 |
Description  |
|
|
BACKGROUND OF THE INVENTION
This invention relates to a means of labeling nucleic acids, preferably
DNA. More particularly, this invention is directed to a process for
preparing labeled nucleic acids by use of alkylating intercalators
containing a label moiety, where the word label is intended to include
moieties which may be detected both directly and indirectly. In addition,
this invention relates to a means for detecting the presence of a nucleic
acid sequence such as a gene using a hybridization probe containing a
complementary nucleic acid sequence.
In biomedical research and recombinant DNA technology it is often useful to
have indicator probes which allow the user to detect, monitor, localize or
isolate nucleic acids when present in any amount. DNA hybridization
probes, for example, contain a nucleic acid sequence complementary to the
nucleic acid sequence or to the gene to be detected. Such probes have been
used to detect the presence of genes coding for antigens responsible for
graft rejection, such as human leukocyte antigen (HLA), or genetic
disease, such as sickle cell anemia. For example, Sood et al., PNAS, 78,
616-620 (1981) describe the isolation of cDNA clones for HLA-B antigens.
These clones were prepared by synthesizing cDNA from an mRNA mix
containing mRNA coding for the desired HLA antigen, inserting the cDNA
into a vector, transforming a bacterial host and isolating transformant
clones that contain the desired DNA segment by probing with an
oligonucleotide probe that is specific for the desired DNA sequence.
Ploegh et al., PNAS, 77, 6081-6085 (1980) have also reported cloning a
cDNA probe for an HLA gene sequence. In addition, U.S. Pat. No. 4,358,535
to Falkow et al. describe a method for detecting infectious
disease-causing microbes using labeled nucleotide probes complementary to
nucleic acid contained by the pathogenic microbe. Until recently, the
materials most sensitive and therefore useful for this purpose were
radioactively labeled nucleic acids such as those labeled with isotopes
of, e.g., hydrogen (.sup.3 H), phosphorus (.sup.32 p) or iodine (.sup.125
I).
Such radioactive compounds, however, suffer from various drawbacks,
including extensive safety precautions, expensive equipment,
health-monitoring services and waste treatment, and high usage costs due
to the instability of the materials. Therefore, there is an increasing
incentive to search for suitable nonradioactive labels for nucleic acids
which would provide sensitive probes.
Already known is that haptens can initiate an immune response if bound to a
carrier, so as to be useful for labeling and identification. Thus, for
example, hapten-labeled DNA can be detected with antibodies.
It is also known that biotin interacts with streptavidin or avidin, a
68,000 dalton glycoprotein from egg white, to form a tightly held
non-covalent complex which has been recently used to develop methods for
visually localizing specific proteins, lipids or carbohydrates on or
within cells. For example, Manning et al., Chromosoma, 53, 107 (1975) have
determined the chromosomal location of ribosomal genes by election
microscopy using a biotinized protein, cytochrome C, chemically
crosslinked to RNA as a hybridization probe. Langer et al., Proc. Natl.
Acad. Sci. USA, 78, 6633-6637 (1981) describe a method for labeling DNA by
enzymatic incorporation of nucleotide analogs containing functional groups
such as biotin via DNA polymerase I, and Leary et al., Proc. Natl. Acad.
Sci. USA, 80, 4045-4049 (1982) have used this method to label DNA probes
with biotinylated nucleotides. European Patent Publication No. 0,063,879
published Nov. 3, 1982 to D. Ward et al. describes nucleotide derivatives
which contain biotin, iminobiotin, lipoic acid and other labels attached
covalently to the pyrimidine or purine ring which will interact with
proteins such as avidin or antibodies. When biotin is bound specifically
by an avidin-linked enzyme complex, detection is seen as a color change in
a chromogenic substrate. When an avidin-alkaline phosphatase complex is
used to detect biotinylated DNA probes after hybridization, sensitivity
has been shown to approach that of autoradiography used to detect .sup.32
p labeled probes.
The method of Ward et al. for nonradioactive labeling results in labeling
of the hybridizing region of the probe, thus causing significant
interference with hybridization.
U.S. patent application Ser. No. 444,438 filed Nov. 24, 1982 to Letsinger
et al. describes bifunctional intercalaters containing a phenanthridium
moiety as an agent for introducing markers (e.g., fluorescent probes) at
specified regions in polynucleotides.
Various methods exist for attaching chemical labels to DNA. For example, it
is well known how to attach chemical moieties to pyrimidine and purine
rings using an acetoxymercuration reaction whereby covalently bound
mercury atoms are introduced into the 5-position of the pyrimidine ring,
the C-8 position of the purine ring, or the C-7 position of a
7-diazapurine ring. European Patent Publication 0,063,879 supra describes
the preparation of a nucleotide derivative by a process where a mercurated
intermediate is formed which reacts with a reactive chemical moiety which
may be the label or which then reacts with the label compound. In these
methods, the labeled nucleotide is then incorporated into DNA so that the
DNA is labeled.
