U.S. patent number 6,911,535 [Application Number 10/050,277] was granted by the patent office on 2005-06-28 for biomolecule/polymer conjugates.
This patent grant is currently assigned to Solvlink Biosciences. Invention is credited to David A. Schwartz.
United States Patent |
6,911,535 |
Schwartz |
June 28, 2005 |
Biomolecule/polymer conjugates
Abstract
The present invention is directed to methods for immobilizing
natural or synthetic biomolecules to surfaces. The methods comprise
covalently linking the natural or synthetic biomolecule to a mono-
or bi-functional polymer and covalently and/or electrostatically
immobilizing the biomolecule/polymer conjugate to an unmodified or
modified surface. The biomolecule is an oligonucleotide, a
polynucleotide, a protein, a glycoprotein, a peptide or a
carbohydrate that has been modified to incorporate a single or
plurality of nucleophilic groups. These groups comprise an
aliphatic or aromatic amino, thiol, hydrazine, thiosemicarbazide,
hydrazide, thiocarbazide, carbazide, aminooxy, a derivative of
2-hydrazinopyridine or aminoxyacetic acid or a single or plurality
of electrophilic groups. The electrophilic groups comprise an
aliphatic or aromatic aldehyde, ketone, epoxide, isocyanate,
isothiocyanate, succinimidyl ester or cyanuric chloride or a
linkable aromatic aldehyde or ketone. The surface has been modified
to possess either neutral, cationic or anionic groups or a
combination neutral, anionic and/or cationic moieties.
Inventors: |
Schwartz; David A. (Encinitas,
CA) |
Assignee: |
Solvlink Biosciences (San
Diego, CA)
|
Family
ID: |
26728092 |
Appl.
No.: |
10/050,277 |
Filed: |
January 15, 2002 |
Current U.S.
Class: |
530/402; 435/180;
530/395; 530/815; 530/816; 536/22.1 |
Current CPC
Class: |
C07D
207/452 (20130101); C07F 7/1804 (20130101); C07D
213/82 (20130101); C07D 401/12 (20130101); C07K
1/1077 (20130101); C07K 14/005 (20130101); C07K
14/315 (20130101); C07K 14/52 (20130101); C07K
14/70503 (20130101); C07K 16/2863 (20130101); C08G
65/329 (20130101); C08G 65/336 (20130101); C12N
9/96 (20130101); G01N 33/54353 (20130101); A61K
47/645 (20170801); A61K 47/61 (20170801); A61K
47/62 (20170801); A61K 47/643 (20170801); A61K
47/6801 (20170801); C07D 207/46 (20130101); C07K
2319/00 (20130101); C08L 2203/02 (20130101); C12N
2710/16722 (20130101); Y10S 530/815 (20130101); Y10S
530/816 (20130101) |
Current International
Class: |
A61K
47/48 (20060101); C07F 7/18 (20060101); C07K
1/00 (20060101); C07D 401/00 (20060101); C07K
14/005 (20060101); C07K 14/06 (20060101); C12N
9/96 (20060101); C07K 1/107 (20060101); C07F
7/00 (20060101); C08G 65/00 (20060101); C07D
207/00 (20060101); C07D 207/46 (20060101); C07D
401/12 (20060101); C08G 65/336 (20060101); C07D
207/452 (20060101); C07K 14/705 (20060101); C08G
65/329 (20060101); C07D 213/00 (20060101); C07K
14/435 (20060101); C07K 14/315 (20060101); C07K
16/18 (20060101); C07K 14/52 (20060101); C07K
14/195 (20060101); C07K 16/28 (20060101); C07D
213/82 (20060101); G01N 33/543 (20060101); C07K
001/00 (); C07K 017/00 (); C08H 001/00 (); C12N
011/08 (); C07H 021/00 () |
Field of
Search: |
;530/402,815,816,395
;435/180,181 ;536/22.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Naff; David M.
Attorney, Agent or Firm: David B. Waller & Assoc.
Parent Case Text
RELATED APPLICATIONS
This application is related to co-owned U.S. utility application
entitled "TRIPHOSPHATE OLIGONUCLEOTIDE MODIFICATION REAGENTS AND
USES THEREOF", to Schwartz et al., filed Aug. 1, 2000, patent
application Ser. No.: 09/630,627, now U.S. Pat. No. 6,686,461 B1.
This application is related to co-owned U.S. utility application,
entitled "FUNCTIONAL BIOPOLYMER MODIFICATION REAGENTS AND USES
THEREOF", to Schwartz et al., filed Aug. 1, 2000, patent
application Ser. No.: 09/630,060 and to patent application Ser. No.
09/815,978, entitled "HYDRAZINE-BASED AND CARBONYL-BASED
BIFUNCTIONAL CROSSLINKING REAGENTS", to Schwartz, filed Mar. 22,
2001, now U.S. Provisional Application Ser. No. 60/191,186 filed
Mar. 22, 2000. The above-referenced applications are incorporated
herein by reference in their entirety.
Claims
We claim:
1. A biomolecule/polymer conjugate wherein said biomolecule is
conjugated to said polymer by a hydrazone bond, wherein said
biomolecule is a protein, a glycoprotein or a peptide and wherein
said polymer is a poly-L-lysine, poly-L-ornithine or
polyethyleneimine.
2. A biomolecule/polymer conjugate wherein said biomolecule is
conjugated to said polymer by a oxime bond, wherein said
biomolecule is a protein, a glycoprotein or a peptide and wherein
said polymer is a poly-L-lysine, poly-L-ornithine or
polyethyleneimine.
Description
BACKGROUND OF THE INVENTION
Proteomic and genomic microarray technology allows researchers to
perform multiple experiments simultaneously, "multiplexing", as
hundreds to thousands of proteins or genes are immobilized on a
surface and exposed to target ligands to determine the proclivity
of the immobilized protein for its ligand or for an oligonucleotide
to hybridize to its complement in competition with other targets.
The production of both protein and poly/oligonucleotide microarrays
is currently very inefficient due to the sub-optimal properties of
bioconjugation methods available to immobilize protein and
poly/oligonucleotides. This invention describes novel, efficient
and simple methods for immobilization of oligonucleotides, proteins
and other biomolecules to surfaces.
Poly/oligonucleotide immobilization: There are two technologies
being employed to produce oligonucleotide microarrays (see page 6,
DNA Microarrays, M. Schena, Oxford University Press. Oxford,
England OX2 6DP). One is direct synthesis of oligonucleotides on
surfaces via photolithography (www.affymetrix.com, Affymetrix,
Inc., Sunnyvale, Calif.) or use of reagent-directed devices such as
inkjet spotters. A second method is covalent attachment of an
oligonucleotide modified to possess a reactive functionality
bonding to its reactive partner that has been immobilized on the
surface.
The direct synthesis method allows for very dense microarrays, i.e.
up to 250K oligos/sq. centimeter on a slide. This method however
has significant shortcomings in that failure sequences remain on
the chip, yields of each coupling step is low, .about.95%, leading
to both poor yield of full length product and the inability to
produce oligonucleotides longer than 20-25 mers. The presence of
failure sequences could also lead to false positive hybridization
results. Also due to the length of the sequence immobilized on the
surface, <25 mers, multiple sequences to each gene must be
immobilized on the surface to overcome the inherent wide difference
in Tm, melting temperatures, of 25 mers. Sequences must be designed
carefully and a deconvoluting step is required in the analysis of
the results.
Covalent attachment methods also have limitations due to poor
stability of surface chemistry immobilization of the first reaction
partner also due to the small spotting volumes, .about.1 nL, there
is fast drying of the spot which does not allow sufficient time for
covalent chemistry to occur. Poor water stability of one or both of
the reactive components of the bioconjugation couples such as
maleimido/thiol, succinimidyl ester/amino will also significantly
reduce the efficiency of coupling.
Preparation of PCR- and cDNA-based arrays is routinely accomplished
by electrostatic immobilization of the anionic polynucleotide to a
surface that has been modified to incorporate cationic species. The
two major cationic-based slides are produced from aminopropylsilane
or cationic polymers such as poly-1-lysine or polyethyleneimine.
While arrays produced by electrostatic immobilization on cationic
surface gives a product that has yielded good results, the
immobilization efficiency of the polynucleotides is poor <20%.
This poor yield is of importance due to the cost of producing PCR
or cDNA products. Also the reproducibility of slides is
capricious.
The optimal polynucleotide microarray for gene expression analysis
would be an oligonucleotide-based microarray consisting of
oligonucleotides of 60-80 mer oligonucleotides immobilized on
surfaces. This type of array would be preferred over cDNA arrays as
the cost of producing 60-80 mer oligonucleotides would be far
cheaper, more easily reproducible, automatable and oligonucleotides
can be more easily scaled up to produce larger- almost unlimited-
quantities of product. The optimal length of oligonucleotides for
SNP (single nucleotide polymorphism) analysis on microarrays is
20-25 oligonucleotides while oligonucleotides of 60-80 bases are
preferred for gene expression analysis.
The power of the microarray is the massive number of experiments
that can be performed simultaneously in a small area requiring
minimal amounts of reagents and samples. However to prepare 10-100
.mu.m.sup.2 spots incorporating the biomolecule small volumes
(<10 nL) are spotted. These small volumes evaporate too quickly
and therefore reaction time for covalent chemistry to occur between
the biomolecule and the surface is insufficient leading to
extremely poor yields and poor reproducibility. Devices to retain
humidity during spotting have been developed but do not solve the
evaporation problem completely.
In the case of direct oligonucleotide immobilization there is
single point attachment between the oligonucleotide and the
surface. The reaction kinetics in a two-phase system is slower than
solution phase kinetics. The formation of a covalent bond in water
is slow as well as the competing hydrolysis of the electrophilic
partner in the reaction. The competing electrostatic interaction
between the oligonucleotide and a glass surface will also interfere
with the covalent chemistry.
Protein Immobilization: Current methods to immobilize proteins to
surface include direct binding of proteins to poly-cationic glass
surfaces (B. B. Haab, M. J. Dunham and P. O. Brown, Genome Biol. 1,
(2000)). This method functioned well for immobilization of families
of proteins such as antibodies but when different proteins or
antigens were immobilized the binding was not as efficient or
consistent.
Macbeath and Schreiber (Science 289, 1760 (2000)) also describe
direct immobilization of protein on modified surfaces. They
directly immobilized proteins on aldehyde coated glass surfaces via
multiple imine bonds. They also describe pre-coating glass slides
with bovine serum albumin (BSA) and subsequently reacting the amino
groups on the protein with homo-bifunctional succinimidyl esters.
The succinimidyl groups on the protein are subsequently used to
react with amino groups on the desired protein to form an
immobilized conjugate.
Thus, due to the limitations of currently available methods as
described above, there is a need for efficient methods for
producing both protein and poly/oligonucleotide-based microarrays.
