U.S. patent application number 16/530742 was filed with the patent office on 2019-12-12 for incorporation of unnatural nucleotides and methods thereof.
The applicant listed for this patent is THE SCRIPPS RESEARCH INSTITUTE, SYNTHORX, INC.. Invention is credited to Hans AERNI, Carolina CAFFARO, Vivian T. DIEN, Aaron W. FELDMAN, Emil C. FISCHER, Jerod PTACIN, Floyd E. ROMESBERG, Yorke ZHANG.
Application Number | 20190376054 16/530742 |
Document ID | / |
Family ID | 65002471 |
Filed Date | 2019-12-12 |
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United States Patent
Application |
20190376054 |
Kind Code |
A1 |
PTACIN; Jerod ; et
al. |
December 12, 2019 |
INCORPORATION OF UNNATURAL NUCLEOTIDES AND METHODS THEREOF
Abstract
Disclosed herein are methods, compositions and kits for the
synthesis of proteins which comprises unnatural amino acids that
utilize a mutant tRNA.
Inventors: |
PTACIN; Jerod; (La Jolla,
CA) ; CAFFARO; Carolina; (La Jolla, CA) ;
AERNI; Hans; (La Jolla, CA) ; ZHANG; Yorke;
(La Jolla, CA) ; FISCHER; Emil C.; (La Jolla,
CA) ; FELDMAN; Aaron W.; (La Jolla, CA) ;
DIEN; Vivian T.; (La Jolla, CA) ; ROMESBERG; Floyd
E.; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SYNTHORX, INC.
THE SCRIPPS RESEARCH INSTITUTE |
La Jolla
La Jolla |
CA
CA |
US
US |
|
|
Family ID: |
65002471 |
Appl. No.: |
16/530742 |
Filed: |
August 2, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2018/041509 |
Jul 10, 2018 |
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16530742 |
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62531325 |
Jul 11, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12P 21/02 20130101;
C12N 15/113 20130101; C12N 15/11 20130101; C12N 9/93 20130101; C12N
15/90 20130101; C12Y 601/01026 20130101 |
International
Class: |
C12N 9/00 20060101
C12N009/00; C12P 21/02 20060101 C12P021/02 |
Claims
1. A cell comprising: (a) a nucleoside triphosphate transporter
from Phaeodactylum tricornutum; (b) a tRNA from Methanosarcina
mazei; and (c) a pyrrolysine aminoacyl tRNA synthetase from
Methanosarcina barkeri.
2. The cell of claim 1, wherein the cell comprises an
oligonucleotide encoding the tRNA.
3. The cell of claim 1, wherein the cell further comprises an
oligonucleotide encoding the pyrrolysine aminoacyl tRNA
synthetase.
4. The cell of claim 1, wherein the cell further comprises a single
oligonucleotide encoding the tRNA and the pyrrolysine aminoacyl
tRNA synthetase.
5. The cell of claim 4, wherein the cell further comprises an
oligonucleotide comprising one or more unnatural nucleobases.
6. The cell of claim 5, wherein the one or more unnatural
nucleobases comprises a structure selected from and
##STR00007##
7. The cell of claim 5, wherein the oligonucleotide comprises a
first unnatural nucleobase having a structure ##STR00008## and a
second unnatural nucleobase having a structure ##STR00009## and
wherein the first unnatural nucleobase and the second unnatural
nucleobase form a base pair in the oligonucleotide.
8. The cell of claim 1, wherein the tRNA from Methanosarcina mazei
comprises one or more anti-codons selected from GYT and GYC,
wherein Y is an unnatural nucleobase.
9. The cell of claim 8, wherein Y is selected from ##STR00010##
10. The cell of claim 1, wherein the cell is E. coli.
11. A method of producing a protein in a cell, wherein the protein
comprises an unnatural amino acid, the method comprising: (a)
providing a cell comprising: (i) a nucleoside triphosphate
transporter from Phaeodactylum tricornutum that transports a
nucleotide into the cell; (ii) a tRNA from Methanosarcina mazei;
(iii) a pyrrolysine aminoacyl tRNA synthetase from Methanosarcina
barkeri; and (iv) an unnatural amino acid; and (b) synthesizing a
protein in the cell by a process of: (i) transporting a nucleotide
into the cell by the nucleoside triphosphate transporter; (ii)
incorporating the nucleotide into a double-stranded
oligonucleotide; (iii) translating the protein from the
double-stranded oligonucleotide utilizing the tRNA, the tRNA
synthetase, and the unnatural amino acid.
12. The method of claim 11, further comprising expressing the tRNA
from a plasmid in the cell.
13. The method of claim 11, further comprising expressing the
pyrrolysine aminoacyl tRNA synthetase from a plasmid in the
cell.
14. The method of claim 11, further comprising expressing the tRNA
and the pyrrolysine aminoacyl tRNA synthetase from a single plasmid
in the cell.
15. The method of claim 11, wherein the nucleotide comprises an
unnatural nucleobase.
16. The method of claim 15, wherein the unnatural nucleobase is
##STR00011##
17. The method of claim 11, further comprising: (a) providing a
first nucleotide comprising a nucleobase having a structure
##STR00012## and a second nucleotide comprising a nucleobase having
a structure ##STR00013## and (b) forming an unnatural base pair in
the cell with the first nucleotide and the second nucleotide in the
double-stranded oligonucleotide.
18. The method of claim 11, wherein the tRNA from Methanosarcina
mazei comprises one or more anti-codons selected from GYT and GYC,
wherein Y comprises an unnatural nucleobase.
19. The method of claim 18, wherein Y is selected from
##STR00014##
20. The method of claim 11, wherein the cell is E. coli.
Description
CROSS-REFERENCE
[0001] This application is a continuation of International
Application No. PCT/US2018/041509, filed on Jul. 10, 2018, which
claims the benefit of U.S. provisional patent application No.
62/531,325 filed on Jul. 11, 2017, both of which are incorporated
herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] Oligonucleotides and their applications have revolutionized
biotechnology. However, the oligonucleotides including both DNA and
RNA each includes only the four natural nucleotides of adenosine
(A), guanosine (G), cytosine (C), thymine (T) for DNA, and the four
natural nucleotides of adenosine (A), guanosine (G), cytosine (C),
and uridine (U) for RNA, and which significantly restricts the
potential functions and applications of the oligonucleotides.
[0003] The ability to sequence-specifically synthesize/amplify
oligonucleotides (DNA or RNA) with polymerases, for example by PCR
or isothermal amplification systems (e.g., transcription with T7
RNA polymerase), has revolutionized biotechnology. In addition to
all of the potential applications in nanotechnology, this has
enabled a diverse range of new technologies such as the in vitro
evolution via SELEX (Systematic Evolution of Ligands by Exponential
Enrichment) of RNA and DNA aptamers and enzymes. See, for example,
Oliphant A R, Brandl C J & Struhl K (1989), Defining the
sequence specificity of DNA-binding proteins by selecting binding
sites from random-sequence oligonucleotides: analysis of yeast GCN4
proteins, Mol. Cell Biol., 9:2944-2949; Tuerk C & Gold L
(1990), Systematic evolution of ligands by exponential enrichment:
RNA ligands to bacteriophage T4 DNA polymerase, Science,
249:505-510; Ellington A D & Szostak J W (1990), In vitro
selection of RNA molecules that bind specific ligands, Nature,
346:818-822.
[0004] In some aspects, these applications are restricted by the
limited chemical/physical diversity present in the natural genetic
alphabet (the four natural nucleotides A, C, G, and T in DNA, and
the four natural nucleotides A, C, G, and U in RNA). Disclosed
herein is an additional method of generating nucleic acids that
contains an expanded genetic alphabet.
SUMMARY OF THE INVENTION
[0005] Disclosed herein, in certain embodiments, are methods of
producing a protein containing an unnatural amino acid, the method
comprising: preparing a mutant tRNA wherein the mutant tRNA
comprises a mutant anticodon sequence selected from Table 1 or 2;
preparing a mutant mRNA wherein the mutant mRNA comprises a mutant
codon sequence selected from Table 1 or 2; and synthesizing the
protein containing an unnatural amino acid utilizing the mutant
tRNA and the mutant mRNA. In some instances, the protein is
synthesized in a cell-free translation system. In some instances,
the protein is synthesized in a cell (semi-synthetic organism or
SSO). In some instances, the semi-synthetic organism comprises a
microorganism. In some instances, the semi-synthetic organism
comprises a bacterium. In some instances, the semi-synthetic
organism comprises an Escherichia coli. In some instances, the
mutant anticodon of the mutant tRNA pairs with a mutant codon
selected from Tables 1-3. In some instances, the unnatural amino
acid comprises at least one unnatural nucleotide. In some
instances, the unnatural nucleotide comprises an unnatural
nucleobase. In some instances, the unnatural base of the unnatural
nucleotide is selected from the group consisting of
2-aminoadenin-9-yl, 2-aminoadenine, 2-F-adenine, 2-thiouracil,
2-thio-thymine, 2-thiocytosine, 2-propyl and alkyl derivatives of
adenine and guanine, 2-amino-adenine, 2-amino-propyl-adenine,
2-aminopyridine, 2-pyridone, 2'-deoxyuridine,
2-amino-2'-deoxyadenosine 3-deazaguanine, 3-deazaadenine,
4-thio-uracil, 4-thio-thymine, uracil-5-yl, hypoxanthin-9-yl (I),
5-methyl-cytosine, 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 5-bromo, and 5-trifiuoromethyl uracils and cytosines;
5-halouracil, 5-halocytosine, 5-propynyl-uracil, 5-propynyl
cytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted
pyrimidines, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil,
5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine
arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil,
5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil,
5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and
5-iodouracil, 6-alkyl derivatives of adenine and guanine,
6-azapyrimidines, 6-azo-uracil, 6-azo cytosine, azacytosine,
6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine,
7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine,
7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and
guanines; N4-ethylcytosine, N-2 substituted purines, N-6
substituted purines, 0-6 substituted purines, those that increase
the stability of duplex formation, universal nucleic acids,
hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded
nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines,
phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps,
phenoxazine cytidine
(9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido [3',2':4,5]pyrrolo [2,3-d]pyrimidin-2-one),
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyl
aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methythio-N6-isopentenyladenine,
uracil-5oxyacetic acid, wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxacetic acid methylester,
uracil-5-oxacetic acid, 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine and those in which the purine or pyrimidine base
is replaced with a heterocycle. In some instances, the unnatural
nucleotide is selected from the group consisting of (only
nucleobase portion shown, ribose and phosphate backbone omitted for
clarity)
##STR00001##
[0006] In some instances, the unnatural nucleotide is selected from
the group consisting of (only nucleobase portion shown, ribose and
phosphate backbone omitted for clarity)
##STR00002## ##STR00003##
[0007] In some instances, the unnatural nucleotide further
comprises an unnatural sugar moiety. In some instances, the
unnatural sugar moiety of the unnatural nucleotide is selected from
the group consisting of a modification at the 2' position: OH;
substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,
SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2F; O-alkyl,
S-alkyl, N-alkyl; O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl,
S-alkynyl, N-alkynyl; O-alkyl-O-alkyl, 2'-F, 2'-OCH.sub.3,
2'--O(CH.sub.2).sub.2OCH.sub.3 wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1-C.sub.10,
alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl,
--O[(CH2).sub.nO].sub.mCH.sub.3, --O(CH.sub.2).sub.nOCH.sub.3,
--O(CH.sub.2).sub.nNH.sub.2, --O(CH.sub.2).sub.nCH.sub.3,
--O(CH.sub.2).sub.n--ONH.sub.2, and
--O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and
m are from 1 to about 10; and/or a modification at the 5' position:
5'-vinyl, 5'-methyl (R or S), a modification at the 4' position,
4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
any combination thereof. In some instances, the mutant anticodon or
the mutant codon further comprises an unnatural backbone. In some
instances, the mutant anticodon and the mutant codon further
comprises an unnatural backbone. In some instances, the unnatural
nucleotides are recognized by a DNA polymerase, an RNA polymerase,
or a reverse transcriptase. In some instances, an unnatural
nucleotide is incorporated by the RNA polymerase into the mRNA
during transcription to generate a mutant mRNA containing a mutant
codon. In some instances, an unnatural nucleotide is incorporated
by the RNA polymerase into the tRNA during transcription to
generate a mutant tRNA containing a mutant anticodon. In some
instances, an unnatural nucleotide is incorporated by the RNA
polymerase into the mRNA during transcription to generate a mutant
mRNA. In some instances, an unnatural nucleotide is incorporated by
the RNA polymerase into the tRNA during transcription to generate a
mutant tRNA. In some instances, the mutant tRNA is charged with an
unnatural amino acid residue. In some instances, a protein
containing an unnatural amino acid is generated during translation
utilizing the mutant tRNA and the mutant mRNA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Various aspects of the disclosure are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present disclosure will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the disclosure
are utilized, and the accompanying drawings of which:
[0009] FIG. 1A illustrates the chemical structure of the dNaM-dTPT3
UBP and a natural dA-dT base pair.