Methods also exist for studying the structure of DNA. For example,
psoralens, which are a class of planar furocoumarin molecules capable of
intercalating into double-stranded DNA in the presence of single-stranded
DNA, will covalently bond to and crosslink DNA when activated by long-wave
(>350 nm) UV light. Covalent bonding involves a two-step process: (1)
intercalating the planar-structured psoralen between the base pairs in the
double helix structure of the nucleic acid to produce a psoralen-nucleic
acid complex and (2) irradiating the complex with light of the proper
wavelength to form covalent bonds between the psoralen molecules and
pyrimidine nucleotides which occur as integral entities of nucleic acid
strands.
This covalent bonding enables the study in vivo of secondary structures of
DNA such as packaging of nucleic acid within viruses. Use of 4'-adducts of
4,5',8-trimethylpsoralen to bond DNA covalently is described in U.S. Pat.
No. 4,124,598 to Hearst et al. Hearst, Rapoport and others have
extensively studied the incorporation of psoralens into DNA and RNA. Brown
et al., Gene, 20, 139-144 (1982) teaches stabilizing radioactive RNA-DNA
hybridization probes using trimethylpsoralen.
Saffran et al., Proc. Natl. Acad. Sci. U.S.A., 79, 4594-4598 (1982)
describes site-directed psoralen crosslinking of DNA to enable structural
analysis using a psoralen derivative containing a thiol group. In this
process the plasmid DNA molecule has mercurated nucleotides incorporated
near a restriction site so that the psoralen is directed to the bases
through a Hg-S linkage.
In addition to their use in studying nucleic acid secondary structure,
commercial applications of the psoralen derivatives include their use in
treating certain dermatological disorders and for viral inactivation to
produce vaccine.
Use of mercurated compounds in reaction sytheses involves extra expense and
necessitates safety precautions in view of the toxicity of mercury.
Another use for compounds which label DNA is in chromosome banding or
staining. An example described in the literature is the use of the Giemsa
reagent to stain regions or bands of chromosomes differentially, as
described in the article by V. G. Dev et al., Lancet (England) 1, 1285
(June 10, 1972). Because chromosomes have characteristic banding patterns,
this procedure can be used to distinguish chromosomes. This ability to
distinguish chromosomes has been very useful in the study of chromosome
anomalies. For example, Down's syndrome can be diagnosed by determining
that the individual is trisomic for chromosome 21.
SUMMARY OF THE INVENTION
To obviate the disadvantages associated with the labeled probes presently
existing, the present invention provides a means for producing stable
labeled nucleic acid hybridization probes which, rather than using
mercurated intermediates or enzymes, employs specific labeling compounds
capable of both intercalation and alkylation to introduce label moieties
into double-stranded nucleic acids. Because the labeling reagents herein
are selective for double-stranded nucleic acids, the hybridizing region of
the probe is not labeled, as is the hybridizing region of the probes
prepared by the nick translation method of incorporating biotinylated
nucleotides, which is the most common method. The advantage of this
feature is that the present probes in which the hybridizing region is free
from obstruction and the separate labeled region is stabilized by covalent
bonding are therefore relatively insensitive to more stringent
hybridization conditions as compared to probes in which the nucleotide
labels with protruding groups are incorporated into the hybridizing
region. More stringent hybridization conditions such as higher
temperatures and lower salt content in the media result in more specific
hybridization and lower background. In addition, higher temperatures may
increase the rate of hybridization.
As a further advantage the method herein is preferably employed to label
nucleic acids nonradioactively to avoid the disadvantages of radioactive
labeling.
As yet a further advantage, the labeling reagents of the present invention
may also have application in chromosome banding because these compounds
bind nucleic acids specifically and can have fluorescent or chromogenic
label moieties. The advantage of using the labeling compounds herein over
other dyes to stain DNA or other nucleic acids is that the compounds
herein are covalently attached to the DNA so that the labeled DNA can be
treated more harshly in the wash procedure required to remove
unincorporated dye.
Specifically, the present invention provides as a reagent a novel labeling
composition of the formula:
[A][B]L
wherein A is an alkylating intercalation moiety, B is a divalent organic
moiety (hereinafter referred to as spacer arm) having a straight chain of
at least two carbon atoms, and L is a monovalent label moiety capable of
producing a detectable signal. Preferably A is a 4'-methylene-substituted
psoralen moiety, and most preferably a
4'-methylene-substituted-4,5',8-trimethylpsoralen moiety. In addition, the
preferred spacer arm is a straight chain compound having at least seven
carbon atoms.
