U.S. patent application number 09/846603 was filed with the patent office on 2001-10-18 for sample processing to release nucleic acids for direct detection.
Invention is credited to Dattagupta, Nanibhushan, Sridhar, C. Nagaraja, Wu, Whei-Kuo.
Application Number | 20010031473 09/846603 |
Document ID | / |
Family ID | 22518038 |
Filed Date | 2001-10-18 |
United States Patent
Application |
20010031473 |
Kind Code |
A1 |
Dattagupta, Nanibhushan ; et
al. |
October 18, 2001 |
Sample processing to release nucleic acids for direct detection
Abstract
The present invention describes compositions and methods for
releasing nucleic acids from cells in a form that is suitable for
labeling/capture, amplification, or detection in a single reagent
addition step. The compositions include a lipid, membrane
fluidizing compound, enzyme for degrading cell structure, metal
chelators, or one or more nucleic acid probes or primers
complementary to the nucleic acid to be detected. The compositions
are non-denaturing and non-inhibitory of enzymes or proteins that
are used in nucleic acid release, amplification, labeling or
detection. The invention also provides kits for performing the
above methods.
Inventors: |
Dattagupta, Nanibhushan;
(San Diego, CA) ; Sridhar, C. Nagaraja; (San
Diego, CA) ; Wu, Whei-Kuo; (San Diego, CA) |
Correspondence
Address: |
Peng Chen
Morrison & Foerster LLP
Suite 500
3811 Valley Centre Drive
San Diego
CA
92130-2332
US
|
Family ID: |
22518038 |
Appl. No.: |
09/846603 |
Filed: |
April 30, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09846603 |
Apr 30, 2001 |
|
|
|
09385624 |
Aug 26, 1999 |
|
|
|
6242188 |
|
|
|
|
60146579 |
Jul 30, 1999 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
435/6.1 |
Current CPC
Class: |
C12N 15/1003
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
We claim:
1. A composition for releasing nucleic acid from a sample in a form
suitable for directly detecting the nucleic acid, said composition
comprising: an aqueous solution comprising one or more lipids for
releasing nucleic acid from the sample and further comprising one
or more of: i) an enzyme(s) to degrade cell structure; ii) a
non-ionic membrane fluidizing compound(s); and iii) a metal
chelator(s); wherein said aqueous solution is non-denaturing and
non-inhibitory of enzymes or proteins used in nucleic acid release,
amplification, labeling or detection.
2. The composition of claim 1, further comprising one or more
nucleic acid probes or primers complementary to the nucleic acid to
be detected.
3. The composition of claim 1, wherein at least a portion of said
lipid(s) is in the form of liposomal vesicles having an aqueous
solution encapsulated therein.
4. The composition of claim 1, further comprising reagents for
labeling nucleic acid, said reagents comprising: a binding ligand
comprising a chemical moiety that binds to a nucleic acid that when
activated by light, forms at least one covalent bond therewith; and
a label comprising a detectable moiety.
5. The composition of claim 4, further comprising a binding
enhancer, wherein said binding enhancer comprises a chemical moiety
that has a specific affinity for nucleic acids.
6. The composition of claim 1, wherein said one or more lipids is
3-(2-aminopropyl-1,3-dihexadecyloxypropyl) hexadecyl ether.
7. The composition of claim 1, wherein said or more lipids is
3-(2aminopropyl-1-octadecyloxy-3-benzyloxypropyl) benzyl
sulfide.
8. The composition of claim 1, wherein said one or more lipids is
bis(3-benzyloxypropyl-1-octadecyloxy-3-benzyloxy-2-propyl
amine)polyethyleneglycol.
9. The composition of claim 8, wherein said polyethylene glycol is
pentaoxaheptadecane.
10. A composition for releasing nucleic acid from a sample in a
form suitable for directly detecting the nucleic acid, said
composition comprising: an aqueous solution of a non-ionic membrane
fluidizing compound(s), and further one or more of: i) an enzyme(s)
to degrade cell structure; ii) a lipid(s); and iii) a metal
chelator(s); wherein said aqueous solution is non-denaturing and
non-inhibitory of enzymes or proteins used in nucleic acid release,
amplification, labeling or detection.
