypeptide being produced can be formed by activation
of an activatible group or via a reactive group in the tRNA (e.g.,
the Y base) or mRNA.
[0012] In one embodiment, the tRNA analogue is produced by
replacing the 3' terminal nucleotide (e.g., the 3' terminal A) of
yeast tRNA.sup.phe by 3'-amino-3'-deoxyadenosine and charging the
tRNA with phenylalanine. In another embodiment, the tRNA analogue
is produced by replacing the 3' terminal nucleotide (e.g., the 3'
terminal A) of yeast tRNA.sup.phe by puromycin. Yeast tRNA.sup.phe
is useful because it naturally contains a modified guanine base
(the Y base) at position 37 that serves as an activatible group
that becomes reactive when exposed to UV light, coupling the tRNA
analogue to an mRNA. After aminoacylation with phenylalanine, the
3'-amino-3'-deoxyadenosine substitution transforms the yeast
tRNA.sup.phe into a tRNA analogue (a 3'-amino-3'-deoxyadenosine
substituted tRNA) that contains phenylalanine linked to the RNA 3'
terminus by an amide bond. In this embodiment, the amino acid
moiety is the phenylalanine moiety of the
3'-amino-3'-deoxyadenosine-substituted tRNA. Alternatively,
puromycin substitution transforms the yeast tRNA.sup.phe into a
tRNA analogue (a puromycin-substituted tRNA) that contains
methoxytyrosine linked to the RNA 3' terminus by an amide bond. In
this embodiment, the amino acid moiety is the methoxytyrosine
moiety of the puromycin-substituted tRNA. In both embodiments, the
amide linkage between the RNA and the phenylalanine or
methoxytyrosine prevents the peptidyl-tRNA analogue from being a
suitable donor substrate for ribosome-directed peptidyl transfer
and, thus, when a polypeptide is transferred to the amino-acid
moiety of the tRNA analogue, the polypeptide becomes stably
connected to the tRNA analogue.
[0013] FIGS. 1a and 1b are schematic representations of these
embodiments of the present invention. FIGS. 1a and 1b show tRNA
analogues that contain an amide bond (very thick line) stably
linking the tRNA portion and the amino-acid moiety of the analogue.
In FIG. 1a, the tRNA analogue is 3'-amino-3'-deoxyadenosine
substituted and the amino acid moiety is a phenylalanine moiety. In
FIG. 1b, the tRNA analogue is puromycin substituted and the amino
acid moiety is a methoxytyrosine moiety. It also shows the Y base,
which is a modified nucleotide which can be used to couple the tRNA
analogue to mRNA when the base is activated by UV irradiation. The
tRNA portion of the molecule is represented schematically by a
thick line. FIG. 2 illustrates the action of the tRNA analogue of
this embodiment of the present invention. During translation, when
the ribosome reaches a phenylalanine codon (UUU or UUC) in the
mRNA, the methoxytyrosine portion (amino-acid moiety) of the tRNA
analogue is used as an acceptor substrate and is joined to the
carboxy terminus of the nascent polypeptide; because the
methoxytyrosine is joined to the tRNA portion of the tRNA analogue
by an amide bond, translation stalls. The stalled
polypeptide-tRNA.sup.phe can be crosslinked to the mRNA (through
the Y base of the tRNA.sup.phe that was used to make the tRNA
analogue) by mild UV irradiation.
[0014] The present invention also relates to a diverse library or
collection of encoded polypeptides which each comprise three
components that are fused into a single molecule that can be
produced and manipulated in vitro. Each encoded polypeptide
molecule of the library, referred to as a polypeptide-tRNA
analogue-mRNA fusion, comprises a polypeptide of any length, an
mRNA encoding the polypeptide and a tRNA analogue that is located
between the polypeptide and its encoding mRNA and forms a link
between these two components. The mRNA component of each fusion can
be an mRNA or mRNA-like molecule (of any sequence or length) that
is translated by the ribosome. The mRNA component contains elements
required for its expression and elements (e.g., primer binding
sites) that permit it to be reverse transcribed and then amplified
and/or cloned. The tRNA analogue component of each fusion is as
described above. That is, the tRNA analogue component, comprises 1)
a tRNA or tRNA-like molecule; 2) an amino acid moiety which can act
as an acceptor substrate, but not as a donor substrate, for
ribosome-directed peptidyl transfer and 3) an activatible moiety
that mediates the covalent coupling of the tRNA analogue to
messenger RNA.