Therefore, it is an object herein to provide ternary systems based
on both novel bifunctional polymers and biomolecule/bifunctional
polymer conjugates and methods to immobilize these conjugates on
modified and unmodified surfaces for the efficient and reproducible
production of both polynucleotide and oligonucleotide-based
microarrays.
SUMMARY OF THE INVENTION
Ternary systems B/P/S comprised of biomolecule (B)/polymer (P)
conjugates, B/P, linked electrostatically and/or covalently to
modified and unmodified surfaces (S) to produce biomolecule
microarrays are provided. Methods to produce both polynucleotide
and oligonucleotide arrays as well as protein and peptide arrays
are given. The general scheme for these systems is schematically
represented in FIG. 1.
The first component of the ternary B/P/S is a biomolecule modified
with a reactive moiety that does not interfere with the function of
the biomolecule. The second component is a polymer modified with a
reactive moiety that reacts with the reactive moiety on the
biomolecule to form a covalent linkage. The polymer may also
possess a third type of reactive moiety that does not react with
the reactive moiety on the biomolecule. The third component is a
solid surface such as silica-based surfaces, i.e. glass, silica
beads or fibre optic bundles. Other third component solids surfaces
include plastic, latex beads, membranes such as cellulose or
nitrocellulose or metal such as gold that is unmodified or modified
with the same reactive moiety as incorporated on the biomolecule.
The surface may also possess a reactive moiety that forms a
covalent linkage with the second reactive component on the
polymer.
The optimal bioconjugation reaction chemistry for enablement of
this invention comprises use of reactive moieties following
incorporation onto any of the components are stable and form stable
covalent linkages with good kinetics. This invention provides such
chemistries and methods.
A major advantage of the ternary BPS systems is that covalent
attachment of the biomolecule to the polymer occurs in solution
without reaction time limitations. In contrast to direct single
point attachment of the biomolecule to the surface this method
provides multiple point covalent attachment points between the
modified polymer and the surface as well as electrostatic
interactions. This method produces higher yielding more stable
immobilzation and better reproducibility than the previous
described methods.
Charged and uncharged polymers modified to incorporate reactive
functionalities are provided for covalent attachment of
biomolecules, especially poly/oligonucleotides and
peptides/proteins. Methods to conjugate these biomolecules to the
polymers are provided as well as methods for electrostatic and/or
covalent attachment to modified and unmodified surfaces are
provided.
The most direct, simple and efficient preparation of an
oligonucleotide/polymer conjugate would have the following
properties: (1) direct incorporation of the first reactive
component of the bioconjugate couple directly on the
oligonucleotide or peptide during solid phase synthesis without the
requirement for any post-synthetic activation, (2) indefinite
stability of both reactive components of the bioconjugate couple
following incorporation on either the biomolecule or the polymer,
(3) good kinetics of covalent bond formation between the modified
oligonucleotide and the modified polymer without the need for a
reagent-mediated reaction or competing reactions on the
bioconjugate couple moieties such as hydrolysis, (4) simple
incorporation of the first reactive component of the bioconjugate
couple on the surface of choice, (5) fast kinetic of immobilization
of the biomolecule/polymer conjugate on the modified surface, (6)
long term stability of the biomolecule/polymer conjugate on the
surface.
One enablement of this invention describes and demonstrates the
immobilization of oligonucleotide/polymer conjugates to modified
and unmodified glass surfaces in a more efficient manner than
current direct spotting of modified oligonucleotides on modified
surfaces.
The oligonucleotide is modified to incorporate the first reactive
component of a bioconjugate couple on the 3' or 5' end of the
oligonucleotide. Alternatively the first reactive component can be
incorporated on any internal position of the oligonucleotides.
Internal positions include but are not limited to a position on the
base of the oligonucleotide such as the 5 position of a pyrimidine
or on the 2' position on the sugar. A polymer is modified to
incorporate the second reactive component of the bioconjugate
couple. Subsequently the modified biomolecule is reacted with the
modified polymer to form the desired biomolecule/polymer
conjugate.
A preferred enablement of this invention requires incorporation of
the first reactive component of the bioconjugate couple or a
reactive moiety with similar reactivity on the surface. For example
if the first reactive component is an electrophile any
electrophilic reactive moiety may be incorporated on the surface
that forms a covalent bond with the second reactive component on
the polymer. A further preferred embodiment is immobilization of
the oligonucleotide/polymer conjugate on a silica-based surface.
The most preferred silica-based surfaces are glass, fiber optic
wires and silica-based beads. Therefore incorporation of the first
reactive component of the bioconjugate couple or a reactive
component of similar reactivity on the surface is required.
The preferred bioconjugate couple to be employed in the enablement
of the ternary oligonucleotide/polymer/surface system is the
hydrazine/carbonyl or aminooxy/carbonyl couple (see FIG. 2).
Oligonucleotide monomers containing hydrazino, oxyamino, or
carbonyl groups that can be incorporated into an oligonucleotide
chain during solid phase oligonucleotide synthesis are provided.
Methods for immobilization and conjugation of biopolymer first
components, particularly oligonucleotides, containing hydrazino,
oxyamino, or carbonyl modifications are provided. The resulting
first reactive components can then be used for any purpose for
which oligonucleotides are used. They are particularly suitable for
conjugation to a second reactive component for immobilization on a
surface. The monomers provided herein are readily incorporated into
oligonucleotide chains, hence can be used in any application that
involves or uses an oliogonucleotide
Ternary B/P/S systems wherein the biopolymer is a polynucleotide
can also be prepared in a variety of ways as described in the
Detailed Description section. In short, a single or a plurality of
functional moieties can be incorporated either terminally or
internally on the polynucleotide. The modified polynucleotide is
conjugated to a polymer modified to incorporate the second reactive
component of a bioconjugation couple and the polynucleotide/polymer
conjugate is immobilized on a surface in an identical manner
described above for oligonucleotides.
Also described are methods for preparing protein/polymer/surface
and peptide/polymer/surface ternary systems. For proteins and
peptides one component of a bioconjugate couple is incorporated on
the protein or peptide and conjugated to a polymer modified to
incorporate the second component of a bioconjugate couple. The
conjugate is immobilized on a surface in an identical manner
described for oligonucleotides wherein the conjugate is reacted on
a surface possessing a functional group that forms a covalent
and/or electrophilic bond to the reactive moiety on the
polymer.
DESCRIPTION OF THE FIGURES
FIG. 1: General scheme outlining the protocol to prepare a BPS
system.
FIG. 2: Generic hydrazine (aminooxyl/carbonyl bioconjugation couple
that is described for the preparation of BPS system.
FIG. 3: Structures of the hydrazine, aminooxy and carbonyl moieties
that are suitable for use as partners in the hydrazine
(aminooxyl/carbonyl bioconjugation couple.
FIG. 4: Scheme used to prepare both HyNic-modified poly-l-lysine
and conjugation to a 5'-aldehyde-modified oligonucleotide.
FIG. 5: Matrix experiment (see Example 1) demonstrating the
covalent nature of the immobilization of a 5'-aldehyde
oligo//HyNic/poly-l-lysine (polyK) conjugate on an aldehyde
modified glass slide following hybidization to its fluorescent
complement.
FIG. 6: Matrix experiment (see Example 2) demonstrating the
covalent nature of the immobilization of a 5'-hydrazino
oligo//SCHO/poly-l-lysine (polyK) conjugate on an amino modified
glass slide following hybridization to its fluorescent
complement.
FIG. 7: Schematic demonstrating the steps in the preparation of a
polynucleotide/polymer/surface system (see Example 7).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Definitions
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 this invention belongs. All patents,
patent applications and publications referred to throughout the
disclosure herein are incorporated by reference in their entirety.
In the event that there are a plurality of definitions for a term
herein, those in this section prevail.
Polynucleotide is defined as DNA or RNA>50 bases derived from
natural sources, PCR or reverse transcriptase methods
Oligonucleotides are defined as any combination or DNA, RNA or PNA
(peptide nucleic acids) sequences<100 bases synthesized via
standard solid phase oligonucleotide methods incorporating natural
or unnatural bases and natural or unnatural sugars.
A "probe" is the immobilized biomolecule such as nucleic acid with
known sequence or protein or peptide or carbohydrate.
A "target" is the free biomolecule/sample whose identity/abundance
is being detected.
As used herein polymers include, but are not limited to natural and
synthetic polymers. As used herein polymers can be derived from
biological monomers such as amino acids, nucleotides or
carbohydrates or any combination thereof. As used herein polymers
may be neutral, cationic or anionic or any combination thereof.
As used herein synthetic polymers may be modified to incorporate
reactive moieties used in bioconjugate chemistry.
As used herein bioconjugate couples include, but are not limited to
maleimido/thiol, a-bromoacetamido/thiol, succinimidyl ester/amino,
avidin/biotin, hydrazine/carbonyl and pyridyldisulfide/thiol.
As used herein, "hydrazino groups" include, but are not limited to,
hydrazines, hydrazides, semicarbazides, carbazides,
thiosemicarbazides, thiocarbazides, hydrazine carboxylates and
carbonic acid hydrazines (see, e.g., FIG. 1).
As used herein, hydrazone linkages include, but are not limited to,
hydrazones, acyl hydrazones, semicarbazones, carbazones,
thiosemicarbazones, thiocarbazones, hydrazone carboxylates and
carbonic acid hydrazones.
As used herein, an oxyamino group has the formula --O--NH.sub.2. An
oxime has the formula --O--N.dbd.R.
As used herein, a protected hydrazino or a protected oxyamino group
refers to a hydrazino or oxyamino group that has been derivatized
as a salt of the hydrazino or oxyamino group, including but not
limited to, mineral acids salts, such as but not limited to
hydrochlorides and sulfates, and salts of organic acids, such as
but not limited to acetates, lactates, malates, tartrates,
citrates, ascorbates, succinates, butyrates, valerates and
fumarates; or with any amino or hydrazino protecting group known to
those of skill in the art (see, e.g., Greene et al. (1999)
Protective Groups in Organic Synthesis (3rd Ed.) (J. Wiley Sons,
Inc.)). Preferred amino and hydrazino protecting groups herein
include, but are not limited to, amino or hydrazino protecting
groups useful in the synthesis of oligonucleotides, more preferably
monomethoxytrityl (MMT), dimethoxytrityl (DMT),
9-fluorenylmethoxycarbonyl (FMOC), acetyl, trifluoroacetyl,
benzoyl, or a hydrazone or oxime that is cleaved under mild acidic
conditions (e.g., 100 mM acetate, pH 4.5-5.5) including, but not
limited to, a hydrazone or oxime formed from a lower aliphatic
aldehyde or ketone, preferably from acetone, propanal,
cyclohexanone or 2-butanone.
As used herein surfaces including, but not limited to, plastics
such as molded plastic or latex beads, modified or unmodified
glass, metals such as gold or silver.
