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Description  |
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FIELD OF THE INVENTION
This invention relates generally to polynucleotide probes, and in
particular relates to a polynucleotide probe containing at least one
labeled, modified nucleotide.
DESCRIPTION OF RELEVANT LITERATURE
Meinkoth and Wahl, Anal. Biochem., (1984) 138:267-284, provide a review
article of hybridization techniques. See also Leary et al., Proc. Natl.
Acad. Sci. USA (1983) 80:4045-4049, for a description of the dot blot
assay. Sandwich hybridization is described by Ranki et al., Curr. Top.
Microbiol. Immunology (1983) pp. 308ff. See also Ranki et al., Gene (1983)
21:77-85, Virtanen et al., Lancet (1983) 381-383, and U.S. Pat. No.
4,486,539. EPA 123,300 describes biotin-avidin complexes for use in
detecting nucleic acid sequences. Sung, in Nucl. Acids Res.
9(22):6139-6151 (1981) and in J. Org. Chem. 47:3623-3628 (1982), discusses
the synthesis of a modified nucleotide and application of the modified
structure in oligonucleotide synthesis. Modified nucleotides are also
discussed in Draper, Nucleic Acids Res. 12:2:989-1002 (1984), wherein it
is suggested that cytidine residues in RNA be modified so as to bind to
reporter molecules. Later work suggests similar modification of cytidine
residues in DNA (Anal. Biochem. 157(2):199 (1986). European Patent
Application 063879, filed Apr. 6, 1982, and PCT Application No.
PCT/US84/00279 also describe modified nucleotides and applications
thereof.
BACKGROUND OF THE INVENTION
The increasing ease of cloning and synthesizing DNA sequences has greatly
expanded opportunities for detecting particular nucleic acid sequences of
interest. No longer must one rely on the use of immunocomplexes for the
detection of pathogens, ligands, antigens, and the like. Rather than
detecting particular determinant sites, one can detect DNA sequences or
RNA sequences associated with a particular cell. In this manner, diseases
can be diagnosed, phenotypes and genotypes can be analyzed, as can
polymorphisms, relationships between cells, and the like.
Analyses of DNA sequences typically involve the binding of an analyte
sequence to a solid support and hybridization of a complementary sequence
to the bound sequence. The annealing and complexing steps usually involve
an extended period of time and require careful washing to minimize
non-specific background signals. Applicants' co-pending application Ser.
No. 07,624, now U.S. Pat. No. 4,868,105 describes new techniques for
analyzing nucleic acid sequences which are faster, minimize the number of
manipulative steps, and provide for an increased signal to noise ratio.
This application, a continuation-in-part of Ser. No. 807,624 now U.S. Pat.
No. 4,868,105, the disclosure of which is incorporated by reference
herein, is directed in particular to novel polynucleotide probes useful,
inter alia, in the techniques described in applicants' co-pending parent
application.
The majority of polynucleotide probes in current use are radioactively
labeled, e.g. with isotopes of hydrogen (.sup.3 H), phosphorus (.sup.32
P), carbon (.sup.14 C) or iodine (.sup.125 I). These materials are
relatively simple to synthesize by direct inclusion of the radioactive
moieties, e.g. by kinasing with .sup.32 P-labeled ATP, equilibrating with
tritiated water, or the like. As is well known, however, use of such
radioactive labels has drawbacks, and other detectable species which are
not radioactive are preferred.
In order to incorporate other, non-radioactive types of detectable species
in a nucleotide, some sort of chemical modification of the nucleotide is
required. It is widely recognized that nucleotide modification is a
difficult and sensitive procedure, as any modification reaction has to be
mild enough to leave the RNA or DNA molecules intact, while giving a
modified nucleotide product which can participate in normal base pairing
and stacking interactions. These considerations typically limit nucleotide
substitution positions to the 5-position of a pyrimidine and the
8-position of a purine, as noted in the literature (see, e.g., European
Patent Application 063879, cited supra).
Other considerations must also be taken into account. Base pairing may be
hindered during hybridization if the detectable label is at one end of the
nucleotide chain rather than present at some point within it. Further, it
has proved difficult to provide even non-radioactively labeled probes
which may be inexpensively synthesized in large quantity. Thus, many known
probes are limited in their potential applications.
SUMMARY OF THE INVENTION
Accordingly, it is an object of the present invention to overcome the
aforementioned disadvantages of the prior art.
It is another object of the present invention to provide a novel modified
nucleotide useful in the synthesis of labeled polynucleotide probes.
It is still another object of the present invention to provide a nucleotide
modified at the 4-position of a pyrimidine base so as to include an
alkylamine or other reactive moiety which is derivatizable with a
detectable label.
It is yet another object of the present invention to provide a nucleotide
so modified at the 4-position which is further modified at the 5-position.