A compound useful as an intermediate in preparing these labeling reagents
has the formula: [B]L, where B is a divalent organic moiety (spacer arm)
having a straight chain comprising two or more --(CH.sub.2).sub.x O--
moieties where x is a number from 1 to 4 inclusive, preferably no more
than 2, and L is a monovalent label moiety capable of producing a
detectable signal.
The labeling reagent herein, and preferably the one comprising a
4'-methylene-substituted-4,5',8-trimethylpsoralen moiety attached to the
spacer arm via the 4'-methylene group, can be used to label nucleic acids
by a two-step process comprising:
(a) contacting an at least partially double-stranded nucleic acid with one
or more of the labeling reagents described above so as to cause the
alkylating intercalation moiety of the reagent to intercalate into the
nucleic acid to form a complex; and
(b) activating the complex so as to induce the alkylating intercalation
moiety of the reagent to bond covalently to one or more of the nucleic
acid strands.
The above process can be used more specifically to prepare labeled nucleic
acid hybridization probes for detecting nucleic acid sequences by:
(a) contacting a nucleic acid comprising a double-stranded nucleic acid
region adjacent to a single-stranded region capable of hybridization with
the nucleic acid sequence to be detected, with one or more of the reagents
described above, the contacting causing the alkylating intercalation
moiety of the reagent to intercalate into the nucleic acid to form a
complex; and
(b) activating the complex so as to induce the alkylating intercalation
moiety of the reagent to bond covalently to the double-stranded nucleic
acid region, via either one or both of the nucleic acid strands.
Preferably these processes are carried out using nonradioactively labeled
reagents.
The invention also includes the hybridization probe itself comprising a
nucleic acid which comprises a double-stranded nucleic acid region
adjacent to a single-stranded region where the double-stranded region is
covalently bound to one or more alkylating intercalation moieties via one
or both nucleic acid strands and where the alkylating intercalation moiety
is bound to the spacer arm which is in turn bound to the label moiety.
In a further aspect the invention provides a process for detecting the
probe comprising exposing the probe to a means by which the label moiety
of the probe is capable of being identified, and identifying the label
moiety using an appropriate identification technique. Examples of such
techniques include spectroscopic, radioisotopic, photochemical, chemical,
immunochemical or biochemical means as by using a polypeptide, lectin or
antibody capable of forming a complex with the label moiety of the probe.
Using the preferred biochemical means, the probe is contacted with a
polypeptide, lectin or antibody capable of forming a complex therewith
under suitable conditions so as to form the complex, said polypeptide,
lectin or antibody being capable of or including a label which can be
detected when the complex is formed, and the complex is detected using an
appropriate detection technique.
In yet another embodiment of the invention, one or more nucleic acid
sequences, preferably those characteristic of a pathogenic microbe, which
are contained in a sample suspected of containing the sequence or
sequences are detected by a process comprising:
(a) contacting the sample containing the nucleic acid(s) to be detected,
and generally consisting of cells, body fluid or viral or tissue sample,
with an effective amount of reagent sufficient to open the cells, body
fluid, viral capsids, or tissue of the sample and separate the strands of
the nucleic acid(s);
(b) depositing the sample before, during, or after step (a) on an inert
support;
(c) contacting the deposited sample with an effective amount of reagent
sufficient to affix a substantially single-stranded form of the nucleic
acid(s) on the support;
(d) contacting the affixed nucleic acid single-stranded form with an
effective amount of the hybridization probe as described above under
hybridization conditions; and
(e) detecting hybridization of the single-stranded nucleic acid sequences
by means of the label moiety on the probe.
In still another aspect of the invention, certain labeled reagents herein
can be used to label specific regions of chromosomes in an improved
process wherein a reagent detectable by fluorescent or chromogenic
detection means is used to stain the chromosome regions differentially so
as to distinguish the chromosomes and the reagent is then detected by the
detection means.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 represents a restriction map of pCHL2 Chlamydia trachomatis plasmid
cloned into the BamHI site of pBR 322. The M13mp9CHL2.1 subclone of pCHL2
is an approximately 1500 base pair insert between the XmaI and PstI
restriction sites as indicated.
FIG. 2 represents a restriction map of M13mp9CHL2.1 containing the insert
from the plasmid pCHL2.
FIG. 3 represents the cloning region of M13mp9 (FIG. 3A) and the cloning
region of M13mp10 (FIG. 3B).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The following terms as used in the specification and claims have the
following definitions:
"Spacer arm" refers to a divalent organic moiety which is chemically
non-reactive with the alkylating intercalation moiety and label moiety
emloyed herein and is of sufficient length (at least two carbon atoms in
the main chain) to permit detection of the reagent by the necessary
detection means. Its purpose is to connect the alkylating intercalation
moiety with the label moiety while avoiding interaction between the two so
that the label moiety can be readily detected.
