U.S. patent application number 16/542121 was filed with the patent office on 2020-01-16 for linking methods, compositions, systems, kits and apparatuses.
The applicant listed for this patent is Life Technologies Corporation. Invention is credited to John DAVIDSON, Guobin LUO, Theo NIKIFOROV.
Application Number | 20200017846 16/542121 |
Document ID | / |
Family ID | 45809592 |
Filed Date | 2020-01-16 |
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United States Patent
Application |
20200017846 |
Kind Code |
A1 |
DAVIDSON; John ; et
al. |
January 16, 2020 |
LINKING METHODS, COMPOSITIONS, SYSTEMS, KITS AND APPARATUSES
Abstract
In some embodiments, the disclosure relates generally to methods
as well as related compositions, systems, kits and apparatus
comprising linking proteins to target compounds and/or to locations
of interest using tethers. For example, the tether can be used to
link the protein to a target compound, for example, to link an
enzyme to a substrate. Similarly, the tether can be used to link
the protein at or near a desired location on a surface. In one
group of embodiments, the tether includes a polynucleotide and the
target compound or location on the surface includes another
polynucleotide that is capable of hybridizing to the tether. In
such embodiments, the tether can be used to link the protein to the
target compound or location using nucleic acid hybridization.
Inventors: |
DAVIDSON; John; (Guilford,
CT) ; NIKIFOROV; Theo; (Carlsbad, CA) ; LUO;
Guobin; (Oceanside, CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Life Technologies Corporation |
Carlsbad |
CA |
US |
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|
Family ID: |
45809592 |
Appl. No.: |
16/542121 |
Filed: |
August 15, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15786223 |
Oct 17, 2017 |
10385329 |
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16542121 |
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13984346 |
Sep 13, 2013 |
9868945 |
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PCT/US12/24266 |
Feb 8, 2012 |
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15786223 |
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61440723 |
Feb 8, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 11/10 20130101;
G01N 2333/9126 20130101; C12N 9/1241 20130101; C12Q 1/68
20130101 |
International
Class: |
C12N 11/10 20060101
C12N011/10; C12N 9/12 20060101 C12N009/12 |
Claims
1. A composition comprising a protein tethered to a substrate.
2. The composition of claim 1, wherein the tether comprises a first
polynucleotide having a first polynucleotide sequence hybridized to
a second polynucleotide having a second polynucleotide
sequence.
3. The composition of claim 1, wherein the first polynucleotide is
attached to the protein.
4. The composition of claim 1, wherein the second polynucleotide is
attached to the substrate.
5. The composition of claim 1, wherein the protein comprises an
enzyme.
6. The composition of claim 1, wherein the protein comprises a
polymerase enzyme.
7. The composition of claim 1, wherein the protein comprises a DNA
polymerase enzyme.
8. A composition comprising a protein linked to a tether, the
tether including a first polynucleotide and the first
polynucleotide including a first polynucleotide sequence, wherein
the linking further includes contacting the tethered protein with a
second polynucleotide, wherein the second polynucleotide is linked
to a surface.
9. A method for conducting a nucleotide incorporation reaction
using the composition of claim 1.
10. A method for conducting a nucleotide incorporation reaction
using the composition of claim 8.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/786,223, filed Oct. 17, 2017 which is
hereby incorporated by reference in its entirety, which is a
divisional of U.S. patent application Ser. No. 13/984,346, filed
Sep. 13, 2013, now, U.S. Pat. No. 9,868,945, which is a U.S. 371
national stage entry of international application No.
PCT/US2012/24266, filed Feb. 8, 2012, which claims the benefit
under 35 U.S.C. 119(e) of U.S. Provisional Application No.
61/440,723, filed Feb. 8, 2011.
BACKGROUND
[0002] The ability of enzymes to catalyze biological reactions is
is fundamental to life. A range of biological applications use
enzymes to synthesize various biomolecules in vitro. One
particularly useful class of enzymes are the polymerases, which can
catalyze the polymerization of biomolecules (e.g., nucleotides or
amino acids) into biopolymers (e.g., nucleic acids or peptides).
For example, polymerases that can polymerize nucleotides into
nucleic acids, particularly in a template-dependent fashion, are
useful in recombinant DNA technology and nucleic acid sequencing
applications. Many nucleic acid sequencing methods monitor
nucleotide incorporations during in vitro template-dependent
replication of a target nucleic acid molecule by a polymerase.
[0003] When using an enzyme to catalyze a biological reaction of
interest, it can be useful to confine the enzyme so that it is
co-localized with its substrate. Such co-localization can increase
the rate or efficiency of enzymatic catalysis, thereby increasing
the enzymatic activity and/or product yield under a given set of
reaction conditions. Various methods of co-localizing enzymes with
substrates, typically by immobilizing the enzyme on a support that
is then contacted with a solution including the substrate, have
been reported. However, such methods typically cause a reduction in
enzyme activity and succeed only at low efficiencies. Such methods
typically also require modification of the enzyme prior to or after
enzyme immobilization, which can be time-consuming and technically
challenging to perform. There remains a need in the art for simple,
efficient and reliable methods to tether enzymes to, or in the
vicinity of, their target substrates so as to increase the rate or
product yield of the enzymatic reaction, as well as more generally
to tether proteins to desired locations.
DETAILED DESCRIPTION OF THE DRAWINGS
[0004] FIGS. 1A-1D depict exemplary embodiments of steps in
constructing a tethered polymerase. FIG. 1A depicts a tether
structure including a polynucleotide sequences, a primary amine at
the 5' end and a FAM group at the 3' end. FIG. 1B depicts the
tether structure and SMCC. FIG. 1C depicts the tether+SMCC reaction
and the resulting product. FIG. 1D depicts tethers linked to two
cysteine residues of Bst polymerase.
[0005] FIG. 2 depicts exemplary results of labeling polymerases
with AF647 maleimide.
[0006] FIG. 3 depicts exemplary results of SMCC activation of
FAM-labeled tether oligonucleotides and a
maleimide-oligonucleotide-FAM product.
[0007] FIG. 4 depicts a tethering reaction and gel analysis of
exemplary reaction products with Klenow fragment (KF) and Bst
polymerases.
[0008] FIG. 5 depicts an embodiment of a reaction using DTT to
reduce disulfide bonds of the polymerase.
[0009] FIG. 6 depicts an embodiment of an activation reaction of
FAM-oligonucleotide+SMCC to form a derivatized FAM-oligonucleotide
product.
[0010] FIG. 7 depicts an embodiment of a tethering reaction of a
polymerase+derivatized FAM-oligonucleotide tether to form a
polymerase linked to oligonucleotide tethers.