As used herein, an oligonucleotide is a nucleic acid, including,
but not limited to, a ribonucleic acid (RNA), a deoxyribonucleic
acid (DNA), and analogs thereof such as a protein nucleic acid
(PNA), of any length, including chromosomes and genomic material,
such as PCR products or sequencing reaction products, preferably
DNA including double and single stranded forms. Single stranded
forms of the oligonucleotides are also provided.
As used herein, a conjugate is a compound containing two components
covalently linked together. For example, a first component, e.g.,
an oligonucleotide, is conjugated through a covalent hydrazone
linkage to a second component, as defined herein, to form a
conjugate.
As used herein, carbonyl derivatives include, but are not limited
to, ketones and aldehydes.
As used herein, complementary reactive groups are those that, when
reacted together, form a covalent linkage, including, but not
limited to, a hydrazone or oxime linkage. Thus, a hydrazino group,
as defined herein, is complementary to a carbonyl derivative. An
oxyamino group is also complementary to a carbonyl derivative.
As used herein, "phosphorous based coupling group" refers to any
phosphorous-containing group known to those of skill in the art to
be useful in oligonucleotide synthesis including, but not limited
to, phosphorodithioate, phosphorothioate, phosphoramidate,
phosphonate, phosphodiester, phosphotriester, thiophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester or
boranophosphate. As is appreciated by those of skill in the art, a
number of chemistries, including, but not limited to,
phosphoramidite, phosphonamide, H-phosphonate and phosphotriester
chemistries, have been developed for the stepwise solid phase
synthesis of oligonucleotides (DNA or RNA) and modified analogs
(i.e., 2' modified RNA, methylphosphonates, 3'-5' phosphoramidites,
and phosophorothioates). All phosphorous based coupling groups
known to those of skill in the art are contemplated for use in the
reagents and methods provided herein. See, e.g., Glen Research
Catalog of Products for DNA Research, Glen Reserach, Sterling, Va.
Exemplary phosphorous based coupling groups herein include
.beta.-cyanoethyl-N,N-diisopropylphosphoramidites.
As used herein, a biopolymer is any compound found in nature, or
derivatives thereof, made up of monomeric units. Biopolymers
include, but are not limited to, oligonucleotides, peptides,
peptide nucleic acids (PNAs), glycoproteins and oligosaccharides.
Thus, the monomeric units include, but are not limited to,
nucleotides, nucleosides, amino acids, PNA monomers,
monosaccharides, and derivatives thereof.
As used herein, a macromolecule refers to a molecule of colloidal
size (i.e., of high molecular weight), including, but not limited
to, proteins, polynucleic acids, polysaccharides and
carbohydrates.
As used herein, a reporter molecule refers to a molecule, such as
an enzyme or indicator, which is capable of generating a detectable
signal (e.g., by colorimetric, chemiluminescent, bioluminescent,
fluorescent, or potentiometric means) when contacted with a
suitable substrate under appropriate reaction conditions. Exemplary
reporter enzymes include, but are not limited to, alkaline
phosphatase, horseradish peroxidase, .beta.-galactosidase, aryl
esterase, sulfatase and urease.
As used herein, a nucleobase is a heterocyclic moiety that is found
in naturally occurring oligonucleotides, including ribonucleic
acids (RNA) and deoxyribonucleic acids (DNA), and analogs thereof,
including deaza analogs. Preferred nucleobases include, but are not
limited to, cytosines, uracils, adenines, guanines and thymines,
and analogs thereof including deaza analogs.
As used herein, a fluorophore refers to a fluorescent compound.
Fluorescence is a physical process in which light is emitted from
the compound following absorption of radiation. Generally, the
emitted light is of lower energy and longer wavelength than that
absorbed. Preferred fluorophores herein are those whose
fluorescence can be detected using standard techniques.
As used herein, a derivative of a compound includes a salt, ester,
enol ether, enol ester, solvate or hydrate thereof that can be
prepared by those of skill in this art using known methods for such
derivatization. Salts include, but are not limited to, amine salts,
such as but not limited to N,N'-dibenzylethylenediamine,
chloroprocaine, choline, ammonia, diethanolamine and other
hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine,
N-benzylphenethylamine,
1-para-chlorobenzyl-2-pyrrolidin-1'-ylmethylbenzimidazole,
diethylamine and other alkylamines, piperazine and
tris(hydroxymethyl)aminomethane; alkali metal salts, such as but
not limited to lithium, potassium and sodium; alkali earth metal
salts, such as but not limited to barium, calcium and magnesium;
transition metal salts, such as but not limited to zinc; and other
metal salts, such as but not limited to sodium hydrogen phosphate
and disodium phosphate; and also including, but not limited to,
salts of mineral acids, such as but not limited to hydrochlorides
and sulfates; and salts of organic acids, such as but not limited
to acetates, lactates, malates, tartrates, citrates, ascorbates,
succinates, butyrates, valerates and fumarates. Esters include, but
are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl,
aralkyl, heteroaralkyl, cycloalkyl and heterocyclyl esters of
acidic groups, including, but not limited to, carboxylic acids,
phosphoric acids, phosphinic acids, sulfonic acids, sulfinic acids
and boronic acids. Enol ethers include, but are not limited to,
derivatives of formula C.dbd.C(OR) where R is hydrogen, alkyl,
alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl,
cycloalkyl ar heterocyclyl. Enol esters include, but are not
limited to, derivatives of formula C.dbd.C(OC(O)R) where R is
hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl,
heteroaralkyl, cycloalkyl ar heterocyclyl. Solvates and hydrates
are complexes of a compound with one or more solvent or water
molecule, preferably 1 to about 100, more preferably 1 to about 10,
most preferably one to about 2, 3 or 4, solvent or water
molecules.
It is to be understood that the compounds provided herein can
contain chiral centers. Such chiral centers can be of either the
(R) or (S) configuration, or can be a mixture thereof. Thus, the
compounds provided herein may be enantiomerically pure, or be
stereoisomeric or diastereomeric mixtures. In the case of amino
acid residues, such residues may be of either the L- or D-form. The
preferred configuration for naturally occurring amino acid residues
is L.
As used herein, alkyl, alkenyl and alkynyl carbon chains, if not
specified, contain from 1 to 20 carbons, preferably 1 to 16
carbons, and are straight or branched. Alkenyl carbon chains of
from 2 to 20 carbons preferably contain 1 to 8 double bonds, and
the alkenyl carbon chains of 1 to 16 carbons preferably contain 1
to 5 double bonds. Alkynyl carbon chains of from 2 to 20 carbons
preferably contain 1 to 8 triple bonds, and the alkynyl carbon
chains of 2 to 16 carbons preferably contain 1 to 5 triple bonds.
Exemplary alkyl, alkenyl and alkynyl groups herein include, but are
not limited to, methyl, ethyl, propyl, isopropyl, isobutyl,
n-butyl, sec-butyl, tert-butyl, isopentyl, neopentyl, tert-penytyl
and isohexyl. The alkyl, alkenyl and alkynyl groups, unless
otherwise specified, can be optionally substituted, with one or
more groups, preferably alkyl group substituents that can be the
same or different. As used herein, lower alkyl, lower alkenyl, and
lower alkynyl refer to carbon chains having less than about 6
carbons. As used herein, "alk(en)(yn)yl" refers to an alkyl group
containing at least one double bond and at least one triple
bond.
As used herein, an "alkyl group substituent" includes halo,
haloalkyl, preferably halo lower alkyl, aryl, hydroxy, alkoxy,
aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio,
carboxy alkoxycarbonyl, oxo and cycloalkyl.
As used herein, "aryl" refers to cyclic groups containing from 5 to
19 carbon atoms. Aryl groups include, but are not limited to
groups, such as fluorenyl, substituted fluorenyl, phenyl,
substituted phenyl, naphthyl and substituted naphthyl, in which the
substituent is lower alkyl, halogen, or lower alkoxy.
As used herein, an "aryl group substituent" includes alkyl,
cycloalkyl, cycloalkylalkyl, aryl, heteroaryl optionally
substituted with 1 or more, preferably 1 to 3, substituents
selected from halo, halo alkyl and alkyl, aralkyl, heteroaralkyl,
alkenyl containing 1 to 2 double bonds, alkynyl containing 1 to 2
triple bonds, alk(en)(yn)yl groups, halo, pseudohalo, cyano,
hydroxy, haloalkyl and polyhaloalkyl, preferably halo lower alkyl,
especially trifluoromethyl, formyl, alkylcarbonyl, arylcarbonyl
that is optionally substituted with 1 or more, preferably 1 to 3,
substituents selected from halo, halo alkyl and alkyl,
heteroarylcarbonyl, carboxy, alkoxycarbonyl, aryloxycarbonyl,
aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,
arylaminocarbonyl, diarylaminocarbonyl, aralkylaminocarbonyl,
alkoxy, aryloxy, perfluoroalkoxy, alkenyloxy, alkynyloxy,
arylalkoxy, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl,
arylaminoalkyl, amino, alkylamino, dialkylamino, arylamino,
alkylarylamino, alkylcarbonylamino, arylcarbonylamino, azido,
nitro, mercapto, alkylthio, arylthio, perfluoroalkylthio,
thiocyano, isothiocyano, alkylsulfinyl, alkylsulfonyl,
arylsulfinyl, arylsulfonyl, aminosulfonyl, alkylaminosulfonyl,
dialkylaminosulfonyl and arylaminosulfonyl.
As used herein, "aralkyl" refers to an alkyl group in which one of
the hydrogen atoms of the alkyl is replaced by an aryl group.
As used herein, "heteroaralkyl" refers to an alkyl group in which
one of the hydrogen atoms of the alkyl is replaced by a heteroaryl
group.
As used herein, "cycloalkyl" refers to a saturated mono- or
multicyclic ring system, preferably of 3 to 10 carbon atoms, more
preferably 3 to 6 carbon atoms; cycloalkenyl and cycloalkynyl refer
to mono- or multicyclic ring systems that respectively include at
least one double bond and at least one triple bond. Cycloalkenyl
and cycloalkynyl groups can preferably contain 3 to 10 carbon
atoms, with cycloalkenyl groups more preferably containing 4 to 7
carbon atoms and cycloalkynyl groups more preferably containing 8
to 10 carbon atoms. The ring systems of the cycloalkyl,
cycloalkenyl and cycloalkynyl groups can be composed of one ring or
two or more rings which can be joined together in a fused, bridged
or spiro-connected fashion, and can be optionally substituted with
one or more alkyl group substituents. "Cycloalk(en)(yn)yl" refers
to a cylcoalkyl group containing at least one double bond and at
least one triple bond.