[0015] The diverse library or collection of encoded polypeptides is
produced by combining the tRNA analogue with an in vitro
translation mixture that contains a diverse mixture of mRNA
sequences (such as a randomized pool of mRNAs) and incubating the
mixture under conditions appropriate for in vitro translation of
mRNA sequences and formation of the linkage between the tRNA
analogue and the mRNA. Translation of the mRNAs is stalled as a
result of the presence of the stably linked amino acid moiety of
the tRNA analogue. At first, members of the diverse collection of
mRNAs are translated normally: When a codon is reached, a matching
aminoacyl tRNA is selected by the ribosome and bound to the A site.
The previously synthesized portion of the nascent polypeptide
(which is attached to a P-site bound tRNA) is transferred to the
amino acid on the newly selected aminoacyl tRNA (effectively
extending the length of the encoded polypeptide by one amino acid)
and the tRNA previously bound to the P site is replaced by the
newly selected tRNA, which now becomes the P-site bound tRNA. The
ribosome uses the now empty A site to match another codon and
aminoacyl tRNA. Repetition of this cycle translates mRNA sequences
into their corresponding polypeptide sequences. For members of the
mRNA of the library this normal translation ends when the tRNA
analogue (instead of a normal aminoacyl tRNA) is selected by the
ribosome and the ribosome binds the tRNA analogue to the A site.
The polypeptide is then transferred to the amino-acid moiety of the
tRNA analogue. The stable linkage between the amino-acid moiety and
the RNA portion of the tRNA analogue prevents the polypeptide from
being further transferred and, thus, translation is stalled with
the polypeptide stably attached to the tRNA analogue, which is
bound to the mRNA that encodes the polypeptide. Prior to,
concurrent with, or after transfer of the polypeptide to the tRNA
analogue, the second functionality of the tRNA analogue is used to
covalently couple the tRNA analogue to the mRNA. In this way the
bifunctional tRNA analogue is used to generate the desired library
of polypeptide-tRNA analogue-mRNA fusions.
[0016] In one embodiment, the tRNA analogue can be yeast
tRNA.sup.phe, where the 3' terminal nucleotide (e.g., the 3'
terminal A of yeast tRNA.sup.phe) is replaced by
3'-amino-3'-deoxyadenosine and then charged with phenylalanine. In
another embodiment, the tRNA analogue can be yeast tRNA.sup.phe,
where the 3' terminal nucleotide (e.g., the 3' terminal A of yeast
tRNA.sup.phe) is replaced by puromycin. The resulting tRNA
analogues are bifunctional and during in vitro protein synthesis,
they can form a stable link (as a result of ribosome directed
peptidyl transfer) with the carboxy-terminal amino acid of a
polypeptide being produced and a stable link with the mRNA encoding
that polypeptide (by the action of mild UV irradiation). FIG. 2 is
a schematic representation of one embodiment of the present
invention. It represents steps which occur during in vitro
translation that link the growing polypeptide to the tRNA analogue
(top panel) and that link the tRNA analogue to the mRNA (bottom
panel). As illustrated (top panel), a normal polypeptidyl
containing tRNA is located in the P site and a tRNA analogue (e.g.,
PHE-N-tRNA in which 3'-amino-3'-deoxyadenosine is boxed), is
located in the A site of a ribosome. Peptidyl transfer (represented
by small arrows in the top panel) occurs, resulting in formation of
a polypeptide-tRNA fusion by transfer of the peptide being produced
to the amino acid moiety of the tRNA analogue (the amide link
between the amino-acid moiety and the RNA portion of the tRNA
analogue is shown as a very thick line). This amide link stalls
protein synthesis, presumably after translocation of the
polypeptide-tRNA analogue from the A site to the P site (bottom
panel). Exposure of the stalled complexes to UV irradiation results
in crosslinking of the polypeptide-tRNA analogue to the mRNA
through the Y base of the tRNA analogue, thus generating the
desired polypeptide-tRNA analogue-mRNA fusion.
[0017] The present invention also relates to the encoded
polypeptides (polypeptide-tRNA analogue-mRNA fusions) which
comprise the diverse libraries or collections; methods of
generating or producing the diverse libraries; a method of
identifying and, optionally, amplifying members of the encoded
polypeptide library (referred to as target members) which have
desired characteristics; target members of the library identified
by the method and fragments of the target members (e.g.,
polypeptides, fragments of polypeptides, or fragments of
polypeptide-tRNA analogue-mRNA fusions.)