[0011] FIG. 8 depicts exemplary results of oligonucleotide
tethering reactions performed with varying salt concentrations.
[0012] FIG. 9 depicts exemplary results of sequencing reactions
using an untethered sequencing polymerase.
[0013] FIG. 10 depicts exemplary results of sequencing reactions
using a sequencing polymerase tethered with an oligonucleotide.
DETAILED DESCRIPTION
[0014] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as is commonly understood by one
of ordinary skill in the art to which these inventions belong. All
patents, patent applications, published applications, treatises and
other publications referred to herein, both supra and infra, are
incorporated by reference in their entirety. If a definition and/or
description is explicitly or implicitly set forth herein that is
contrary to or otherwise inconsistent with any definition set forth
in the patents, patent applications, published applications, and
other publications that are herein incorporated by reference, the
definition and/or description set forth herein prevails over the
definition that is incorporated by reference.
[0015] The practice of the disclosure will employ, unless otherwise
indicated, conventional techniques of molecular biology,
microbiology and recombinant DNA techniques, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, Sambrook, J., and Russell, D. W.,
2001, Molecular Cloning: A Laboratory Manual, Third Edition;
Ausubel, F. M., et al., eds., 2002, Short Protocols In Molecular
Biology, Fifth Edition.
[0016] As used herein, the terms "a," "an," and "the" and similar
referents used herein are to be construed to cover both the
singular and the plural unless their usage in context indicates
otherwise. Accordingly, the use of the word "a" or "an" when used
in the claims or specification, including when used in conjunction
with the term "comprising", may mean "one," but it is also
consistent with the meaning of "one or more," "at least one," and
"one or more than one."
[0017] As used herein, the terms "link", "linked", "linkage" and
variants thereof comprise any type of fusion, bond, adherence or
association that is of sufficient stability to withstand use in the
particular biological application of interest. Such linkage can
comprise, for example, covalent, ionic, hydrogen, dipole-dipole,
hydrophilic, hydrophobic, or affinity bonding, bonds or
associations involving van der Waals forces, mechanical bonding,
and the like. Optionally, such linkage can occur between a
combination of different molecules, including but not limited to:
between a nanoparticle and a protein; between a protein and a
label; between a linker and a functionalized nanoparticle; between
a linker and a protein; between a nucleotide and a label; and the
like. Some examples of linkages can be found, for example, in
Hermanson, G., Bioconjugate Techniques, Second Edition (2008);
Aslam, M., Dent, A., Bioconjugation: Protein Coupling Techniques
for the Biomedical Sciences, London: Macmillan (1998); Aslam, M.,
Dent, A., Bioconjugation: Protein Coupling Techniques for the
Biomedical Sciences, London: Macmillan (1998).
[0018] In some embodiments, the disclosure relates generally to
methods (as well as related compositions, systems, kits and
apparatus) comprising link proteins to target compounds and/or to
locations of interest using tethers. For example, the tether can be
used to link the protein to a target compound, for example, to link
an enzyme to a substrate. Similarly, the tether can be used to link
the protein at or near a desired location on a surface. In one
group of embodiments, the tether includes a polynucleotide and the
target compound or location on the surface includes another
polynucleotide that is capable of hybridizing to the tether. In
such embodiments, the tether can be used to link the protein to the
target compound or location using nucleic acid hybridization.
[0019] Linking proteins to desired targets or locations by
exploiting the ability of nucleic acid molecules to selectively
hybridize to each other has several advantages. For example,
methods of affixing nucleic acids to surfaces, optionally in array
format, are well-developed and can easily be adapted for use in
protein-based assays. Similarly, the use of nucleic acid
hybridization as a tethering mechanism is simple to perform, can be
reversed at will via appropriate adjustment of reaction conditions,
can be employed using native (i.e., unmodified) nucleic acid
molecules and eliminates the need to use binding catalysts or other
reactants. Furthermore, by exploiting the ability of
polynucleotides to hybridize with each other in a sequence-specific
manner, reactions can be performed in multiplex format where
different groups of protein are selectively linked to different
targets or locations using the same linking conditions.
[0020] The use of tethering mechanisms can also be useful in
decreasing the cost or effort associated with perfoming
protein-based applications. For example, many such applications
(e.g., enzyme reactions) can consume large amounts of protein,
which can be costly and time-consuming to prepare. This problem is
aggravated when using proteins in methods that monitor and detect
aggregate signals from a population of protein molecules acting
upon a population of targets, either in asynchronous (e.g., single
molecule) or synchronous format. This problem can be especially
intractable when multiple rounds of washing or reagent exchange are
involved. In such methods, it can be very costly to repeatedly
provide proteins for each fresh round of reaction, particularly at
sufficiently high concentrations to permit reaction of the protein
with a target. In such situations, the use of tethers to link the
protein to the target or to a surface can eliminate loss of
proteins during reaction washes, thereby reducing or eliminating
the need to replenish the protein in the reaction following each
wash. For example, multiple reaction and wash cycles can be
performed without consuming large amounts of expensive protein
reagents in each wash.
[0021] Tethering can also effectively increase the local
concentration of the protein within the zone of reaction with a
target, thus effectively increase the rate of reaction and/or
increasing the total amount of product formed within a given amount
of time. For example, tethering of an enzyme can increase the rate
of an enzyme-catalyzed reaction. Typically, such rate is limited by
enzyme concentration; tethering limits the ability of the enzyme to
diffuse away from the reaction site, effectively increasing the
localized protein concentration without requiring the use of very
large amounts of protein.
[0022] Finally, use of tethered proteins can sometimes increase the
spectrum of reaction conditions available to the user. For example,
protein tethering can permit the use of reaction conditions that
enhance protein activity but would otherwise cause loss of
untethered proteins from the reaction mixture. Described further
herein are some exemplary embodiments that further illustrate the
various advantages of tethered proteins. For example, use of a
tethered polymerase in nucleotide incorporation and/or primer
extension applications, such as cyclical ("step-wise")
sequencing-by-synthesis reactions, can reduce the amount of
polymerase consumed in each extension step, can increase the
reaction rate and/or the total amount of product formed under given
reaction conditions and can also permit the use of high-salt
reaction conditions without significant loss of polymerase between
washes, thus effectively increasing the amount of signal obtained
from each extension and reducing the amount of incomplete
extensions at each step.