As used herein, "heteroaryl" refers to a monocyclic or multicyclic
ring system, preferably of about 5 to about 15 members where one or
more, more preferably 1 to 3 of the atoms in the ring system is a
heteroatom, that is, an element other than carbon, for example,
nitrogen, oxygen and sulfur atoms. The heteroaryl can be optionally
substituted with one or more, preferably 1 to 3, aryl group
substituents. The heteroaryl group can be optionally fused to a
benzene ring. Exemplary heteroaryl groups include, for example,
furyl, imidazinyl, pyrrolidinyl, pyrimidinyl, tetrazolyl, thienyl,
pyridyl, pyrrolyl, N-methylpyrrolyl, quinolinyl and isoquinolinyl,
with pyridyl and quinolinyl being preferred.
As used herein, "heterocyclic" refers to a monocyclic or
multicyclic ring system, preferably of 3 to 10 members, more
preferably 4 to 7 members, even more preferably 5 to 6 members,
where one or more, preferably 1 to 3 of the atoms in the ring
system is a heteroatom, that is, an element other than carbon, for
example, nitrogen, oxygen and sulfur atoms. The heterocycle can be
optionally substituted with one or more, preferably 1 to 3 aryl
group substituents. Preferred substituents of the heterocyclic
group include hydroxy, amino, alkoxy containing 1 to 4 carbon
atoms, halo lower alkyl, including trihalomethyl, such as
trifluoromethyl, and halogen. As used herein, the term heterocycle
includes reference to heteroaryl.
As used herein, the nomenclature alkyl, alkoxy, carbonyl, etc. are
used as is generally understood by those of skill in this art. For
example, as used herein alkyl refers to saturated carbon chains
that contain one or more carbons; the chains are straight or
branched or include cyclic portions or be cyclic.
As used herein, alicyclic refers to aryl groups that are
cyclic.
For purposes herein, where the number of any given substituent is
not specified (e.g., "haloalkyl"), there can be one or more
substituents present. For example, "haloalkyl" includes one or more
of the same or different halogens. As another example, "C.sub.1-3
alkoxyphenyl" can include one or more of the same or different
alkoxy groups containing one, two or three carbons.
As used herein, "halogen" or "halide" refers to F, Cl, Br or I.
As used herein, pseudohalides are compounds that behave
substantially similar to halides. Such compounds can be used in the
same manner and treated in the same manner as halides (X.sup.-, in
which X is a halogen, such as Cl or Br). Pseudohalides include, but
are not limited to, cyanide, cyanate, thiocyanate, selenocyanate,
trifluoromethoxy, trifluoromethyl and azide.
As used herein, "haloalkyl" refers to a lower alkyl radical in
which one or more of the hydrogen atoms are replaced by halogen
including, but not limited to, chloromethyl, trifluoromethyl,
1-chloro-2-fluoroethyl and the like.
As used herein, "haloalkoxy" refers to RO-- in which R is a
haloalkyl group.
As used herein, "sulfinyl" or "thionyl" refers to --S(O)--. As used
herein, "sulfonyl" or "sulfuryl" refers to --S(O).sub.2 --. As used
herein, "sulfo" refers to --S(O).sub.3 --.
As used herein, "carboxy" refers to a divalent radical,
--C(O)O--.
As used herein, "aminocarbonyl" refers to --C(O)NH.sub.2.
As used herein, "alkylaminocarbonyl" refers to --C(O)NHR in which R
is hydrogen or alkyl, preferably lower alkyl. As used herein
"dialkylaminocarbonyl" as used herein refers to --C(O)NR'R in which
R' and R are independently selected from hydrogen or alkyl,
preferably lower alkyl; "carboxamide" refers to groups of formula
--NR'COR.
As used herein, "diarylaminocarbonyl" refers to --C(O)NRR' in which
R and R' are independently selected from aryl, preferably lower
aryl, more preferably phenyl.
As used herein, "aralkylaminocarbonyl" refers to --C(O)NRR' in
which one of R and R' is aryl, preferably lower aryl, more
preferably phenyl, and the other of R and R' is alkyl, preferably
lower alkyl.
As used herein, "arylaminocarbonyl" refers to --C(O)NHR in which R
is aryl, preferably lower aryl, more preferably phenyl.
As used herein, "alkoxycarbonyl" refers to --C(O)OR in which R is
alkyl, preferably lower alkyl.
As used herein, "aryloxycarbonyl" refers to --C(O)OR in which R is
aryl, preferably lower aryl, more preferably phenyl.
As used herein, "alkoxy" and "alkylthio" refer to RO-- and RS--, in
which R is alkyl, preferably lower alkyl.
As used herein, "aryloxy" and "arylthio" refer to RO-- and RS--, in
which R is aryl, preferably lower aryl, more preferably phenyl.
As used herein, "alkylene" refers to a straight, branched or
cyclic, preferably straight or branched, bivalent aliphatic
hydrocarbon group, preferably having from 1 to about 20 carbon
atoms, more preferably 1 to 12 carbons, even more preferably lower
alkylene. The alkylene group is optionally substituted with one or
more "alkyl group substituents." There can be optionally inserted
along the alkylene group one or more oxygen, sulphur or substituted
or unsubstituted nitrogen atoms, where the nitrogen substituent is
alkyl as previously described. Exemplary alkylene groups include
methylene (--CH.sub.2 --), ethylene (--CH.sub.2 CH.sub.2 --),
propylene (--(CH.sub.2).sub.3 --), cyclohexylene (--C.sub.6
H.sub.10 --), methylenedioxy (--O--CH.sub.2 --O--) and
ethylenedioxy (--O--(CH.sub.2).sub.2 --O--). The term "lower
alkylene" refers to alkylene groups having 1 to 6 carbons.
Preferred alkylene groups are lower alkylene, with alkylene of 1 to
3 carbon atoms being particularly preferred.
As used herein, "alkenylene" refers to a straight, branched or
cyclic, preferably straight or branched, bivalent aliphatic
hydrocarbon group, preferably having from 2 to about 20 carbon
atoms and at least one double bond, more preferably 1 to 12
carbons, even more preferably lower alkenylene. The alkenylene
group is optionally substituted with one or more "alkyl group
substituents." There can be optionally inserted along the
alkenylene group one or more oxygen, sulphur or substituted or
unsubstituted nitrogen atoms, where the nitrogen substituent is
alkyl as previously described. Exemplary alkenylene groups include
--CH.dbd.CH--CH.dbd.CH-- and --CH.dbd.CH--CH.sub.2 --. The term
"lower alkenylene" refers to alkenylene groups having 2 to 6
carbons. Preferred alkenylene groups are lower alkenylene, with
alkenylene of 3 to 4 carbon atoms being particularly preferred.
As used herein, "alkynylene" refers to a straight, branched or
cyclic, preferably straight or branched, bivalent aliphatic
hydrocarbon group, preferably having from 2 to about 20 carbon
atoms and at least one triple bond, more preferably 1 to 12
carbons, even more preferably lower alkynylene. The alkynylene
group is optionally substituted with one or more "alkyl group
substituents." There can be optionally inserted along the
alkynylene group one or more oxygen, sulphur or substituted or
unsubstituted nitrogen atoms, where the nitrogen substituent is
alkyl as previously described. Exemplary alkynylene groups include
--C.ident.C--C.ident.C--, --C.ident.C-- and --C.ident.C--CH.sub.2
--. The term "lower alkynylene" refers to alkynylene groups having
2 to 6 carbons. Preferred alkynylene groups are lower alkynylene,
with alkynylene of 3 to 4 carbon atoms being particularly
preferred.
As used herein, "alk(en)(yn)ylene" refers to a straight, branched
or cyclic, preferably straight or branched, bivalent aliphatic
hydrocarbon group, preferably having from 2 to about 20 carbon
atoms and at least one triple bond, and at least one double bond;
more preferably 1 to 12 carbons, even more preferably lower
alk(en)(yn)ylene. The alk(en)(yn)ylene group is optionally
substituted with one or more "alkyl group substituents." There can
be optionally inserted along the alkynylene group one or more
oxygen, sulphur or substituted or unsubstituted nitrogen atoms,
where the nitrogen substituent is alkyl as previously described.
Exemplary alk(en)(yn)ylene groups include
--C.dbd.C--(CH.sub.2).sub.n --C.ident.C--, where n is 1 or 2. The
term "lower alk(en)(yn)ylene" refers to alk(en)(yn)ylene groups
having up to 6 carbons. Preferred alk(en)(yn)ylene groups are lower
alk(en)(yn)ylene, with alk(en)(yn)ylene of 4 carbon atoms being
particularly preferred.
As used herein, "arylene" refers to a monocyclic or polycyclic,
preferably monocyclic, bivalent aromatic group, preferably having
from 5 to about 20 carbon atoms and at least one aromatic ring,
more preferably 5 to 12 carbons, even more preferably lower
arylene. The arylene group is optionally substituted with one or
more "alkyl group substituents." There can be optionally inserted
around the arylene group one or more oxygen, sulphur or substituted
or unsubstituted nitrogen atoms, where the nitrogen substituent is
alkyl as previously described. Exemplary arylene groups include
1,2-, 1,3- and 1,4-phenylene. The term "lower arylene" refers to
arylene groups having 5 or 6 carbons. Preferred arylene groups are
lower arylene.
As used herein, "heteroarylene" refers to a bivalent monocyclic or
multicyclic ring system, preferably of about 5 to about 15 members
where one or more, more preferably 1 to 3 of the atoms in the ring
system is a heteroatom, that is, an element other than carbon, for
example, nitrogen, oxygen and sulfur atoms. The heteroarylene group
are optionally substituted with one or more, preferably 1 to 3,
aryl group substituents.
As used herein, "alkylidene" refers to a bivalent group, such as
.dbd.CR'R", which is attached to one atom of another group, forming
a double bond. Exemplary alkylidene groups are methylidene
(.dbd.CH.sub.2) and ethylidene (.dbd.CHCH.sub.3). As used herein,
"aralkylidene" refers to an alkylidene group in which either R' or
R" is and aryl group.
As used herein, "amido" refers to the bivalent group --C(O)NH--.
"Thioamido" refers to the bivalent group --C(S)NH--. "Oxyamido"
refers to the bivalent group --OC(O)NH--. "Thiaamido" refers to the
bivalent group --SC(O)NH--. "Dithiaamido" refers to the bivalent
group --SC(S)NH--. "Ureido" refers to the bivalent group
--HNC(O)NH--. "Thioureido" refers to the bivalent group
--HNC(S)NH--.
As used herein, "semicarbazide" refers to --NHC(O)NHNH--.
"Carbazate" refers to the bivalent group --OC(O)NHNH--.
"Isothiocarbazate" refers to the bivalent group --SC(O)NHNH--.
"Thiocarbazate" refers to the bivalent group --OC(S)NHNH--.
"Sulfonylhydrazide" refers to the group --SO.sub.2 NHNH--.
"Hydrazide" refers to the bivalent group --C(O)NHNH--. "Azo" refers
to the bivalent group --N.dbd.N--. "Hydrazinyl" refers to the
bivalent group --NH--NH--.