11. The composition of claim 10, further comprising one or more
nucleic acid probes or primers complementary to the nucleic acid to
be detected.
12. The composition of claim 10, further comprising reagents for
labeling nucleic acid, said reagents comprising: a binding ligand
comprising a chemical moiety that binds to a nucleic acid that when
activated by light, forms at least one covalent bond therewith; and
a label comprising a detectable moiety.
13. The composition of claim 10, further comprising a binding
enhancer, wherein said binding enhancer comprises a chemical moiety
that has a specific affinity for nucleic acids.
14. The composition of claim 10, wherein at least a portion of said
lipids are in the form of liposomal vesicles and wherein the
aqueous solution is encapsulated therein.
15. The composition of claim 10, wherein said non-ionic membrane
fluidizing compound is 3-(2-aminopropyl-1,3-dihexadecyloxypropyl)
hexadecyl ether.
16. The composition of claim 10, wherein said non-ionic membrane
fluidizing compound is
3-(2aminopropyl-1-octadecyloxy-3-benzyloxypropyl) benzyl
sulfide.
17. The composition of claim 10, wherein said non-ionic membrane
fluidizing compound is
bis(3-benzyloxypropyl-1-octadecyloxy-3-benzyloxy-2- -propyl
amine)-polyethyleneglycol.
18. A method for detecting the presence of a nucleotide sequence in
nucleic acid of a sample, said method comprising the steps of: (a)
providing an aqueous solution comprising one or more lipids for
releasing nucleic acid from the sample, said solution further
comprising one or more of: i) an enzyme(s) to degrade cell
structure; ii) a non-ionic membrane fluidizing compound(s); and
iii) a metal chelator(s); wherein said aqueous solution is
non-denaturing and non-inhibitory of enzymes or proteins used in
nucleic acid release, amplification, labeling or detection; (b)
contacting the sample with the aqueous solution of step a) under
conditions suitable for releasing the nucleic acid from the cells;
(c) contacting the nucleic acid with one or more nucleic acid
probes or primers that are complementary to the nucleic acid to be
detected: i) under conditions suitable for the one or more nucleic
acid probes to hybridize to the nucleic acid to form a hybridized
product; or ii) under conditions suitable for amplification of the
nucleic acid to form an amplified product; and (d) detecting the
hybridized product by capture or separation from unhybridized
nucleic acid probe and nucleic acid of the sample or detecting the
amplified nucleotide sequence, whereby the presence of a nucleotide
sequence in nucleic acid of a sample is determined.
19. The method of claim 18, wherein at least a portion of said
lipids are in the form of liposomal vesicles having the aqueous
solution encapsulated therein.
20. The method of claim 18, wherein said amplification reaction is
selected from the group consisting of: polymerase chain reaction,
ligase chain reaction, transcription based amplification reaction,
nucleic acid sequence based amplification reaction and strand
displacement amplification reaction.
21. The method of claim 18, wherein said aqueous solution further
comprises a nucleic acid labeling reagent to label the nucleic acid
from the cells to facilitate detection of the nucleic acid of the
sample subsequent to hybridization, wherein the nucleic acid
labeling reagent comprises: a binding ligand comprising a chemical
moiety that binds to a nucleic acid and that, when activated by
light, forms at least one covalent bond therewith and a label
comprising a detectable moiety; and exposing the nucleic acid
labeling reagent and nucleic acid to light of an appropriate length
of time and wavelength to cause the binding ligand to become
covalently attached to the nucleic acid.
22. The method of claim 21, wherein said nucleic acid labeling
reagent further comprises a binding enhancer, wherein said binding
enhancer comprises a chemical moiety that has a specific affinity
for nucleic acids.
23. The method of claim 21, wherein said aqueous solution further
comprises the nucleic acid probes or primers and the nucleic acid
labeling reagent, whereby release of nucleic acid and labeling is
performed by a single addition of the aqueous solution.