"Label moiety" refers to a monovalent moiety which is capable of producing
a detectable signal, i.e., which can be detected in small quantities by
detection means which generate a signal. Examples of suitable such means
includes spectroscopic or photochemical means, e.g., fluorescence or
luminescence, or biochemical, immunochemical, or chemical means such as
changes in physical, biochemical, immunochemical or chemical properties on
contact with a detector analysis compound or reaction with a polypeptide
or polypeptide/enzyme mixture to form a detectable complex. Thus, as used
herein the term "label" is intended to include both moieties that may be
detected directly, such as radioisotopes or fluorochromes, and reactive
moieties that are detected indirectly via a reaction which forms a
detectable product, such as enzymes that are reacted with substrate to
form a product that may be detected spectrophotometrically. It is noted
that the labeling reagent may contain a radioactive label moiety such as a
radioisotope, but the preferred hybridization probe herein is
nonradioactively labeled to avoid the disadvantages associated with
radioactivity analysis.
"Body fluid" refers to human or animal body fluid such as, e.g., blood
serum, cerebrospinal fluid, amniotic fluid, urine, and the like.
"Tissue" refers to biological tissue extract material which is not
necessarily cellular by definition.
"Intercalation" refers to the non-covalent insertion of the labeling
composition herein between the base pairs in the nucleic acid double helix
structure. "Alkylating intercalation moiety" refers to moieties which
initially intercalate with the nucleic acid, are specific to
double-stranded nucleic acids, and upon activation covalently bond to one
or both of the nucleic acid strands. The preferred compounds are those
which will irreversibly crosslink the nucleic acid, i.e., bond to both of
the nucleic acid strands.
Examples of suitable alkylating intercalation compounds from which the
moieties are derived include mitomycin C as described by Lown et al., Can.
J. Biochem., 54, 110ff (1976), carzinophilin A as described by Lown et
al., J.A.C.S., 104, 3213-3214 (1982),
3,5-diazido-5-ethyl-6-phenylphenanthridinium as described by Woolley et
al., Biochemistry, 22, 3226-3231 (1983), psoralen compounds and
derivatives thereof, and other compounds which can be devised which have
structures allowing intercalation and alkylation, preferably irreversible
crosslinking of nucleic acids, to occur. The preferred such moieties
herein are 4'-methylene-substituted psoralen moieties such as those
derived from psoralen compounds described and sold by HRI Associates,
Inc., of Emeryville, Calif. via their Oct. 1, 1983 price schedule. These
are preferred because their planar structure allows ready intercalation
and they are able to crosslink the nucleic acids covalently. The
4'-methylene group is present to act as a link with the spacer arm.
Examples of suitable psoralen moieties include 4'-methylene-substituted
psoralen, 4'-methylene-substituted-5-methylangelicin,
4'-methylene-substituted-5-methoxypsoralen, and
4'-methylene-substituted-4,5',8-trimethylpsoralen. The most preferred
psoralen moiety herein is
4'-methylene-substituted-4,5',8-trimethylpsoralen due to its enhanced
intercalating efficiency.
"Activation" of the complex of alkylating intercalation moiety and nucleic
acid refers to means used to induce the alkylating intercalation moiety of
the labeling reagent to bond covalently to the double-stranded region of
the nucleic acid. The appropriate activation means will depend mainly on
the type of alkylating intercalation moiety being employed. For example,
the psoralen moieties and the moiety derived from
3,5-diazido-5-ethyl-6-phenylphenanthridinium will require activation by
irradiation with ultraviolet light. Mitomycin C will form a complex
activated by reduction thereof. Carzinophilin A will form a complex
activated by protonation (acid activation) thereof. Thus, any means
appropriate to the type of moiety employed for the reagent may be utilized
for this purpose.
The purpose of the spacer arm is to provide a chemical linkage between the
alkylating intercalation moiety and the label moiety so that the label can
readily interact without interference with such detection means as
antibodies, other detector polypeptides, or chemical reagents. The number
of atoms in the straight (main) chain of the spacer arm generally depends
on the particular label moiety employed. The chain must be sufficiently
long to permit access of the detector molecule to the binding site, i.e.,
to avoid interference by the nucleic acid. Generally, depending mainly on
the nature of the label moiety, the straight chain will contain at least
two carbon atoms. This signifies that the direct chain extending between
the alkylating intercalation moiety and the label moiety consists of at
least two chain carbon atoms, excluding the atoms which are directly
attached to the chain atoms or contained in branches off the chain atoms.
Thus, e.g., a spacer arm which has the formula:
##STR1##
contains a straight chain having six carbon atoms and eight total atoms.
Preferably the straight chain will contain at least seven total atoms.