As used herein, the term "amino acid" refers to .alpha.-amino acids
which are racemic, or of either the D- or L-configuration. The
designation "d" preceding an amino acid designation (e.g., dAla,
dSer, dVal, etc.) refers to the D-isomer of the amino acid. The
designation "dl" preceding an amino acid designation (e.g., dlPip)
refers to a mixture of the L- and D-isomers of the amino acid.
As used herein, when any particular group, such as phenyl or
pyridyl, is specified, this means that the group is unsubstituted
or is substituted. Preferred substituents where not specified are
halo, halo lower alkyl, and lower alkyl.
As used herein, a composition refers to any mixture of two or more
products or compounds. It can be a solution, a suspension, liquid,
powder, a paste, aqueous, non-aqueous or any combination
thereof.
As used herein, a combination refers to any association between two
or more items.
As used herein, fluid refers to any composition that can flow.
Fluids thus encompass compositions that are in the form of
semi-solids, pastes, solutions, aqueous mixtures, gels, lotions,
creams and other such compositions.
As used herein, substantially identical to a product means
sufficiently similar so that the property of interest is
sufficiently unchanged so that the substantially identical product
can be used in place of the product.
As used herein, the abbreviations for any protective groups, amino
acids and other compounds, are, unless indicated otherwise, in
accord with their common usage, recognized abbreviations, or the
IUPAC-IUB Commission on Biochemical Nomenclature (see, Biochem.
1972, 11, 942).
A. Functional Polymers and Functional Polymer/Biomolecule
Conjugates and Immobilization to Surfaces.
Biomolecules incorporating a first reactive component and
functional polymers incorporating a second reactive component that
forms a covalent bond when reacted with first reactive component on
the biomolecule are provided. Surfaces that are not modified but
form electrostatic or hydrophobic interactions with
polymer/biomolecule conjugates are provided as well as surfaces
that are modified to incorporate the first reactive component or a
reactive component of similar reactivity. In the case where the
surface is modified with the first component the
polymer/bioconjugate possesses available second components to form
a covalent bond between the polymer and the surface (FIG. 1).
The preparation of the ternary system requires a covalent reaction
scheme wherein the biomolecule/polymer/surface conjugate is
mediated by electrophilic/nucleophilic/electrophilic or
nucleophilic/electrophilic/nucleophilic covalent chemistries. In
the example of an electrophilic/nucleophilic/electrophilic system
the biomolecule is modified to incorporate a electrophile such as
an aldehyde, the polymer is modified to incorporate a nucleophile
that reacts with the electrophilic first component on the
biomolecule, such as a hydrazine or aminooxy moiety and the surface
is modified to incorporate an electrophile, such as an aldehyde,
succinimidyl ester or isothiocyanante that reacts with the
nucleophile incorporated on the surface. The electrophile on the
surface may be the same or different as incorporated on the
biomolecule. An overview of this method is described in the scheme
below: ##STR1##
A most preferred enablement of this invention includes the use of a
linking chemistry between all three components of the invention
that incorporates moieties that are stable when incorporated on any
of the three components and when required form covalent and/or
electrostatic or other linkage rapidly and in most cases
irreversibly. It is of utmost importance that any modification does
not interfere with the biological properties of the biomolecule.
There may be certain instance wherein reversibility of the linkage
is important however.
It is an embodiment of the invention that the biomolecules are
modified to incorporate a first reactive component of a
bioconjugate couple. Bioconjugate couples include, but are not
limited to maleimido/thiol, .alpha.-bromoacetamido/thiol,
succinimidyl ester/amino, avidin/biotin, hydrazine/carbonyl and
pyridyldisulfide/thiol. In a preferred embodiment the polymers are
modified to possess a protected or unprotected hydrazino, a
protected or unprotected oxyamino, or a carbonyl group for
formation of a hydrazone or oxime linkage with an appropriately
modified surface or biopolymer. The hydrazino moiety can be an
aliphatic, aromatic or heteroaromatic hydrazine, semicarbazide,
carbazide, hydrazide, thiosemicarbazide, thiocarbazide, carbonic
acid dihydrazine or hydrazine carboxylate (see, FIG. 2). The
protecting groups are salts of the hydrazino or oxyamino group, or
any amino or hydrazino protecting groups known to those of skill in
the art (see, e.g., Greene et al. (1999) Protective Groups in
Organic Synthesis (3rd Ed.) (J. Wiley Sons, Inc.)). The carbonyl
moiety can be any carbonyl-containing group capable of forming a
hydrazone linkage with one or more of the above hydrazino moieties.
Thus, in particularly preferred embodiments include
6-hydrazinonicotinamide moiety or 4-thiosemicarbazidobenzamide
moiety. Preferred carbonyl moieties include aldehydes and ketones.
Particularly preferred embodiments for irreversible linkages are
hydrazones formed from aromatic ketones or aldehydes and aromatic
hydrazines due to their increased stability or oximes formed from
ketones and aminooxy moieties.
The design/selection of the polymer to be used in any specific
application is dependent upon a variety of criteria. These criteria
include the properties of the surface, the conditions of the
protocols that the polymer will be exposed both before and after
immobilization and to avoid deleterious interactions with the
biomolecule. Therefore it is anticipated that cationic, anionic or
neutral polymers may be used in this invention. Polymers may be
prepared by modification of pre-synthesized polymers or reactive
function or their protected forms may be incorporated on monomers
prior to polymerization. Polymers may be homopolymers, copolymers
or block co-polymers. Polymers made be composed on synthetic or
natural polymers or any combination thereof. The specific
application will dictate the desired properties of the polymer.
A preferred embodiment of the invention for immobilization on
silica/glass surfaces includes cationic polymers derived from
natural or unnatural polyaminoacids wherein one or more monomer
possesses a cationic moiety such as amino or guanidinium groups.
The monomers of natural polyaminoacids include lysine, ornithine or
guanidine or any combination thereof. Cationic polymers prepared
from non-natural sources include cationic groups including primary
or secondary amines. A preferred synthetic cationic polymers
includes but are not limited to polyethyleneimine. Polymers may be
composed of a single monomer unit to form a homo-polymer or
multiple monomers to form hetero-polymers tertiary amino or
quaternary ammonium groups may also be included in the polymer to
incorporate a cationic non-nucleophilic function. Preferred
cationic polymers include poly-1-lysine and polyethyleneimine.
It is also anticipated for modification of silica surfaces that
cationic polymers may be modified with reactive functionalities and
subsequently coated on the surface. Current methods describe the
immobilization of unmodified cationic polymers such as
poly-1-lysine on glass slides for the electrostatic immobilization
of polynucleotides. In a preferred embodiment of this invention
amine-containing cationic polymers such as poly-1-lysine is
modified with the first component of a bioconjugate couple and
subsequently immobilized on the glass surface. In a most preferred
embodiment poly-1-lysine is modified to possess hydrazine, aminooxy
or carbonyl groups. The immobilized functionalized polymer is then
reacted directly with a biomolecule possessing the second component
of the bioconjugate couple for example carbonyl-containing
biomolecule for covalent attachment to a hydrazine or aminooxy
modified poly-1-lysine or a hydrazine or aminooxy modified
biomolecule for covalent attachment to a carbonyl-modified
poly-1-lysine. The most preferred hydrazine, aminooxy and carbonyl
moieties are similar to those described below.
In another embodiment glass immobilized with functionalized
cationic polymers can themselves react covalently as well as
eletrostatically to anionic polymers possessing both the first
component of a bioconjugate couple and covalently linked to
biomolecules.
Polymers may be modified to possess any one component of a
bioconjugate couple. Bioconjugate couples include, but are not
limited to maleimido/thiol, a-bromoacetamido/thiol, succinimidyl
ester/amino, avidin/biotin, hydrazine/carbonyl and
pyridyldisulfide/thiol. In a preferred embodiment the polymers are
modified to possess a protected or unprotected hydrazino, a
protected or unprotected oxyamino, or a carbonyl group. for
formation of a hydrazone or oxime linkage with an appropriately
modified surface or biopolymer. The hydrazino moiety can be an
aliphatic, aromatic or heteroaromatic hydrazine, semicarbazide,
carbazide, hydrazide, thiosemicarbazide, thiocarazide, carbonic
acid dihydrazine or hydrazine carboxylate (see, FIG. 1). The
protecting groups are salts of the hydrazino or oxyamino group, or
any amino or hydrazino protecting groups known to those of skill in
the art (see, e.g., Greene et al. (1999) Protective Groups in
Organic Synthesis (3rd Ed.) (J. Wiley Sons, Inc.)). The carbonyl
moiety can be any carbonyl containing group capable of forming a
hydrazone linkage with one or more of the above hydrazino moieties.
Preferred carbonyl moieties include aldehydes and ketones.
It is an object of the invention that biomolecules conjugated to
polymers include synthetic peptides, proteins, synthetic
oligonucleotides, polynucleotides derived from natural sources or
synthesized via enzyme-mediated reactions including polymerases and
reverse transcriptases. Biomolecules also include carbohydrates and
glycoproteins.
It is an object of this invention to use unmodified surfaces to
immobilize the polymer/biomolecule conjugate via electrostatic
and/or hydrophobic interaction. In a preferred embodiment with
unmodified glass a cationic polymer/biomolecule conjugate is
immobilized via electrostatic interaction between the cationic
polymer and the unmodified glass.
It is a further embodiment of the invention to employ surfaces with
reactive moieties to form a covalent linkage with the second
reactive component incorporated on the polymer of the
polymer/biomolecule couple. The component incorporated on the
polymer is chosen from one component of a bioconjugate couple.
Bioconjugate couples include, but are not limited to
maleimido/thiol, a-bromoacetamido/thiol, succinimidyl ester/amino,
avidin/biotin, hydrazine/carbonyl and pyridyldisulfide/thiol. In a
preferred embodiment the polymers are modified to possess a
protected or unprotected hydrazino, a protected or unprotected
oxyamino, or a carbonyl group for formation of a hydrazone or oxime
linkage with an appropriately modified surface or biopolymer. The
hydrazino moiety can be an aliphatic, aromatic or heteroaromatic
hydrazine, semicarbazide, carbazide, hydrazide, thiosemicarbazide,
thiocarazide, carbonic acid dihydrazine or hydrazine carboxylate
(see, FIG. 1). The protecting groups are salts of the hydrazino or
oxyamino group, or any amino or hydrazino protecting groups known
to those of skill in the art (see, e.g., Greene et al. (1999)
Protective Groups in Organic Synthesis (3rd Ed.) (J. Wiley Sons,
Inc.)). The carbonyl moiety can be any carbonyl-containing group
capable of forming a hydrazone linkage with one or more of the
above hydrazino moieties. Thus, in particularly preferred
embodiments include 6-hydrazinonicotinamide moiety or
4-thiosemicarbazidobenzamide moiety. Preferred carbonyl moieties
include aldehydes and ketones. A particularly preferred embodiment
is aromatic ketones and aldehydes.