[0010] FIG. 1B illustrates the gene cassette used to express
sfGFP(AXC).sup.151 and tRNA(GYT).sup.Ser. P.sub.T7 and T.sub.T7
denote the T7 RNAP promoter and terminator, respectively. In
controls where sfGFP is expressed in the absence of serT, the
sequence following the sfGFP T7 terminator is absent.
[0011] FIG. 1C illustrates a graph of fluorescence of cells
expressing sfGFP and tRNA.sup.Ser with the indicated position
151-codon and anticodon, respectively. Minus sign denotes the
absence of serT in the expression cassette. t=0 corresponds to the
addition of IPTG to induce expression of T7 RNAP and tRNA.sup.Ser
(if present); aTc was added at t=0.5 h to induce expression of
sfGFP. AGT, natural Ser codon; TAG, amber stop codon; CTA amber
suppressor anticodon. Data shown as mean.+-.s.d., n=4 cultures,
each propagated from an individual colony.
[0012] FIG. 1D illustrates a graph of growth of cells expressing
sfGFP and tRNA.sup.Ser with the indicated position 151-codon and
anticodon, respectively. Minus sign denotes the absence of serT in
the expression cassette. t=0 corresponds to the addition of IPTG to
induce expression of T7 RNAP and tRNA.sup.Ser (if present); aTc was
added at t=0.5 h to induce expression of sfGFP. AGT, natural Ser
codon; TAG, amber stop codon; CTA amber suppressor anticodon. Data
shown as mean.+-.s.d., n=4 cultures, each propagated from an
individual colony.
[0013] FIG. 1E illustrates a Western blot of lysates (normalized by
OD.sub.600) from cells collected at the last time point shown in
FIG. 1C and FIG. 1D, probed with an .alpha.-GFP antibody
(N-terminal epitope).
[0014] FIG. 1F illustrates a graph of the relative abundance of the
amino acids (indicated by their single letter codes in the figure
legend) detected at position 151 of sfGFP purified from cells
expressing sfGFP(AGT).sup.151 or sfGFP(AXC).sup.151 and
tRNA.sup.Ser(GYT), as determined by LC-MS/MS and precursor ion
intensity based quantitation (amino acids detected at <0.1% (on
average, for both codons) are not shown; see Methods for details
and Table 4 for a complete list of amino acids detected). Data
shown as mean with individual data points, n=4 purified sfGFP
samples, each from a culture propagated from an individual colony
and collected at the last time point shown in FIG. 1C and FIG.
1D.
[0015] FIG. 2A illustrates a graph of fluorescence of cells
expressing sfGFP with the indicated position 151-codon, in the
presence (+) or absence (-) of a tRNA.sup.Pyl with a cognate
anticodon, PylRS, or 20 mM PrK
(N.sup.6-[(2-propynyloxy)carbonyl]-L-lysine) in the media.
Fluorescence was determined at the last time point shown in FIG.
2B. Asterisk denotes the absence of tRNA.sup.Pyl in cells
expressing sfGFP(TAC).sup.151. TAC, natural Tyr codon; TAG, amber
stop codon; n.d., not determined. Data shown as mean with
individual data points, each propagated from an individual
colony.
[0016] FIG. 2B illustrates a timecourse analysis of a subset of the
data shown in FIG. 2A. Plus and minus signs denote the presence or
absence, respectively, of 20 mM PrK in the media. t=0 corresponds
to the addition of IPTG to induce expression of PylRS, T7 RNAP, and
tRNA.sup.Pyl; aTc was added at t=1 h to induce expression of sfGFP.
Data shown as mean.+-.s.d., n=4 cultures, each propagated from an
individual colony.
[0017] FIG. 2C illustrates Western blots of sfGFP purified from
cells expressing sfGFP and tRNA.sup.Pyl with the indicated
position-151 codon and anticodon, respectively, with or without
click conjugation of TAMRA and/or addition of 20 mM PrK to the
media. tRNA.sup.Pyl is absent (-) in cells expressing
sfGFP(TAC).sup.151. sfGFP was purified from cultures collected at
the last time point shown in FIG. 2B. Western blots were probed
with an .alpha.-GFP antibody and imaged to detect both sfGFP and
the conjugated TAMRA.
[0018] FIG. 2D illustrates a graph of the relative abundance of
amino acids (indicated by their single letter codes in the figure
legend) at position 151 of sfGFP purified from cells (collected at
the last time point shown in FIG. 2B) expressing sfGFP(TAC).sup.151
or sfGFP and tRNA.sup.Pyl with the indicated position-151 codon and
a cognate anticodon, respectively, as determined by LC-MS/MS and
precursor ion intensity based quantitation (amino acids detected at
<0.1% (on average, for all codons) are not shown; see Methods
for details and Table 4 for a complete list of amino acids
detected). Data shown as mean with individual data points, n=4
purified sfGFP samples, each from a culture propagated from an
individual colony.
[0019] FIG. 3A illustrates a graph of fluorescence of cells
expressing sfGFP(TAC).sup.151 or sfGFP and tRNA.sup.pAzF with the
indicated position-151 codon and a cognate anticodon, respectively,
in the presence (+) or absence (-) of 5 mMpAzF in the media. t=0
corresponds to the addition of IPTG to induce expression ofpAzFRS,
T7 RNAP, and tRNA.sup.pAZF; aTc was added at t=0.5 h to induce
expression of sfGFP. TAC, natural Tyr codon; TAG, amber stop codon.
Data shown as mean.+-.s.d., n=4 cultures, each propagated from an
individual colony. The fluorescence observed with
sfGFP(AXC).sup.151 in the absence ofpAzF is attributed to charging
of tRNA.sup.pAzF(GYT) with a natural amino acid (likely Tyr).
[0020] FIG. 3B illustrates a Western blot of sfGFP purified from
cells expressing sfGFP and tRNA.sup.pAzF with the indicated
position-151 codon and anticodon, respectively, with or without
click conjugation of TAMRA and/or addition of 5 mM pAzF to the
media. Where indicated, the minus sign denotes the absence of
tRNA.sup.pAzF in cells expressing sfGFP(TAC).sup.151. sfGFP was
purified from cultures collected at the last time point shown in
FIG. 3A. Western blots were probed with an .alpha.-GFP antibody and
imaged to detect both sfGFP and the conjugated TAMRA.
[0021] FIG. 4 illustrates fluorescence of cells expressing sfGFP
with various codons at position 151. Cells carrying a sfGFP plasmid
with the indicated position-151 codons were grown to an
OD.sub.600.about.0.5 and induced with IPTG and aTc. Fluorescence
measurements were taken after 3 h of induction. Data shown as mean
with individual data points, n=3 cultures split from a single
colony and grown in parallel.
[0022] FIG. 5A illustrates decoding of the AXC codon with natural
near-cognate anticodons, with a graph of fluorescence of cells
expressing sfGFP(AXC).sup.151 with or without tRNA.sup.Ser with the
indicated anticodon. Cells were induced as described in FIG. 1C and
FIG. 1D and fluorescence measurements correspond to the last time
point shown in FIG. 1C. Values for the GYT anticodon and in the
absence of tRNA.sup.Ser (-tRNA) correspond to the same values in
FIG. 1c,d. Data shown as mean.+-.s.d., n=4 cultures, each
propagated from an individual colony.
[0023] FIG. 5B illustrates decoding of the AXC codon with natural
near-cognate anticodons, with a graph of growth of cells expressing
sfGFP(AXC).sup.151 with or without tRNA.sup.Ser with the indicated
anticodon. Cells were induced as described in FIG. 1C and FIG. 1D
and fluorescence measurements correspond to the last time point
shown in FIG. 1C. Values for the GYT anticodon and in the absence
of tRNA.sup.Ser (-tRNA) correspond to the same values in FIG. 1C
and FIG. 1D. Data shown as mean.+-.s.d., n=4 cultures, each
propagated from an individual colony.
[0024] FIG. 6A illustrates Western blots and growth of cells
decoding AXC and GXC codons with tRNA.sup.Pyl. Western blot of
lysates (normalized by OD.sub.600) from cells expressing sfGFP with
the indicated position 151-codon, in the presence (+) or absence
(-) of a tRNA.sup.Pyl with a cognate anticodon, PylRS, or 20 mM PrK
in the media. Blots were probed with an .alpha.-GFP antibody
(N-terminal epitope). Cells were induced and collected at an
equivalent time point as described in FIG. 2B.
[0025] FIG. 6B illustrates growth of cultures analyzed in FIG. 6A.
The fold change in OD.sub.600 between induction of sfGFP (t=1 h)
and the final time point is greatest when all components necessary
for aminoacylating tRNA.sup.Pyl are present. Variations in the
absolute value of OD.sub.600 are due to small variations in cell
density at the start of T7 RNAP (and if present tRNA.sup.Pyl)
induction (t=0). Data shown as mean.+-.s.d., n=4 cultures, each
propagated from an individual colony.
[0026] FIG. 7A illustrates decoding AXC and GXC codons with
tRNA.sup.Pyl and cell growth as a function of added unnatural
ribotriphosphates. Fluorescence of purified sfGFP (lower panel)
from cells expressing sfGFP and tRNA.sup.Pyl with the position
151-codon/anticodon indicated, in the presence (+) or absence (-)
of each unnatural ribotriphosphate in the media, and with or
without 20 mM PrK. Cells were induced as described in FIG. 2B and
fluorescence measurements were taken at the end of induction
(.about.3.5 h), prior to collecting the cells and purifying the
sfGFP protein for click conjugation of TAMRA and western
blotting.
[0027] FIG. 7B illustrates a gel of decoding AXC and GXC codons
with tRNA.sup.Pyl as a function of added unnatural
ribotriphosphates. Western blots were probed with an .alpha.-GFP
antibody and imaged to detect both sfGFP and the conjugated TAMRA;
all lanes correspond to sfGFP purified from cells grown with added
PrK. Data shown as mean with individual data points, n=3 cultures,
each propagated from an individual colony; n.d., not
determined.
[0028] FIG. 7C, illustrates graphs of fluorescence and growth of
cells expressing sfGFP(TAC).sup.151 in the presence (+) or absence
(-) of both unnatural deoxyribotriphosphates and each unnatural
ribotriphosphate. t=0 corresponds to the addition of IPTG to induce
expression of T7 RNAP; aTc was added at t=1 h to induce expression
of sfGFP. Data shown as mean.+-.s.d., n=3 cultures, each propagated
from an individual colony. At the concentrations used (see
Methods), dNaMTP and dTPT3TP do not inhibit cell growth, whereas
both unnatural ribotriphosphates, particularly TPT3TP, show some
inhibition of growth.
[0029] FIG. 7D illustrates a graph of cell growth corresponding to
the cultures with added PrK (20 mM) whose fluorescence is shown in
FIG. 2B. Cells expressing sfGFP with natural codons were grown
without any unnatural triphosphates, whereas cells expressing sfGFP
with unnatural codons were grown with both unnatural deoxy- and
ribotriphosphates. Data shown as mean.+-.s.d., n=4 cultures, each
propagated from an individual colony.
[0030] FIG. 8A illustrates a gel of decoding AXC and GXC codons
with tRNA.sup.Pyl as a function of PrK concentration in the media.
Western blots of sfGFP purified from cells expressing sfGFP and
tRNA.sup.Pyl with the indicated position-151 codon/anticodon, with
click conjugation of TAMRA and the addition of PrK to the media at
the indicated concentrations. sfGFP was induced and purified from
cells collected as described in FIG. 2B. Western blots were probed
with an .alpha.-GFP antibody and imaged to detect both sfGFP and
the conjugated TAMRA.
[0031] FIG. 8B illustrates a graph of decoding AXC and GXC codons
with tRNA.sup.Pyl as a function of PrK concentration in the media.
Fluorescence of cells (measured at the last time point shown in c)
expressing sfGFP and tRNA.sup.Pyl with the indicated position-151
codon and anticodon, respectively, as a function of PrK
concentration in the media. Fluorescence values for 0 and 20 mM PrK
are the same as the (-) and (+) PrK values, respectively, shown in
FIG. 2B. Data shown as mean.+-.s.d., n=4 cultures, each propagated
from an individual colony.
[0032] FIG. 8C illustrates a timecourse analysis of fluorescence.
For clarity, only one representative culture is shown for each
codon/anticodon pair and PrK concentration. Without being bound by
theory, we attribute the low level of sfGFP produced in the absence
of PrK to decoding by endogenous tRNAs and loss of UBP retention in
sfGFP (Table 5). However, the relative amount of sfGFP that
contains PrK (FIG. 8A) and absolute amount of sfGFP expressed (FIG.