When biotin is the label moiety, the straight chain will preferably
contain at least 10 total atoms. The molecular weight limits of the spacer
arm will be determined by the types of atoms contained therein and by
solubility considerations. As the molecular weight of, for example,
polyethylene glycol increases, the spacer arm derived therefrom becomes
less water soluble at room temperature and becomes more waxy. Thus, it is
less useful in the present invention. The maximum molecular weight for the
spacer arm is generally about 1000 to ensure adequate water solubility and
fluidity thereof.
The atoms which may be employed in the straight chain include carbon,
oxygen, nitrogen, sulfur and other substitutes therefor which will be
inert to the detection means. The linkage of these atoms may include any
well known bonds such as, e.g., carbon-carbon single bonds, carbon-carbon
double bonds, carbon-nitrogen single bonds, carbon-oxygen single bonds and
carbon-sulfur single bonds. Generally, however, the straight chain will
consist of a hydrocarbon chain of --(CH.sub.2)-- groups or olefin groups
or of --(CH.sub.2).sub.x Y-- groups where Y is a polar group containing N,
O, or S atoms, including amide groups, and x is an integer of at least 1.
Preferably, the straight chain contains polar groups to ensure that the
spacer arm will be hydrophilic so that it will be extended rather than
coiled in aqueous solution. Also preferably, the straight chain is a
series of --(CH.sub.2).sub.x O-- groups where x is a number from 1 to 4,
more preferably 1 to 3, and most preferably for solubility, x is no more
than 2. If biotin is the label moiety, preferably the chain also contains
an amide group through which the biotin will be attached. Although the
main chain may contain one or more branches such as alkyl groups, it is
preferred that the entire spacer arm be a straight chain to avoid steric
hindrance and solubility problems. If the chain is too branched, the
spacer arm will make the labeling reagent less able to intercalate into
the DNA. Particularly toward the end of the chain attached to the
alkylating intercalation moiety (and preferably the 4'-methylene portion
of a psoralen moiety) the chain is preferably straight. The terminal
groups of the spacer arm may be, e.g., amino groups or carboxy groups and
are preferably, before connection with the label moiety and alkylating
intercalation group, primary or secondary amino groups. More preferably
these amino groups are primary amino groups so that the chain will be
straight, without pendent intervening groups attached to the nitrogen
atoms which cause steric hindrance. Most preferably the spacer arm is of
the formula:
##STR2##
where R is either --H or a formyl group and R" is --H, x is a number from
1 to 4, preferably 2, and y is a number from 2 to 4, preferably 2. It is
understood that other modifiable functionalities such as thiol, amide,
hydroxy, carboxylic acid and epoxide groups may be incorporated in the
chain.
The label moiety pf the labeling reagent herein is capable of producing a
signal which can be detected by detection means. Examples of such
detection means include spectroscopy, such as fluorescence and
luminescence, photochemistry, radioactivity, biochemical means,
immunochemical means, chemical means, and the like. Preferred means
include forming a detectable complex with a polypeptide, lectin or
antibody in the presence or absence of an enzyme associated with the
polypeptide, lectin or antibody. Depending on the label moiety employed,
an example of a polypeptide useful for this purpose is avidin complexed
with an enzyme when biotin is the label moiety. Suitable antibodies would
include, e.g., antibiotin antibodies or antidinitrophenol antibodies if
the label moiety is dinitrophenol. Lectins, which are glycoproteins, would
be employed as the detection means if carbohydrates are used as label
moieties.
The detection means, if it is an antibody, a lectin, or some other
polypeptide capable of complexing with the label moiety, would be linked
to an entity capable of generating a detectable change. Examples of such
entities include enzymes such as, e.g., alkaline phosphatase, which has
chromogenic or fluorogenic substrates, or luciferase, which can generate
luminescence. The label moiety may be any group possessing the detection
properties described above, including haptens, which are only immunogenic
when attached to a suitable carrier, but are capable of interacting with
appropriate antibodies to produce detectable complexes.
Examples of suitable label moieties include those of the formulae:
##STR3##
As label moieties containing aromatic groups tend to intercalate into the
nucleic acid(s), the preferred label moiety is nonaromatic, and the most
preferred label moiety is biotin.
Examples of preferred labeling reagents of this invention include:
##STR4##
where R and R' are independently --H;
##STR5##
where R and R' are as defined above;
##STR6##
where n is an integer of at least 2, preferably 2 to 10, inclusive, and
most preferably 2. The compounds where n is 2 are the most preferred,
because they are easy to prepare, have a positive charge at neutral pH
which imparts both enhanced solubility and stabilization to the
intercalated compound, lack steric hindrance to interaction with the
stacked base pairs of double-stranded nucleic acids, and exhibit improved
incorporation into DNA. The formylated derivative where n is 2 is
particularly preferred.