[0023] In some embodiments, the disclosure relates generally to
methods, compositions, systems, apparatuses and kits for linking a
protein to a surface, comprising: contacting a tethered protein
with a surface, where the tethered protein includes a tether linked
to protein, and where the tether of the tethered protein includes a
surface-reactive moiety and the surface includes a tether-reactive
moiety that is capable of reacting with the surface-reactive
moiety; and forming a linkage between the surface-reactive moiety
with the tether-reactive moiety, thereby linking the tethered
protein to the surface. In some embodiments, the tether reactive
moiety and the surface-reactive moiety each comprise one of two
complementary members of a binding pair. The binding pair can be
selected from a group consisting of: a biotin moiety and an avidin
moiety, an antigenic epitope and an antibody or immunogically
reactive fragment thereof, an antibody and a hapten, a digoxigen
moiety and an anti-digoxigen antibody, a fluorescein moiety and an
anti-fluorescein antibody, an operator and a repressor, a nuclease
and a nucleotide, a lectin and a polysaccharide, a steroid and a
steroid-binding protein, an active compound and an active compound
receptor, a hormone and a hormone receptor, an enzyme and a
substrate, an immunoglobulin and protein A, and two polynucleotides
that are complementary to each other over at least some portion of
their respective lengths (where complementarity is optionally
defined according to conventional Watson-Crick base pairing rules
or alternatively according to some other base-pairing
paradigm).
[0024] In some embodiments, the surface-reactive moiety of the
tether includes a first polynucleotide having a first
polynucleotide sequence, and the tether-reactive moiety of the
surface includes a second polynucleotide having a second
polynucleotide sequence, where the first and second polynucleotide
sequences are at least 80% complementary to each other (where
complementarity is optionally defined according to conventional
Watson-Crick base pairing rules or alternatively according to some
other base-pairing paradigm). In some embodiments, the first and
second polynucleotide sequences are at least 85%, at least 90%, at
least 95%, at least 97% or at least 99% complementary to each
other.
[0025] In some embodiments, the disclosure relates generally to
methods (and related compositions, systems, apparatuses and kits)
for linking a protein to a surface, comprising: binding a tethered
protein to a surface, where the tethered protein includes a protein
linked to a tether, the tether including a first polynucleotide and
the first polynucleotide including a first polynucleotide sequence;
where the binding further includes contacting the tethered protein
with a second polynucleotide, wherein the second polynucleotide is
linked to a surface and includes a second polynucleotide sequence
that is at least 80% complementary to the first polynucleotide
sequence, and hybridizing the first polynucleotide sequence to the
second polynucleotide sequence, thereby linking the tethered
polymerase to the surface. In some embodiments, the first and
second polynucleotide sequences are at least 80% complementary to
each other (where complementarity is optionally defined according
to conventional Watson-Crick base pairing rules or alternatively
according to some other base-pairing paradigm). In some
embodiments, the first and second polynucleotide sequences are at
least 85%, at least 90%, at least 95%, at least 97% or at least 99%
complementary to each other.
[0026] In some embodiments, the disclosure relates generally to
methods, compositions, systems, apparatuses and kits useful for
co-localizing an enzyme and its substrate using a tether to link
the enzyme to, or in the vicinity of, the substrate. In some
embodiments, the colocalization can be performed prior to, or
during, the reaction of the enzyme with the substrate. Such
co-localization can increase the rate of the enzymatic reaction
and/or increase the the product yield.
[0027] In some embodiments, the disclosure relates generally to
methods of co-localizing an enzyme with a substrate by linking an
enzyme (or any biologically active fragment thereof) to a substrate
using a tether, thereby forming an tethered enzyme-substrate
complex that includes the enzyme (or biologically active fragment)
linked to the substrate through the tether. Typically, the linking
is done in such a manner that the enzyme (or biologically active
fragment) retains enzymatic activity and can still react with the
substrate to form a product after the linking is complete. In one
exemplary embodiment, the enzyme-reactive moiety of the tether can
be linked to the enzyme (or biologically active fragment) to form a
tethered enzyme. The tethered enzyme can be contacted with the
substrate under conditions where the substrate-reactive moiety of
the tether in the tethered enzyme binds to the substrate, thereby
forming a tethered enzyme-substrate complex that includes the
enzyme linked to the substrate through the tether. Optionally, the
tethered enzyme-substrate complex retains enzymatic activity. For
example, the enzyme (or biologically active fragment) of the
tethered enzyme-substrate complex can bind to the substrate within
the tethered enzyme-substrate complex, or to any other substrate
molecule within the reaction mixture, and can catalyze the
enzyme-substrate reaction. In some embodiments, the substrate is
linked to a surface, such that formation of the tethered
enzyme-substrate complex effectively localizes the enzyme (or
biologically active fragment) to the surface.
[0028] In some embodiments, the tether is not used to link the
enzyme to the substrate, but is instead used to link the enzyme to
a surface. For example, the tether can link the enzyme to to the
surface, thereby forming a surface-linked tethered enzyme, while
the substrate can be free-floating or independently attached to the
same or different surface. In some embodiments, the substrate is
linked to the same surface as the the tethered enzyme. For example,
the enzyme and substrate can each be independently linked to the
same surface at attachment sites that are sufficiently close to
each other, and where the tether is sufficiently flexible, to
permit reaction of the enzyme with the substrate.
[0029] In some embodiments, the disclosure relates generally to
methods (and related compositions, systems, kits and apparatuses)
for co-localizing an enzyme and its substrate comprising: forming a
tethered enzyme by linking a tether to the enzyme (or biologically
active fragment thereof), where the tethered enzyme retains
enzymatic activity; and binding the tethered enzyme to a substrate.
Optionally, the tether includes an enzyme-reactive moiety, and the
linking further includes reacting the enzyme-reactive moiety with
the enzyme, thereby forming an enzyme-tether linkage that links the
tether to the enzyme. The linkage can be a covalent linkage, an
electrostatic linkage or any other linkage suitable for linking the
enzyme (or biologically active fragment) to the tether. In some
embodiments, the tether includes a substrate-reactive moiety, and
the disclosed methods (and related compositions, systems, kits and
apparatuses) further involve linking the tether to the substrate by
reacting the substrate-reactive moiety with the substrate, and the
linking further includes reacting the substrate-reactive moiety
with the substrate, thereby forming an substrate-tether linkage
that links the tether to the substrate. In some embodiments, the
enzyme in the tethered enzyme selected from the group consisting
of: a polymerase, a ribosome, a helicase, a pyrophosphatase and an
apyrase.