It is a further object of the present invention to provide a labeled
polynucleotide probe, at least one pyrimidine nucleotide of which is
modified at the 4-position so as to be derivatizable with a detectable
label.
It is still a further object of the invention to provide methods of making
derivatizable alkylamine nucleotides.
It is another object of the invention to provide a method of using a probe
labeled according to the method of the present invention to detect the
presence of known nucleic acid sequences in a sample.
Additional objects, advantages and novel features of the invention will be
set forth in part in the description which follows, and in part will
become apparent to those skilled in the art on examination of the
following, or may be learned by practice of the invention.
In one aspect of the invention, a modified, derivatizable nucleotide is
provided having the structure of Formula 1:
##STR2##
wherein R.sup.1 is a reactive group derivatizable with a detectable label,
which reactive group may be amine, carboxyl or thiol and further may be
protected for various synthetic manipulations, R.sup.2 is an optional
linking moiety such as those typically used to label proteins, and
includes an amide, thioether or disulfide linkage or a combination
thereof, R.sup.3 is selected from the group consisting of hydrogen,
methyl, bromine, fluorine and iodine, R.sup.4 is hydrogen, an anchoring
group which covalently binds the structure to a solid support, or a
blocking group such as dimethoxytrityl or pixyl, which blocking group is
generally base-stable and acid-sensitive, R.sup.5 is hydrogen, an
anchoring group which covalently binds the structure to a solid support,
or a phosphorus derivative enabling addition of nucleotides at the 3'
position, and may be, for example, PO.sub.3 H.sub.2, a phosphotriester, a
phosphodiester, a phosphite, a phosphoramidite, H-phosphonate or a
phosphorothioate, and R.sup.6 is H, OH, or OR where R is a functional
group useful as a protecting moiety in RNA synthesis, and x is an integer
in the range of 1 and 8 inclusive.
In another aspect of the invention, a method of making the above modified
nucleotide is provided including the step of derivatizing the R.sup.1
moiety with a detectable label.
In still another aspect, a polynucleotide probes is provided using one or
more of the above modified nucleotides. The probe can be used to screen a
sample containing a plurality of single-stranded or double-stranded
polynucleotide chains, and will label the desired sequence, if present, by
hybridization.
DETAILED DESCRIPTION OF THE INVENTION
"Derivatizable" nucleotides are nucleotides modified so as to include at
the 4-position of a pyrimidine a functional group which can react with a
detectable label. An example of a derivatizable nucleotide is one which
has been modified at the 4-position with an alkylamine moiety so that a
free amine group is present on the structure.
"Derivatized" nucleotides are nucleotides in which the derivatizable
functional group at the 4-position of the pyrimidine is bound, covalently
or otherwise, directly or indirectly, to a detectable label.
"Alkylamine nucleotides" are nucleotides having an alkylamine group at the
4-position of a pyrimidine, bound to the structure in such a way as to
provide a free amine group at that position.
A "polynucleotide" is a nucleotide chain structure containing at least two
nucleotides. The "polynucleotide probe" provided herein is a nucleotide
chain structure, as above, containing at least two nucleotides, at least
one of which includes a modified nucleotide which has substantially the
same structure as that given by Formula 1.
"Detectable label" refers to a moiety which accounts for the detectability
of a complex or reagent. In general, the most common types of labels are
fluorophores, chromophores, radioactive isotopes, and enzymes.
"Fluorophore" refers to a substance or portion thereof which is capable of
exhibiting fluorescence in the detectable range. Typically, this
fluorescence is in the visible region, and there are common techniques for
its quantitation. Examples of fluorophores which are commonly used include
fluorescein (usually supplied as fluorescein isothiocyanate [FITC] or
fluorescein amine), rhodamine, dansyl and umbelliferone.
The nucleotide numbering scheme used herein is illustrated by Formulae 2-5.
##STR3##
In a preferred embodiment, the substituents of the modified nucleotide of
Formula 1 are as follows.
R.sup.1, which is a reactive group derivatizable with a detectable label,
is preferably --NH.sub.2, --COOH or --SH.
R.sup.2 is an optional linker moiety which contains an amide, thioether or
disulfide linkage, or a combination thereof. R.sup.2 is preferably a
heterobifunctional linker such as those typically used to bind proteins to
labels. In most cases, a free amino group on a protein or other structure
will react with a carboxylic acid or activated ester moiety of the unbound
R.sup.2 compound so as to bind the linker via an amide linkage. Other
methods of binding the linker to the nucleotide are also possible.
Examples of particularly preferred linkers include
##STR4##
wherein x is an integer in the range of 1 and 8 inclusive.
As may be seen in Formula 1, the linker, if present, is attached to the
nucleotide structure through an alkylamine functionality
--NH--(CH.sub.2).sub.x -- wherein x is an integer in the range of 1 and 8
inclusive, and the alkylamine functionality is present at the 4-position
of the pyrimidine base.