8B and FIG. 8C) increased in a dose-dependent manner with
increasing PrK in the media, ultimately resulting in nearly full
incorporation of PrK, suggesting that endogenous read-through of
the AXC and GXC codons can be efficiently suppressed with
sufficient concentrations of charged PrK-tRNA.sup.Pyl(GYT) or
PrK-tRNA.sup.Pyl(GYC).
[0033] FIG. 8D illustrates a timecourse analysis of cell growth at
various concentrations of PrK for the experiment shown in FIG.
8C.
[0034] FIG. 9 illustrates cell growth of the cultures whose
fluorescence is shown in FIG. 3A. Data shown as mean.+-.s.d., n=4
cultures, each propagated from an individual colony.
[0035] Table 4|Relative abundance of amino acids at position 151 in
sfGFP for experiments described in FIG. 1F and FIG. 2D. sfGFP
purified from cells expressing sfGFP with or without tRNAs with the
indicated position-151 codon and anticodon, respectively, were
analyzed by LC-MS/MS. The extracted MS 1 ion intensities for the
reporter peptides LEYNFNSHNVX.sup.151ITADK (X=PrK or any identified
natural amino acid except K or R) and LEYNFNSHNVX.sup.151 (if X=K
or R) are expressed as a percentage of the sum of ion intensities
for all observable reporter peptides. The table of values
corresponds to the mean relative abundances and 95% CIs of all
amino acids detected at position 151 of sfGFP, n=4 purified sfGFP
samples, each from a culture propagated from an individual colony.
Values <0.1% (on average, for the codons indicated in the
respective figures) are excluded from the data presented in FIG. 1F
and FIG. 2D.
[0036] Table 5|UBP retention. Retention of the UBP(s) in plasmids
with the indicated position-151 codons of sfGFP and anticodons of
the indicated tRNAs were determined for a time point prior to sfGFP
induction and at the end of induction, as described in Methods. The
reported values are the mean UBP retention over the course of the
induction (calculated from the retentions at these two time
points)+95% CI, n=4 cultures, each propagated from an individual
colony, except for values indicated with an asterisk, for which
n=3. n/a, not applicable (because the relevant sequence is natural
or absent). All plasmids were isolated from cultures grown in the
presence of 20 mM PrK or 5 mMpAzF (except for Ser decoding
experiments). SerRS indicates charging with the endogenous E. coli
synthetase. Minus sign denotes the absence of PylRS in cells with
tRNA.sup.Pyl or the absence of an ectopically expressed tRNA.
Retentions in rows indicated with .sctn. correspond to cultures
from which sfGFP was also purified and analyzed by LC-MS/MS and/or
western blot of TAMRA-conjugated sfGFP (see FIG. 1F (Ser), FIG. 2D
(PrK), and FIG. 3B (pAzF)); rows with an asterisk correspond to the
cultures analyzed in FIGS. 7A-D. Despite the fact that all four
unnatural triphosphates enter the cell through the same transporter
and thus competitively inhibit one another's import, no differences
in UBP retention were observed with the presence (+) or absence (-)
of NaMTP and/or TPT3TP in the media. These data, and the
requirement of both unnatural ribotriphosphates for high levels of
sfGFP expression with high-fidelity PrK incorporation (FIGS. 7A-D),
collectively demonstrate that the expression level of the PtNTT2
transporter in YZ3 imports the requisite levels of unnatural
triphosphates necessary to sustain UBP replication and
transcription.
[0037] Table 6|Yields of sfGFP protein expressed in Ser, Prk and
pAzF incorporation experiments. Yields were calculated from the
total amount of protein purified and the volume of culture used for
purification (see Methods). Data are mean.+-.s.d. (n=4 sfGFP
samples, each purified from a culture propagated from an individual
colony) and were determined from the same cultures analyzed in FIG.
1F (for SerRS) and FIG. 2D (for PylRS), as well as the cultures
corresponding to the (+) pAzF samples in FIG. 3A (forpAzFRS).
Yields of purified sfGFP are comparable to the mean total
fluorescence (not normalized to OD.sub.600) of the cultures from
which they were purified. Fluorescence values correspond to the
time point at which cells were collected for sfGFP purification;
see FIG. 1C (Ser), FIG. 2B (PrK), and FIG. 3A (pAzF).
DETAILED DESCRIPTION OF THE INVENTION
Certain Terminology
[0038] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of skill in the art to which the claimed subject matter belongs. It
is to be understood that the foregoing general description and the
following detailed description are exemplary and explanatory only
and are not restrictive of any subject matter claimed. In this
application, the use of the singular includes the plural unless
specifically stated otherwise. It must be noted that, as used in
the specification and the appended claims, the singular forms "a,"
"an" and "the" include plural referents unless the context clearly
dictates otherwise. In this application, the use of "or" means
"and/or" unless stated otherwise. Furthermore, use of the term
"including" as well as other forms, such as "include", "includes,"
and "included," is not limiting.
[0039] As used herein, ranges and amounts can be expressed as
"about" a particular value or range. About also includes the exact
amount. Hence "about 5 .mu.L" means "about 5 .mu.L" and also "5
.mu.L." Generally, the term "about" includes an amount that would
be expected to be within experimental error.
[0040] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
Overview
[0041] The information of life is encoded by a four letter genetic
alphabet, which is made possible by the selective formation of two
base pairs: (d)G-(d)C and (d)A-dT/U. A third, unnatural base pair
(UBP) formed between two synthetic nucleotides expands this system,
thereby increasing the potential for information storage, and has
profound academic and practical implications. Of the wide variety
of synthetic nucleotide analogs that have been reported, several
pair stably with one another within an otherwise natural DNA
duplex, but are not recognized by polymerases, and indicating that
the forces governing stable pairing in duplex DNA are not the same
as those governing polymerase-mediated replication. As a result,
different approaches have been taken to develop replicable UBPs,
for example, UBPs that are designed to interact via complementary
hydrogen bonding (H-bonding) patterns not employed by the natural
nucleotides. Although the natural base pairs form via H-bonding,
there is no reason to assume a priori that H-bonding is the only
force sufficient to underlie the storage (or retrieval) of genetic
information. For example, it has been demonstrated that the Klenow
fragment of E. coli DNA polymerase I (Kf) pairs dA with the
unnatural nucleotide dF, whose difluorotoluene nucleobase is a
shape mimic of thymine that is incapable of significant H-bonding.
This supports a "geometrical selection" mechanism of DNA
replication and suggests that forces other than H-bonding also
contribute to replication.
[0042] The development of UBPs that are replicated, transcribed,
and translated into protein in vitro provide insights into the
forces underlying the storage and retrieval of natural information,
and also enable wide ranging applications in chemical and synthetic
biology. However, the ultimate goal of many efforts to develop UBPs
is their in vivo use as the foundation of a semi-synthetic organism
(SSO)--an organism that stably stores and retrieves increased
(un-natural or synthetic, meaning man made) information. Moreover,
such an SSO has revolutionary practical applications, including for
human health. Most notably, an SSO revolutionizes the growing field
of protein therapeutics. However, compared to traditional small
molecule therapeutics, protein therapeutics are severely limited in
their molecular properties due to the finite chemical diversity
available with the twenty natural amino acids.
[0043] We recently reported the creation of an E. coli SSO that by
virtue of a nucleoside triphosphate transporter from Phaeodactylum
tricornutum (PtNTT2), imports the requisite unnatural triphosphates
from the media and then uses them to replicate a plasmid containing
the UBP dNaM-dTPT3. We have since shown that DNA containing the UBP
may be transcribed in the SSO by T7 RNA polymerase, and that when
an unnatural nucleotide is incorporated into the codon of an mRNA,
different tRNAs charged with ncAAs and containing the cognate
unnatural nucleotide in their anticodon, can efficiently and
selectively decode the unnatural codon. Because the UBP may be
combined at different positions of different codons, this suggests
that the UBP may be used to encode proteins with multiple,
different ncAAs.
[0044] Disclosed herein in certain embodiments are methods,
compositions, and kits for the synthesis of proteins which
comprises unnatural amino acids that utilizes a mutant tRNA. In
some instances, the protein is synthesized in a cell-free
translation system. In some instances, the protein is synthesized
in a cell or semi-synthetic organism (SSO). In some instances, the
semi-synthetic organism comprises a microorganism. In some
instances, the semi-synthetic organism comprises a bacterium. In
some instances, the semi-synthetic organism comprises an
Escherichia coli. In some instances, the mutant tRNA contains a
mutant anticodon sequence. In some instances, the mutant anticodon
sequence is an anticodon sequence illustrated in Table 1. In some
instances, the mutant anticodon sequence is an anticodon sequence
illustrated in Table 2. In some instances, the mutant anticodon
sequence is an anticodon sequence illustrated in Table 3.
TABLE-US-00001 TABLE 1 GGY GYG YGG GAY GYA YGA GCY GYC YGC GUY GYU
YGU CAY CYA YCA CGY CYG YCG CUY CYU YCU CCY CYC YCC AAY AYA YAA AGY
AYG YAG ACY AYC YAC AUY AYU YAU UUY UYU YUU UAY UYA YUA UGY UYG YUG
UCY UYC YUC GYY YGY YYG CYY YCY YYC AYY YAY YYA UYY YUY YYU YYY
TABLE-US-00002 TABLE 2 GGX GXG XGG GAX GXA XGA GCX GXC XGC GUX GXU
XGU CAX CXA XCA CGX CXG XCG CUX CXU XCU CCX CXC XCC AAX AXA XAA AGX
AXG XAG ACX AXC XAC AUX AXU XAU UUX UXU XUU UAX UXA XUA UGX UXG XUG
UCX UXC XUC GXX XGX XXG CXX XCX XXC AXX XAX XXA UXX XUX XXU XXX
TABLE-US-00003 TABLE 3 GXY GYX XYG YXG XGY YGX AXY AYX XYA YXA XAY
YAX CXY CYX XYC YXC XCY YCX UXY UYX XYU YXU XUY YUX XYY XXY YXX YXX
YXY XYX
[0045] In some instances, the mutant anticodon of the mutant tRNA
pairs with a mutant codon. In some embodiments, the mutant codon is
a mutant codon illustrated in Table 1. In some embodiments, the
mutant codon is a mutant codon illustrated in Table 2. In some
embodiments, the mutant codon is a mutant codon illustrated in
Table 3.
[0046] In some embodiments, the Y and X illustrated in Table 1,
Table 2, and Table 3 represent unnatural bases of the unnatural
nucleotide. In some embodiments, the unnatural base is selected
from the group consisting of 2-aminoadenin-9-yl, 2-aminoadenine,
2-F-adenine, 2-thiouracil, 2-thio-thymine, 2-thiocytosine, 2-propyl
and alkyl derivatives of adenine and guanine, 2-amino-adenine,
2-amino-propyl-adenine, 2-aminopyridine, 2-pyridone,
2'-deoxyuridine, 2-amino-2'-deoxyadenosine 3-deazaguanine,
3-deazaadenine, 4-thio-uracil, 4-thio-thymine, uracil-5-yl,
hypoxanthin-9-yl (I), 5-methyl-cytosine, 5-hydroxymethyl cytosine,
xanthine, hypoxanthine, 5-bromo, and 5-trifiuoromethyl uracils and
cytosines; 5-halouracil, 5-halocytosine, 5-propynyl-uracil,
5-propynyl cytosine, 5-uracil, 5-substituted, 5-halo, 5-substituted
pyrimidines, 5-hydroxycytosine, 5-bromocytosine, 5-bromouracil,
5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine
arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil,
5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil,
5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and
5-iodouracil, 6-alkyl derivatives of adenine and guanine,
6-azapyrimidines, 6-azo-uracil, 6-azo cytosine, azacytosine,
6-azo-thymine, 6-thio-guanine, 7-methylguanine, 7-methyladenine,
7-deazaguanine, 7-deazaguanosine, 7-deaza-adenine,
7-deaza-8-azaguanine, 8-azaguanine, 8-azaadenine, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, and 8-hydroxyl substituted adenines and
guanines; N4-ethylcytosine, N-2 substituted purines, N-6
substituted purines, 0-6 substituted purines, those that increase
the stability of duplex formation, universal nucleic acids,
hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded
nucleic acids, fluorinated nucleic acids, tricyclic pyrimidines,
phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps,
phenoxazine cytidine
(9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido [3',2':4,5]pyrrolo [2,3-d]pyrimidin-2-one),
5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)
uracil, 5-carboxymethyl aminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methythio-N6-isopentenyladenine,
uracil-5oxyacetic acid, wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxacetic acid methylester,
uracil-5-oxacetic acid, 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and
2,6-diaminopurine and those in which the purine or pyrimidine base
is replaced with a heterocycle.