[0030] In some embodiments, the disclosure relates generally to
methods (and related compositions, systems, apparatuses and kits)
for linking polymerases to target compounds, or to desired
locations of interest. In some embodiments, the polymerase can
include that is capable of catalyzing the incorporation of one or
more nucleotides into a nucleic acid molecule. Typically but not
necessarily such nucleotide incorporation can occur in a
template-dependent fashion. The polymerase can be a
naturally-occurring polymerase, or any subunit or truncation
thereof, a mutant, variant, derivative, recombinant, fusion,
modified, chemically modified, or otherwise engineered form of any
polymerase, a synthetic molecule or assembly that can catalyze
nucleotide incorporation, as well as analogs, derivatives or
fragments thereof that retain the ability to catalyze such
nucleotide incorporation. Optionally, the polymerase can be a
mutant polymerase comprising one or more mutations involving the
replacement of one or more amino acids with other amino acids, the
insertion or deletion of one or more amino acids, or the linkage of
parts of two or more polymerases. Typically, the polymerase
comprises one or more active sites at which nucleotide binding
and/or catalysis of nucleotide polymerization can occur. Some
exemplary polymerases include without limitation DNA polymerases
including both DNA-dependent and RNA-dependent DNA polymerases
(such as for example Bst DNA polymerase, TherminatorTM polymerase,
KOD polymerase, Phi-29 DNA polymerase, reverse transcriptases and
E. coli DNA polymerase) and RNA polymerases. In some embodiments,
the polymerase is a fusion proteins comprising at least two
portions linked to each other, where the first portion comprises a
peptide that can catalyze the polymerization of nucleotides into a
nucleic acid strand and is linked to a second portion that
comprises a second polypeptide, such as, for example, a reporter
enzyme or a processivity-enhancing domain. In one exemplary
embodiment, the polymerase is Phusion.RTM. DNA polymerase (New
England Biolabs), which comprises a Pyrococcus-like polymerase
fused to a processivity-enhancing domain as described, for example,
in U.S. Pat. No. 6,627,424.
[0031] Typically, the polymerase includes a nucleic acid binding
site and a polymerase active site. The polymerase active site can
be a site of polymerase activity. The polymerase activity can
comprise any in vivo or in vitro enzymatic activity of the
polymerase that relates to catalyzing the incorporation of one or
more nucleotides into a nucleic acid molecule, for example, primer
extension activity and the like. Typically, but not necessarily
such nucleotide polymerization occurs in a template-dependent
fashion. In addition to such polymerase activity, the polymerase
can possess other activities such as DNA binding activity, 3' to 5'
or 5' to 3' exonuclease activity, and the like.
[0032] In some embodiments, the disclosure relates generally to
methods (and related compositions, systems, kits and apparatuses)
for linking a polymerase to a surface, comprising: contacting a
tethered polymerase with a surface, where the tethered polymerase
includes polymerase linked to a tether including a first
polynucleotide, where the polynucleotide has a first polynucleotide
sequence, and the surface includes a second polynucleotide
including a second polynucleotide sequence, where the first and
second polynucleotide sequences are at least 80% complementary to
each other; and hybridizing the first polynucleotide sequence to
the second polynucleotide sequence, thereby linking the tethered
polymerase to the surface.
[0033] In some embodiments, the first polynucleotide of the tether
is covalently linked to an amino acid residue of the polymerase.
For example, the first polynucleotide can be covalently linked to a
cysteine residue of the polymerase. The first polynucleotide can be
covalently linked to the cysteine residue through a flexible
linker.
[0034] Typically, the tethered polymerase has polymerase activity,
and can catalyze the incorporation of one or more nucleotides into
a nucleic acid molecule.
[0035] In some embodiments, the disclosed methods (and related
compositions, systems, kits and apparatuses) can further include
contacting the tethered polymerase with one or more
nucleotides.
[0036] In some embodiments, the disclosed methods (and related
compositions, systems, kits and apparatuses) can further include
incorporating one or more nucleotides into the second
polynucleotide using the tethered polymerase. For example, the
tethered polymerase can polymerize the one or more nucleotides onto
the 3' end of the second polynucleotide.
[0037] In some embodiments, the surface includes a third
polynucleotide, and the disclosed methods (and related
compositions, systems, kits and apparatuses) can further include
incorporating one or more nucleotides into the third polynucleotide
using the tethered polymerase. For example, the tethered polymerase
can polymerize the one or more nucleotides onto the 3' end of the
third polynucleotide.
[0038] In some embodiments, the disclosure relates generally to
compositions (as well as related methods, systems, kits and
apparatus) comprising tethered proteins, where the tether can be
fixed to a site of interest. For example, the tether can link the
protein to a substrate or to a surface.
[0039] In some embodiments, the disclosure relates generally to
compositions (and related methods, systems, kits and apparatuses)
comprising tethered enzymes. Typically, the tethered enzyme
includes an enzyme linked to a tether, where the tethered enzyme
has enzymatic activity.
[0040] In some embodiments, the tether is a linear tether. In some
embodiments, the tether can include a linear polymer, which can be
rigid or flexible. In some embodiments, the tether includes a
polynucleotide. In some embodiments, the tether includes a flexible
linker. In some embodiments, the flexible linker can have a
persistence length of up to about 200 nm; for example between about
0.5 nm and about 1500 nm; typically between about 1.0 nm and about
15 nm or between about 1.5 nm and about 10 nm; even more typically
between about 1.5 nm and 5.0 nm, where the persistence length is
measured under any reaction conditions that are suitable for primer
extension (for example, in a buffer comprising between 0 and about
2M salt (e.g., NaCl or MgCl.sub.2)), even more typically in a
buffer comprising between 0 and about 100 mM salt (e.g., NaCl or
MgCl.sub.2). Methods of measuring persistence length of
polynucleotides in solution are known in the art; see, e.g., Murphy
et al., "Probing Single Stranded DNA Conformational Flexibility
Using Fluorescence Spectroscopy", Biophys J. 2004 April; 86(4):
2530-2537. In some embodiments, the flexible linker includes a
polynucleotide. In some embodiments, the flexible linker includes a
polypeptide. In some embodiments, the flexible linker includes
polyethylene glycol.
[0041] Optionally, the tether can include one or more labels. The
one or more labels can be detected using any suitable detection
system. For example the one or more labels can include fluorescent
labels, luminescent labels, chemically detectable labels, or
magnetically detectable labels.