In one method, the reagents herein may be prepared in two steps wherein the
alkylating intercalation moiety is attached to the spacer arm, and the
resultant compound is reacted to attach the label moiety thereto. For
example, the precursor:
##STR7##
(where the alkylating intercalation moiety is
4'-methylene-4,5',8-trimethylpsoralen) may be prepared by methods which
depend on what the spacer arm is.
If the spacer arm contains terminal amino groups
##STR8##
and no other reactive functional groups, it may be attached to the
4'-methylene group of the 4,5',8-trimethylpsoralen derivative by the
two-step method described by Saffran et al., Proc. Natl. Acad. Sci. USA,
79, 4594 (1982). In that method, a hydrocarbon or polyether hydrocarbon
chain terminated on each end with halide groups, preferably chloride
groups, which is either commercially available or readily prepared, is
reacted with methylamine to form the corresponding chain with methylamino
groups on both ends instead of halide groups. This compound is then
reacted with chloromethyltrioxsalen, which is commercially available, to
form the desired precursor.
For example, the precursor:
##STR9##
is prepared by reacting Cl--(CH.sub.2 CH.sub.2 O).sub.2 --CH.sub.2
CH.sub.2 Cl with methylamine at 85.degree. C. for 3-5 days to form
CH.sub.3 NH(CH.sub.2 CH.sub.2 O).sub.2 --CH.sub.2 CH.sub.2 --NHCH.sub.3,
which is in turn reacted with chloromethyltrioxsalen at 110.degree. C. for
15 hours to form the precursor.
In the second step of this first method for preparing the novel labeling
reagents of this invention the precursor as described above is reacted
with the appropriate label moiety. This is accomplished by reacting a
labeled compound which has a terminal group reactive with the terminal
group of the spacer arm, and thus the reaction conditions will depend on
the particular spacer arm and label moiety employed. For example, if the
precursor has a terminal amino moiety it will react with an active ester
of d-biotin, such as d-biotin p-nitrophenyl ester, when contacted
therewith at temperatures of from about 20.degree. to 40.degree. C.,
preferably room temperature, for about 60 to 120 minutes, preferably 60 to
90 minutes, to release alcohol as a byproduct. A suitable solvent such as
dichloromethane is typically employed. Reaction progress may be monitored
using thin-layer chromatography where the plate is sprayed with ninhydrin
to detect disappearance of the starting material.
As another example, a precursor with the terminal amino moiety (e.g.,
--NHCH.sub.3) will react with fluorescein isothiocyanate in mixed 1:1
THF:pyridine or solvents of similar polarity to insert thereon a label
moiety detectable by fluorescence, at temperatures of from about
20.degree. to 40.degree. C., preferably room temperature, for about 8 to
12 hours, or until the product is detected. The compound selected to
introduce the label moiety into the precursor is preferably such that it
makes the final reagent capable of covalently crosslinking the
double-stranded nucleic acid when inserted therein. This is the case when
active esters of d-biotin or fluorescein isothiocyanate are employed.
If the spacer arm contains internal groups which may be reactive with the
alkylating intercalation moiety, these groups may be masked during
preparation of the precursor before the label moiety is introduced. Thus,
for example, one compound herein containing a spacer arm with an amide
group and biotin as the label moiety may be prepared by a series of steps
where 4,9-dioxa-1,12-dodecanediamine is reacted with di-t-butyl
dicarbonate in methanol to produce the compound:
##STR10##
which is masked with a tert-butyloxy carbonyl group. This compound is in
turn reacted with succinic anhydride to form the compound:
##STR11##
This compound is reacted with aminomethyl trioxsalen hydrochloride so as
to yield:
##STR12##
which, in the presence of an excess amount of formic acid, becomes the
precursor by losing the masking carbamate (or t-BOC) group, and thereby
terminating with a primary amine. This precursor may then be reacted with
an active ester of d-biotin such as d-biotin p-nitrophenyl ester under
conditions as described above or with another label moiety which is
reactive with a terminal --NH.sub.2 group to form the final compound.
The most preferred compound herein, N-biotinyl,N'-(4'-methylene
trioxsalen)-3,6,9-trioxaundecane-1,11-diamine, is prepared by a similar
method wherein tetraethylene glycol, or the appropriate polyethylene
glycol for higher homolog chains, is reacted with paratoluene sulfonyl
chloride in pyridine to yield the bis-tosylate, which when heated with
lithium azide affords the corresponding bis-azide, which in turn is
reduced to the corresponding diamine. The diamine is then converted to the
mono-tert-butyloxy carbonyl protected derivative using di-tert-butyl
dicarbonate in methanol. The protected derivative is then reacted with
chloromethyl trioxsalen hydrochloride at elevated temperature (over
30.degree. C.) to yield the psoralen derivative:
##STR13##
where n is 2 if tetraethylene glycol was employed initially. This
compound, in the presence of an excess amount of formic acid, becomes the
primary amine terminated precursor as described above, which can then be
reacted with, e.g., the N-hydroxysuccinimide ester of biotin or another
appropriate label.