24. The method of claim 18, wherein said nucleic acid probe is
labeled to facilitate detection of the nucleotide sequence
subsequent to hybridization.
25. The method of claim 18, wherein said sample is a clinical
specimen.
26. The method of claim 18, wherein said nucleotide sequence to be
detected in the clinical specimen is diagnostic of infectious
disease, cancer, a human genetic disorder, or defines genetic
profile for forensic, paternity or transplantation purposes.
27. A kit for releasing nucleic acid from a sample in a form
suitable for directly detecting the nucleic acid, said kit
comprising: a vial containing an aqueous solution comprising one or
more lipids for releasing nucleic acid from the cells and further
comprising one or more of: i) an enzyme(s) to degrade cell
structure; ii) a non-ionic membrane fluidizing compound(s); and
iii) a metal chelator(s); wherein said aqueous solution is
non-denaturing and non-inhibitory of enzymes or proteins used in
nucleic acid release, amplification, labeling or detection.
28. The kit of claim 27, wherein said kit further comprises or more
nucleic acid probes or primers complementary to the nucleic acid to
be detected, wherein said probes or primers are contained in the
vial with the aqueous solution or are contained in one or more
separate vials.
29. The composition of claim 27, further comprising a means to
prepare liposomes with the reagents supplied with the kit.
30. The kit of claim 27, further comprising reagents for labeling
nucleic acid, wherein said reagents are contained in the vial with
the aqueous solution or are contained in one or more separate
vials.
31. A kit for releasing nucleic acid from a sample and for
detecting nucleic acid from the sample having a specific nucleotide
sequence, said kit comprising: a vial containing an aqueous
solution comprising a non-ionic membrane fluidizing compound(s),
said aqueous solution being non-denaturing and non-inhibitory of
enzymes or proteins used in nucleic acid release, application,
labeling or detection, and further comprising one or more of: i) an
enzyme(s) to degrade cell structure; ii) a lipid(s); and iii) a
metal chelator(s); wherein said aqueous solution is non-denaturing
and non-inhibitory of enzymes or proteins used in nucleic acid
release, amplification, labeling or detection.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of nucleic acid
detection and, more specifically, to the processing of samples to
release nucleic acids in a condition suitable for direct
detection.
BACKGROUND OF THE INVENTION
[0002] Nucleic acid detection through modern molecular biological
techniques has revolutionized diagnosis of infections, cancer,
inborn genetic errors, HLA typing, and forensic and paternity
testing. Methods to detect nucleic acids commonly requires several
sample processing steps, including use of a lysis reagent to lyse
cells and release the nucleic acids contained within the cells.
Lysis reagents typically consist of a strong detergent such as
sodium dodecyl sulfate and alkaline pH conditions.
[0003] The need for multiple processing steps when using a lysis
reagent, such as one containing a strong detergent, primarily
results from inhibitors of later nucleic acid detection steps that
are present or associated with the lysis reagent. The inhibitors
must be neutralized or removed before amplification or other
additional steps in nucleic acid detection can proceed. These
additional steps result in increased labor and materials costs for
the clinical laboratory. Use of a lysis reagent for nucleic acid
detection also is detrimental because it can, under some
circumstances, degrade the nucleic acids, thereby decreasing
sensitivity in some assay formats. Thus, a need exists for an
approach to isolate nucleic acids from a cell sample that avoids
the additional steps associated with lysis reagents and allows for
release and detection from a single reagent addition step.
SUMMARY OF THE INVENTION
[0004] Accordingly, it is an object of the present invention to
eliminate the additional processing steps and degradation
associated with nucleic acid lysis procedures. This is achieved by
using lipids that are non-denaturing for enzymes and proteins
required in further processing steps.
[0005] It is also an object of the present invention to provide
compositions for releasing nucleic acid from cells or samples that
include reagents for labeling or performing amplification such that
release and detection of nucleic acid can be performed by a single
reagent addition step.