[0018] In the method of generating libraries of encoded
polypeptides (diverse collections of polypeptide-tRNA analogue-mRNA
fusions), mRNA, tRNA analogues (e.g., PHE-N-tRNA) and an
appropriate in vitro translation mixture (e.g., bacterial
translation mixtures, such as from E. coli; eucaryotic translation
mixtures, such as mixtures from wheat germ and rabbit
reticulocytes, or translation mixtures from other organisms) are
combined to produce a combination. The resulting combination is
maintained under conditions appropriate for translation of the
mRNAs, formation of stable peptide-tRNA analogue fusions (linkage
between the terminal amino acid residue of the polypeptide being
expressed and the stably linked amino-acid moiety, e.g., the
phenylalanine of PHE-N-tRNA), and formation of a covalent linkage
fusing the tRNA analogue with the mRNA. The in vitro translation
mixture is a combination of biological reagents and cellular
components which translate mRNAs to produce the polypeptides they
encode. If the tRNA portion of the tRNA analogue is yeast
tRNA.sup.phe then crosslinking is effected by subjecting the
ribosome-bound, stalled polypeptide-tRNA-mRNA complex to UV
irradiation.
[0019] Once a diverse encoded polypeptide library has been
produced, it can be screened, using known methods, to identify
target members which are fusions with desired characteristics. A
key advantage of the encoded polypeptide library of the present
invention, which is comprised of polypeptide-tRNA analogue-mRNA
fusions, is that even members which occur in small numbers (rare
members) and are of interest because of desired biological or
biochemical properties (e.g., binding to a particular ligand,
enzymatic activity) can be enriched and then identified by
amplification, cloning and sequencing of their respective
mRNAs.
[0020] A diverse library of encoded polypeptides can be enriched in
molecules with the desired properties using known methods, to
identify target members. Methods by which target members of the
library can be enriched include affinity enrichment using
immobilized ligand or binding partner and, for enzymatic activity,
affinity to a product of a reaction in which the enzyme has
modified itself (with, for example, a mechanism-based inhibitor) or
a substrate to which it is attached (e.g., Williams, K. P. and D.
P. Bartel, "In Vitro Selection of Catalytic RNA", pp. 367-381 In:
Catalytic RNA, (Fritz Eckstein and David M. J. Lilley, Ed.),
Springer, (1996)).
[0021] Furthermore, libraries enriched in target members can be
amplified and subjected to additional enrichment. For example, a
library of fusions that has been enriched for a desired activity
(an enriched encoded polypeptide library) can be reverse
transcribed, producing the cDNAs of the mRNA components. The cDNAs
can then be amplified (e.g., by PCR or other amplification
methods). The resulting PCR products are subjected to in vitro
transcription, resulting in production of an amplified pool of
mRNAs that encode the members of the enriched fusion library. In
vitro translation of this pool in the presence of the tRNA analogue
links the mRNAs to their translation products, producing an
amplified version of the enriched encoded polypeptide library.
Fusions amplified in this way are subjected to further enrichment
and amplification, which is repeated as necessary until target
members are enriched to the desired extent (e.g., enriched to a
level where they are present in sufficient numbers to be detected
by binding to a ligand of interest or catalyzing a reaction of
interest). After sufficient enrichment, mRNAs of target members are
cloned and individual fusions can be screened for the desired
function. The translation product of the mRNA or a fragment of the
translation product can also be screened for activity without
attachment to the mRNA.