In a second method the labeling reagents herein may be prepared by reacting
the spacer arm with the label moiety to form a precursor which is then
reacted with a reagent supplying the alkylating intercalation moiety
(e.g., a substituted trioxsalen reagent such as chloromethyl- or
aminomethyltrioxsalen) to form the final compound. For example, the
preferred biotin-containing compound containing an amide group in the
spacer arm may not only be prepared as described above, but also may be
prepared by reacting the intermediate:
##STR14##
prepared as described above, with d-biotin in a solvent at about
70.degree.-90.degree. C. until the reaction is complete, then allowing the
product to stand at room temperature with an excess amount of formic acid
to remove the carbamate (t-BOC) group. The product, terminating with a
primary amine group, is then reacted with succinic anhydride to introduce
the amide functionality and a carboxylic acid group at the end. The
resulting precursor, with the formula:
##STR15##
is finally reacted with aminomethyltrioxsalen hydrochloride at room
temperature in the presence of a water-soluble carbodiimide to form the
final product. Alternatively, the carboxyl group may be converted to an
active ester (such as N-hydroxysuccinimide) and be subsequently reacted
with aminomethyltrioxsalen hydrochloride. This latter method of synthesis
is preferred, at least when psoralen reagents are employed, because less
of the costly aminomethyltrioxsalen compound is expended. This latter
method also gives rise to novel intermediate compounds of the formula:
[R]L where B is a spacer arm with a straight chain comprising two or more
--(CH.sub.2).sub.x --O-- moieties where x is a number from 1 to 4
inclusive, preferably no more than 2, and L is a monovalent label moiety
capable of producing a detectable signal, as described above.
The practitioner will recognize that other labeling compounds of this
invention can be prepared by similar techniques, depending on the
particular reagents employed, by adapting the chemistry appropriately.
One useful specific application of the labeling reagents described above
which are detectable by fluorescent or chromogenic detection means is in
labeling specific regions or bands of chromosomes by staining the regions
with the reagent and detecting the reagent, using the known chromosome
banding technique described in the art for the Giemsa reagent. By thus
distinguishing between or among chromosomes one can study chromosome
anomalies such as Down's syndrome.
Another useful application of the reagents described above is in labeling
nucleic acids. Generally this technique involves two steps: (1) contacting
the nucleic acid with one or more of the reagents in such a way to cause
the alkylating intercalation moiety thereof to intercalate into the
nucleic acid to form a complex between the two, and (2) activating the
complex in such a manner that the alkylating intercalation moiety of the
reagent bonds covalently to one or both of the nucleic acid strands.
Basically, the first step (intercalation) is preferably carried out by
incubating the nucleic acid with the reagent(s) at about 0.degree. to
50.degree. C., preferably about 4.degree. to 20.degree. C., in a medium
containing a buffer and having a pH of between about 6 and 9, preferably
about 6 and 8. The incubation generally will not require more than about
10 minutes. The buffer may consist of any buffer useful for this purpose
such as, e.g., 10 mM Tris--HCl at pH 7.0 and 0.1 mM EDTA.
After the nucleic acid has been incubated for a sufficient period of time
to intercalate the alkylating intercalation agent, the nucleic acid
containing complex is, in the same medium, activated by such means as,
e.g., reduction, irradiation, protonation or the like, depending on the
alkylating intercalation moiety, for a sufficient period of time and under
suitable conditions to ensure covalent bonding. The skilled practitioner
will recognize what particular conditions are necessary given a particular
moiety whose alkylating properties are described in the art. If the moiety
is a psoralen derivative, for example, the complex is irradiated with UV
light, preferably at about 300 to 390 nm wavelength, and more preferably
at about 300 to 370 nm, at 1 to 100 mWatts per cm.sup.2 for from 1 minute
to 24 hours, to ensure covalent bonding. The wavelength must be at least
300 nm to induce crosslinking of double-stranded nucleic acids.
The resultant nucleic acids will be labeled so that the label moiety can be
detected by, for example, spectroscopic, photochemical, chemical,
immunochemical or biochemical means. Thus, the labeled nucleic acid(s) may
be subjected to, e.g., ultraviolet light to stimulate fluorescence or
contacted with a polypeptide, lectin or antibody depending on the label
moiety in the labeling reagent. In addition, the detection means may
consist of a combination of an absorber-emitter moiety and a
chemiluminescent catalyst in sufficiently close proximity to each other to
permit non-radioactive energy transfer, in conjunction with
chemiluminescent reagents suitable for inducing a light response in the
presence of the chemiluminescent catalyst, as described in European Patent
Publication No. 0,070,686 published Jan. 26, 1983 and in European Patent
Publication No. 0,070,685 published Jan. 26, 1983. Preferably the
detection means is non-radioactive to obviate the difficulties associated
with radioactive probes.