[0006] To accomplish these and other objectives, there has been
provided, according to one aspect of the present invention, a
composition comprising an aqueous solution for releasing nucleic
acid from a sample for direct detection, comprising one or more
lipids and, one or more of: i) an enzyme(s) to degrade cell
structure; ii) a non-ionic membrane fluidizing compound(s); and
iii) a metal chelator(s). The aqueous solution is non-inhibitory of
enzymes or proteins that are used in nucleic acid release,
amplification, labeling or detection, and can include one or more
nucleic acid probes or primers complementary to the nucleic acid to
be detected.
[0007] According to one embodiment of the present invention, the
lipids of the aqueous solution comprise lipids in the form of
liposomal vesicles or other structure for encapsulating the aqueous
solution.
[0008] According to another embodiment of the present invention,
the aqueous solution includes reagents for labeling nucleic acid.
Such reagents comprise a compound comprising a photoactivatible
binding ligand, a label comprising a detectable moiety and,
optionally, a nucleic acid binding enhancer moiety.
[0009] According to yet another embodiment of the present
invention, the aqueous solution further comprises one or more
nucleic acid probes or primers complementary to the nucleic acid to
be detected.
[0010] According to still yet another embodiment of the present
invention, the one or more lipids of the aqueous solution comprise
3-(2-aminopropyl-1,3-dihexadecyloxypropyl) hexadecyl ether,
3-(2-aminopropyl-1-octadecyloxy-3-benzyloxypropyl) benzyl sulfide,
or bis(3-benzyloxypropyl-1-octadecyloxy-3-benzyloxy-2-propyl
amine)-polyethyleneglycol.
[0011] In another aspect of the present invention, there is
provided a composition comprising an aqueous solution comprising
one or more membrane fluidizing compounds for releasing nucleic
acid and one or more of: i) an enzyme(s) to degrade cell structure;
ii) a lipid(s); and iii) a metal chelator(s). The aqueous solution
is non-denaturing and non-inhibitory of enzymes or proteins that
are used in nucleic acid release, amplification, labeling or
detection.
[0012] According to one embodiment of the present invention, the
lipids of the aqueous solution comprise lipids in the form of
liposomal vesicles or other structure for encapsulating the aqueous
solution.
[0013] According to another embodiment of the present invention,
the aqueous solution includes reagents for labeling nucleic acid.
Such reagents comprise a compound comprising a photoactivatible
binding ligand, a label comprising a detectable moiety and,
optionally, a nucleic acid binding enhancer moiety.
[0014] According to yet another embodiment of the present
invention, the aqueous solution further comprises one or more
nucleic acid probes or primers complementary to the nucleic acid to
be detected.
[0015] In accordance with another another aspect of the present
invention, methods are provided for detecting the presence of a
nucleotide sequence in nucleic acid of a sample using the aqueous
solutions comprising a lipid or membrane fluidizing compound
containing compositions of the present invention. Such methods are
applicable to clinical specimens and are useful for diagnosing a
variety of diseases and conditions.
[0016] In accordance with still yet another aspect of the present
invention, kits are provided for releasing nucleic acid from a
sample in a form suitable for directly detecting the nucleic acid.
The kit comprises a vial containing an aqueous solution comprising
one or more lipids for releasing nucleic acid from the cells and
further comprising one or more of an enzyme(s) to degrade cell
structure, a non-ionic membrane fluidizing compound(s) and a metal
chelator(s). The aqueous solution is non-denaturing and
non-inhibitory of enzymes or proteins used in nucleic acid release,
amplification, labeling or detection.
[0017] In one embodiment, the kit further comprises or more nucleic
acid probes or primers complementary to the nucleic acid to be
detected, wherein said probes or primers are contained in the vial
with the aqueous solution or are contained in one or more separate
vials.
[0018] In another embodiment, the kit includes a means to prepare
liposomes with the reagents supplied with the kit. In another
embodiment, the kit further includes reagents for labeling nucleic
acid, wherein said reagents are contained in the vial with the
aqueous solution or are contained in one or more separate
vials.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention provides novel compositions and
methods for processing cell samples under conditions such that
nucleic acids in cells or otherwise inaccessible to detection are
released in a form suitable for direct nucleic acid detection
assays. The compositions of the invention are designed to release
nucleic acid from cells under conditions that do not result in
denaturation of enzymes or proteins used in nucleic acid release,
amplification, labeling or detection.