[0047] In some instances, the unnatural nucleotide is selected from
the group consisting of (only nucleobase portion shown, ribose and
phosphate backbone omitted for clarity)
##STR00004##
[0048] In some instances, the unnatural nucleotide is selected from
the group consisting of (only nucleobase portion shown, ribose and
phosphate backbone omitted for clarity)
##STR00005## ##STR00006##
[0049] In some instances, the unnatural nucleotide further
comprises an unnatural sugar moiety. In some instances, the
unnatural sugar moiety is selected from the group consisting of a
modification at the 2' position: OH; substituted lower alkyl,
alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl,
Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3,
ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2F; O-alkyl, S-alkyl, N-alkyl;
O-alkenyl, S-alkenyl, N-alkenyl; O-alkynyl, S-alkynyl, N-alkynyl;
O-alkyl-O-alkyl, 2'-F, 2'-OCH.sub.3, 2'--O(CH.sub.2).sub.2OCH.sub.3
wherein the alkyl, alkenyl and alkynyl may be substituted or
unsubstituted C.sub.1-C.sub.10, alkyl, C.sub.2-C.sub.10 alkenyl,
C.sub.2-C.sub.10 alkynyl, --O[(CH2)nO]mCH.sub.3,
--O(CH.sub.2).sub.nOCH.sub.3, --O(CH.sub.2)nNH.sub.2,
--O(CH.sub.2)n CH.sub.3, --O(CH.sub.2)n-ONH.sub.2, and
--O(CH.sub.2).sub.nON[(CH.sub.2)n CH.sub.3)].sub.2, where n and m
are from 1 to about 10; and/or a modification at the 5' position:
5'-vinyl, 5'-methyl (R or S), a modification at the 4' position,
4'-S, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
any combination thereof.
[0050] In some instances, the mutant anticodon or the mutant codon
further comprises an unnatural backbone. In some instances, the
mutant anticodon further comprises an unnatural backbone. In some
instances, the mutant codon further comprises an unnatural
backbone. In some instances, the unnatural backbone is selected
from the group consisting of a phosphorothioate, chiral
phosphorothioate, phosphorodithioate, phosphotriester,
aminoalkylphosphotriester, C.sub.1-C.sub.10 phosphonates,
3'-alkylene phosphonate, chiral phosphonates, phosphinates,
phosphoramidates, 3'-amino phosphoramidate,
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates.
[0051] In some instances, the unnatural nucleotides are recognized
by a polymerase. In some instances, the polymerase is a DNA
polymerase, an RNA polymerase, or a reverse transcriptase. In some
instances, the polymerase comprises .PHI.29, B103, GA-1, PZA,
.PHI.15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4,
PR5, PR722, L17, ThermoSequenase.RTM., 9.degree. Nm.TM.,
Therminator.TM. DNA polymerase, Tne, Tma, Tfl, Tth, TIi, Stoffel
fragment, Vent.TM. and Deep Vent.TM. DNA polymerase, KOD DNA
polymerase, Tgo, JDF-3, Pfu, Taq, T7 DNA polymerase, T7 RNA
polymerase, PGB-D, UlTma DNA polymerase, E. coli DNA polymerase I,
E. coli DNA polymerase III, archaeal DP 1I/DP2 DNA polymerase II,
9.degree. N DNA Polymerase, Taq DNA polymerase, Phusion.RTM. DNA
polymerase, Pfu DNA polymerase, SP6 RNA polymerase, RB69 DNA
polymerase, Avian Myeloblastosis Virus (AMV) reverse transcriptase,
Moloney Murine Leukemia Virus (MMLV) reverse transcriptase,
SuperScript.RTM. II reverse transcriptase, and SuperScript.RTM. III
reverse transcriptase.
[0052] In some instances, the polymerase is DNA polymerase 1-Klenow
fragment, Vent polymerase, Phusion.RTM. DNA polymerase, KOD DNA
polymerase, Taq polymerase, T7 DNA polymerase, T7 RNA polymerase,
Therminator.TM. DNA polymerase, POLB polymerase, SP6 RNA
polymerase, E. coli DNA polymerase I, E. coli DNA polymerase III,
Avian Myeloblastosis Virus (AMV) reverse transcriptase, Moloney
Murine Leukemia Virus (MMLV) reverse transcriptase,
SuperScript.RTM. II reverse transcriptase, or SuperScript.RTM. III
reverse transcriptase.
[0053] In some instances, an unnatural nucleotide is incorporated
by the polymerase into the mRNA during transcription to generate a
mutant mRNA containing a mutant codon. In some instances, an
unnatural nucleotide is incorporated by the polymerase into the
mRNA during transcription to generate a mutant mRNA.
[0054] In some instances, an unnatural nucleotide is incorporated
by the polymerase into the tRNA during transcription to generate a
mutant tRNA containing a mutant anticodon. In some instances, an
unnatural nucleotide is incorporated by the polymerase into the
tRNA during transcription to generate a mutant tRNA.
[0055] In some instances, the mutant tRNA represents an unnatural
amino acid residue. In some instances, an unnatural amino acid
residue is a non-natural amino acid such as those described in Liu
C. C., Schultz, P. G. Annu. Rev. Biochem. 2010, 79, 413.
[0056] In some instances, a protein containing an unnatural amino
acid is generated during translation utilizing the mutant tRNA and
the mutant mRNA. In some instances, the protein containing an
unnatural amino acid is generated under a cell free translation
system. In some instances, the protein is synthesized in a cell or
semi-synthetic organism (SSO). In some instances, the
semi-synthetic organism comprises a microorganism. In some
instances, the semi-synthetic organism comprises a bacterium. In
some instances, the semi-synthetic organism comprises an
Escherichia coli.
Nucleic Acids
[0057] A nucleic acid (e.g., also referred to herein as target
nucleic acid, target nucleotide sequence, nucleic acid sequence of
interest or nucleic acid region of interest) can be from any source
or composition, such as DNA, cDNA, gDNA (genomic DNA), RNA, siRNA
(short inhibitory RNA), RNAi, tRNA or mRNA, for example, and can be
in any form (e.g., linear, circular, supercoiled, single-stranded,
double-stranded, and the like). Nucleic acids can comprise
nucleotides, nucleosides, or polynucleotides. Nucleic acids can
comprise natural and unnatural nucleic acids. A nucleic acid can
also comprise unnatural nucleic acids, such as DNA or RNA analogs
(e.g., containing base analogs, sugar analogs and/or a non-native
backbone and the like). It is understood that the term "nucleic
acid" does not refer to or infer a specific length of the
polynucleotide chain, thus polynucleotides and oligonucleotides are
also included in the definition. Exemplary natural nucleotides
include, without limitation, ATP, UTP, CTP, GTP, ADP, UDP, CDP,
GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP,
dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural
deoxyribonucleotides include dATP, dTTP, dCTP, dGTP, dADP, dTDP,
dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Exemplary natural
ribonucleotides include ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP,
AMP, UMP, CMP, and GMP. For RNA, the uracil base is uridine. A
nucleic acid sometimes is a vector, plasmid, phage, autonomously
replicating sequence (ARS), centromere, artificial chromosome,
yeast artificial chromosome (e.g., YAC) or other nucleic acid able
to replicate or be replicated. An unnatural nucleic acid can be a
nucleic acid analogue.
Unnatural Nucleic Acids
[0058] A nucleotide analog, or unnatural nucleotide, comprises a
nucleotide which contains some type of modification to either the
base, sugar, or phosphate moieties. A modification can comprise a
chemical modification. Modifications may be, for example, of the
3'OH or 5'OH group, of the backbone, of the sugar component, or of
the nucleotide base. Modifications may include addition of
non-naturally occurring linker molecules and/or of interstrand or
intrastrand cross links. In one aspect, the modified nucleic acid
comprises modification of one or more of the 3'OH or 5'OH group,
the backbone, the sugar component, or the nucleotide base, and/or
addition of non-naturally occurring linker molecules. In one aspect
a modified backbone comprises a backbone other than a
phosphodiester backbone. In one aspect a modified sugar comprises a
sugar other than deoxyribose (in modified DNA) or other than ribose
(modified RNA). In one aspect a modified base comprises a base
other than adenine, guanine, cytosine or thymine (in modified DNA)
or a base other than adenine, guanine, cytosine or uracil (in
modified RNA).
[0059] The nucleic acid may comprise at least one modified base.
Modifications to the base moiety would include natural and
synthetic modifications of A, C, G, and T/U as well as different
purine or pyrimidine bases. In some embodiments, a modification is
to a modified form of adenine, guanine cytosine or thymine (in
modified DNA) or a modified form of adenine, guanine cytosine or
uracil (modified RNA).
[0060] A modified base of a unnatural nucleic acid includes but is
not limited to uracil-5-yl, hypoxanthin-9-yl (I),
2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine,
5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,
5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol,
8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo particularly 5-bromo, 5-trifiuoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain
unnatural nucleic acids, such as 5-substituted pyrimidines,
6-azapyrimidines and N-2 substituted purines, N-6 substituted
purines, 0-6 substituted purines, 2-aminopropyladenine,
5-propynyluracil, 5-propynylcytosine, 5-methylcytosine, those that
increase the stability of duplex formation, universal nucleic
acids, hydrophobic nucleic acids, promiscuous nucleic acids,
size-expanded nucleic acids, fluorinated nucleic acids,
5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6
substituted purines, including 2-aminopropyl adenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl
and other alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine,
5-propynyl (--C.ident.C-CI1/4) uracil, 5-propynyl cytosine, other
alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil,
6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-trifluoromethyl, other 5-substituted uracils and
cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine,
2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine,
7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic
pyrimidines, phenoxazine
cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps,
phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those
in which the purine or pyrimidine base is replaced with other
heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine,
2-pyridone, azacytosine, 5-bromocytosine, bromouracil,
5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine
arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil,
5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil,
5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and
5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine,
4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine,
7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine,
2'-deoxyuridine, 2-amino-2'-deoxyadenosine, and those described in
U.S. Pat. Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882;
5,093,232; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941;
5,750,692; 5,763,588; 5,830,653 and 6,005,096; WO 99/62923;
Kandimalla et al. (2001) Bioorg. Med. Chem. 9:807-813; The Concise
Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I.,
Ed., John Wiley & Sons, 1990, 858-859; Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613; and
Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,
Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
Additional base modifications can be found for example in U.S. Pat.
No. 3,687,808, Englisch et al., Angewandte Chemie, International
Edition, 1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense
Research and Applications, pages 289-302, Crooke, S. T. and Lebleu,
B. ed., CRC Press, 1993.
[0061] Unnatural nucleic acids comprising various heterocyclic
bases and various sugar moieties (and sugar analogs) are available
in the art, and the nucleic acid can include one or several
heterocyclic bases other than the principal five base components of
naturally-occurring nucleic acids. For example, the heterocyclic
base may include uracil-5-yl, cytosin-5-yl, adenin-7-yl,
adenin-8-yl, guanin-7-yl, guanin-8-yl, 4-aminopyrrolo [2.3-d]
pyrimidin-5-yl, 2-amino-4-oxopyrolo [2, 3-d] pyrimidin-5-yl,
2-amino-4-oxopyrrolo [2.3-d] pyrimidin-3-yl groups, where the
purines are attached to the sugar moiety of the nucleic acid via
the 9-position, the pyrimidines via the 1-position, the
pyrrolopyrimidines via the 7-position and the pyrazolopyrimidines
via the 1-position.
[0062] Nucleotide analogs can also be modified at the phosphate
moiety. Modified phosphate moieties include but are not limited to
those that can be modified so that the linkage between two
nucleotides contains a phosphorothioate, chiral phosphorothioate,
phosphorodithioate, phosphotriester, aminoalkylphosphotriester,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonate and chiral phosphonates, phosphinates, phosphoramidates
including 3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphorami dates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates. It is understood
that these phosphate or modified phosphate linkage between two
nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and
the linkage can contain inverted polarity such as 3'-5' to 5'-3' or
2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are
also included. Numerous United States patents teach how to make and
use nucleotides containing modified phosphates and include but are
not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is
herein incorporated by reference.
[0063] Unnatural nucleic acids can include
2',3'-dideoxy-2',3'-didehydro-nucleosides (PCT/US2002/006460),
5'-substituted DNA and RNA derivatives (PCT/US2011/033961; Saha et
al, J. Org Chem., 1995, 60, 788-789; Wang et al, Bioorganic &
Medicinal Chemistry Letters, 1999, 9, 885-890; and Mikhailov et al,
Nucleosides & Nucleotides, 1991, 10(1-3), 339-343; Leonid et
al, 1995, 14(3-5), 901-905; and Eppacher et al, Helvetica Chimica
Acta, 2004, 87, 3004-3020; PCT/JP2000/004720; PCT/JP2003/002342;
PCT/JP2004/013216; PCT/JP2005/020435; PCT/JP2006/315479;
PCT/JP2006/324484; PCT/JP2009/056718; PCT/JP2010/067560), or
5'-substituted monomers made as the monophosphate with modified
bases (Wang et al, Nucleosides Nucleotides & Nucleic Acids,
2004, 23 (1 & 2), 317-337).