As noted above, R.sup.3 is hydrogen, methyl, bromine, fluorine or iodine.
Thus, the base of the nucleotide is a pyrimidine optionally substituted at
the 5-position with the aforementioned R.sup.3 substituents.
R.sup.4 is typically hydrogen, if the modified nucleotide is a terminal 5'
structure, or a suitable blocking group useful in polynucleotide
synthesis. Examples of suitable blocking groups include substituted and
unsubstituted aralkyl compounds, where the aryl is, e.g., phenyl,
naphthyl, furanyl, biphenyl and the like, and where the substituents are
from 0 to 3, usually 0 to 2, and include any non-interfering stable
groups, neutral or polar, electron-donating or withdrawing, generally
being of 1 to 10, usually 1 to 6 atoms and generally of from 0 to 7 carbon
atoms, and may be an aliphatic, alicyclic, aromatic or heterocyclic group,
generally aliphatically saturated, halohydrocarbon, e.g., trifluoromethyl,
halo, thioether, oxyether, ester, amide, nitro, cyano, sulfone, amino,
azo, etc.
In one or more steps during nucleotide chain synthesis, it may be desirable
to replace the hydrogen atom or blocking group at the R.sup.4 position
with a more stable, "capping" group. Suitable capping groups include acyl
groups which provide for stable esters. The acyl groups may be organic or
inorganic, including carboxyl, phosphoryl, pyrophosphoryl, and the like.
Of particular interest are alkanoic acids, more particularly
aryl-substituted alkanoic acids, where the acid is at least 4 carbon atoms
and not more than about 12 carbon atoms, usually not more than about 10
carbon atoms, with the aryl, usually phenyl, substituted alkanoic acids
usually of from 8 to 12 carbon atoms. Various heteroatoms may be present
such as oxygen (oxy), halogen, nitrogen, e.g., cyano, etc. For the most
part, the carboxylic acid esters will be base labile, while mild acid
stable, particularly at moderate temperatures below about 50.degree. C.,
more particularly, below about 35.degree. C. and at pHs greater than about
2, more particularly greater than about 4.
The modified nucleotide may also be attached to a support through the
R.sup.4 position so as to facilitate addition of labeled or unlabeled
nucleotides at the 3' (R.sup.5) position. In such a case, R.sup.4 is an
anchoring group as will be described below. Covalent attachment to a
support is also preferred during sample screening, as the time and
complexity of separating the hybridized nucleotide chains from the sample
is substantially reduced. When the modified nucleotide of Formula 1 is
bound to one or more additional nucleotides at the 5' position, the
R.sup.4 substituent is replaced with such additional nucleotides which are
bound through their 3' phosphate groups.
R.sup.5, as noted, is hydrogen or a phosphorus derivative such as PO.sub.3
H.sub.2, a phosphotriester, a phosphodiester, a phosphite, a
phosphoramidite, an H-phosphonate or a phosphorothioate suitable for
polynucleotide synthesis, which derivative enables sequential addition of
nucleotides at the 3' position. More generally, such phosphorus
derivatives are given by Formula 9 and Formula 10:
##STR5##
wherein X is preferably hydrogen or an aliphatic group, particularly a
saturated aliphatic group, a .beta.-heterosubstituted aliphatic group,
where the .beta.-substituent is an electron-withdrawing group which
readily participates in .beta.-elimination, either as the leaving group or
the proton-activating group, substituted methylene, where the substituent
may vary widely and supports a negative charge on the methylene through
inductive or resonating effects; aryl; and aralkyl. Depending on the
nature of the phosphorus functionality, one group may be chosen over
another. Thus, depending upon whether a phorphorchloridite,
phosphoramidite, phosphate, thiophosphate, phosphite, or the like, is
employed, particular phosphoro ester groups will be preferred.
Similarly, the groups employed for Y will depend upon the nature of the
phosphorus derivative employed for oligomerization. When the
phosphoramidite is employed, Y will have the formula --NT.sup.1 T.sup.2,
where T.sup.1 and T.sup.2 may be the same or different and may be
hydrocarbon or have from 0 to 5, usually 0 to 4 heteroatoms, primarily
oxygen as oxy, sulfur as thio, or nitrogen as amino, particular
tert.-amino, NO.sub.2 or cyano. The two T's may be taken together to form
a mono- or polyheterocyclic ring having a total of from 1 to 3, usually 1
to 2 heteroannular members and from 1 to 3 rings. Usually, the two T's
will have a total of from 2 to 20, more usually 2 to 16 carbon atoms,
where the T's may be aliphatic (including alicyclic), particularly
saturated aliphatic, monovalent, or, when taken together, divalent
radicals, defining substituted or unsubstituted heterocyclic rings. The
amines include a wide variety of saturated secondary amines such as
dimethylamine, diethylamine, diisopropylamine, dibutylamine,
methylpropylamine, methylhexylamine, methylcyclopropylamine,
ethylcyclohexylamine, methylbenzylamine, methylcyclohexylmethylamine,
butylcyclohexylamine, morpholine, thiomorpholine, pyrrolidine, piperidine,
2,6-dimethylpiperidine, piperazine and similar saturated monocyclic
nitrogen heterocycles.