The degree of incorporation of these labeling reagents into nucleic acids
can be measured by introducing a tritium atom into the compound as
described in the experimental section. Nucleic acid incorporation by
tritiated reagents can be determined by liquid scintillation counting or
by autoradiography, detection techniques which are known in the art.
The nucleic acid itself which may be labeled by this technique may be any
nucleic acid which can be subjected to intercalation and activation of
alkylation, such as DNA, RNA, hybrids of DNA and RNA, and the like.
Preferably the nucleic acid is DNA. For the purpose of labeling, the
nucleic acid, no matter what type it is, must contain a double-stranded
region to intercalate. More than one type of double-stranded nucleic acid
may be present in the incubation broth for intercalation, and the presence
of proteins in the broth will not interfere with intercalation. Thus, for
example, the preferred psoralen derivative herein is capable, depending on
the label moiety, of crosslinking, for example, one DNA strand to another
DNA strand or one RNA strand to another RNA strand, or one RNA strand to
one DNA strand. The types of double-stranded nucleic acids which may be
employed include, for example, double-stranded nucleic acids, or nucleic
acids containing both double- and single-stranded regions.
A particularly useful application for the labeling reagents herein is in
preparing a labeled nucleic acid hybridization probe (preferably
non-radioactively labeled) for detecting nucleic acid sequences (RNA
and/or DNA) such as, e.g., those characteristic of a pathogenic microbe or
those responsible for or linked to a genetic disease. Pathogens would
include infectious disease causing microorganisms or microorganisms
involved in food spoilage. With such a probe, the method of probe
preparation will be the same as described below for the M13 probe, but the
nucleic acid will comprise a double-stranded region adjacent to a
single-stranded hybridizing region which will act to detect by
hybridization the nucleic acid sequence desired. A DNA of this description
may be prepared by the method described by Brown et al., Gene, 20, 139-144
(1982) where the DNA of the hybridizing region complementary to the
sequence to be detected is inserted into the double-stranded form of a
virus known as M13, which is publicly available. After transformation, a
single-stranded form of M13 can be prepared containing the hybridizing
region which is complementary to the sequence to be detected. The
recombinant M13 is then rendered partially double-stranded by primed
synthesis using a synthetic oligonucleotide primer complementary to a
region 5' to the cloning site and DNA polymerase I. The M13 probes then
obtained are separated from impurities, including free triphosphates, by
chromatography. More specific details can be found in the Materials and
Methods section of the Brown et al. article, supra, the entire disclosure
of which is incorporated herein by reference. The probes thus obtained are
then subjected to intercalation and irradiation with the labeling reagent
as described above. Other probes obtained by different methods may be
employed as the nucleic acid to be treated, provided that they contain a
double-stranded region adjacent to a single-stranded region capable of
hybridization with the complementary nucleic acid sequence which is to be
detected by the probe.
It is preferred herein that the alkylating intercalation moiety of the
reagent bond covalently to both strands of the double-stranded nucleic
acid region, i.e., that the nucleic acid be irreversibly crosslinked.
Crosslinking is preferred because it renders the hybridization probe more
stable to stringent hybridization conditions such as high temperatures
and/or low salt content in the media which are desirable in detecting the
nucleic acid sequences. The trimethylpsoralen compounds are preferred
herein because of their crosslinking efficiency.
The probes herein can be used to detect specific nucleotide sequence of
bacterial, viral, fungal, yeast, mammal, or parasite origin in clinical
samples, whether located in chromosomes, fixed cells, body fluids, viral
samples, or tissue sections. When the presence of a specific nucleic acid
molecule is ascertained by the probe, one can diagnose nucleic
acid-containing etiological agents in a patient. Examples of organisms
which might be detected by the probe herein include Chlamydia trachomatis,
Neisseria gonorhoeae, toxicogenic E. coli organisms, etc. The probe herein
also provides a method for screening bacteria to determine antibiotic
resistance.
The process herein can also be used to detect genetic diseases such as
HLA-linked diseases, thalassemias and sickle cell anemia. The
deoxyribonucleic DNA sequence whose presence or absence (in the case of
some thalassemias) is associated with the disorder can be detected
following hybridization with the probe herein which is detectable using
polypeptides, based on complex formation with the detectable polypeptide.
Hybridization can also be used to determine paternity.
In addition, the probes herein may be used for gene mapping (cytogenetics)
by in situ hybridization methods.
In addition, the probe herein represents a useful research tool in
analyzing target nucleic acids, especially DNA. The details of these
various methods to which the probe may be applied are described further in
European Patent Application No. 0,063,879 to Ward et al.
The method by which the nucleic acid sequences are detected may be any
method utilizing hybridization in con | | |