[0020] General Definitions:
[0021] Oligonucleotide: Low molecular weight deoxyribo-, ribo-,
copolymers of deoxyribo- and ribonucleic acids of chain lengths
between 3 and 150. Such oligonucleotides can have modified
nucleotide residues such as -O- methoxy, phosphorothio-,
methylphosphonates and others known in art.
[0022] Primers: Usually oligonucleotides which are used for
extension reaction by a nucleic acid polymerase after a template
primer hybrid is formed. Such primers can carry sequences specific
for transcription by an RNA polymerase.
[0023] Nucleic Acid Probe: Nucleic acid with substantially
complementary sequences to the target nucleic acids for detection
or capture from a mixture. Such probes can be labeled for detection
or immobilized onto a solid support to enrich the target by
capture. A probe can be an single stranded or partially double
stranded and can be an oligonucleotide or a larger nucleic
acid.
[0024] Membrane fluidizing compound: A chemical substance that
renders a cell membrane fluid or flexible to facilitate release of
cellular material into solution or uptake of extracellular
contents. Compounds that induce pinocytosis in addition to
fluidizing the membrane also are included within the meaning of a
membrane fluidizing compound as used herein. A membrane fluidizing
compound can be a lipid or a non-lipid and can be ionic or
non-ionic. Membrane fluidizing compounds generally do not cause
cell death at lower concentrations that effect membrane fluidity,
however, cell death typically results at higher concentrations of
the compound.
[0025] Lipid: Any of various substances that are soluble in
non-polar organic solvents (such as chloroform and ether), that
with proteins and carbohydrates constitute the principal structural
components of living cells, and that include fats, waxes,
phosphatides, cerebrosides, and related and derived compounds.
[0026] Liposome vesicles: A vesicle composed of one or more
concentric phospholipid bilayers. The structure of the liposomes
may be as a multilamellar vesicle (MLV), a small unilamellar
vesicle (SUV), a large unilamellar vesicle (LUV). A liposome is
formed from a single lipid or combination of lipids (i.e.,
lipsosmal formulation) and optionally other compounds.
[0027] Thiocationic lipid: A lipid molecule with sulfur
substitution and which is positively charged at neutral pH.
[0028] Photoreagent or photoactive reagents: Reagents which under
appropriate wavelengths of light exposure form a covalent bond with
nucleic acid.
[0029] Preferred Embodiments:
[0030] A composition of the present invention for releasing nucleic
acid from a cell sample in a form suitable for directly detecting
the nucleic acid comprises an aqueous solution comprising one or
more lipids for releasing nucleic acid from the cells. As used
herein, an aqueous solution is a water and/or other water miscible
solvent and further includes a buffer to stabilize the pH between 4
and 11, with the ultimate pH depending on the stability of the
nucleic acid to be released.
[0031] The aqueous solution comprising one or more lipids includes
those lipids suitable for releasing cellular or otherwise
inaccessible nucleic acid without denaturation. Liposomal
formulations containing cationic lipids that have been used for
delivery of oligonucleotides and other agents to target cells are
useful for releasing nucleic acid from cells without denaturation
as provided herein. PCT WO 96/40627 and U.S. Pat. Nos. 5,851,548,
5,759,519, 5,756,352, and 5,739,271 teach liposomal formulations
containing cationic lipids.
[0032] The lipids used in the present compositions for releasing
nucleic acid from cells include complex mixtures of different
lipophilic substituents. Such complex mixtures allow for
optimization of the physical properties of the liposomes, such as
pH sensitivity, temperature sensitivity and size. For example, in
certain embodiments, dioleoylphosphatidylethanolamine ("DOPE"), and
other pH sensitive amphiphilic compounds can be used to formulate
liposomes which destabilize at acidic pH. This promotes fusion of
the liposome with endosomal membranes when exposed to the
degradative acidic pH and enzymatic contents of the endosome,
resulting in release of the contents of the endosome into the
cytoplasm. (Ropert, et al., Biochem. Biophys. Res. Comm.