[0042] In some embodiments, the tether can include one or more
reactive moieties. In some embodiments, the tether includes an
enzyme-reactive moiety. The enzyme-reactive moiety can react with
the enzyme under suitable conditions. Reaction of the
enzyme-reactive moiety with the enzyme can result in the formation
of an enzyme-tether linkage that links the tether to the enzyme,
thereby forming a tethered enzyme. The enzyme-tether linkage can
include one or more bonds selected from the group consisting of: a
covalent bond, an electrostatic bond, an affinity-based interaction
and a hydrogen bond. In some embodiments, the enzyme-reactive
moiety of the tether reacts with a sulfhydryl group of a cysteine
residue of the enzyme and forms a covalent bond with a sulfur atom
of the sulfhydryl group of the cysteine. In some embodiments, the
enzyme-reactive moiety of the tether reacts with an amino group of
an amino acid residue of the enzyme. The amino group can be located
on an N-terminal amino acid residue of the enzyme. In some
embodiments, the enzyme-reactive moiety of the tether reacts with a
carboxyl group of an amino acid residue of the enzyme. The carboxyl
group can be located on a C-terminal amino acid residue of the
enzyme.
[0043] In some embodiments, the enzyme includes a reactive
sulfhydryl group (for example, a sulfhydryl group of a cysteine
residue, that is optionally a surface cysteine) and the
enzyme-reactive moiety of the tether includes a linking group that
is capable of reacting with the sulfhydryl group of the cysteine
reside. For example, the linking group can include a reactive amine
that can be activated by suitable treatment (e.g., reaction with
SMCC) to form a reactive maleimide. The tether including the
reactive maleimide can then be contacted with the enzyme under
suitable conditions where the reactive maleimide group reacts with
the sulfhydryl group of the cysteine, forming a covalent linkage,
typically a linkage comprising a thioether bond.
[0044] In some embodiments, the tether includes a
substrate-reactive moiety. The substrate-reactive moiety can react
with the substrate under suitable conditions. For example, the
substrate-reactive moiety can react or bind selectively to the
substrate when contacted with the substrate Reaction of the
substrate-reactive moiety with the substrate can result in the
formation of a substrate-tether linkage that links the tether to
the substrate. The substrate-tether linkage can include one or more
bonds selected from the group consisting of: a covalent bond, an
electrostatic bond, an affinity-based interaction and a hydrogen
bond. In one exemplary embodiment, the substrate-reactive moiety of
the tether comprises a first polynucleotide including a first
polynucleotide sequence, and the substrate includes, e.g., is
linked to, a second polynucleotide including a second
polynucleotide sequence, where the first and second polynucleotide
sequences are at least 80% complementary to each other. When the
tether and substrate are contacted with each other under
hybridization conditions, then first and second polynucleotide
sequences hybridize to each other, thereby forming a nucleic acid
duplex that links the tether to the substrate.
[0045] In some embodiments, the tether of the tethered enzyme
includes a surface-reactive moiety. The surface-reactive moiety can
react with a surface under suitable conditions. Reaction of the
surface-reactive moiety with the surface can result in the
formation of a surface-tether linkage that links the tether to the
surface. The surface-tether linkage can include one or more bonds
selected from the group consisting of: a covalent bond, an
electrostatic bond, an affinity-based interaction and a hydrogen
bond.
[0046] In some embodiments, the surface-reactive moiety of the
tethered enzyme includes a first polynucleotide, and the surface
includes a second polynucleotide. The first and second
polynucleotides may be capable of hybridizing to each other over at
least a portion of their respective lengths. In some embodiments,
the first polynucleotide includes a first polynucleotide sequence
and the second polynucleotide includes a second polynucleotide
sequence, where the first and second polynucleotide sequences are
at least 80% complementary to each other. In some embodiments, the
first and second polynucleotide sequences are at least 80%
complementary, at least 90% complementary, at least 95%
complementary, at least 97% complementary, or at least 99%
complementary to each other. Typically, complementarity can be
defined according to conventional Watson-Crick base pairing rules
(e.g., A base pairs with T and C base pairs with G); alternatively
complementarity can be defined according to non-Watson Crick base
pairing paradigms as well. In some embodiments, the first
polynucleotide sequence and the second polynucleotide sequence are
hybridized to each other.
[0047] In some embodiments, the tether is linked to any one or more
members of the group consisting of: enzyme, substrate and surface.
For example, the tether can first be linked to the enzyme through
the enzyme-reactive moiety of the tether, thereby forming a
tethered enzyme, and the tether of the tethered enzyme can then be
linked to a substrate using the substrate-reactive moiety of the
tether, or alternatively linked directly to a surface using a
surface-reactive moiety of the tether. Alternatively, the tether
can first be linked to a substrate, or to a surface, and then be
linked to the enzyme.
[0048] In a typical embodiment, the enzyme comprises a
polymerase.
[0049] In some embodiments, the disclosure relates generally to a
composition comprising a tethered polymerase including a polymerase
linked to a tether, where the tethered polymerase has polymerase
activity. The polymerase-tether linkage can include one or more
bonds selected from the group consisting of: a covalent bond, an
electrostatic bond, an affinity-based interaction and a hydrogen
bond. In some embodiments, the polymerase can be covalently linked
to the tether. The tether can be linked to an amino acid residue of
the polymerase. In some embodiments, the tether is linked to a
sulfhydryl group of a cysteine residue of the polymerase. In some
embodiments, the tether is linked to an amino group of an amino
acid residue of the polymerase. In some embodiments, the tether is
linked to a carboxyl group of an amino acid residue of the
polymerase. The tether can include a polynucleotide. The polymerase
can be covalently linked to the polynucleotide. The polynucleotide
can be covalently linked to a cysteine residue of the polymerase.
In some embodiments, a 5' end of the polynucleotide is covalently
linked to an amino acid residue of the polymerase. The covalent
linkage between the tether and the polymerase can include a series
of atoms, each atom in the series being linked covalently to the
next atom in the series such that the tether and the polymerase are
ultimately linked to each other through a series of atoms linked
through covalent bonds.
[0050] In some embodiments, the polymerase of the tethered
polymerase is linked to a first member of a binding pair, the
tether is linked to a second member of the binding pair, and where
the first member and the second member are linked to each other to
form the tethered polymerase. The binding pair can be selected from
a group consisting of: a biotin moiety and an avidin moiety, an
antigenic epitope and an antibody or immunogically reactive
fragment thereof, an antibody and a hapten, a digoxigen moiety and
an anti-digoxigen antibody, a fluorescein moiety and an
anti-fluorescein antibody, an operator and a repressor, a nuclease
and a nucleotide, a lectin and a polysaccharide, a steroid and a
steroid-binding protein, an active compound and an active compound
receptor, a hormone and a hormone receptor, an enzyme and a
substrate, an immunoglobulin and protein A, and two polynucleotides
that are complementary to each other over at least some portion of
their respective lengths (where complementarity can optionally be
defined according to conventional Watson-Crick base pairing rules
or alternatively according to some other base-pairing
paradigm).