[0022] The method of the present invention of identifying members
of a diverse library of encoded polypeptides which exhibit a
desired activity is carried out as follows: A diverse library,
whose members are polypeptide-tRNA anlogue-mRNA fusions, is
produced by combining: 1) mRNAs which encode polypeptides; 2) tRNA
analogues, which each comprise: a) a tRNA; b) an amino acid moiety
which can act as an acceptor substrate, but not as a donor
substrate, for ribosome-directed peptidyl transfer and c) a
reactive or activatible moiety near or within the anticodon
stem-loop that can mediate (mediates) the covalent coupling of the
tRNA analogue to mRNA; and 3) an appropriate in vitro translation
mixture, thereby producing a combination. The resulting combination
is maintained under conditions appropriate for translation of the
mRNAs to produce the encoded polypeptides and formation of a stable
amino acid-tRNA analogue bond between the terminal amino acid
residue of a polypeptide produced and the amino acid moiety present
in the tRNA analogue, to form polypeptide-tRNA analogue fusions,
thereby producing a mixture which contains stalled ribosomes that
contain polypeptide-tRNA analogue fusions. The mixture which
contains stalled ribosomes that contain the fusions is exposed to
conditions which favor the crosslinking the tRNA analogue and the
mRNA which encodes the polypeptide of the polypeptide-tRNA analogue
fusion. As a result, polypeptide-tRNA analogue-mRNA fusions are
produced, thereby producing a diverse library of encoded
polypeptides. The diverse library of encoded polypeptides is
enriched for members which exhibit a desired activity, thereby
producing an enriched diverse library comprised of polypeptide-tRNA
analogue-mRNA fusions. The resulting enriched diverse library is
amplified by: reverse transcribing the mRNA components of the
fusions, thereby producing the corresponding cDNA; amplifying and
transcribing in vitro the corresponding cDNA, thereby producing a
pool of amplified, enriched mRNA from the corresponding cDNA;
combining the pool of amplified, enriched mRNA with an appropriate
in vitro translation mixture and tRNA analogues (as described
above), thereby producing a combination; maintaining the
combination under conditions appropriate for translation of the
mRNA to produce the encoded polypeptides and formation of a stable
amino acid-tRNA analogue bond between the terminal amino acid
residue of a polypeptide produced and the amino acid moiety present
in the tRNA analogue, to form polypeptide-tRNA analogue fusions,
thereby producing an amplified enriched mixture which contains
stalled ribosomes that contain polypeptide-tRNA analogue fusion;
and exposing the amplified enriched mixture which contains stalled
ribosomes that contain polypeptide-tRNA analogue fusions to
conditions which favor crosslinking of the tRNA analogue and the
mRNA which encodes the polypeptide of the polypeptide-tRNA analogue
fusion. The steps of enriching the diverse library and amplifying
the enriched diverse library are repeated, as necessary, until
members which exhibit the desired activity are present in
sufficient number to be detected. Subsequently, members which
exhibit the desired activity are detected. As a result, members of
the diverse library which exhibit the desired activity are
identified.
[0023] The translation products of the enriched mRNA (the
polypeptide component of the target members), such as polypeptides
which display activities of interest (e.g., ligand binding or
catalytic activity), as well as engineered derivatives of these
translation products which display activities of interest, are
referred to as target polypeptide fragments. These target
polypeptide fragments are also the subject of this invention.
Target polypeptide fragments can be released or separated from
target members in which they occur, using known methods (e.g.,
enzymes which cleave RNA), or they can be synthesized without
attachment to the mRNA (e.g., using chemical synthesis or mRNA
translation in the absence of the tRNA analogue. They can be used,
for example, as diagnostic or therapeutic reagents (e.g.,
single-chain monoclonal antibodies), protein catalysts, members of
binding pairs, receptors or their ligands, enzymes or enzyme
substrates. Once a polypeptide fragment which has desired
characteristics has been identified, it can be produced using known
methods (e.g., production in an appropriate expression system,
chemical synthesis).
[0024] Ribonucleoprotein fragments of the target members are also
the subject of this invention. They can be used, for example, as
enzymes or ligands.
[0025] The present invention is illustrated by the following
examples, which are not intended to be limiting in any way.
EXAMPLES
Example 1
[0026] Crosslinking of Yeast tRNAphe and Peptidyl Analogues Yeast
tRNAphe to mRNA
[0027] Ribosome Preparation
[0028] Tight couple ribosomes were prepared from Escherichia
(E.)coli strain MRE 600. Two liters of LB broth were innoculated
with 20 ml of a fresh overnight culture and incubated at 37.degree.
C. with vigorous agitation until an absorbance of 0.6 (550 nm) was
reached. Culture flasks were then transferred to an ice water bath
and incubated for 30 minutes. Cells were pelleted by centrifugation
at 8000 rpm in a Sorvall GSA rotor, for 10 minutes, at 4.degree. C.