183(2):879-895 (1992); Juliano, et al., Antisense Res. and Dev.
2:165-176 (1992)). Although not wishing to be bound by any
particular theory, it is believed that pH controlled degradation of
liposomes in the cytoplasm of the cell enhances release of nucleic
acids.
[0033] Lipids used in the present compositions for releasing
nucleic acid from cells also can include sterols to enhance
stability of liposomal vesicles both in vitro and in vivo. In
particular, organic acid derivatives of sterols, such as
cholesterol or vitamin D3, which have been reported to be easier to
formulate than their non-derivatized water-insoluble equivalents
(U.S. Pat. Nos. 4,721,612 and 4,891,208), are useful in preparing
liposomal formulations as described herein.
[0034] Preferred lipids for use in the present compositions and
methods are cationic lipids (i.e., derivatives of glycerolipids
with a positively charged ammonium or sulfonium ion-containing
headgroup), including those useful in liposomal formulations for
the intracellular delivery of negatively charged biomolecules such
as oligonucleotides. The usefulness of cationic lipids may be
derived from the ability of their positively charged headgroups to
interact with negatively charged cell surfaces, although this is
not known for certain. The cationic lipid
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTMA") as described by Felgner, et al., Proc. Natl. Acad. Sci.
(USA) 84:7413-7417 (1987) (U.S. Pat. No. 4,897,355) is a cationic
lipid with an ammonium group that can be used in liposomal
formulations present in the compositions of the invention. In such
formulations, DOTMA may bind to DNA through an ionic lipid-DNA
complex that assists in releasing nucleic acid from a cell. Other
ammonium ion-containing cationic lipid formulations that can be
used in the nucleic acid releasing compositions of the present
invention include the DOTMA analog,
1,2-bis(oleoyloxy)-3(trimethylammonio)propane ("DOTAP")
(Stamatatos, et al., Biochem., 27:3917-3925 (1988)); the lipophilic
derivative of spermine (Behr, et al., Proc. Natl. Acad. Sci. (USA),
86:6982-6986 (1989)); and cetyltrimethylammonium bromide
(Pinnaduwage, et al., Biochem. Biophys. Acta, 985:33-37 (1989);
Leventis, et al., Biochem. Biophys. Acta, 1023:124-132 (1990);
Zhou, et al., Biochem. Biophys. Acta, 1065:8-14 (1991); Farhood, et
al., Biochem. Biophys. Acta, 1111:239-246 (1992); and Gao, et al.,
Biochem. Biophys. Res. Comm., 179:280-285 (1991)).
[0035] Cationic lipids are commercially available including DOTMA
(Gibco BRL, Bethesda, Md.), DOTAP (Boehringer Mannheim, Germany),
and 1,2-diacyl-3-trimethylarnmonium propane ("TAP") (Avanti Polar
Lipids, Alabaster, Ala.).
[0036] Cationic lipids containing sulfonium ions (i.e.,
thiocationic lipids) also can be used in the present nucleic acid
releasing compositions. Sulfonium ions have entirely different
physical properties than ammonium ions, which provides sulfonium
cationic lipids with some unique properties. Ammonium
ion-containing compounds are classified as hard bases, because the
nitrogen atom possesses high electronegativity, is difficult to
polarize and oxidize, and the valence electrons are held tightly by
the nucleus. This characteristic may account for some of the
toxicity associated with ammonium ion-containing lipid
formulations. In contrast, sulfonium ion-containing compounds are
classified as soft bases, because the sulfur atom possesses low
electronegativity, is easy to polarize and oxidize, and the valence
electrons are held more loosely by the nucleus. This decreased
charge density exhibited by sulfonium ion-containing (i.e.
"thiocationic") lipids may effectuate an enhanced interaction with
negatively charged cellular membranes, as well as a decreased
toxicity, leading to compositions with increased ability to release
cell nucleic acid in a non-denatured form.