[0051] As used herein, the term "nucleotide" and its variants
comprises any compound that can bind selectively to, or can be
polymerized by, a polymerase. Typically, but not necessarily,
selective binding of the nucleotide to the polymerase is followed
by polymerization of the nucleotide into a nucleic acid strand by
the polymerase; occasionally however the nucleotide may dissociate
from the polymerase without becoming incorporated into the nucleic
acid strand, an event referred to herein as a "non-productive"
event. Such nucleotides include not only naturally-occurring
nucleotides but also any analogs, regardless of their structure,
that can bind selectively to, or can be polymerized by, a
polymerase. While naturally-occurring nucleotides typically
comprise base, sugar and phosphate moieties, the nucleotides of the
disclosure can include compounds lacking any one, some or all of
such moieties. In some embodiments, the nucleotide can optionally
include a chain of phosphorus atoms comprising three, four, five,
six, seven, eight, nine, ten or more phosphorus atoms. In some
embodiments, the phosphorus chain can be attached to any carbon of
a sugar ring, such as the 5' carbon. The phosphorus chain can be
linked to the sugar with an intervening O or S. In one embodiment,
one or more phosphorus atoms in the chain can be part of a
phosphate group having P and O. In another embodiment, the
phosphorus atoms in the chain can be linked together with
intervening O, NH, S, methylene, substituted methylene, ethylene,
substituted ethylene, CNH.sub.2, C(O), C(CH.sub.2),
CH.sub.2CH.sub.2, or C(OH)CH.sub.2R (where R can be a 4-pyridine or
1-imidazole). In one embodiment, the phosphorus atoms in the chain
can have side groups having O, BH.sub.3, or S. In the phosphorus
chain, a phosphorus atom with a side group other than O can be a
substituted phosphate group. Some examples of nucleotide analogs
are described in Xu, U.S. Pat. No. 7,405,281. In some embodiments,
the nucleotide comprises a label (e.g., reporter moiety) and
referred to herein as a "labeled nucleotide"; the label of the
labeled nucleotide is referred to herein as a "nucleotide label".
In some embodiments, the label can be in the form of a fluorescent
dye attached to the terminal phosphate group, i.e., the phosphate
group or substitute phosphate group most distal from the sugar.
Some examples of nucleotides that can be used in the disclosed
methods and compositions include, but are not limited to,
ribonucleotides, deoxyribonucleotides, modified ribonucleotides,
modified deoxyribonucleotides, ribonucleotide polyphosphates,
deoxyribonucleotide polyphosphates, modified ribonucleotide
polyphosphates, modified deoxyribonucleotide polyphosphates,
peptide nucleotides, metallonucleosides, phosphonate nucleosides,
and modified phosphate-sugar backbone nucleotides, analogs,
derivatives, or variants of the foregoing compounds, and the like.
In some embodiments, the nucleotide can comprise non-oxygen
moieties such as, for example, thio- or borano-moieties, in place
of the oxygen moiety bridging the alpha phosphate and the sugar of
the nucleotide, or the alpha and beta phosphates of the nucleotide,
or the beta and gamma phosphates of the nucleotide, or between any
other two phosphates of the nucleotide, or any combination
thereof.
[0052] As used herein, the term "nucleotide incorporation" and its
variants comprise polymerization of one or more nucleotides to form
a nucleic acid strand including at least two nucleotides linked to
each other, typically but not necessarily via phosphodiester bonds,
although alternative linkages may be possible in the context of
particular nucleotide analogs.
[0053] The following non-limiting examples are provided purely by
way of illustration of exemplary embodiments, and in no way limit
the scope and spirit of the present disclosure. Furthermore, it is
to be understood that any inventions disclosed or claimed herein
encompass all variations, combinations, and permutations of any one
or more features described herein. Any one or more features may be
explicitly excluded from the claims even if the specific exclusion
is not set forth explicitly herein. It should also be understood
that disclosure of a reagent for use in a method is intended to be
synonymous with (and provide support for) that method involving the
use of that reagent, according either to the specific methods
disclosed herein, or other methods known in the art unless one of
ordinary skill in the art would understand otherwise. In addition,
where the specification and/or claims disclose a method, any one or
more of the reagents disclosed herein may be used in the method,
unless one of ordinary skill in the art would understand
otherwise.
EXAMPLES
Example 1: Construction of a Tethered Polymerase
[0054] A tethered polymerase was constructed by covalently linking
a tether including a first polynucleotide sequence to a cysteine
residue of a variant Bst polymerase ("Ion Sequencing Polymerase")
having the following amino acid sequence:
TABLE-US-00001 MAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNERGRFF
LRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLL
LAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAE
HLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKV
DTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLP
VLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVV
RPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSES
DWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSED
EVTPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYFQSFPG
VKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTP
IQGSAADIIKKAMIDLNARLKEERLQAHLLLQVHDELILEAPKEEMERLC
RLVPEVMEQAVTLRVPLKVDYRYGSTWYDAK
[0055] The tether included a polynucleotide sequence complementary
to the sequence of a sequencing primer P1 (described further below)
and a flexible linker including a poly-A stretch, as well as a
primary amine at the 5' end of tether. The 3' end of the tether was
blocked using a FAM group. The structure of the tether is depicted
in FIG. 1A.
[0056] The primary amine of the tether can be activated via
conversion to a maleimide group using SMCC, as shown in FIG. 1B.
The reaction and resulting product is depicted in FIG. 1C.
[0057] The tether of FIG. 1 was then covalently linked to two
cysteine residues of the Bst Polymerase to obtain the tethered
polymerase depicted in FIG. 1D according to the methods described
below.
[0058] Activation of the Tether
[0059] The following reagents were used:
[0060] The tether including a 49 base-pair oligonucleotide with a
tB30 amino tether and FAM; MW=16115.9; 105.7 nm=1.7 mg (IDT DNA).
The structure and primary amino acid sequence of the tether can be
depicted as follows:
TABLE-US-00002 5AmMC12/AA AAA AAA AAA AAA AAA AAA GAC TGC CAA GGC
ACA CAG GGG ATA GGA AA/36-FAM
[0061] Bst; Ion Torrent lot 7 sequencing polymerase, .about.1
mg/mL
[0062] Klenow (KF) exo-wt (106.9 .mu.M; 7.6 mg/mL; Starlight
source)
[0063] AlexaFluor 647 maleimide (1 mg; A20347; MW.about.1300)
[0064] AlexaFluor 647 cadaverine (1 mg; A30679; MW.about.1000)
[0065] SMCC (Molecular Probes; S1534; Lot: 38985A; MW=334.33)
[0066] sulfo-SMCC (Molecular Biosciences; MW=463.37)
[0067] sodium bicarbonate (J.T. BAKER; 3506-1; FW 84.01)
[0068] The Bst polymerase was then labeled with maleimide dye
(AF647 maleimide).