R.sup.5 may also represent a point of attachment for one or more additional
nucleotides at the 3' position. In that case R.sup.5 is phosphate, as such
additional nucleotides are typically bound through a phosphate group.
As at the 5' position, the modified nucleotide may be attached to a support
through the 3' position, i.e. through R.sup.5. When the nucleotide thus
attached to a support, R.sup.5 is an anchoring group as will be described
below.
R.sup.6, in the case of deoxyribose, is H; in the case of ribose, is OH;
and, during RNA synthesis, is a suitable blocking group which protects the
--OH moiety from modification. Blocking groups useful here generally
include those given above for R.sup.4, and the specific choice of blocking
group will be apparent to one skilled in the art. Examples of blocking
groups which are preferred at the R.sup.6 position during RNA synthesis
include silyl ethers such as t-butyldimethylsilyl, substituted methyl
ethers, o-nitrobenzyl ether, esters such as levulinic ester, and the
following pyranyl structures given by Formula 11 (tetrahydropyranyl) and
Formula 12 (4-methoxytetrahydropyranyl):
##STR6##
A particularly preferred blocking group is ortho-nitrobenzyl. Additional
examples of suitable blocking groups may be found in Green, T.W.,
Protective Groups in Organic Synthesis, New York: Wiley & Sons, 1981.
The modified nucleotide will normally be derivatized with a label in a
manner which will allow for detection of complex formation. A wide variety
of labels may be used, and one or another label may be selected depending
upon the desired sensitivity, the equipment available for measuring, the
particular protocols employed, ease of synthesis, and the like. Labels
which have found use include enzymes, fluorescers, chemiluminescers,
radionuclides, enzyme substrates, cofactors or suicide inhibitors,
specific binding pair members, particularly haptens, or the like. The
molecule involved with detection may be covalently bound to the modified
nucleotide or indirectly bound through the intermediacy of a specific
binding pair, i.e. ligand and receptor. Examples of ligands and receptors
include biotin-avidin, hapten-antibody, ligand-surface membrane receptor,
metal-chelate, etc.
As suggested above, it is preferred that the modified nucleotide be
covalently bound to a support at either the R.sup.4 or R.sup.5 positions
for oligonucleotide synthesis. A wide variety of supports may be used,
including silica, Porasil C, polystyrene, controlled pore glass (CPG),
kieselguhr, poly(dimethylacrylamide), poly(acrylmorpholide), polystyrene
grafted onto poly(tetrafluoroethylene), cellulose, Sephadex LH-20,
Fractosil 500, etc.
Depending on the nature of the support, different functionalities will
serve as anchors. As noted above, these "anchoring" groups are at either
the 3' or the 5' position, i.e. at either the R.sup.5 R.sup.4 positions,
respectively. For silicon-containing supports, such as silica and glass,
substituted alkyl or aryl silyl compounds will be employed to form a
siloxane or siloximine linkage. With organic polymers, ethers, esters,
amines, amides, sulfides, sulfones and phosphates may find use. For aryl
groups, such as polystyrene, halomethylation can be used for
functionalization, where the halo group may then be substituted by oxy,
thio (which may be oxidized to sulfone), amino, phospho (as phosphine,
phosphite or phosphate), silyl or the like. With a diatomaceous earth
element (e.g., kieselguhr), activation may be effected by a polyacrylic
acid derivative and the active functionality reacted with amino groups to
form amine bonds. Polysaccharides may be functionalized with inorganic
esters, e.g. phosphate, where the other oxygen serves to link the chain.
With polyacrylic acid derivatives, the carboxyl or side chain
functionality, e.g., N-hydroxyethyl acrylamide, may be used in
conventional ways for joining the linking group.
The modified nucleotide of Formula 1, as previously suggested, can be used
as a substrate for synthesis of polynucleotide probes. Additional
nucleotides may be sequentially added at the 5' position by, for example,
the phosphoramidite method of Beaucage and Caruthers, Tetrahedron Lett.
22(20):1859-62 (1981) or the phosphotriester method of Itakura, J. Biol.
Chem. 250:4592 (1975), or the like, or at the 3' position by the method of
Belagaje and Brush, Nuc. Acids Research 10:6295 (1982), or both. The
nucleotides which are sequentially added may be unlabeled, or they may be
modified according to Formula 1 and derivatized with a label at the
R.sup.1 moiety. Accordingly, one or more labels may be present within a
polynucleotide chain rather than at one end.