A. Oligo/polymer/surface Systems
It is an additional object of the present invention to provide
ternary oligo/polymer/surface embodiments for genomic screening by
hybridization. The use of such ternary systems provides more cost
effective, reproducible microarrays than that currently produced.
The use of these systems allows the immobilization of 5-150 mer
oligonucleotides. This invention will allow direct capture of solid
phase synthesized full length oligonucleotides from a crude cleaved
oligonucleotide mixture as only the terminally functionalized
oligonucleotide will form a covalent linkage with the polymer and
the unmodified capped failure sequences will be washed away. Also
the covalent linkage will be performed in solution unlike spotting
techniques wherein a modified oligonucleotide is contacted with a
modified surface and allowed to react in a solid/liquid two-phase
system. This latter method is sub-optimal as very small volumes,
.rho.Ls to .eta.Ls of oligonucleotide, are used which dry quickly
inefficient immobilization. The method of this invention is
preferable over synthesis of oligonucleotide on the chip as failure
sequences will not be immobilized and longer sequences>25 mers
can be efficiently immobilized. The longer oligonucleotides are
preferred for genetic expression analysis as differences in the
melting temperature, Tm, between sequences is less variable leading
to better results.
The most direct, simple and efficient preparation of an
oligonucleotide/polymer conjugate would have the following
properties: (1) direct incorporation of the first component of the
bioconjugate couple directly on the oligonucleotide during solid
phase synthesis without the requirement for any post-synthetic
activation, (2) indefinite stability of both components of the
bioconjugate couple following incorporation on either the
oligonucleotide or the polymer, (3) good kinetics of covalent bond
formation between the modified oligonucleotide and the modified
polymer without the need for a reagent-mediated reaction or
competing reactions on the bioconjugate couple moieties, (4) simple
incorporation of the first component of the bioconjugate couple on
the surface of choice, (5) fast kinetic of immobilization of the
oligonucleotide/polymer conjugate on the modified surface, (6) long
term stability of the oligonucleotide/polymer conjugate on the
surface.
It is a specific object of the invention that the first reactive
component of the bioconjugate couple is incorporated on the 3', 5'
or an internal position of the oligonucleotide to be conjugated to
the polymer. It is a most preferred embodiment that the first
reactive component is incorporated on either the 3' or 5' end of
the oligonucleotide. In a preferred embodiment the polymer is
modified to possess a protected or unprotected hydrazino, a
protected or unprotected oxyamino, or a carbonyl group for
formation of a hydrazone or oxime linkage with an appropriately
modified surface and/or biopolymer. The hydrazino moiety can be an
aliphatic, aromatic or heteroaromatic hydrazine, semicarbazide,
carbazide, hydrazide, thiosemicarbazide, thiocarazide, carbonic
acid dihydrazine or hydrazine carboxylate (see, FIG. 3). The
protecting groups are salts of the hydrazino or oxyamino group, or
any amino or hydrazino protecting groups known to those of skill in
the art (see, e.g., Greene et al. (1999) Protective Groups in
Organic Synthesis (3rd Ed.) (J. Wiley Sons, Inc.)). The carbonyl
moiety can be any carbonyl-containing group capable of forming a
hydrazone linkage with one or more of the above hydrazino moieties.
Thus, in a particularly preferred embodiment includes
6-hydrazinonicotinamide moiety or 4-thiosemicarbazidobenzamide
moiety. Another preferred embodiment includes the use of carbonyl
moieties, i.e. aldehydes and ketones. A particularly preferred
embodiment is aromatic ketones and aldehydes.
Surfaces modified to possess the first reacting group of a
bioconjugate couple are provided. In one embodiment glass surfaces
can be modified to possess the first component of the bioconjugate
couple by a treatment with a silane possessing the first component
or a reactive component of similar reactivity. The first component
can also be incorporated on a cationic polymer including but not
limited to poly-1-lysine or polyethyleneimine and the modified
polymer immobilized on the glass surface via electrostatic
interactions. Silica-based surfaces such as glass, silica beads or
fiber optic cables modified to possess aldehyde or
hydrazine/aminoxy functionalities are preferred embodiments.
Plastic, polystyrene (latex) surfaces modified to possess the first
reactive group are further described. Either carbonyls or
hydrazines as the first component of the hydrazine/carbonyl
bioconjugate couple are preferred embodiments wherein the second
component is incorporated on the polymer. Other surface similarly
functionalized such as metals, including gold or silver, are
further preferred embodiments.
B. Polynucleotide/polymer/surface
Polynucleotides may be functionalized in a several ways to possess
first reactive component. It is mandatory that the functional group
incorporated via modified triphosphates on the polynucleotide be
stable to conditions used in the polymerase chain reaction or
reverse transcriptase reaction. First components of bioconjugate
couples that are suitable for this purpose include amino, hydrazine
and carbonyl moieties. These functionalities are preferred
embodiments of this invention.
In one enablement any of the three moieties may be incorporated at
any position of a the base of a nucleoside triphosphate such that
the modification does not interfere with both the incorporation of
the triphosphate in the enzymic incorporation of the nucleoside in
the growing polynucleotide and does not interfere with the hydrogen
bonding properties of the synthesized polynucleotide. A variety of
positions on all the natural nucleosides have been described
including the 5-position of cytidine, thymidine and uridine. The
N-4 position of thymidine and uridine has also been described.
Functional moieties have also been incorporated on unnatural
nucleosides such as deaza purines have been described and we
anticipate that amino, hydrazine or carbonyl groups incorporated on
these moieties would similarly function as described herein to
produce desired conjugates when reacted with polymers possessing
the second functionality.
In another embodiment one primer of the primer set is immobilized
on a polymer and this multiple primer/polymer conjugate is combined
with the second primer during polynucleotide synthesis. The PCR or
reverse transcriptase product is immobilized on an unmodified or
appropriately modified surface and subsequently heated to melt of
the non-conjugated strand. One extremely important criterion is
that the bond linking the primer to the polymer must be stable to
the conditions required for elongation of the primers. Preferred
embodiments include primers modified to possess a hydrazine or
carbonyl moiety and the polymer is cationic and modified to possess
a carbonyl or hydrazine moiety to react with its reaction partner
respectively. If the product is to be immobilized via covalent
and/or electrostatic interactions on a silica surface it is a
preferred embodiment that the polymer be cationic. It may also be
advantageous that the polymer be neutral and the reactive groups be
neutral as the polynucleotide increases in length the charge of the
conjugate will be increasingly negative and if a cationic polymer
is used there is a possibility that the complex will be neutral and
it may precipitate during elongation. Therefore a second preferred
embodiment would employ a neutral polymer modified to incorporate
either a hydrazine or carbonyl group that would react with a primer
modified on the 5'-end with a carbonyl or hydrazine that would
covalently link the oligonucleotide to the polymer via a hydrazone
linkage. The hydrazone linkage is stable to elevated temperatures
at physiological pH. It is a further embodiment that the linkage
between the polymer and the oligonucleotide could be an amide bond
formed by carbodiimide-mediated couple between amine and carboxyl
groups either the polymer or the oligonucleotide.
A third method to immobilize a polynucleotide/polymer on a surface
would be to incorporate on the 5'-end of a primer one or more first
components and use this modified primer in a PCR or reverse
transcriptase polynucleotide amplification reaction. Subsequently
the product would be conjugated to a polymer that possesses the
second reactive component that will form a covalent linkage to
polynucleotide the first component. The polynucleotide/polymer
would be immobilized on a surface and heated to melt the
non-conjugated strand off the surface. It is extremely advantageous
to remove the non-conjugated strand so it will not compete with the
target during the hybridization step. Currently no methods are able
to selectively immobilize only one strand. Preferred functional
groups to be incorporated on the 5'-end on a primer include amines,
hydrazines, oxyamines or aldehydes. To capture amine modified
polynucleotides carbodiimide mediated amide formation to an
carboxyl-containing polymer is anticipated. Other electrophilic
surfaces such as epoxy, succinimidyl ester or isothiocyanates would
similar react with the amino group. A preferred embodiment is to
incorporate hydrazine or aminooxy groups on the primer and capture
the reactive polynucleotide on a carbonyl containing polymer. Also
incorporation of a carbonyl group on the primer followed by capture
on a hydrazine or aminooxy containing polymer would form the
desired polynucleotide/polymer conjugate. A cationic polymer
incorporating the second component of the reaction is the preferred
embodiment for immobilization on silica-based surfaces.
One can also envisage the incorporation of multiple first
components on the 5'-end of the primer in dendrimeric or polymeric
constructions. The use of multiple first components would increase
the likelihood of covalent attachment to the polymer and the
formation of multiple covalent attachments to the polymer. Cationic
dendrimers themselves modified to incorporate multiple primers are
a further embodiment of this invention. Preferred functionalities
include those describe above for single first component modified
primers.
C. Protein/polymer/surface
The production of "protein chips" for proteomic purposes is also
anticipated using this invention.
The general process for immobilization of proteins or peptides as
anticipated by this invention includes the following steps: (1)
incorporation of a first reactive component on the protein or
peptide, (2) incorporation of a second reactive component on a
polymer that forms a covalent linkage with the first reactive
component on the protein, (3) reacting the modified protein with
the modified polymer to form a covalently linked protein/polymer
conjugate, (5) preparation of a modified or unmodified surface for
immobilization of the protein/peptide conjuate and (5) contacting
the peptide/polymer conjugate to unmodified or modified
surface.
Examples of first and second reactive components include but are
not limited to bioconjugate couples standardly used by one skilled
in-the-art including maleimido/thiols, a-haloacetamides/thiol,
amines/succinimidyl esters and hydrazine or aminooxy/carbonyl
couples. It is extremely advantageous to use first and second
components that have extended (>1 month) to indefinite stability
following incorporation of the protein/peptide or polymer. Thus a
preferred embodiment of this invention is the use of the
hydrazine/carbonyl or aminooxy/carbonyl couples as incorporation of
any of these moieties on protein/peptides have indefinite stability
in neutral aqueous conditions.
Modification of the protein or peptide with carbonyl groups can be
accomplished with carbonyl heterobifunctional succinimidyl esters
including succinimidyl 4-formylbenzoate or succinimidyl
4-acetylbenzoate to incorporate aromatic carbonyl groups or
succinimidyl levulinate to incorporate aliphatic ketone groups. It
is well known to those skilled in the art that glycoproteins can be
oxidized with sodium periodate to cleave 1,2-diol groups on
carbohydrates to produce aldehydes. These oxidized glycoproteins
are also suitably reactive for this purpose.
In a preferred embodiment proteins or peptides can be directly
modified to incorporate hydrazine or hydrazide groups using the
previously described bifunctional hydrazine, hydrazide or
thiosemicarbazide reagents (as described in Schwartz et al., U.S.