Cells were washed by resuspension in approximately 50 ml of buffer
A (50 mM Tris-HCl, pH 7.5; 10 mM MgCl.sub.2; 100 mM NH.sub.4Cl, 6
mM 2-mercaptoethanol, 0.5 mM EDTA) and pelleted by centrifugation
for 5 minutes at 8000 rpm in a Sorvall SS-34 rotor. The pelleted
cells were then transferred to a large mortar at 4.degree. C. Lysis
of cells was performed in the 4.degree. C. cold room by slow
addition of 4.degree. C. alumina (2.5 grams for every gram of
cells) while grinding with a pestle. Typically, lysis took about
twenty minutes. When lysis was complete, buffer A was added to
dilute the grinding mixture to a total volume of about 30 ml. This
was divided evenly between two Sorval SS-34 centrifuge tubes and
the alumina was cleared from the solution by centrifugation for 5
minutes at 5000 rpm. The supernatant (cell lysate) was removed and
clarified from cellular debris by centrifugation for 20 minutes at
15,000 rpm in a Sorvall SS-34 rotor; the clarification was then
repeated. The NH.sub.4Cl concentration of the clarified supernatant
was adjusted to 500 mM and the ribosomes were pelleted by
centrifugation for 4 hours, at 4.degree. C., at 40,000 rpm in a
Beckman Ti60 rotor. The ribosome pellet was washed with
approximately 5 ml of buffer B (50 mM Tris-HCl, pH 7.5; 6 mM
MgCl.sub.2; 100 mM NH.sub.4Cl, 6 mM 2-mercaptoethanol, 0.5 mM EDTA)
and then resuspended in 4 ml of buffer B by adding a small stir bar
directly to the centrifuge tube and mounting the tube above a stir
plate in the 4.degree. C. coldroom. (It is important to maintain
gentle stirring throughout resuspension of the ribosomes.) When the
ribosomes were completely resuspended (20-60 minutes) 1 ml aliquots
were layered onto 36 ml, 10-50% sucrose gradients (in buffer B) and
centrifuged for 13 hours, at 4.degree. C., at 20,000 rpm in a
Beckman SW28 rotor. The gradients were fractionated and the 70S
ribosome peak was collected. The magnesium concentration was
adjusted to 10 mM and the ribosomes were pelleted from solution by
centrifugation for 4 hours, at 4.degree. C., at 60,000 rpm in a
Beckman Ti60 rotor. The ribosome pellet was washed with 5 ml of
buffer C (50 mM Tris-HCl, pH 7.5; 6 mM MgCl.sub.2; 100 mM
NH.sub.4Cl, 0.5 mM EDTA) and resuspended as before in 1 ml of
buffer C. The concentration of the ribosomes was determined at 260
nm (1 O.D. of 70S ribosomes=23 pmol) and buffer C was added to
obtain a final concentration of 10 .mu.M. The ribosomes were then
aliquoted (10 .mu.l), frozen in liquid nitrogen and stored at
-70.degree. C.
[0029] Preparation of S100 Extract
[0030] Twenty grams of E. coli cells were lysed and the cellular
debris removed as described above. Ribosomes were removed from the
clarified cell lysate by centrifugation for 4 hours, at 4.degree.
C., at 40,000 rpm in a Beckman Ti60 rotor and the supernatant was
diluted two fold with buffer D (10 mM Tris-HCl pH 7.5; 30 mM
NH.sub.4Cl; 10 mM MgCl.sub.2; and 6 mM 2-mercaptoethanol). This
solution was stirred with 12 g of dry DEAE-cellulose that was
equilibrated with buffer D, washed with distilled water, and then
dried. The slurry was filtered on a scintered glass funnel and
washed with 1-2 liters of buffer D. The DEAE-cellulose cake was
resuspended in buffer D and packed in a column. The S100 extract
was then eluted with buffer D containing 250 mM NH.sub.4Cl (the
desired fractions elute as a sharp band and can usually be
identified by eye as they have a pale yellow color. Small aliquots
(100 .mu.l) of the purified S100 extract were frozen in liquid
nitrogen and stored at -70 .degree. C. The tRNA charging efficiency
of S100 was determined by monitoring TCA-precipitable counts in the
presence of tRNA.sup.phe and radiolabeled phenylalanine [S100 is
incubated for 10 minutes at 37.degree. C. with tRNA.sup.phe (1
.mu.M), 14C-phenylalanine (20 .mu.M), and ATP (2 mM) in 30 mM
Tris-HCl pH 7.5, 15 mM 1M MgCl.sub.2, 25 mM KCl, 4 mM DTT].