[0064] Unnatural nucleic acids can include modifications at the
5'-position and the 2'-position of the sugar ring (PCT/US94/02993),
such as 5'-CH.sub.2 substituted 2'-O-protected nucleosides (Wu et
al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et al.
Bioconjugate Chem. 1999, 10, 921-924). Unnatural nucleic acids can
include amide linked nucleoside dimers have been prepared for
incorporation into oligonucleotides wherein the 3' linked
nucleoside in the dimer (5' to 3') comprises a 2'-OCH.sub.3 and a
5'-(S)--CH.sub.3 (Mesmaeker et al, Synlett, 1997, 1287-1290).
Unnatural nucleic acids can include 2'-substituted 5'-CH.sub.2 (or
O) modified nucleosides (PCT/US92/01020). Unnatural nucleic acids
can include 5'methylenephosphonate DNA and RNA monomers, and dimers
(Bohringer et al, Tet. Lett., 1993, 34, 2723-2726; Collingwood et
al, Synlett, 1995, 7, 703-705; and Hutter et al, Helvetica Chimica
Acta, 2002, 85, 2777-2806). Unnatural nucleic acids can include
5'-phosphonate monomers having a 2'-substitution (US 2006/0074035)
and other modified 5'-phosphonate monomers (WO 97/35869). Unnatural
nucleic acids can include 5'-modified methylenephosphonate monomers
(EP614907 and EP629633). Unnatural nucleic acids can include
analogs of 5' or 6'-phosphonate ribonucleosides comprising a
hydroxyl group at the 5' and or 6' position (Chen et al,
Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786; Jung et al,
Bioorg. Med. Chem., 2000, 8, 2501-2509, Gallier et al, Eur. J. Org.
Chem., 2007, 925-933 and Hampton et al, J. Med. Chem., 1976, 19(8),
1029-1033). Unnatural nucleic acids can include 5'-phosphonate
deoxyribonucleoside monomers and dimers having a 5'-phosphate group
(Nawrot et al, Oligonucleotides, 2006, 16(1), 68-82). Unnatural
nucleic acids can include nucleosides having a 6'-phosphonate group
wherein the 5' or/and 6'-position is unsubstituted or substituted
with a thio-tert-butyl group (SC(CH.sub.3).sub.3) (and analogs
thereof); a methyleneamino group (CH.sub.2NH.sub.2) (and analogs
thereof) or a cyano group (CN) (and analogs thereof) (Fairhurst et
al, Synlett, 2001, 4, 467-472; Kappler et al, J. Med. Chem., 1986,
29, 1030-1038 and J. Med. Chem., 1982, 25, 1179-1184; Vrudhula et
al, J. Med. Chem., 1987, 30, 888-894; Hampton et al, J. Med. Chem.,
1976, 19, 1371-1377; Geze et al, J. Am. Chem. Soc, 1983, 105(26),
7638-7640 and Hampton et al, J. Am. Chem. Soc, 1973, 95(13),
4404-4414)
[0065] Unnatural nucleic acids can also include modifications of
the sugar moiety. Nucleic acids of the invention can optionally
contain one or more nucleosides wherein the sugar group has been
modified. Such sugar modified nucleosides may impart enhanced
nuclease stability, increased binding affinity, or some other
beneficial biological property. In certain embodiments, nucleic
acids comprise a chemically modified ribofuranose ring moiety.
Examples of chemically modified ribofuranose rings include, without
limitation, addition of substitutent groups (including 5' and/or 2'
substituent groups; bridging of two ring atoms to form bicyclic
nucleic acids (BNA); replacement of the ribosyl ring oxygen atom
with S, N(R), or C(R.sub.1)(R.sub.2) (R.dbd.H, C.sub.1-C.sub.12
alkyl or a protecting group); and combinations thereof. Examples of
chemically modified sugars can be found in WO 2008/101157, US
2005/0130923, and WO 2007/134181.
[0066] A modified nucleic acid may comprise modified sugars or
sugar analogs. Thus, in addition to ribose and deoxyribose, the
sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose,
glucose, arabinose, xylose, lyxose, and a sugar "analog"
cyclopentyl group. The sugar can be in pyranosyl or in a furanosyl
form. The sugar moiety may be the furanoside of ribose,
deoxyribose, arabinose or 2'-O-alkylribose, and the sugar can be
attached to the respective heterocyclic bases either in [alpha] or
[beta] anomeric configuration. Sugar modifications include, but are
not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs,
2'-fluoro-DNA, and 2'-alkoxy- or amino-RNA/DNA chimeras. For
example, a sugar modification may include, 2'-O-methyl-uridine and
2'-O-methyl-cytidine. Sugar modifications include
2'-O-alkyl-substituted deoxyribonucleosides and 2'-O-ethyleneglycol
like ribonucleosides. The preparation of these sugars or sugar
analogs and the respective "nucleosides" wherein such sugars or
analogs are attached to a heterocyclic base (nucleic acid base) is
known. Sugar modifications may also be made and combined with other
modifications.
[0067] Modifications to the sugar moiety include natural
modifications of the ribose and deoxy ribose as well as unnatural
modifications. Sugar modifications include but are not limited to
the following modifications at the 2' position: OH; F; O-, S-, or
N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or
O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be
substituted or unsubstituted C.sub.1 to C.sub.10, alkyl or C.sub.2
to C.sub.10 alkenyl and alkynyl. 2' sugar modifications also
include but are not limited to --O[(CH.sub.2).sub.n O].sub.m
CH.sub.3, --O(CH.sub.2).sub.nOCH.sub.3, --O(CH.sub.2)n NH.sub.2,
--O(CH.sub.2).sub.n CH.sub.3, --O(CH.sub.2).sub.n--ONH.sub.2,
and--O(CH.sub.2).sub.nON[(CH.sub.2)n CH.sub.3)J.sub.2, where n and
m are from 1 to about 10.
[0068] Other modifications at the 2' position include but are not
limited to: C.sub.1 to C.sub.10 lower alkyl, substituted lower
alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3,
OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3,
ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted
silyl, an RNA cleaving group, a reporter group, an intercalator, a
group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic
properties of an oligonucleotide, and other substituents having
similar properties. Similar modifications may also be made at other
positions on the sugar, particularly the 3' position of the sugar
on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides
and the 5' position of 5' terminal nucleotide. Modified sugars
would also include those that contain modifications at the bridging
ring oxygen, such as CH.sub.2 and S. Nucleotide sugar analogs may
also have sugar mimetics such as cyclobutyl moieties in place of
the pentofuranosyl sugar. There are numerous United States patents
that teach the preparation of such modified sugar structures such
as U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;
5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,658,873; 5,670,633; 4,845,205; 5,130,302; 5,134,066;
5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;
5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;
5,614,617; 5,681,941; and 5,700,920, each of which is herein
incorporated by reference in its entirety, which detail and
describe a range of base modifications. Each of these patents is
herein incorporated by reference.
[0069] Examples of nucleic acids having modified sugar moieties
include, without limitation, nucleic acids comprising 5'-vinyl,
5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH.sub.3, and
2'-O(CH.sub.2).sub.2OCH.sub.3 substituent groups. The substituent
at the 2' position can also be selected from allyl, amino, azido,
thio, O-allyl, O--C C.sub.1O alkyl, OCF.sub.3,
O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2--O--N(R.sub.m)(R.sub.n), and
O--CH.sub.2--C(.dbd.O)--N(R.sub.m)(R.sub.n), where each R.sub.m and
R.sub.n is, independently, H or substituted or unsubstituted
C.sub.1-C.sub.10 alkyl.
[0070] In certain embodiments, nucleic acids of the present
invention include one or more bicyclic nucleic acids. In certain
such embodiments, the bicyclic nucleic acid comprises a bridge
between the 4' and the 2' ribosyl ring atoms. In certain
embodiments, nucleic acids provided herein include one or more
bicyclic nucleic acids wherein the bridge comprises a 4' to 2'
bicyclic nucleic acid. Examples of such 4' to 2' bicyclic nucleic
acids include, but are not limited to, one of the formulae:
4'-(CH.sub.2)--O-2' (LNA); 4'-(CH.sub.2)--S-2';
4'-(CH.sub.2).sub.2--O-2' (ENA); 4'-CH(CH.sub.3)--O-2' and
4'-CH(CH.sub.2OCH.sub.3)--O-2',and analogs thereof (see, U.S. Pat.
No. 7,399,845, issued on Jul. 15, 2008);
4'-C(CH.sub.3)(CH.sub.3)--O-2' and analogs thereof, (see
WO2009/006478, WO2008/150729, US2004/0171570, U.S. Pat. No.
7,427,672, Chattopadhyaya, et al, J. Org. Chem., 2 09, 74,
118-134), and WO 2008/154401, published on Dec. 8, 2008). Also see,
for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin
et al, Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc.
Natl. Acad. Sci. U.S.A, 2000, 97, 5633-5638; Kumar et al., Bioorg.
Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem.,
1998, 63, 10035-10039; Srivastava et al, J. Am. Chem. Soc, 129(26)
8362-8379 (Jul. 4, 2007); Elayadi et al, Curr. Opinion Invens.
Drugs, 2001, 2, 558-561; Braasch et al, Chem. Biol, 2001, 8, 1-7;
Oram et al, Curr. Opinion Mol Ther., 2001, 3, 239-243; U.S. Pat.
Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133,
6,525,191, 6,670,461, and 7,399,845; International applications WO
2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181;
U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and
US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574,
61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and
61/099,844; and PCT International Applications Nos.
PCT/US2008/064591, PCT US2008/066154, and PCT US2008/068922,
PCT/DK98/00393; and U.S. Pat. Nos. 4,849,513; 5,015,733; 5,118,800;
and 5,118,802.
[0071] In certain embodiments, nucleic acids can comprise linked
nucleic acids. Nucleic acids can be linked together using any inter
nucleic acid linkage. The two main classes of inter nucleic acid
linking groups are defined by the presence or absence of a
phosphorus atom. Representative phosphorus containing inter nucleic
acid linkages include, but are not limited to, phosphodiesters,
phosphotriesters, methylphosphonates, phosphoramidate, and
phosphorothioates (P.dbd.S). Representative non-phosphorus
containing inter nucleic acid linking groups include, but are not
limited to, methylenemethylimino
(--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--), thiodiester
(--O--C(O)--S--), thionocarbamate (--O--C(O)(NH)--S--); siloxane
(--O--Si(H).sub.2--O--); and N,N*-dimethylhydrazine
(--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--). In certain embodiments,
inter nucleic acids linkages having a chiral atom can be prepared a
racemic mixture, as separate enantiomers, e.g., alkylphosphonates
and phosphorothioates. Unnatural nucleic acids can contain a single
modification. Unnatural nucleic acids can contain multiple
modifications within one of the moieties or between different
moieties.
[0072] Backbone phosphate modifications to nucleic acid include,
but are not limited to, methyl phosphonate, phosphorothioate,
phosphoramidate (bridging or non-bridging), phosphotriester,
phosphorodithioate, phosphodithioate, and boranophosphate, and may
be used in any combination. Other non-phosphate linkages may also
be used.
[0073] In some embodiments, backbone modifications (e.g.,
methylphosphonate, phosphorothioate, phosphoroamidate and
phosphorodithioate internucleotide linkages) can confer
immunomodulatory activity on the modified nucleic acid and/or
enhance their stability in vivo.
[0074] A phosphorous derivative (or modified phosphate group) can
be attached to the sugar or sugar analog moiety in and can be a
monophosphate, diphosphate, triphosphate, alkylphosphonate,
phosphorothioate, phosphorodithioate, phosphoramidate or the like.
Exemplary polynucleotides containing modified phosphate linkages or
non-phosphate linkages can be found in Peyrottes et al. (1996)
Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al. (1996) Nucleic
Acids Res. 24:2318-2323; and Schultz et al. (1996) Nucleic Acids
Res. 24:2966-2973; Matteucci (1997) "Oligonucleotide Analogs: an
Overview" in Oligonucleotides as Therapeutic Agents, (DJ. Chadwick
and G. Cardew, ed.) John Wiley and Sons, New York, N.Y.; (Zon
(1993) "Oligonucleoside Phosphorothioates" in Protocols for
Oligonucleotides and Analogs, Synthesis and Properties (Agrawal,
ed.) Humana Press, pp. 165-190); (Miller et al. (1971) JACS
93:6657-6665); (Jager et al. (1988) Biochem. 27:7247-7246), (Nelson
et al. (1997) JOC 62:7278-7287) (U.S. Pat. No. 5,453,496);
Micklefield, J. 2001, Current Medicinal Chemistry 8: 1157-1179.