This polynucleotide probe includes at least one modified nucleotide having
substantially the same structure as that given by Formula 1, i.e.
including at least one modified nucleotide having the structure given by
Formula 13:
##STR7##
wherein R.sup.1 is a reactive group derivatized with a detectable label,
R.sup.2 is an optional linking moiety including an amide, thioether or
disulfide linkage or a combination thereof, R.sup.3 is selected from the
group consisting of hydrogen, methyl, bromine, fluorine and iodine,
R.sup.6 is H, OH, or OR where R is an acid-sensitive, base-stable
protecting group and x is an integer in the range of 1 and 8 inclusive.
The polynucleotide probe may have a single label or a plurality of labels,
depending upon the nature of the label and the mechanism for detection.
Where the label is fluorescent, for example, a distance of at least 3 to
12 Angstroms should be maintained between fluorescent species to avoid any
fluorescence quenching.
Such labeled polynucleotide probes may be used in the assays described in
applicants' co-pending application Ser. No. 807,624, now U.S. Pat. No.
4,868,105, or in any number of other applications, including conjugation
with enzymes, antibodies and solid supports. An example of one such use of
applicants' novel oligonucleotide probes is in the detection of a known
sequence of DNA. The probe may be prepared so as to be attached, for
example, to a standard latex solid support or to an avidin support in the
case of biotin-labeled probes. Sample containing single-stranded or
double-stranded DNA sequences to be analyzed is caused to contact the
probe for a time sufficient for hybridized nucleic acid complexes to form,
and any such complexes are detected by means of the fluorescent, biotin or
otherwise detectable label.
Synthesis of the modified nucleotide: The present invention also relates to
a method of synthesizing the novel modified nucleotide of Formula 1. In
the preferred embodiment, a pyrimidine nucleotide is provided which has
the structure of Formula 14 or Formula 15:
##STR8##
wherein R.sup.3 is as given above, R.sup.4 and R.sup.5 are hydrogen, and
R.sup.6 is OH or H. The 5' position of the sugar ring--and the 2' position
as well if the sugar is ribose rather than deoxyribose--is then protected
against modification during subsequent reaction steps by addition of a
dimethoxytrityl group (see Example 3) or other suitable protecting group,
the addition reaction allowed to proceed for a time sufficient to ensure
substantial completeness. Similarly, the 3' hydroxyl group is protected
with a silyl or other suitable functionally (see Example 4).
Examples of particularly suitable protecting groups include those set forth
above as "R.sup.6 ", i.e., substituted methyl ethers, esters, pyranyls and
the like.
When the nucleoside is thymine or uracil, or uracil modified at the
5-position by an R.sup.3 substituent, i.e. a pyrimidine or substituent
pyrimidine which has an oxy rather than an amino substituent at the
4-position, the carbonyl is converted to an amine moiety by, for example,
reaction with an activating agent such as
1-(mesitylene-2-sulfonyl)-tetrazole (MS-tet) or other suitable condensing
reagent. Activating agents for use herein also include other sulfonyl
compounds given by the formula E.sub.1 -SO.sub.2 -E.sub.2 wherein E.sub.1
is tetrazoyl, nitrotriazoyl, triazoyl, imidazoyl, nitroimidazoyl, or the
like, and E.sub.2 is an aryl or substituted aryl group such as mesitylene,
etc. Another class of suitable activating agents is given by Formula 16:
##STR9##
wherein E.sub.1 is as defined above. In Formula 16b, E.sub.1 is present in
a solution containing the activating agent but is not bound thereto, and X
is a halogen substituent, preferably chlorine. In general, any activating
agent may be used and may include one or more halogen substituents,
preferably chlorine, on the ring structure which after reaction can be
displaced by ethylene diamine or like reagent. This conversion is followed
by reaction with an alkyldiamine such as ethylenediamine to give a
nucleotide having a --NH--(CH.sub.2).sub.x NH.sub.2 functionality at the
4-position of the pyrimidine ring (see Examples 5, 6). The free amine
group so provided is then optionally reacted with caproic acid, an
activated caproic acid ester, or with a caproic acid derivative such as
6-aminocaproic acid, in order to ensure sufficient spacing between the
nucleotide and the detectable label to be attached at the R.sup.1 moiety.
The caproic acid or related compound may be labeled prior to attachment
(see Example 7) or subsequently.
When the nucleoside is cytosine or a 5-modified cytosine, i.e. substituted
with an R.sup.3 other than hydrogen, the exocyclic amino functionality can
be converted to an N.sup.4 -aminoalkyl or N.sup.4 -aminoaryl cytosine by
reaction with an aryl sulfonyl chloride followed by reaction with an
alkyl- or aryldiamine (Scheme I). See, e.g., Markiewicz, W. T. and R.