[0031] Preparation of tRNAs and mRNA
[0032] Yeast tRNA.sup.phe was purchased from Sigma. Elongator
methionine tRNA was transcribed by T7 polymerase from DNA obtained
by PCR amplification of E. coli chromosomal DNA using the (+)
strand primer TAA-TAC-GAC-TCA-CTA-TAG-GCT-ACG-TAG-CTC-AGT-TGG (SEQ
ID NO.: 1) and the (-) strand primer TGG-TGG-CTA-CGA-CGG-GAT-TC
(SEQ ID NO.: 2). A portion of the T4 gene-32 mRNA was transcribed
by T7 polymerase from a PCR amplified section (-55 to +85) of
plasmid pKsK12A (Krisch and Allet, PNAS (USA), 79:4937-4941 (1982))
using the (+) strand primer
TAA-TAC-GAC-TCA-CTA-TAG-GTA-AAG-TGT-CAT-TAG-C (SEQ ID NO.: 3) and
the (-) strand primer CTT-TAT-CTT-CAG-AAG-AAA-AAC-C (SEQ ID NO.:
4). The mRNA was labeled with yeast polyA polymerase and .sup.32P
labeled cordycepin.
[0033] Amino Acylation and Acetylation of Yeast tRNAphe
[0034] Amino acylation of yeast tRNA.sup.phe (1 .mu.M) with
phenylalanine (20 .mu.M) was performed by incubation for 10 minutes
at 37.degree. C. with ATP (2 mM) and the appropriate amount of S100
in 30 mM Tris-HCl pH 7.5, 15 mM 1M MgCl2, 25 mM KCl, 4 mM DTT. To
stop the reaction and recover the acylated tRNA (PHE-tRNA.sup.phe;
typically 70% yield) one-tenth volume of 3M NaOAc pH 5.0 was added,
and the reaction was extracted two times with acid phenol, two
times with chloroform, and precipitated with 2.5 volumes of
ethanol. The PHE-tRNA.sup.phe was resuspended at a concentration of
10 .mu.M in 0.2M NaOAc pH 5.0 and the amine of the phenylalanine
was acetylated (to produce the peptidyl analogue
N-acetyl-PHE-tRNA.sup.phe) by adding one-hundredth volume of acetic
anhydride followed by incubation on ice for 30 minutes. A fresh
aliquot of acetic anhydride was added and the incubation continued
for another 30 minutes. The reaction was stopped and the
N-acetyl-PHE-tRNA.sup.phe) isolated by adding 2.5 volumes of
ethanol and precipitating. N-acetyl-PHE-tRNAphe was resuspended at
a concentration of 10 .mu.M in 10 mM NaOAc pH 5.0 and stored at
-20.degree. C.
[0035] Binding of mRNA and tRNA to the Ribosome and Crosslinking of
N-acetyl-PHE-tRNAphe to the mRNA
[0036] Ribosomal complexes with tRNA.sup.met bound to the P site of
the ribosome and the first codon (AUG) of the labeled gene-32 mRNA
were formed by incubating ribosomes (0.5 .mu.M), tRNA.sup.met (1
.mu.M), and mRNA (5 .mu.M) in 25 .mu.l of buffer (Tris-HCl ph 7.5,
30 mM NH.sub.4Cl, 20 mM MgCl.sub.2) for 10 minutes at 37.degree. C.
Deacylated yeast tRNA.sup.phe was bound to the A site
(corresponding to the second codon (UUU) of the mRNA) by adding 25
pmol of the tRNA and continuing the incubation for an additional 10
minutes. Alternatively, N-acetyl-PHE-tRNA.sup.phe was bound to the
P site by adding 25 pmol of N-acetyl-PHE-tRNA.sup.phe and
continuing the incubation for an additional 10 minutes; the tRNA
initially binds to the A site but spontaneous translocation should
move it into the P site of the ribosome. The phenylalanine tRNA in
either type of complex was crosslinked to the mRNA by spotting the
reaction on a petri dish and irradiating for 45 minutes (at a
distance of 10 cm) with a 450 watt, medium pressure, mercury vapor
lamp (ACE glass). The samples were maintained at 4.degree. C.
during the irradiation and short wavelength UV light was blocked by
a suitable filter (the top of a plastic petri dish). The extent of
crosslinking between the mRNA and tRNA (typically .about.20% of the
ribosomes contain crosslinked, tRNA-mRNA fusions) was determined by
polyacrylamide gel electrophoresis.
Example 2
[0037] Synthesis of a Bifunctional tRNA and its Use in Generating
Encoded Peptide Libraries
[0038] PHE-N-tRNA
[0039] A method to produce PHE-N-tRNA involves replacement of the
3'-terminal A of a tRNA with 3'-amino-3'-deoxyadenosine and
charging of the 3'-amino-3'-deoxyadenosine substituted tRNA with
phenylalanine (Fraser, T. H. and Rich, A., Proc. Nat. Acad. Sci.