Pat. Nos. 5,753,520, 5,679,772, 5,420,285 and 5,206,370) including
succinimidyl 6-hydraziniumnicot hydrochloride, succinimidyl
4-hydrazidiumbenzoate hydrochloride or succinimidyl
4-thiosemicarbazidium benzoate hydrochloride respectively.
The level of modification of either aldehyde or hydrazine groups
can be controlled by controlling the stoichiometry of addition of
the heterobifunctional reagents to the protein during the
modification reaction. It is desirable to incorporate sufficient
groups on the protein so that the kinetics of conjugation is
appropriate and that overmodification does not compromise the
biological function of the protein or lead to precipitation or
other unwanted interactions of the protein.
Similar hydrazine or carbonyl containing polymers described above
for the preparation of oligonucleotide/polymer conjugates are used
to form conjugates with appropriately modified proteins or
peptides. Addition of the carbonyl or hydrazine modified protein to
the hydrazine or carbonyl modified polymer respectively leads to a
covalently linked conjugate.
Surfaces described for immobilization of oligonucleotide/polymer
conjugates can be used for immobilization of the above-described
protein/polymer conjugates. In one preferred embodiment amino
polymers including but not limited to poly-1-lysine or ornithine or
polyethyleneimine are modified to incorporate hydrazine or carbonyl
moieties and subsequently reacted with the appropriately modified
proteins. The protein/polymer conjugate is then immobilize on glass
surfaces that have been treated with aqueous ethanolic hydroxide
solution followed by water washing and drying. The polycationic
nature of the protein/polymer conjugate forms a stable
electrostatic linkage. Alternately as described for the
immobilization of the oligonucleotide/polymer conjugate the silica
based surface can be modified to include the first component of the
bioconjugate couple that will lead to both covalent and
electrostatic linking of the polymer to the surface.
D. Peptide/polymer/surface Ternary System
Immobilization of synthetic peptides to surface via electrostatic
interactions is not feasible due to varying charge and
hydrophobic/hydrophilic nature of each peptide. Therefore it is an
embodiment of this invention that one component of a bioconjugate
couple is incorporated on the peptide either during solid phase
synthesis or post-synthetically. The modified peptide is
subsequently covalently linked to a polymer possessing the second
component of the bioconjugate couple. The peptide/polymer conjugate
is subsequently immobilized on a surface that possesses the first
component of the bioconjugate couple that was incorporated on the
peptide. In a most preferred embodiment of this enablement the
hydrazine or aminooxy and carbonyl bioconjugate couple is employed.
Carbonyl groups are incorporated on the C or N terminus of a
peptide during solid phase synthesis using reagents such as
succinimidyl 4-formylbenzoate or succinimidyl levulinate. Hydrazine
groups are incorporated using appropriately protected forms of
succinimidyl 6-hydrazinonicotinate.
The modified peptides are immobilized on the appropriately modified
polymers to produce the peptide/polymer conjugate. The conjugate is
subsequently immobilized on a surface containing the first
component of the bioconjugate couple. In a most preferred
embodiment a cationic polymer is immobilized on a glass surface
possessing the first component.
EXAMPLES
Example 1 (see FIG. 4)
Preparation of 5'-aldehyde modified oligonucleotides: 5'-aldehyde
modified oligonucleotide 5'-OHC-aryl-TTT TTT TAG CCT AAC TGGA TGC
CAT G-3' was obtained from Solulink, Inc (San Diego, CALIF.). The
5'-aldehyde was incorporated using Solulink's proprietary aldehyde
phosphoramidite linker (Schwartz, filed Aug. 1, 2000
Preparation of HyNic::polyethyeleneimine: A solution of
polyethyleneimine (50% by weight; 1 g; Sigma Chemicals, St. Louis,
Mo.) in DI water (3 mL) was prepared and the pH lowered to 7.4 with
concentrated hydrochloric acid (1.5 mL). 10.times. Conjugation
buffer (1 M phosphate, 1.5 M NaCl, pH 7.4; 0.45 mL) was added. A
solution of SANH (27.4 mg) was dissolved in DMF (200 uL). Four 1.0
mL aliquots were treated with SANH/DMF solution (0, 17.5, 35.0 and
52.5 uL) respectively. On addition the reaction mixtures became
cloudy and were allowed to stand at room temperature for 2 hours.
The reaction mixtures were centrifuged to remove any precipitate.
Four NAP-25 columns (Apbiotech, Piscataway, N.J.) were
pre-equilibrated with 0.1 M MES, 0.9% NaCl, pH 4.7. Fractions were
analyzed by spotting 0.5 uL on a F490 silica gel TLC plate (Merck)
and visualized using a short wavelength fluorescent lamp. UV
positive fractions were combined and analyzed for hydrazine
modification using a colorimetric assay by addition of an aliquot
(2 uL) to a 0.5 mM solution of p-nitrobenzaldehyde in MES, pH 4.7
(98 uL) and recording A390 and quantifying using the molar
extinction coefficient of the hydrazone formed (22000).
Preparation of HyNic::poly-1-lysine: A solution of poly-1-lysine
(10 mg; Sigma Chemicals, St. Louis, Mo.; cat. #P-7890) was
dissolved in conjugation buffer, 0.1 M phosphate, 0.15 M NaCl, pH
7.4 (1 mL). A solution of succinimidyl 6-hydrazinonicotinate
acetone hydrazone (SANH; 1.3 mg) was dissolved in DMSO (13 uL). To
two poly-1-lysine aliquots (200 uL) were added the SANH/DMSO
solution (2.85 uL (10 equivalents) and 5.7 uL (20 equivalents)).
The reaction mixtures were vortexed and incubated at room
temperature for 2 h. The modified polymer was isolated by gel
filtration on a NAP-25 column (Pharamacia) pre-equilibrated with
0.1 M MES, 0.9% NaCl, pH 4.7 buffer. Fractions (1 mL) were
collected and analyzed by UV (A260). Fractions containing UV active
product were combined to yield the desired product. The product was
analyzed colorimetrically for hydrazine content by dissolving an
aliquot (2 uL) in a 0.5 mM solution of p-nitrobenzaldehyde (98 uL)
and incubating at 37.degree. C. for 1 h.followed by taking A390
readings (extinction coefficient 22000). The HyNic:poly-1-lysine
polymer was used directly in the conjugation step. The
amine/hydrazine content was determined using the TNBSA assay
(trinitrobenzenesulfonic acid; Pierce Chemical, Inc., Rockville,
Ill.)
Preparation of oligonucleotide/HyNic:poly-1-lysine conjugate: In an
initial preparation of oligonucleotide/polymer conjugates
5'-amino-oligonucleotide (2.6 uL of a 0.4715 OD/uL solution in
water) was added to both unmodified poly-1-lysine (10 uL of a 5
mg/mL solution in PBS buffer) and to HyNic:poly-1-lysine (10 uL)
solution in PBS buffer) containing 6M urea to a final volume of 200
uL) and the mixture was incubated at room temperature for 1 hour.
After incubation an aliquot of each reaction mixture was diluted
with PBS (95 uL) and their spectrum scanned (200-440 nm).
Ultraviolet scans (200-400 nm) showed that the aldehyde
oligonucleotide/HyNic:poly-1-lysine conjugate displayed an
absorption at A360 indicating the formation of a HyNic/benzaldehyde
hydrazone. The other combinations did not display any absorption
other than oligonucleotide A260.
In a separate experiment both the amino and aldehyde modified
oligonucleotides were reacted with the HyNic:poly-1-lysine polymer.
Solutions of both amino and aldehyde oligos (0.1, 0.2, 0.4, 0.6,
0.8 and 1.0 ug/uL) were prepared in 0.1 M MES, 0.9% NaCl, pH 4.7
buffer. A solution of HyNic:poly-1-lysine (1 uL) in 8 uL 0.1 M MES,
0.9% NaCl, pH 4.7 buffer (80 uL) was prepared. An aliquot of each
oligo concentration (1 uL) was added to the polymer solution (9 uL)
and incubated at room temperature for 10 min.
Immobilization of oligonucleotide/polymer conjugate on various
glass surfaces Aliquots of the oligonucleotide/HyNic:polymer
solutions (0.3 uL) were spotted on unmodified glass surface,
aminopropylsilane modified glass surface (Corning, Corning, N.Y.)
and aldehyde modified glass surfaces (Cel Associates, Supplier:
TeleChem (www.arrayit.com, Sunnyvale, Calif.) and allowed to dry.
Following drying the plate was washed in 2.times.SSC, 0.01% SDS for
10 min and then allowed to dry.
Hybridization to immobilized oligonucleotide/polymer/surface
system: The hybridization step was performed by preparing a
solution of 5'-fluorescein labeled complementary oligonucleotide
(sequence: 5'-FAM-CAT GGC ATC AGT TAG GCT-3'; 0.6 OD dissolved in
1.0 mL 2.times.SSC, 0.01% SDS and the solution was applied to the
plate containing the immobilized oligo/polymer conjugate for 1 min.
The plate was subsequently washed (2.times.SSC, 0.01% SDS) and
dried. The plate was visualized initially by placing the slide on a
long wavelength fluorescent lamp and obtaining a digital picture.
The plate was also examined for fluorescence using a GSI Lumonics
5000 MicroArray Slide reader using fluorescein filters. Results
indicated saturated signal down to 10 ng/uL and a signal within
limits of detection at 5 ug/uL for the aldehyde modified
oligonucleotide formulations. The amino modified oligonucleotides
had very weak signals at all concentrations. Direct spotting of
amino or aldehyde modified oligonucleotides on any surface gave no
signal.
Titration of the 5'-OHC-oligo/polyK-HyNic conjugate: To test the
minimal oligo (5'-TTT TTT TAG CCGT AAC TGA TGC CAT G-3')
concentration conjugated to polyK-HyNic that will give a signal
following hybridization to its fluorescently labeled complementary
was performed. Decreasing concentrations (22, 11, 5.5, 2.3, 1.2,
0.6 and 0.0 uM) of both 5'-H2N-oligo and 5'-OHC-oligo were reacted
with polyK/HyNic. Aliquots (0.3 uL) were spotted on unmodified
glass, amino glass (Cel Sci) and aldehyde glass (Cel Sci) and
allowed to dry. The plates were washed with 2.times.SSC (twice) and
the hybridization was performed as above. Surfaces were visualized
using a GSI Lumonics 5000 MicroArray reader.
Results indicate (1) no signal on 0.0 uM spot, (2) signals above
the detection limit of the instrument were obtained down to 2.3 uM,
(3) strong signals within the limits of the instrument were
obtained at 1.2 and 0.6 uM and (4) there were equal signals on all
three surfaces (5) the 5'-OHC-oligo/polyK-HyNic conjugate signals
were much stronger than the weak 5'-H2N-oligo/polyK-HyNic signals
and (6) direct spotting of both the amino and aldehyde oligos on
any of the surfaces gave no signal.