[0075] Backbone modification may comprise replacing the
phosphodiester linkage with an alternative moiety such as an
anionic, neutral or cationic group. Examples of such modifications
include: anionic internucleoside linkage; N3' to P5'
phosphoramidate modification; boranophosphate DNA;
prooligonucleotides; neutral internucleoside linkages such as
methylphosphonates; amide linked DNA; methylene(methylimino)
linkages; formacetal and thioformacetal linkages; backbones
containing sulfonyl groups; morpholino oligos; peptide nucleic
acids (PNA); and positively charged deoxyribonucleic guanidine
(DNG) oligos, Micklefield, J. 2001, Current Medicinal Chemistry 8:
1157-1179. A modified nucleic acid may comprise a chimeric or mixed
backbone comprising one or more modifications, e.g. a combination
of phosphate linkages such as a combination of phosphodiester and
phosphorothioate linkages.
[0076] Substitutes for the phosphate can be for example, short
chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts. Numerous United States patents
disclose how to make and use these types of phosphate replacements
and include but are not limited to U.S. Pat. Nos. 5,034,506;
5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;
5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;
5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;
5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;
5,677,437; and 5,677,439, each of which is herein incorporated by
reference. It is also understood in a nucleotide substitute that
both the sugar and the phosphate moieties of the nucleotide can be
replaced, by for example an amide type linkage (aminoethylglycine)
(PNA). U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262 teach how
to make and use PNA molecules, each of which is herein incorporated
by reference. (See also Nielsen et al., Science, 1991, 254,
1497-1500). Conjugates can be chemically linked to the nucleotide
or nucleotide analogs. Such conjugates include but are not limited
to lipid moieties such as a cholesterol moiety (Letsinger et al.,
Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid
(Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a
thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. K Y.
Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med.
Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et
al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain,
e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,
EMSOJ, 1991, 10, 1111-1118; Kabanov et al, FEBS Lett., 1990, 259,
327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium
1-di-O-hexadecyl-rac-glycero-S--H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochem. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937. Numerous United States
patents teach the preparation of such conjugates and include, but
are not limited to U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941, each of which is herein incorporated by
reference.
Polymerase
[0077] A particularly useful function of a polymerase is to
catalyze the polymerization of a nucleic acid strand using an
existing nucleic acid as a template. Other functions that are
useful are described elsewhere herein. Examples of useful
polymerases include DNA polymerases and RNA polymerases.
[0078] The ability to improve specificity, processivity, or other
features of polymerases unnatural nucleic acids would be highly
desirable in a variety of contexts where, e.g., unnatural nucleic
acid incorporation is desired, including amplification, sequencing,
labeling, detection, cloning, and many others. The present
invention provides polymerases with modified properties for
unnatural nucleic acids, methods of making such polymerases,
methods of using such polymerases, and many other features that
will become apparent upon a complete review of the following.
[0079] In some instances, disclosed herein includes polymerases
that incorporate unnatural nucleic acids into a growing template
copy, e.g., during DNA amplification. In some embodiments,
polymerases can be modified such that the active site of the
polymerase is modified to reduce steric entry inhibition of the
unnatural nucleic acid into the active site. In some embodiments,
polymerases can be modified to provide complementarity with one or
more unnatural features of the unnatural nucleic acids.
Accordingly, the invention includes compositions that include a
heterologous or recombinant polymerase and methods of use
thereof.
[0080] Polymerases can be modified using methods pertaining to
protein engineering. For example, molecular modeling can be carried
out based on crystal structures to identify the locations of the
polymerases where mutations can be made to modify a target
activity. A residue identified as a target for replacement can be
replaced with a residue selected using energy minimization
modeling, homology modeling, and/or conservative amino acid
substitutions, such as described in Bordo, et al. J Mol Biol 217:
721-729 (1991) and Hayes, et al. Proc Natl Acad Sci, USA 99:
15926-15931 (2002).
[0081] Any of a variety of polymerases can be used in a method or
composition set forth herein including, for example, protein-based
enzymes isolated from biological systems and functional variants
thereof. Reference to a particular polymerase, such as those
exemplified below, will be understood to include functional
variants thereof unless indicated otherwise. In some embodiments, a
polymerase is a wild type polymerase. In some embodiments, a
polymerase is a modified, or mutant, polymerase.
[0082] Polymerases, with features for improving entry of unnatural
nucleic acids into active site regions and for coordinating with
unnatural nucleotides in the active site region, can also be used.
In some embodiments, a modified polymerase has a modified
nucleotide binding site.
[0083] In some embodiments, a modified polymerase has a specificity
for an unnatural nucleic acid that is at least about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the
specificity of the wild type polymerase toward the unnatural
nucleic acid. In some embodiments, a modified or wild type
polymerase has a specificity for an unnatural nucleic acid
comprising a modified sugar that is at least about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the
specificity of the wild type polymerase toward a natural nucleic
acid and/or the unnatural nucleic acid without the modified sugar.
In some embodiments, a modified or wild type polymerase has a
specificity for an unnatural nucleic acid comprising a modified
base that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild
type polymerase toward a natural nucleic acid and/or the unnatural
nucleic acid without the modified base. In some embodiments, a
modified or wild type polymerase has a specificity for an unnatural
nucleic acid comprising a triphosphate that is at least about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%,
99.99% the specificity of the wild type polymerase toward a nucleic
acid comprising a triphosphate and/or the unnatural nucleic acid
without the triphosphate. For example, a modified or wild type
polymerase can have a specificity for an unnatural nucleic acid
comprising a triphosphate that is at least about 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the
specificity of the wild type polymerase toward the unnatural
nucleic acid with a diphosphate or monophosphate, or no phosphate,
or a combination thereof.
[0084] In some embodiments, a modified or wild type polymerase has
a relaxed specificity for an unnatural nucleic acid. In some
embodiments, a modified or wild type polymerase has a specificity
for an unnatural nucleic acid and a specificity to a natural
nucleic acid that is at least about 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of
the wild type polymerase toward the natural nucleic acid. In some
embodiments, a modified or wild type polymerase has a specificity
for an unnatural nucleic acid comprising a modified sugar and a
specificity to a natural nucleic acid that is at least about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%,
99.99% the specificity of the wild type polymerase toward the
natural nucleic acid. In some embodiments, a modified or wild type
polymerase has a specificity for an unnatural nucleic acid
comprising a modified base and a specificity to a natural nucleic
acid that is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the specificity of the wild
type polymerase toward the natural nucleic acid.
[0085] Absence of exonuclease activity can be a wild type
characteristic or a characteristic imparted by a variant or
engineered polymerase. For example, an exo minus Klenow fragment is
a mutated version of Klenow fragment that lacks 3' to 5'
proofreading exonuclease activity.
[0086] The method of the invention may be used to expand the
substrate range of any DNA polymerase which lacks an intrinsic 3 to
5' exonuclease proofreading activity or where a 3 to 5' exonuclease
proofreading activity has been disabled, e.g. through mutation.
Examples of DNA polymerases include polA, polB (see e.g. Parrel
& Loeb, Nature Struc Biol 2001) polC, polD, polY, polX and
reverse transcriptases (RT) but preferably are processive,
high-fidelity polymerases (PCT/GB2004/004643). In some embodiments
a modified or wild type polymerase substantially lacks 3' to 5'
proofreading exonuclease activity. In some embodiments a modified
or wild type polymerase substantially lacks 3' to 5' proofreading
exonuclease activity for an unnatural nucleic acid. In some
embodiments, a modified or wild type polymerase has a 3' to 5'
proofreading exonuclease activity. In some embodiments, a modified
or wild type polymerase has a 3' to 5' proofreading exonuclease
activity for a natural nucleic acid and substantially lacks 3' to
5' proofreading exonuclease activity for an unnatural nucleic
acid.
[0087] In some embodiments, a modified polymerase has a 3' to 5'
proofreading exonuclease activity that is at least about 60%, 70%,
80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the proofreading
exonuclease activity of the wild type polymerase. In some
embodiments, a modified polymerase has a 3' to 5' proofreading
exonuclease activity for an unnatural nucleic acid that is at least
about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the
proofreading exonuclease activity of the wild type polymerase to a
natural nucleic acid. In some embodiments, a modified polymerase
has a 3' to 5' proofreading exonuclease activity for an unnatural
nucleic acid and a 3' to 5' proofreading exonuclease activity for a
natural nucleic acid that is at least about 60%, 70%, 80%, 90%,
95%, 97%, 98%, 99%, 99.5%, 99.99% the proofreading exonuclease
activity of the wild type polymerase to a natural nucleic acid. In
some embodiments, a modified polymerase has a 3' to 5' proofreading
exonuclease activity for a natural nucleic acid that is at least
about 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, 99.99% the
proofreading exonuclease activity of the wild type polymerase to
the natural nucleic acid.
[0088] In a related aspect, the invention provides methods of
making a modified polymerase that include structurally modeling a
parental polymerase, e.g., a DNA polymerase, identifying one or
more complex stability or nucleotide interaction feature affecting
complex stability or nucleotide access or binding in the active
site or a complementarity feature for a nucleotide analog at the
active site, and mutating the parental polymerase to include or
remove these features. For example, the polymerase can be mutated
to improve steric access of the unnatural nucleotide to the active
site or to improve charge-charge or hydrophobic interactions
between the unnatural nucleotide and the polymerase. The methods
also include determining whether the resulting modified polymerase
displays an increased incorporation of a nucleotide or unnatural
nucleotide into a growing nucleic acid copy as compared to the
parental polymerase.
[0089] Polymerases can be characterized according to their rate of
dissociation from nucleic acids. In some embodiments, a polymerase
has a relatively low dissociation rate for one or more natural and
unnatural nucleic acids. In some embodiments, a polymerase has a
relatively high dissociation rate for one or more natural and
unnatural nucleic acids. The dissociation rate is an activity of a
polymerase that can be adjusted to tune reaction rates in methods
set forth herein.
[0090] Polymerases can be characterized according to their fidelity
when used with a particular natural and/or unnatural nucleic acid
or collections of natural and/or unnatural nucleic acid. Fidelity
generally refers to the accuracy with which a polymerase
incorporates correct nucleic acids into a growing nucleic acid
chain when making a copy of a nucleic acid template. DNA polymerase
fidelity can be measured as the ratio of correct to incorrect
natural and unnatural nucleic acid incorporations when the natural
and unnatural nucleic acid are present, e.g., at equal
concentrations, to compete for strand synthesis at the same site in
the polymerase-strand-template nucleic acid binary complex. DNA
polymerase fidelity can be calculated as the ratio of
(k.sub.cat/K.sub.m) for the natural and unnatural nucleic acid and
(k.sub.cat/K.sub.m) for the incorrect natural and unnatural nucleic
acid; where k.sub.cat and K.sub.m are Michaelis-Menten parameters
in steady state enzyme kinetics (Fersht, A. R. (1985) Enzyme
Structure and Mechanism, 2nd ed., p 350, W. H. Freeman & Co.,
New York., incorporated herein by reference). In some embodiments,
a polymerase has a fidelity value of at least about 100, 1000,
10,000, 100,000, or 1.times.10.sup.6, with or without a
proofreading activity.
[0091] Polymerases from native sources or variants thereof can be
screened using an assay that detects incorporation of an unnatural
nucleic acid having a particular structure. In one example,
polymerases can be screened for the ability to incorporate an
unnatural nucleic acid or UBP; e.g., d5SICSTP, dNaMTP, or
d5SICSTP-dNaMTP UBP. A polymerase, e.g., a heterologous polymerase,
can be used that displays a modified property for the unnatural
nucleic acid as compared to the wild-type polymerase. For example,
the modified property can be, e.g., K.sub.m, k.sub.cat, V.sub.max,
polymerase processivity in the presence of an unnatural nucleic
acid (or of a naturally occurring nucleotide), average template
read-length by the polymerase in the presence of an unnatural
nucleic acid, specificity of the polymerase for an unnatural
nucleic acid, rate of binding of an unnatural nucleic acid, rate of
product (pyrophosphate, triphosphate, etc.) release, branching
rate, or any combination thereof. In one embodiment, the modified
property is a reduced K.sub.m for an unnatural nucleic acid and/or
an increased k.sub.cat/K.sub.m or V.sub.max/K.sub.m for an
unnatural nucleic acid. Similarly, the polymerase optionally has an
increased rate of binding of an unnatural nucleic acid, an
increased rate of product release, and/or a decreased branching
rate, as compared to a wild-type polymerase.