[0069] Results are depicted in FIG. 2.
[0070] The tether including the FAM-labeled oligonucleotide was
activated with SMCC to generate a maleimide-oligo-FAM product.
Representative results of the activation reaction are depicted in
FIG. 3.
[0071] The maleimide-oligo-FAM product was purified with NAP-5
column
[0072] Two different polymerases, Klenow fragment ("KF") and the
variant Bst polymerase ("Bst") were reacted with
maleimide-oligo-FAM to form tethered polymerases, and the reaction
products were purified on a gel. Representative results are
depicted in FIG. 4. A possible product band was detected using gel
analysis using less than 10% of the reaction.
[0073] Linkage of the Polymerase to the Tether
[0074] The following protocol was used to link the polymerase to
the tether:
[0075] (a) Polymerase DTT Treatment and Purification to Reduce
Disulfide Bonds:
[0076] Reagents (Stock Concentration) and Materials:
[0077] Lot 7 sequencing Bst polymerase (.about.1 mg/mL), DTT (1
M),
[0078] Tris pH 8.5 (1 M), Exchange Buffer (50 mM Mes pH 6.0, 2 mM
EDTA) NAP-5 column (G.E. NAP column, product #17-0853-02),
[0079] Amicon 30K centrifugal filter (Millipore Cat No. UFC503096
Lot No. ROMA72710), 1-1.5 mL Eppendorf Tubes
[0080] Protocol:
[0081] Mix Bst polymerase (25 .mu.L), DTT (1 .mu.L), Tris pH 8.5 (5
.mu.L) and ddH.sub.2O (69 .mu.L). Incubate at 4.degree. C. for 1-12
h. Purify Bst polymerase using a NAP-5 column. Use gravity flow to
prepare and use the NAP-5 column: (1) drain storage buffer (2) add
exchange buffer and pass >15 mL exchange buffer (3) add reaction
mix and drain until all of the mix has entered the matrix and (4)
fill column with exchange buffer and (6) collect 5 drop fractions.
Next use the Nanodrop to determine in which fraction(s) contain the
Bst polymerase. Pool the fractions and concentrate using the Amicon
30K centrifugal filter using the manufacture's instructions.
[0082] (Amicon Protocol: spin for 7 min at 11,000.times.g. Transfer
filter to clean collection tube and insert in an inverted manner.
Spin for 1 min at 1000.times.g). Measure the absorbance of the
concentated Bst polymerase using the Nanodrop and determine the Bst
concentration using the following formula:
.epsilon..sub.280=58000/Mcm.
[0083] The reaction is depicted in FIG. 5.
[0084] (b) Activation of FAM-Oligo with SMCC
[0085] The oligo tether was activated using SMCC to generate a
FAM-oligo derivatized with maleimide
[0086] Reagents:
[0087] SMCC (MW=334.33 g/mol; Molecular Probes; product #: S1534;
Lot:38985A),
[0088] Sodium bicarbonate (J.T. Baker; product #:3506-01)
[0089] FAM-oligo (tB30 Amino tether FAM; MW=16115.9; 105.7 nm=1.7
mg),
[0090] DMSO
[0091] Protocol:
[0092] FAM-oligo was dissolved in ddH.sub.2O to obtain a 500 .mu.M
concentration. Approximately 1 mg of SMCC was weighed and dissolved
with DMSO to prepare a 20 mM concentration. The following reaction
mix was prepared: FAM-oligo (20 .mu.L), NaHCO.sub.3 (20 .mu.L) and
SMCC (10 .mu.L). The reaction was incubated at room temperature for
20-30 min. The product was purified using a NAP-5 column. The same
protocol to purify the polymerase was used to purify the product.
The fractions were identified with UV-Vis (nanodrop). An aliquot of
the identified fraction was diluted with an equal amount of 250 mM
phosphate (pH 9) to measure the absorbance of FAM and use a molar
extinction coeffiecient of .epsilon..sub.510=58000/Mcm to determine
the FAM concentration. Analytical HPLC confirmed that the desired
product. However, an additional peak was also observed. This
corresponding peak is probably the product with a hydrolyzed
maleimide. Reducing the reaction pH and time minimizes the
production of this product.
[0093] The reaction is depicted in FIG. 6.
[0094] (c) Linking the Tether to the Polymerase
[0095] The tether was linked to the polymerase to form a tethered
polymerase as follows:
[0096] Reagents:
[0097] Bst (DTT treated and purified),
[0098] FAM-oligo-Maleimide (freshly prepared)
[0099] Exchange buffer (50 mM MES pH 6.0, 2 mM EDTA or 50 mM ACES
pH 6.8, 2 mM EDTA)
[0100] NaCl
[0101] Protocol:
[0102] Mix Bst and FAM-oligo-maleimide in exchange buffer in the
presence of 0.5-1 M NaCl.
[0103] Incubate reaction at 4.degree. C. for .about.12 h
[0104] The reaction is depicted in FIG. 7.
[0105] As depicted in FIG. 8, the use of varying salt
concentrations in the exchange buffer resulted in different product
yields, with the inclusion of NaCl pushing the reaction yield to
greater than 50%.
Example 2: Use of Tethered Polymerase for Nucleic Acid
Sequencing
[0106] The tethered polymerase of the prior Example, comprising a
Bst polymerase linked to an oligonucleotide tether, was used in an
ion-based sequencing reaction using the Ion Torrent PGM sequencing
platform (Life Technologies).
[0107] Both tethered polymerase and corresponding untethered
polymerase (control) were bound to to ssDNA beads and washed with
high salt. The sequencing primer was then hybridized to the ssDNA
template. Following hybridization, the beads were run in a standard
sequencing reaction on the Ion Torrent PGM Sequencing platform. As
depicted in FIG. 9 and FIG. 10 sequencing using high-salt washes
between successive extensions in the PGM platform was observed to
be supported by the tethered polymerase of Example 1, but not with
the corresponding (control) untethered polymerase. The use of high
salt buffers is advantageous because it increases polymerase
activity.
[0108] As depicted in FIG. 9, the Ion Sequencing Polymerase (in
untethered form) was observed to bind Ion Spheres (beads) with
ssDNA templatein the Ion W2 reagent (6.3 mM MgCl.sub.2, 13 mM NaCl,
0.01% Triton X-100, pH 7.5) with low affinity and could be washed
off with increasing ionic strength. At 1 M NaCl, 100% of Ion
Sequencing Polymerase 1.0 was washed off Ion Spheres.