Kierzek, 7th Intl. Round Table, pp. 32 and 72 (1986). Alternatively,
preparation of N.sup.4 -substituted cytosine may be effected using a
bisulfite-catalyzed exchange reaction. See Schulma, L. H. et al., Nuc.
Acids Res. 9:1203-1217 (1981) and Draper, D. E., Nuc. Acids Res.
12:989-1002 (1984).
##STR10##
In Scheme I, the abbreviation "FMOC" indicates fluoroenylmethylene
oxycarbonyl, while "NiPr.sub.2 " represents diisopropylamine.
##STR11##
Alternatively, where the alkylamine group is more than about 6 carbon atoms
long, the free amine group thereof may directly bond to a suitable
detectable label.
The synthesis may further include removal of the dimethoxytrityl or other
protecting groups with acid, followed by, if desired, phosphorylation or
phosphitylation of the 3' position in preparation for sequential addition
of nucleotides.
It is to be understood that while the invention has been described in
conjunction with the preferred specific embodiments thereof, that the
foregoing description as well as the examples which follow are intended to
illustrate and not limit the scope of the invention, which is defined by
the scope of the appended claims. Other aspects, advantages and
modifications within the scope of the invention will be apparent to those
skilled in the art to which the invention pertains.
EXAMPLES
Examples 1
Labeling of Caproic Acid Derivative
##STR12##
To 1 mmole of fluorescein isothiocyanate in 5 ml of DMF was added 2 mmole
of 6-aminocaproic acid and 540 .mu.l of triethylamine. After 24 h at room
temperature, the product was isolated by preparative thin layer
chromatography (Warner and Legg, Inorg. Chem. 18:1839 (1979)). The dried
product was suspended in 10 ml of 1:1 DMF/THF (v/v) to which 1.5 mmole of
N-hydroxy succinimide and 1 mmole of dicyclohexylcarbodiimide were added.
After 18 h at room temperature the solution was filtered through glass
wool and diluted to a 0.2M final concentration of A with DMF (assuming a
100% yield from step 1).
Example 2
6-N.sup.4 -(2-Aminoethyl)- Deoxycytidine
##STR13##
An alkylated derivative of deoxycytidine, N.sup.4 -(2-aminoethyl)
deoxycytidine (B) was prepared from properly protected deoxyuridine via
the 4-tetrazoyl derivative as described by Reese and Ubasawa, Tetrahedron
Lett. 21:2265 (1984). This latter derivative was converted to B by
displacement of the tetrazoyl moiety with ethylene diamine essentially as
described by Sung, J. Org. Chem. 47:3623 (1982) and Maggio et al.,
Tetrahedron Lett. 25:3195 (1984). The corresponding
5'-DMT-3'-phosphoramidite N.sup.4 -(2-N-trifluoroacetylaminoethyl)
deoxycytidine was prepared by blocking the alkylamine with trifluoroacetic
anhydride and then preparing the corresponding N,N-diisopropyl
phosphoramidite as described (Beaucage and Caruthers, supra; McBride and
Caruthers, Tetrahedron Lett. 24:245 (1983)).
Example 3
5'-Dimethoxytrityl-2'-Deoxyuridine
##STR14##
To 2-Deoxyuridine (10 g, 44 mmole) dried by coevaporation of pyridine and
suspended in pyridine (100 ml) was added 18.4 g (54 mmole)
4.4'-dimethoxytrityl chloride (DMT-Cl). The reaction was allowed to
proceed for 18 h at room temperature, and 100 ml methanol was added to
deactivate excess DMT-Cl. Most of the pyridine was then removed in vacuo,
and the residue, dissolved in 500 ml ethyl acetate, was washed with
saturated aqueous NaHCO.sub.3 (3.times.500 ml). The organic phase was
dried over solid Na.sub.2 SO.sub.4 and evaporated to dryness. The residue
was purified by flash chromatography on silica gel to give 18.0 g (77%) of
5'-dimethoxytrityl-2'-deoxyuridine (C).
Example 4
5'-O-(4,4'-Dimethoxytrityl)-3'-t-Butyldimethylsilyl-2'-Deoxyuridine
##STR15##
To 18 g (34 mmole) of C in 200 ml DMF was added imidazole (5.8 g, 85 mmole)
with rapid stirring to assure complete dissolution. t-Butyldimethylsilyl
chloride (7.65 g, 51 mmole) dissolved in a small volume of DMF was added
dropwise with stirring and the reaction was allowed to proceed in the dark
for 18 h at room temperature. The reaction mixture was diluted with ethyl
acetate (250 ml) and extracted with NaHCO.sub.3 (3.times.250 ml). The
organic phase was dried over Na.sub.2 SO.sub.4 and evaporated to dryness.