[0092] At the same time, a polymerase can incorporate natural
nucleic acids, e.g., A, C, G, and T, into a growing nucleic acid
copy. For example, a polymerase optionally displays a specific
activity for a natural nucleic acid that is at least about 5% as
high (e.g., 5%, 10%, 25%, 50%, 75%, 100% or higher), as a
corresponding wild-type polymerase and a processivity with natural
nucleic acids in the presence of a template that is at least 5% as
high (e.g., 5%, 10%, 25%, 50%, 75%, 100% or higher) as the
wild-type polymerase in the presence of the natural nucleic acid.
Optionally, the polymerase displays a k.sub.cat/K.sub.m or
V.sub.max/K.sub.m for a naturally occurring nucleotide that is at
least about 5% as high (e.g., about 5%, 10%, 25%, 50%, 75% or 100%
or higher) as the wild-type polymerase.
[0093] Polymerases used herein that can have the ability to
incorporate an unnatural nucleic acid of a particular structure can
also be produced using a directed evolution approach. A nucleic
acid synthesis assay can be used to screen for polymerase variants
having specificity for any of a variety of unnatural nucleic acids.
For example, polymerase variants can be screened for the ability to
incorporate an unnatural nucleic acid or UBP; e.g., dTPT3, dNaM
analog, or dTPT3-dNaM UBP into nucleic acids. In some embodiments,
such an assay is an in vitro assay, e.g., using a recombinant
polymerase variant. Such directed evolution techniques can be used
to screen variants of any suitable polymerase for activity toward
any of the unnatural nucleic acids set forth herein.
[0094] Modified polymerases of the compositions described can
optionally be a modified and/or recombinant 129-type DNA
polymerase. Optionally, the polymerase can be a modified and/or
recombinant 129, B103, GA-1, PZA, D15, BS32, M2Y, Nf, G1, Cp-1,
PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.
[0095] Nucleic acid polymerases generally useful in the invention
include DNA polymerases, RNA polymerases, reverse transcriptases,
and mutant or altered forms thereof. DNA polymerases and their
properties are described in detail in, among other places, DNA
Replication 2.sup.nd edition, Kornberg and Baker, W. H. Freeman,
New York, N.Y. (1991). Known conventional DNA polymerases useful in
the invention include, but are not limited to, Pyrococcus furiosus
(Pfu) DNA polymerase (Lundberg et al., 1991, Gene, 108: 1,
Stratagene), Pyrococcus woesei (Pwo) DNA polymerase (Hinnisdaels et
al., 1996, Biotechniques, 20:186-8, Boehringer Mannheim), Thermus
thermophilus (Tth) DNA polymerase (Myers and Gelfand 1991,
Biochemistry 30:7661), Bacillus stearothermophilus DNA polymerase
(Stenesh and McGowan, 1977, Biochim Biophys Acta 475:32),
Thermococcus litoralis (TIi) DNA polymerase (also referred to as
Vent.TM. DNA polymerase, Cariello et al, 1991, Polynucleotides Res,
19: 4193, New England Biolabs), 9.degree. Nm.TM. DNA polymerase
(New England Biolabs), Stoffel fragment, Thermo Sequenase.RTM.
(Amersham Pharmacia Biotech UK), Therminator.TM. (New England
Biolabs), Thermotoga maritima (Tma) DNA polymerase (Diaz and
Sabino, 1998 Braz J Med. Res, 31:1239), Thermus aquaticus (Taq) DNA
polymerase (Chien et al, 1976, J. Bacteoriol, 127: 1550), DNA
polymerase, Pyrococcus kodakaraensis KOD DNA polymerase (Takagi et
al., 1997, Appl. Environ. Microbiol. 63:4504), JDF-3 DNA polymerase
(from thermococcus sp. JDF-3, Patent application WO 0132887),
Pyrococcus GB-D (PGB-D) DNA polymerase (also referred as Deep
Vent.TM. DNA polymerase, Juncosa-Ginesta et al., 1994,
Biotechniques, 16:820, New England Biolabs), UlTma DNA polymerase
(from thermophile Thermotoga maritima; Diaz and Sabino, 1998 Braz
J. Med. Res, 31:1239; PE Applied Biosystems), Tgo DNA polymerase
(from thermococcus gorgonarius, Roche Molecular Biochemicals), E.
coli DNA polymerase I (Lecomte and Doubleday, 1983, Polynucleotides
Res. 11:7505), T7 DNA polymerase (Nordstrom et al, 1981, J Biol.
Chem. 256:3112), and archaeal DP1I/DP2 DNA polymerase II (Cann et
al, 1998, Proc. Natl. Acad. Sci. USA 95:14250). Both mesophilic
polymerases and thermophilic polymerases are contemplated.
Thermophilic DNA polymerases include, but are not limited to,
ThermoSequenase.RTM., 9.degree. Nm.TM., Therminator.TM., Taq, Tne,
Tma, Pfu, Tfl, Tth, TIi, Stoffel fragment, Vent.TM. and Deep
Vent.TM. DNA polymerase, KOD DNA polymerase, Tgo, JDF-3, and
mutants, variants and derivatives thereof. A polymerase that is a
3' exonuclease-deficient mutant is also contemplated. Reverse
transcriptases useful in the invention include, but are not limited
to, reverse transcriptases from HIV, HTLV-I, HTLV-II, FeLV, FIV,
SIV, AMV, MMTV, MoMuLV and other retroviruses (see Levin, Cell
88:5-8 (1997); Verma, Biochim Biophys Acta. 473:1-38 (1977); Wu et
al, CRC Crit Rev Biochem. 3:289-347(1975)). Further examples of
polymerases include, but are not limited to 9.degree.N DNA
Polymerase, Taq DNA polymerase, Phusion.RTM. DNA polymerase, Pfu
DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase, and
VentR.RTM. DNA polymerase Gardner et al. (2004) "Comparative
Kinetics of Nucleotide Analog Incorporation by Vent DNA Polymerase
(J. Biol. Chem., 279(12), 11834-11842; Gardner and Jack
"Determinants of nucleotide sugar recognition in an archaeon DNA
polymerase" Nucleic Acids Research, 27(12) 2545-2553.) Polymerases
isolated from non-thermophilic organisms can be heat inactivatable.
Examples are DNA polymerases from phage. It will be understood that
polymerases from any of a variety of sources can be modified to
increase or decrease their tolerance to high temperature
conditions. In some embodiments, a polymerase can be thermophilic.
In some embodiments, a thermophilic polymerase can be heat
inactivatable. Thermophilic polymerases are typically useful for
high temperature conditions or in thermocycling conditions such as
those employed for polymerase chain reaction (PCR) techniques.
[0096] In some embodiments, the polymerase comprises 129, B103,
GA-1, PZA, D15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5,
Cp-7, PR4, PR5, PR722, L17, ThermoSequenase.RTM., 9.degree. Nm.TM.,
Therminator.TM. DNA polymerase, Tne, Tma, Tfl, Tth, TIi, Stoffel
fragment, Vent.TM. and Deep Vent.TM. DNA polymerase, KOD DNA
polymerase, Tgo, JDF-3, Pfu, Taq, T7 DNA polymerase, T7 RNA
polymerase, PGB-D, UlTma DNA polymerase, E. coli DNA polymerase I,
E. coli DNA polymerase III, archaeal DP1IUDP2 DNA polymerase II,
9.degree. N DNA Polymerase, Taq DNA polymerase, Phusion.RTM. DNA
polymerase, Pfu DNA polymerase, SP6 RNA polymerase, RB69 DNA
polymerase, Avian Myeloblastosis Virus (AMV) reverse transcriptase,
Moloney Murine Leukemia Virus (MMLV) reverse transcriptase,
SuperScript.RTM. II reverse transcriptase, and SuperScript.RTM. III
reverse transcriptase.
[0097] In some embodiments, the polymerase is DNA polymerase
1-Klenow fragment, Vent polymerase, Phusion.RTM. DNA polymerase,
KOD DNA polymerase, Taq polymerase, T7 DNA polymerase, T7 RNA
polymerase, Therminator.TM. DNA polymerase, POLB polymerase, SP6
RNA polymerase, E. coli DNA polymerase I, E. coli DNA polymerase
III, Avian Myeloblastosis Virus (AMV) reverse transcriptase,
Moloney Murine Leukemia Virus (MMLV) reverse transcriptase,
SuperScript.RTM. II reverse transcriptase, or SuperScript.RTM. III
reverse transcriptase.
[0098] Additionally, such polymerases can be used for DNA
amplification and/or sequencing applications, including real-time
applications, e.g., in the context of amplification or sequencing
that include incorporation of unnatural nucleic acid residues into
DNA by the polymerase. In other embodiments, the unnatural nucleic
acid that is incorporated can be the same as a natural residue,
e.g., where a label or other moiety of the unnatural nucleic acid
is removed by action of the polymerase during incorporation, or the
unnatural nucleic acid can have one or more feature that
distinguishes it from a natural nucleic acid.
[0099] Since at least the last common ancestor of all life on
earth, genetic information has been stored in a four-letter
alphabet that is propagated and retrieved by the formation of two
base pairs. The central goal of synthetic biology is to create new
life forms and functions, and the most general route to this goal
is the creation of semi-synthetic organisms (SSOs) whose DNA
harbors two additional letters that form a third, unnatural base
pair (UBP). Previously, our efforts to generate such SSOs
culminated in the creation of a strain of Escherichia coli that by
virtue of a nucleoside triphosphate transporter from Phaeodactylum
tricornutum (PtNTT2), imports the requisite unnatural triphosphates
from the media and then uses them to replicate a plasmid containing
the UBP dNaM-dTPT3 (FIG. 1A). While the SSO stores increased
information, it did not retrieve it, which requires in vivo
transcription of the UBP into mRNA and tRNA, aminoacylation of the
tRNA with an unnatural amino acid, and finally, efficient
participation of the UBP in decoding at the ribosome. Here, we
report the in vivo transcription of DNA containing dNaM and dTPT3
into mRNAs with two different unnatural codons and tRNAs with
cognate unnatural anticodons, and their efficient decoding at the
ribosome to direct the site-specific incorporation of natural or
non-canonical amino acids (ncAAs) into superfolder green
fluorescent protein (sfGFP). The results demonstrate that
interactions other than hydrogen bonding can contribute to every
step of information storage and retrieval. The resulting SSO both
encodes and retrieves increased information and should serve as a
platform for the creation of new life forms and functions.
[0100] Green fluorescent protein and variants such as sfGFP have
served as model systems for the study of ncAA incorporation using
the amber suppression system, including at position Y151, which has
been shown to tolerate a variety of natural and ncAAs (FIG. 4). To
explore the decoding of unnatural codons, we first focused on the
incorporation of Ser at position 151 of sfGFP, as E. coli serine
aminoacyl-tRNA synthetase (SerRS) does not rely on anticodon
recognition for tRNA aminoacylation, thus eliminating the potential
complications of inefficient charging. SSO strain YZ3 was
transformed with a plasmid encoding sfGFP and an E. coli
tRNA.sup.Ser gene (serT), with sfGFP codon 151 (TAC) replaced by
the unnatural codon AXC (sfGFP(AXC).sup.151; X=NaM), and the
anticodon of serT replaced by the unnatural anticodon GYT
(tRNA.sup.Ser(GYT); Y=TPT3) (FIG. 1B). Transformants were grown in
media supplemented with dNaMTP and dTPT3TP, then supplemented
further with NaMTP and TPT3TP, as well as
isopropyl-.beta.-D-thiogalactoside (IPTG) to induce expression of
T7 RNA polymerase (T7 RNAP) and tRNA.sup.Ser(GYT). After a brief
period of tRNA induction, anhydrotetracycline (aTc) was added to
induce expression of sfGFP(AXC).sup.151.
[0101] Following induction, cells transformed with a control
plasmid encoding sfGFP(AXC).sup.151 but lacking tRNA.sup.Ser(GYT)
showed dramatically reduced fluorescence compared to cells
transformed with a plasmid encoding sfGFP with a natural Ser codon
at position 151 (sfGFP(AGT).sup.151; FIG. 1C). Moreover, cell
growth began to plateau upon induction of sfGFP(AXC).sup.151 (FIG.
1D), likely due to the stalling and sequestering of ribosomes.
Lysates of these cells were subjected to western blotting with an
anti-GFP antibody, which revealed a significant reduction in sfGFP
expression and the presence of sfGFP truncated at the position of
the unnatural codon (FIG. 1E). In contrast, cells transformed with
the plasmid encoding both sfGFP(AXC).sup.151 and tRNA.sup.Ser(GYT)
exhibited fluorescence that was nearly equal to that of control
cells expressing sfGFP(AGT).sup.151 (FIG. 1C), cell growth did not
plateau upon induction of sfGFP(AXC).sup.151 (FIG. 1D), and western
blots of lysates from these cells revealed only full-length sfGFP
protein (FIG. 1E). Furthermore, we assessed the ability of all four
natural near-cognate tRNAs (tRNA.sup.Ser(GNT); N=G, C, A, or T),
expressed in an identical fashion, to decode the AXC codon. In each
case, little fluorescence was observed and the growth defect
remained (FIGS. 5A and 5B). These data demonstrate that PtNTT2 is
able to import both the deoxy- and ribotriphosphates of both
unnatural nucleotides, that T7 RNA polymerase is able to transcribe
mRNA and tRNA containing the unnatural nucleotides in vivo, and
that the ribosome only efficiently decodes the unnatural codon with
an unnatural anticodon.