The table below compares the efficiency of immobilization of the
above method and current published methods. The immobilization step
in this method is not reagent mediated unlike the Telechem method
that requires a strong reducing reagent (sodium borohydride) for
immobilization. Also this method does not require special
controlled humidity conditions unlike the Mosaic method that
requires a minimal humidity level to allow reaction to occur.
Unlike the other methods this method results in multiple contact
points, both covalent and electrostatic, to the surface and not a
single point attachment.
Spotting solution (uM) this method 0.6-1.3 Telechem
(www.arrayit.com) 30 Mosaic (www.mosaicbio.com) 20
Demonstration of the Covalency of the Oligonuleotide/polymer
Conjugate to the Aldehyde surface:
A 4.times.6 matrix experiment was designed comparing the
oligonucleotide
5'-TTT TTT TAG CCGT AAC TGA TGC CAT G-3'
with the following modifications:
1) 5'-H2N--
2) 5'-OHC---
3) 5'-H2NHN--
4) 5'-H2NHNCO--
formulated with the following polymers at 2.5 uM oligo
concentration:
1) polyK (poly-1-lysine (20K MW))
2) polyK/.phi.CHO (10.times.)
3) polyK/.phi.CHO (20.times.)
4) polyK/HyNic (100.times.)
5) polyK/HyNic (20.times.)
6) no polymer
The oligonucleotide/polymer conjugates were prepared by incubating
the appropriate concentration of oligonucleotide with the
appropriate polymer (.about.0.03 uM poly-1;-lysine concentration);
in 0.1 M MES, 0.9% NaCl, pH 4.7 for 30 min at room temperature to
make a final 4 uM oligonucleotide solution.
0.3 uL aliquots of oligonucleotide/polymer conjugates were spotted
on amino and aldehyde plates (Cel-Sci, Houston, Tex.). After drying
the plates were washed with hybridization buffer (2.times.SSC, 0.1%
SDS) at room temperature for 15 min by gentle shaking. After drying
5'-fluorescein-labelled complementary oligonucleotide (5'-FAM-CAT
GGC ATC AGT TAG GCT-3') in (2.times.SSC, 0.1% SDS) was placed on
the surface of the glass. Following a 1 minute incubation the
plates were washed with hybridization buffer for 1 min and
fluorescence was visualized under long wavelength UV light and a
digital picture was taken.
The results (FIG. 5) were that only the 5'-OHC--oligo/polyK-HyNic
at both modification levels on the aldehyde plates demonstrated
fluorescence.
Example 2
SFB modification of poly-1-lysine: Two aliquots of poly-1-lysine
(MW 20,700; Sigma Chemicals, St. Louis, Mo.; 200 uL of a 10 mg/mL
solution in 100 mM phosphate, 150 mM NaCl, pH 4.7) were prepared.
Succinimidyl 4-formylbenzoate (SFB; 1.75 mg; 7.1.times.10.sup.-3
mmol) was dissolved in DMF (17.5 uL). Aliquots of the SFB/DMF
solution (2.5 and 5.0 uL) were added to the poly-1-lysine
solutions. The reaction mixtures were incubated at room temperature
for 2 hours. The modified polymers were isolated by gel filtration
using NAP-10 columns (APBiotech, Piscataway, N.J.) pre-equilibrated
with 0.1 mM MES, 0.9% NaCl, pH 4.7. Fractions containing polymer
were identified using the Bradford Protein Assay and combined. The
amino content of the polymers was determined by TNBSA assay
(trinitrobenzenesulfonic acid; Pierce Chemicals (Rockville, Ill.).
The aldehyde content was determined using the 2-hydrazinopyridine
assay as described in example 1 for the quantification of the
aldehyde moiety on the oligonucleotide). The amine concentration
was determined using the TNBSA assay described in Example 1. The
modified polymers were used directly.
Preparation of 5'-hydrazine-modified oligonucleotide: A 25-mer
phosphodiester oligonucleotide modified to incorporate a
C6-aminolinker (Glen Research amino-C6 amidite ) was prepared
(5'-NH2-(CH2)6-TTT TTT TAG CCT AAC TGGA TGC CAT G-3'; MW 7791
g/mol, 229.5 OD/umol; TriLink BioTechnologies, Inc., San Diego,
Calif.). The oligonucleotide was dissolved in conjugation buffer
(100 mM phosphate, 150 mM sodium chloride, pH 7.4) to a
concentration of 0.92 OD/uL. To a solution of oligonucleotide (64
uL; 2 mg) was added DMF (32 uL). A solution of succinimidyl
4-hydrazinonicotinate acetone hydrazone (SANH; (3.8 mg) in DMF (100
uL) was prepared. An aliquot of the SANH/DMF solution (18.8 uL; 10
equivalents) was added to the oligonucleotide solution and the
reaction allowed to incubate at room temperature overnight. The
reaction was monitored by C18 RP-HPLC (solution A: 50 mM
triethylammonium acetate, solution B: acetonitrile-gradient 0-50% A
over 30 min; 50-80% over 10 min; 80-0% over 5 min). The
hydrazine-modified oligonucletide was deprotected and purified
using a Millipore 5K MWCO ultrafree diafiltration device by
diluting the reaction mixture with 100 mM acetate, pH 4.7 and
concentrating in the diafiltration device. The retentate was
further washed with buffer (2.times.400 uL). The oligonucleotides
was quantified by A260 assay and the hydrazine incorporation was
determined using the p-nitrobenzaldehyde assay described in Example
1 and determined to be >95% hydrazine-modified.
Conjugation of hydrazine-modified oligonucleotide to
aldehyde-modified poly-1-lysine: An aliquot of aldehyde-modified
poly-1-lysine (.about.0.03 uM modified poly-1;-lysine
concentration; 1 uL) was diluted with 0.1 M MES, 0.9% NaCl, pH 4.7
(8 uL) and an 1 uL aliquot of the prediluted oligonucleotide
solutions was added and the reaction allowed to incubate at room
temperature for 1 h. The solution was directly spotted on the
glass.
Hybridization to immobilized oligonucleotide/polymer/surface system
was performed in an identical manner as described in Example 1.
Both amino-glass and aldehyde glass were examined in a similar
4.times.6 matrix as described in Example 1. The results (FIG. 6)
demonstrated only the 5'-H2NHN-oligo/polyK- .phi.CHO at both
modification levels on the amino plates demonstrated fluorescence
and
Example 3
Preparation of amino, aldehyde or hydrazine polynucleotides with
appropriate triphosphates: Two primers and template DNA in reaction
buffer containing a 70/30 mixture of dCTP and dCTP modified to
incorporate a aromatic aldehyde group on the 5-position is added to
dGTP, dTTP and dATP in equimolar amounts with heat stable DNA
polymerase. A PCR reaction is performed by cycling of denaturation,
annealing and extension steps. PCR products are purified using
spincolumn [QIAGEN] to remove small molecule impurities.
Conjugation of PCR product to polymer The PCR product incorporating
the aldehyde moiety is added to a solution of poly-1-lysine/HyNic
polymer and incubated at room temperature for 4 h. The solution is
used directly for immobilization.
Immobilization of polynucleotide/polymer to surface: Aliquots of
the polynucleotide/polymer conjugate are spotted on
aldehyde-modified glass surfaces and allowed to dry. The slide is
washed with 2.times.SCC (three times).
Hybridization: The polynucleotide/polymer immobilized slide is used
in a hybridization experiment with a fluorescently labeled cDNA
sample.
Example 4
PCR of primer/polymer conjugate: The 5'-modified aldehyde
primer/HyNic-polyK conjugate is prepared as in example 1 is
combined with second primer and template DNA in reaction buffer
containing dNTPs and heat stable DNA polymerase. A PCR reaction is
performed by cycling of denaturation, annealing and extension
steps. PCR products are purified using spincolumn [QIAGEN] to
remove small molecule impurities.
Immobilization and hybridization of PCR/RT product: The
polynucleotide/polymer conjugate is exchanged into 0.1 M MES, 0.9%
NaCl, pH 4.7 and is spotted on aldehyde modified glass. The spot is
allowed to dry and washed with 2.times.SSC. The second
non-conjugated strand is removed by heat or urea treatment. The
plate is hybridized to fluorescently labeled target and visualized
in a MicroArray fluorescent instrument.
Example 5
PCR of 5'-aldehyde-modified primer: A 5'-modified aldehyde primer
and the second non-modified primer are combined with template DNA
in reaction buffer containing dNTPs and heat stable DNA polymerase.
A PCR reaction is performed by cycling of denaturation, annealing
and extension steps. PCR products are purified using spincolumn
[QIAGEN] to remove small molecule impurities.
Conjugation of terminally labeled PCR product to
HyNic/poly-1-lysine: The purified PCR product is added to a
solution of HyNic/poly-1-lysine in 0.1 M MES, 0.9% NaCl, pH 4.7.
The reaction is allowed to proceed at room temperature for 1-3
hours and the solution used directly. The slide is processed as
described in Example 4 to melt off the second strand and
subsequently hybridize to the immobilized strand.
Example 6
Preparation of aldehyde modified proteins: Anti-DNP polyclonal
antibody (5 mg/mL in conjugation buffer (see Example 1)) is treated
with a solution of succinimidyl 4-formyl benzoate in DMF (15
equiv). The reaction mixture is allowed to stand at room
temperature for 3 h and the protein is isolated using a NAP 10
column pre-equilibrated with 0.1 M MES, 0.9% NaCl, pH 4.7). The
protein containing fractions are combined and the protein
concentration is determined using the BCA assay (Pierce Chemical,
Rockville, Ill.) and the aldehyde concentration determined using
the 2-hydrazinopyridine assay described in Example 2.
Conjugation of aldehyde modified proteins to hydrazine modified
poly-1-lysine: An aliquot of the aldehyde-modified protein above is
added to a solution of poly-1-lysine/HyNic and incubated at room
temperature for 2 h. The solutions is used directly for spotting on
the aldehyde surface.
Immobilization and antigen capture: A serial dilution of the
protein/polymer conjugate is prepared and aliquots (1 nL) of the
protein/polymer conjugate is spotted on aldehyde modified surfaces
and allowed to dry. The surface is washed with conjugation buffer
and challenged with the fluorescently modified antigen.
Example 7
Preparation of hydrazine modified proteins: In a similar manner to
that described in Example 6 the protein is modified with
succinimidyl 6-hydraziniumnicotinate hydrochloride (Schwartz et
al., Bioconjugate Chem 2, 333 (1991) to incorporate hydrazine
groups on the protein. The hydrazine modified protein is
subsequently conjugated to aldehyde modified poly-1-lysine (see
Example 2). The protein/polymer conjugate is immobilized on an
amino or hydrazine or oxyamino surface. The immobilized protein is
similarly challenged with fluorescently labeled antigen as
described in Example 6.
* * * * *