[0109] As depicted in FIG. 10, the Ion Sequencing Polymerase
tethered with an oligo as described in Example 1, where the tether
immobilizes the tethered polymerase to the ssDNA template, was
observed to remain on the Ion beads under high salt conditions as
the tethered polymerase was not washed off in these conditions.
High salt is not expected to harm the polymerase except to wash it
from the ssDNA.
[0110] The performance of the Ion Sequencing Polymerase in both
tethered and untethered (control) forms in an Ion PGM Sequencing
system was measured and compared.
[0111] The untethered form of the Ion Sequencing Polymerase
exhibited no signal using high salt sequencing conditions (20 mM
MgCl2, 200 mM NaCl, 0.01% Triton X-100, pH 7.5), and no observable
sequencing reads were obtained.
[0112] In contrast, the tethered Ion Sequencing Polymerase
demonstrated robust sequencing performance using the high salt
sequencing conditions (20 mM MgCl2, 200 mM NaCl, 0.01% Triton
X-100, pH 7.5), where over 800,000 Q17 reads were obtained and over
30 key signals detected from a single sequencing reaction. As these
results indicate, PGM sequencing at high salt (>200 mM ionic
strength) is supported by the tethered Ion Sequencing Polymerase
but not by the untethered Ion Sequencing Polymerase.
Sequence CWU 1
1
21581PRTArtificial SequenceSynthetic amino acid 1Met Ala Lys Met
Ala Phe Thr Leu Ala Asp Arg Val Thr Glu Glu Met1 5 10 15Leu Ala Asp
Lys Ala Ala Leu Val Val Glu Val Val Glu Glu Asn Tyr 20 25 30His Asp
Ala Pro Ile Val Gly Ile Ala Val Val Asn Glu Arg Gly Arg 35 40 45Phe
Phe Leu Arg Pro Glu Thr Ala Leu Ala Asp Pro Gln Phe Val Ala 50 55
60Trp Leu Gly Asp Glu Thr Lys Lys Lys Ser Met Phe Asp Ser Lys Arg65
70 75 80Ala Ala Val Ala Leu Lys Trp Lys Gly Ile Glu Leu Cys Gly Val
Ser 85 90 95Phe Asp Leu Leu Leu Ala Ala Tyr Leu Leu Asp Pro Ala Gln
Gly Val 100 105 110Asp Asp Val Ala Ala Ala Ala Lys Met Lys Gln Tyr
Glu Ala Val Arg 115 120 125Pro Asp Glu Ala Val Tyr Gly Lys Gly Ala
Lys Arg Ala Val Pro Asp 130 135 140Glu Pro Val Leu Ala Glu His Leu
Val Arg Lys Ala Ala Ala Ile Trp145 150 155 160Glu Leu Glu Arg Pro
Phe Leu Asp Glu Leu Arg Arg Asn Glu Gln Asp 165 170 175Arg Leu Leu
Val Glu Leu Glu Gln Pro Leu Ser Ser Ile Leu Ala Glu 180 185 190Met
Glu Phe Ala Gly Val Lys Val Asp Thr Lys Arg Leu Glu Gln Met 195 200
205Gly Lys Glu Leu Ala Glu Gln Leu Gly Thr Val Glu Gln Arg Ile Tyr
210 215 220Glu Leu Ala Gly Gln Glu Phe Asn Ile Asn Ser Pro Lys Gln
Leu Gly225 230 235 240Val Ile Leu Phe Glu Lys Leu Gln Leu Pro Val
Leu Lys Lys Thr Lys 245 250 255Thr Gly Tyr Ser Thr Ser Ala Asp Val
Leu Glu Lys Leu Ala Pro Tyr 260 265 270His Glu Ile Val Glu Asn Ile
Leu His Tyr Arg Gln Leu Gly Lys Leu 275 280 285Gln Ser Thr Tyr Ile
Glu Gly Leu Leu Lys Val Val Arg Pro Asp Thr 290 295 300Lys Lys Val
His Thr Ile Phe Asn Gln Ala Leu Thr Gln Thr Gly Arg305 310 315
320Leu Ser Ser Thr Glu Pro Asn Leu Gln Asn Ile Pro Ile Arg Leu Glu
325 330 335Glu Gly Arg Lys Ile Arg Gln Ala Phe Val Pro Ser Glu Ser
Asp Trp 340 345 350Leu Ile Phe Ala Ala Asp Tyr Ser Gln Ile Glu Leu
Arg Val Leu Ala 355 360 365His Ile Ala Glu Asp Asp Asn Leu Met Glu
Ala Phe Arg Arg Asp Leu 370 375 380Asp Ile His Thr Lys Thr Ala Met
Asp Ile Phe Gln Val Ser Glu Asp385 390 395 400Glu Val Thr Pro Asn
Met Arg Arg Gln Ala Lys Ala Val Asn Phe Gly 405 410 415Ile Val Tyr
Gly Ile Ser Asp Tyr Gly Leu Ala Gln Asn Leu Asn Ile 420 425 430Ser
Arg Lys Glu Ala Ala Glu Phe Ile Glu Arg Tyr Phe Gln Ser Phe 435 440
445Pro Gly Val Lys Arg Tyr Met Glu Asn Ile Val Gln Glu Ala Lys Gln
450 455 460Lys Gly Tyr Val Thr Thr Leu Leu His Arg Arg Arg Tyr Leu
Pro Asp465 470 475 480Ile Thr Ser Arg Asn Phe Asn Val Arg Ser Phe
Ala Glu Arg Met Ala 485 490 495Met Asn Thr Pro Ile Gln Gly Ser Ala
Ala Asp Ile Ile Lys Lys Ala 500 505 510Met Ile Asp Leu Asn Ala Arg
Leu Lys Glu Glu Arg Leu Gln Ala His 515 520 525Leu Leu Leu Gln Val
His Asp Glu Leu Ile Leu Glu Ala Pro Lys Glu 530 535 540Glu Met Glu
Arg Leu Cys Arg Leu Val Pro Glu Val Met Glu Gln Ala545 550 555
560Val Thr Leu Arg Val Pro Leu Lys Val Asp Tyr Arg Tyr Gly Ser Thr
565 570 575Trp Tyr Asp Ala Lys 580249DNAArtificial
SequenceSynthetic DNA 2aaaaaaaaaa aaaaaaaaaa gactgccaag gcacacaggg
gataggaaa 49
* * * * *