[0102] To assess the fidelity of decoding, we analyzed protein
purified from cells expressing both sfGFP(AXC).sup.151 and
tRNA.sup.Ser(GYT) via LC/MS-MS and relative quantitation via peak
intensities, which revealed a 98.5.+-.0.7% (95% CI, n=4)
incorporation of Ser at position 151, with Ile/Leu being the
predominant contaminant (FIG. 1F, Table 4). Given that the
retention of the UBP in the sfGFP(AXC).sup.151 gene was 98.+-.2%
(95% CI, n=4) (Table 5) and that X.fwdarw.T is typically the major
mutation during replication (which for AXC would result in the Ile
codon ATC), we attribute the majority of the protein not containing
Ser at position 151 to loss of the UBP during replication and
conclude that the fidelity of translation with the unnatural codon
is high.
TABLE-US-00004 TABLE 4 Sample S Y PrK I/L N V K G C M Relative MS1
ion intensities (%) sfGFP(AGT).sup.151 99.80 0.03 0.06 0.00 0.04
0.03 0.00 0.02 0.02 0.00 sfGFP(AXC).sup.151/ 98.47 0.04 0.04 1.23
0.14 0.02 0.00 0.05 0.01 0.00 tRNA.sup.Ser(GYT) sfGFP(TAC).sup.151
0.11 99.71 0.06 0.00 0.05 0.02 0.00 0.02 0.02 0.01
sfGFP(TAG).sup.151/tRNA.sup.Pyl(CTA) 0.06 0.04 99.53 0.00 0.04 0.01
0.29 0.01 0.01 0.00 sfGFP(AXC).sup.151/ 0.25 0.03 96.16 2.06 1.06
0.02 0.37 0.03 0.01 0.00 tRNA.sup.Pyl(GYT) sfGFP(GXC).sup.151/ 0.06
0.04 97.50 0.00 0.01 1.26 0.74 0.37 0.01 0.00 tRNA.sup.Pyl(GYC) 95%
CI (%) sfGFP(AGT).sup.151 0.31 0.04 0.09 0.00 0.06 0.05 0.01 0.03
0.03 0.00 sfGFP(AXC).sup.151/ 0.73 0.04 0.03 0.64 0.04 0.01 0.00
0.04 0.01 0.00 tRNA.sup.Ser(GYT) sfGFP(TAC).sup.151 0.06 0.11 0.05
0.00 0.03 0.02 0.00 0.01 0.02 0.00 sfGFP(TAG).sup.151/ 0.03 0.02
0.11 0.00 0.02 0.02 0.03 0.01 0.01 0.00 tRNA.sup.Pyl(CTA)
sfGFP(AXC).sup.151/ 0.13 0.02 0.25 0.06 0.03 0.01 0.06 0.01 0.02
0.01 tRNA.sup.Pyl(GYT) sfGFP(GXC).sup.151/tRNA.sup.Pyl(GYC) 0.05
0.04 0.70 0.00 0.01 0.24 0.28 0.22 0.01 0.00
TABLE-US-00005 TABLE 5 % UBP Retention Anti. % UBP Retention aaRS
tRNA NaMTP TPT3TP Codon sfGFP codon in tRNA gene SerRS -- + + AXC
98 .+-. 0 -- n/a SerRS.sctn. Ser + + AXC 98 .+-. 2 GYT 89 .+-. 2
SerRS Ser + + AXC 94 .+-. 8 GAT n/a SerRS Ser + + AXC 94 .+-. 2 GGT
n/a SerRS Ser + + AXC 95 .+-. 0 GCT n/a SerRS Ser + + AXC 95 .+-. 1
GTT n/a -- Pyl + + AXC 97 .+-. 1 GYT 89 .+-. 2 PylRS -- + + AXC 97
.+-. 1 -- n/a PylRS Pyl + + TAC n/a GYT 92 .+-. 3 PylRS.sctn. Pyl +
+ AXC 96 .+-. 1 GYT 90 .+-. 2 PylRS* Pyl + + AXC 98 .+-. 0 GYT 95
.+-. 2 PylRS* Pyl + - AXC 98 .+-. 1 GYT 96 .+-. 1 PylRS* Pyl - +
AXC 98 .+-. 1 GYT 95 .+-. 1 PylRS* Pyl - - AXC 97 .+-. 1 GYT 94
.+-. 4 -- Pyl + + GXC 98 .+-. 1 GYC 96 .+-. 3 PylRS -- + + GXC 97
.+-. 3 -- n/a PylRS Pyl + + TAC n/a GYC 96 .+-. 1 PylRS.sctn. Pyl +
+ GXC 97 .+-. 1 GYC 95 .+-. C PylRS* Pyl + + GXC 96 .+-. 3 GYC 97
.+-. 1 PylRS* Pyl + - GXC 96 .+-. 2 GYC 97 .+-. 1 PylRS* Pyl - +
GXC 97 .+-. 2 GYC 97 .+-. 0 PylRS* Pyl - - GXC 96 .+-. 1 GYC 97
.+-. 1 pAzFRS RS.sctn. pAzF + + AXC 98 .+-. 0 GYT 90 .+-. 1 pAzFRS
RS pAzF + + TAC n/a GYT 91 .+-. 1 *Corresponds to the cultures
analyzed in FIGS. 7A-7D.
[0103] To demonstrate the encoding of ncAAs with UBPs, we
constructed plasmids analogous to those used above, but with the
tRNA.sup.Ser gene replaced with the Methanosarcina mazei
tRNA.sup.Pyl(GYT) gene. tRNA.sup.Pyl can be selectively charged by
the Methanosarcina barkeri pyrrolysine aminoacyl tRNA synthetase
(PylRS) with the ncAA N.sup.6-[(2-propynyloxy)carbonyl]-L-lysine
(PrK). In addition to the codon AXC, we also analyzed the codon GXC
and the corresponding tRNA.sup.Pyl(GYC). The SSO, carrying a
separate plasmid encoding an IPTG-inducible PylRS, was transformed
with the required plasmids and grown with or without added PrK. In
control experiments with cells expressing either sfGFP(AXC).sup.151
or sfGFP(GXC).sup.151 in the absence of either PylRS, the cognate
unnatural tRNA.sup.Pyl, or PrK, we observed only low cellular
fluorescence (FIG. 2A), truncation of sfGFP (FIGS. 6A and 6B), and
a plateau in cell growth (FIG. 6B). In contrast, for either
unnatural mRNA with its cognate unnatural tRNA, when PylRS was
present and PrK was added, we observed high fluorescence (64% and
69% of sfGFP(TAC).sup.151 for AXC and GXC, respectively) (FIGS. 2A
and 2B), robust production of full-length sfGFP (FIG. 6A), and
normal growth (FIG. 6B).
[0104] To verify the incorporation of PrK, sfGFP was affinity
purified from cell lysates using a C-terminal Strep-tag II and
subjected to copper-catalyzed click chemistry to attach a
carboxytetramethylrhodamine (TAMRA) dye (TAMRA-PEG.sub.4-N.sub.3),
which was found to shift the electrophoretic mobility of sfGFP
during SDS-PAGE, thus allowing us to assess the fidelity of PrK
incorporation by western blotting (FIG. 2C). We observed strong
TAMRA signal and that virtually all of the sfGFP was shifted when
purified from cells expressing sfGFP(AXC).sup.151 and
tRNA.sup.Pyl(GYT) or sfGFP(GXC).sup.151 and tRNA.sup.Pyl(GYC), and
which had been cultured in media supplemented with PrK (FIG. 2C).
In contrast, little to no TAMRA signal or shifted sfGFP was
observed when NaMTP, TPT3TP, or both were absent (FIGS. 7A and 7B).
Finally, no TAMRA signal or shifted sfGFP was observed in protein
purified from cells expressing sfGFP(TAC).sup.151 with either
unnatural tRNA (FIG. 2C). This data demonstrates that PrK is
specifically incorporated into sfGFP via decoding of the unnatural
codons by tRNAs with an unnatural anticodon.
[0105] With optimal PrK concentrations (FIGS. 8A-8D), we purified
54.+-.4 and 55.+-.6 .mu.g/mL of sfGFP (s.d., n=4, .about.40% of the
sfGFP(TAC).sup.151 control (Table 6) for the AXC and GXC codons,
respectively. Moreover, based on mass spectrometry analysis, the
purity of sfGFP with PrK was 96.2.+-.0.3% (95% CI, n=4) for the AXC
codon and 97.5.+-.0.7% (95% CI, n=4) for the GXC codon (FIG. 2D).
Although the yield of sfGFP protein purified was slightly lower
than with amber suppression (87.+-.6 .mu.g/mL, s.d., n=4 (Table
6)), due to a moderate reduction in growth with addition of the
unnatural ribotriphosphates (FIGS. 7C and 7D), decoding of both
unnatural codons resulted in higher fluorescence than amber
suppression when normalized to cell density (FIGS. 2A and 2B),
implying that decoding with the unnatural codons is more efficient
than amber suppression.
[0106] To explore the encoding of other ncAAs with UBPs, we
examined the encoding of p-azido-phenylalanine (pAzF) with the AXC
codon and an evolved Methanococcus jannaschii TyrRS/tRNA.sup.Tyr
pair (pAzFRS/tRNA.sup.pAzF). With induction of the synthetase and
the addition of pAzF to the growth media, we observed robust
fluorescence equivalent to that of cells expressing natural
sfGFP(TAC).sup.151 and normal growth with sfGFP(AXC).sup.151 and
tRNA.sup.pAzF(GYT) (FIG. 3A, FIG. 9). Full-length sfGFP was
purified (86.+-.6 .mu.g/mL, s.d., n=4; 68% of the
sfGFP(TAC).sup.151 control, Table 6) and subjected to copper-free
click chemistry using a dibenzocyclooctyl (DBCO) group to attach
TAMRA (TAMRA-PEG.sub.4-DBCO). We observed robust TAMRA conjugation
to sfGFP isolated from cells expressing sfGFP(AXC).sup.151 and
tRNA.sup.pAzF(GYT) and cultured in the presence of pAzF (FIG. 3B).
Although we were unable to accurately assess the fidelity of pAzF
incorporation due to decomposition of the azido moiety, .about.93%
of the sfGFP protein was shifted, which compares favorably to the
.about.95% shifted sfGFP produced via amber suppression (FIG.
3B).
TABLE-US-00006 TABLE 6 Relative to Total Yield control Fluor
Relative to Sample aaRS (.mu.g/mL) (%) (a.u.) control (%)
sfGFP(AGT).sup.151 SerRS 100 .+-. 8 100 269 100
sfGFP(AXC).sup.151/tRNA.sup.Ser(GYT) (endogenous) 97 .+-. 9 96 259
96 sfGFP(TAC).sup.151 PylRS 135 .+-. 17 100 400 100
sfGFP(TAG).sup.151/tRNA.sup.Pyl(CTA) 87 .+-. 6 65 242 60
sfGFP(AXC).sup.151/tRNA.sup.Pyl(GYT) 54 .+-. 4 40 153 38
sfGFP(GXC).sup.151/tRNA.sup.Pyl(GYC) 55 .+-. 6 41 166 41
sfGFP(TAC).sup.151 pAzFRS 127 .+-. 15 100 405 100
sfGFP(TAG).sup.151/tRNA.sup.pAzF(CTA) 75 .+-. 9 59 287 71
sfGFP(AXC).sup.151/tRNA.sup.pAzF(GYT) 86 .+-. 6 68 333 82
[0107] Since at least the last common ancestor of all life on
earth, proteins have been produced via the decoding of codons
written solely with the four-nucleotide genetic alphabet. We have
now demonstrated the decoding of two new codons, written with an
expanded genetic alphabet, and used the new codons to
site-specifically incorporate ncAAs into proteins. We find that for
every step of information storage and retrieval, hydrogen bonds, so
obviously central to the natural base pairs, may at least in part
be replaced with complementary packing and hydrophobic forces.
Despite their novel mechanism of decoding, the unnatural codons can
be decoded as efficiently as their fully natural counterparts.
While we have only examined the decoding of two unnatural codons,
the UBP is unlikely to be limited to these, and when combined with
a recently reported Cas9 editing system that reinforces UBP
retention, it will likely make available more codons than can ever
be used. Thus, the reported SSO may be just the first of a new form
of semi-synthetic life that is able to access a broad range of
forms and functions not available to natural organisms.
[0108] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
* * * * *