The residue was purified by flash chromatography on silica gel to give
15.0 g (68% yield) of
5'-O-(4,4'-dimethoxytrityl-3'-t-butyldimethylsilyl-2'-deoxyuridine (D).
Example 5
4-(1,2,3,4-Tetrazol-1-yl)-[5'-(4,4'-Dimethoxytrityl)-3'-t-Butyldimethyl-sil
yl-.beta.-D-2'-Deoxyribosyl] Pyridine-2(lH)-one
##STR16##
To 15.0 g (23 mmole) of D, dried by coevaporation of pyridine and dissolved
in pyridine (50 ml) was added diphenylphosphate (2.9 g, 11.5 mmole)
dissolved in pyridine (5 ml). 1-(Mesitylene-2-sulfonyl)-tetrazole (MS-tet)
(15.5 g, 61.5 mmole) dissolved in pyridine (45 ml) was added and the
reaction mixture allowed to proceed in the dark for 18 h at room
temperature. To the dark brown reaction mixture was added 25 ml water.
After 30 min, the product was concentrated under reduced pressure. The
residue was dissolved in 250 ml methylene chloride, washed with an aqueous
NaHCO.sub.3 solution (3.times.250 ml), dried over Na.sub.2 SO.sub.4, and
the solvent was removed under reduced pressure in the presence of toluene.
The residue was purified by flash chromatography on silica gel to give
10.0 g (62%) of
4-(1,2,3,4-Tetrazol-1-yl)-[5'-(4,4'-dimethoxy-trityl)-3'-t-butyldimethylsi
lyl-.beta.-D-2'-deoxy-ribosyl]-pyridine-2(1H)-one (E).
Example 6
4-N-(2-Aminoethyl)-5'-Dimethoxytrityl-3'-t-Butyldimethylsilyl-2'-Deoxycytid
ine
##STR17##
To a solution of ethylene diamine (9.3 ml, 143 mmole) in dioxane (100 ml)
cooled to 5.degree. C. was added E (10.0 g, 14.3 mmole) and left for one
hour. The solvent was removed at reduced pressure and the residue was
coevaporated with toluene to remove excess ethylene diamine. The product
was purified by chromatography on a silica gel column, eluted with 12-20%
methanol in methylene chloride to give 7.15 g (75%) of
4-N-(2-aminoethyl)-5'-dimethoxytrityl-3'-t-butyldimethylsilyl-2'-deoxycyti
dine (F). The product was shown to react positively with ninhydrin,
confirming the presence of a free amine moiety.
Example 7
N.sup.4
-(N-FMOC-6-Aminocaproyl-2-Aminoethyl)-5'-Dimethyltrityl-3'-t-Butyldimethyl
silyl-2'-Deoxycytidine
##STR18##
To a solution of F (6.5 g, 9.6 mmole) in pyridine (50 ml) was added
N-FMOC-6-aminocaproic acid (4.26 g, 12 mmole) (FMOC represented by
structure H) and DCC (2.96 g, 14.4 mmole). After 3 h, the reaction was
complete as judged by tlc (silica in 10% methanol/methylene chloride).
Pyridine was removed at reduced pressure. The residue was extracted with
ethyl acetate, insoluble dicyclohexylurea (DCHU) filtered off and the
solvent removed. The product was isolated by silica gel chromatography
eluted with 4% methanol in methylene chloride affording 7.3 g (70%) of
N.sup.4
-(N-FMOC-6-amino-caproyl-2-amino-ethyl)-5'-dimethyltrityl-3'-t-butyldi-met
hylsilyl-2'-deoxycytidine (G).
Example 8
##STR19##
A solution of tetrabutylammonium fluoride (15 mmole, 15 ml of a lM solution
in THF) and aqueous HF (1.05 ml of a 50% aqueous solution) were mixed and
dried by coevaporation of pyridine. The residue was dissolved in pyridine
(15 ml) and added to G (7.2 g, 7.3 mmole) which was dissolved by
sonication. After 18 hours at 4.degree. C. the reaction mixture was
diluted with 200 ml methylene chloride. Concentrated aqueous NaHCO.sub.3
was carefully added followed by solid NaHCO.sub.3, added gradually so as
to neutralize the HF/pyridine. After drying over Na.sub.2 SO.sub.4, the
organic phase was concentrated to an oil, which was subjected to silica
gel chromatography. The product N.sup.4
-(N-FMOC-6-aminocaproyl-2-aminoethyl)-5'-dimethoxytri-tyl-2'-deoxycytidine
(I) was eluted with 5-6% methanol in methylene chloride to give an 86%
yield (6.0 g).