USA 70:2671 (1972)). Using this procedure with yeast tRNA.sup.phe
generates PHE-N-tRNA, a tRNA analogue that can act as an acceptor
substrate but not a donor substrate during protein synthesis and
that can be crosslinked to mRNA. First, the 3'-terminal A, of yeast
tRNA.sup.phe is removed (resulting in tRNA(-A)) and then
3'-amino-3'-deoxyadenosine monophosphate is attached to the end of
tRNA(-A) by the action of tRNA nucleotidyl transferase. This
3'-amino-3'-deoxyadenosine substituted tRNA is then charged with
phenylalanine and S100.
[0040] Strategy for Synthesis of a Bifunctional tRNA
(PURO-tRNA.sup.phe)
[0041] A method to produce PURO-tRNA (a tRNA with its 3' terminal A
residue replaced by puromycin) involves replacement of the
3'-terminal C and A of a tRNA with pCpPuromycin (puromycin that has
been extended at its 5' hydroxyl by a 3', 5-cytidine diphosphate).
The 3'-terminal C and A of yeast tRNAphe are removed (resulting in
tRNA(-CA)) and then pCpPuromycin is ligated to the end of the
truncated tRNA to seamlessly fuse the two molecules. Using this
procedure with yeast tRNA.sup.phe generates PURO-tRNA.sup.phe, a
tRNA analogue that can act as an acceptor substrate but not a donor
substrate during protein synthesis and that can be crosslinked to
mRNA.
[0042] Removal of the 3'-Terminal A and Removal of the 3'-Terminal
C and A of Yeast tRNA.sup.phe
[0043] Yeast tRNA.sup.phe (300 .mu.M) and venom phosphodiesterase
(Crotalus atrox) were incubated in 40 mM glycine-NaOH pH 8.7, and
10 mM magnesium acetate for 10 minutes at 37.degree. C. Typically,
0.1 to 10 mg of venom per ml have been used in the literature;
however, it was useful to titrate the venom to identify the
concentration that produced quantitative removal of the C and A.
After incubation, the reaction was brought to 0.3 M sodium acetate,
5 mM EDTA, 0.5% SDS, phenol extracted three times, chloroform
extracted two times, 2.5 volumes of ethanol were added and the tRNA
was precipitated. If tRNA(-A) was the desired product the tRNA was
incubated with tRNA nucleotidyl transferase and CTP in 50 mM
Glycine pH 9.2, 30 mM KCl 12.5 mM MgCl.sub.2, 2.5 mg/ml reduced
glutathione and 0.375 mg/ml BSA for 30 minutes. The reaction was
phenol extracted and tRNA(-A) resuspended in water. If the desired
product was tRNA(-CA) the tRNA was purified by polyacrylamide gel
electrophoresis and was resuspended in water and then adjusted to a
concentration of 10 .mu.M in 70 mM Tris-HCl pH 8.5, 30 mM MgCl2,
and 1.6 mM DTT.
[0044] Synthesis of 3'-amino-3'-deoxyadenosine and Attachment to
tRNA(-A).
[0045] 3'-amino-3'-deoxyadenosine was made by the method of Gerber
and Lechevalier (J. Org. Chem. 27:1731(1962)) and can be
phosphorylated according to Fraser and Rich (Proc. Nat. Acad. Sci.
USA 70:2671 (1973)). 3'-amino-3'-deoxyadenosine triphosphate,
tRNA(-A), and tRNA nucleotidyl transferase were used to generate
yeast N-tRNA (yeast tRNA.sup.phe whose 3'-terminal adenosine has
been substituted with 3'-amino-3'-deoxyadenosin- e) by incubation
at 37.degree. C. for 30 minutes in 50 mM Glycine pH 9.2, 30 mM KCl,
12.5 mM MgCl.sub.2, 2.5 mg/ml reduced glutathione and 0.375 mg/ml
BSA. PHE-N-tRNA was generated by incubating N-tRNA, phenylalanine
and S100 at 37.degree. C. for 30 minutes in 30 mM Tris pH 7.5, 25
mM KCl, 15 mM MgCl.sub.2, and 4 mM DTT. PHE-N-tRNA was purified by
HPLC.