Example 9
##STR20##
To 5.1 g (5.7 mmole) of I in methylene chloride containing
(diisopropylethylamine) was added
##STR21##
(chloro-N,N-diisopropylaminomethoxy phosphine, 1.3 ml [1.2 eq.], K) at
0.degree. C. under argon. After 1 hr, ethyl acetate (200 ml) was added and
washed with 80% saturated aqueous sodium chloride; after drying of the
organic phase over Na.sub.2 SO.sub.4, the product in methylene chloride
was added dropwise to hexane at -40.degree. C. to precipitate 4.43 g (75%)
of J.
Example 10
Probe Preparation (Fluorescein Label)
Synthetic oligonucleotides were prepared by an automated phosphoramidite
method as described in Warner et al., DNA 3:401(1984). Purification was
carried out according to Sanchez-Pescador and Urdea, DNA 3:339 (1984).
The aminoethyl derivative of deoxycytidine as prepared in Example 2 was
incorporated by standard coupling procedures during the oligonucleotide
synthesis and the purified modified oligonucleotides were used for
incorporation of a fluorescein label as follows. To a dried sample (3-5 OD
260 units) of the aminoethyl deoxycytidine containing oligomer were added
50 .mu.l of DMF and 25 .mu.l of the 0.2M stock solution of A described
above. After 18 h at room temperature, the solution was partially purified
by Sephadex G-10 chromatography eluted with water, dried and further
purified by polyacrylamide gel, as above.
Example 11
Probe Preparation (Biotin Label)
Using the probes containing aminoethylcytidine as prepared in the previous
example, biotin labeling was achieved as follows. The oligonucleotide (3-5
OD 260 units) was taken up in 50 .mu.l 0.Ml sodium phosphate, pH 7.0 and
50 .mu.l of DMF to which 100 .mu.l of a DMF solution containing 1 mg of a
"long chain" N-hydroxysuccinimidyl biotin (Pierce Chemical) was added.
After 18 h at room temperature, the biotinylated probe was purified as
described for the fluorescein labeled probe.
Example 12
Synthesis of Horseradish Peroxidase (HRP): DNA Conjugates
Sequence 1 (5'-[LCA]CTGAACGTTCAACCAGTTCA-3') where LCA=N.sup.4
(6-aminocaproyl-2-aminoethyl)-deoxy cytidine) was synthesized chemically
and purified as described elsewhere (Warner, et al. (1984) DNA 3, 401). To
10 OD 260 units dissolved in 50 .mu.l of water were added 10 .mu.l of 1.0M
sodium borate, pH 9.3, and 500 .mu.l of distilled dimethylformamide
containing 20 mg of p-phenylene diisothiocyanate. The solution was
vortexed and set for 2 hr at room temperature in the dark. Approximately 3
ml of n-butanol was then added. After vortexing, adding 3 ml of water, and
vortexing again, the tube was centrifuged and the yellowish upper layer
discarded. The extraction process was repeated with subsequent n-butanol
additions until an final volume of approximately 50 .mu.l was obtained.
The butanol was removed by evacuation, then 10 mg of HRP in 200 .mu.l of
0.1M burate, pH 9.3, was added. The mixture was vortexed, then set at room
temperature overnight in the dark.
Separation of the HRP-DNA conjugate from free enzyme and DNA was achieved
on a 7% polyacrylamide gel. The 250 .mu.l reaction mixture was quenched
with 100 .mu.l of 25% glycerol, 0.5% SDS, 0.5% bromophenol blue, 2.5 mM
EDTA. The solution was then distributed into 10 lanes of a 20.times.20
0.15 cm gel and run at 60 mAmps under standard conditions (Maxam, A., and
Gilbert, W., (1980) Methods in Enzymol 65, 499-560) until the bromophenol
blue was about 2/3 down the gel. The gels were set on Baker F-254 silica
60 plates that had been covered with Saran Wrap (Dow) and examined with a
handheld UV-short wavelength lamp held above. Pictures of the UV-shadowed
bands were taken with a Polaroid MP-4 camera system fitted with a Kodak
No. 59 green filter, after which the bands were cut out with a razor
blade. The bands were put into a 10-ml Bio-Rad polypropylene econo-columns
to which 3 ml of 0.1M sodium phosphate, pH 7.5, was added, then set at
room temperature overnight.
The contents of the column were filtered through the frit at the column
bottom into an Amicon centricon microconcentrator that had been washed
twice with distilled water. The HRP-DNA conjugate was then concentrated by
centrifugation at 3500 rpm and washed twice with 1x PBS also by
centrifugation. The final solution was then stored at 4.degree. C.
Example 13
Assay for HBV DNA Using HRP-DNA Probe and a Biotinylated Probe Bound to an
Avidin Bead
Biotin labeled probe (B': 1000 pmoles in 66.7 .mu.l of water) was combined
with 5 ml of a 0.25% (w/v) solution of 0.8.mu. avidin beads (Pandex
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