[0046] Synthesis of pCpPuromycin and Attachment to the Truncated
tRNA
[0047] Several methods have been used to make CpPuromycin and
CpPuromycin analogues (e.g. Harris, R. J. et al., Can. J. Bioch.,
50:918-926(1972); Nyilas, A. et al., Bioorganic and Medicinal
Chemistry Letters, 3(6):1371-1374(1993); Green and Noller, Science,
1998). CpPuromycin prepared by the method of Green and Noller
(1998) is used. CpPuromycin (10 .mu.M) is phosphorylated by
incubation at 37.degree. C. for 30 minutes with T4 polynucleotide
kinase in 70 mM Tris-HCl pH 7.5, 10 mM MgCl2, 5 mM DTT, and 30
.mu.M ATP. (It is important to keep the concentration of ATP at 2-5
fold over the concentration of CpPuromycin as the subsequent
ligation is sensitive to excessive ATP). The kinase is inactivated
by heating the reaction to 65 .degree. C. for 20 minutes. The
solution is brought to 20 .mu.g/ml BSA, an equal volume of 10 .mu.m
tRNA(-CA) in (70 mM Tris-HCl pH 8.5, 50 mM MgCl2, and 1.6 mM DTT)
is added, and the solution is incubated for 60 minutes at
37.degree. C. with T4 RNA ligase. Ligation of pCpPuromycin to
tRNA(-CA) produces the desired PURO-tRNA.sup.phe product which is
purified by polyacrylamide gel electrophoresis.
Example 3
[0048] Using the bifunctional tRNA to generate an encoded
polypeptide library
[0049] An in vitro translation mixture is combined with a complex
pool of mRNA sequences, and an appropriate amount of a bifunctional
tRNA (PHE-N-tRNA or PURO-tRNA.sup.phe). The translation mixture
contains all of the factors required for in vitro translation
(e.g., initiation factors, the tRNAs, elongation factors, amino
acids, etc.) except for mRNA. Translation mixtures are purchased
(Promega) or they can be made from crude cellular extracts
(extracts from several organisms are used and protocols for making
them are readily available in the literature). The appropriate
concentration of a bifuntional tRNA is added to the translation
mixture. Translation is initiated by the addition of the complex
pool of mRNA sequences. All of the members of the pool of mRNA
sequences have a constant sequence at their 5' end that permits
them to be translated by the ribosome, an internal, randomized,
polypeptide-coding segment that is devoid of stop codons, and a
UUU- and UUC-rich 3' coding segment that recruits a bifunctional
tRNA after translation of the randomized segment is completed. The
bifunctional tRNA concentration in the translation mixture is
carefully adjusted so that during translation, the UUU and UUC
codons that are contained within the randomized coding segment are
decoded by normal phenylalanine tRNA that are present in the
translation mixture. When the UUU- and UUC-rich 3' coding segment
(of each translating mRNA) is reached, there is a high probability
that a bifunctional tRNA will eventually be selected to decode a
UUU or UUC codon. Decoding by a bifunctional tRNA stalls
translation of the mRNA because a bifunctional tRNA can accept the
growing polypeptide chain but cannot transfer the polypeptide chain
to another acylated tRNA. This generates polypeptide-bifunctional
tRNA fusions that are bound to ribosomes. The translation mixture
is concurrently or subsequently irradiated at a distance of 10 cm
with a 450 watt, medium pressure, mercury vapor lamp (ACE glass;
short wavelength UV light is blocked by a suitable filter). This
activates the Y base of the bifunctional tRNA and causes the base
(and therefore the polypeptide-bifunctional tRNA fusion) to become
crosslinked to the mRNA that encodes the polypeptide. After
irradiation, disruption of the ribosomes with EDTA releases the
encoded polypeptide library so that it can be purified and
used.
[0050] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
spirit and scope of the invention as defined by the appended
claims. Those skilled in the art will recognize or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
specifically herein. Such equivalents are intended to be
encompassed in the scope of the claims.
Sequence CWU 1
1
4 1 36 DNA Artificial Sequence Primer for E. Coli Chromosomal DNA 1
taatacgact cactataggc tacgtagctc agttgg 36 2 20 DNA Artificial
Sequence Primer for E. Coli Chromosonal DNA 2 tggtggctac gacgggattc
20 3 34 DNA Artificial Sequence + Strand Primer 3 taatacgact
cactataggt aaagtgtcat tagc 34 4 22 DNA Artificial Sequence - Strand
Primer 4 ctttatcttc agaagaaaaa cc 22
* * * * *