[0037] Cationic lipids with relatively small polar headgroups as
described by Felgner, et al., J. Biol. Chem., 269(4):2550-2561
(1994), can be particularly useful in the present compositions for
releasing nucleic acids. However, the sulfonium ion type cationic
lipid, which has a relatively larger headgroup, also can be useful
because of the physiochemical properties associated with the
sulfonium ion. A lipid headgroup that consists of a sulfur atom
surrounded by adjoining saturated carbon atoms exhibits a diffusion
of charge to the neighboring carbon atoms that can facilitate
interaction of the lipid with cellular membranes, as well as
decrease the toxicity of the lipid (U.S. Pat. No. 5,759,519).
[0038] Liposomal preparations of the present invention can have a
positively charged surface by including in the formulation,
saturated or unsaturated aliphatic amines, including, for example,
stearylamine and oleylamine, sphingosine, phosphatidylethanolamine,
N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammoniumchloride,
cholesterylhemisuccinate,
3.beta.-(N-(N',N'-dimethylaminoethane)carbamoyl- )cholesterol and
cholesteryl(4'-trimethylammonio)butanoate, with preference given to
stearylamine and sphingosine as described in U.S. Pat. No.
5,759,519.
[0039] The present compositions for releasing nucleic acid include
a lipid that can form liposomes or other structures under the
appropriate conditions. Prior methods of forming liposomes and
encapsulating aqueous solution are applicable for preparing the
nucleic acid releasing compositions of the present invention (e.g.,
Olson, et al., Biophys, Acta, 557:9 (1979)). For example, prior art
liposomal formulations used to encapsulate hemoglobin (e.g., U.S.
Pat. No. 4,911,929) are to produce liposomal vesicles as described
herein. Such liposomal formulation contains roughly equivalent
quantities of cholesterol and phosphatidylcholine, with 5 to 10%
negatively charged lipid, such as phosphatidic acid, dicetyl
phosphate, or dimyristoyl phosphatidyl glycerol (DMPG). Hydration
of the dried lipid film results in formation of multilamellar
vesicles (MLV), which can be extruded at low-pressure (e.g., 50-90
psi) through filters of progressively smaller pore size to large
unilamellar vesicles (LUVs). Once the liposomal vesicles are
formed, any unencapsulated aqueous solution can be removed, if
desired, by centrifugation or diafiltration and then recycled.
[0040] Lipid used for the formation of the liposome can be natural
or synthetic and include phospholipids, glycolipids, and lipid
related compounds. Exemplary lipids include, lecithin
(phosphatidylcholine), phosphatidylethanolamine, phosphatidic acid,
phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,
sphingomyelin, cardiolipin, and hydrogenated derivatives thereof,
which can be used either alone or in combination. The glycolipids
include cerebroside, sulfolipid (e.g., sulfatide), and ganglioside.
The structure of the liposomes may be as a multilamellar vesicle
(MLV), a small unilamellar vesicle (SUV), or large unilamellar
vesicle (LUV).
[0041] To stabilize the lipid, an antioxidant such as tocopherol
(vitamin E) can be added to the solution. A suitable amount of an
antioxidant is about 0.01 to 0.5% by weight based on the weight of
the phospholipid. The liposome composition of the invention also
can contain as a stabilizer, a high molecular weight polymer such
as albumin, dextran, vinyl polymers, non-ionic surface active
agents, gelatin, and hydroxyethyl starch.
[0042] Liposomal vesicles that encapsulate aqueous solutions as
used herein can be prepared by a variety of known methods. For
example, conventionally used hydration, reversed phase evaporation,
removal of surfactant, solvent injection, freeze-thawing and
dehydration-rehydration can be employed.
[0043] In the hydration method, the selected lipids are dissolved
in an organic solvent (e.g., chloroform and ether), which is
non-denaturing, and the solvent is evaporated from the resulting
solution yield a thin homogeneous film. The aqueous solution
containing, for example, an enzyme(s), a non-ionic membrane
fluidizing compound(s), a metal chelator(s) or nucleic acid probes
or primers (discussed further below) is added to the thin membrane,
and the mixture is subjected to agitation and sonication to yield a
liposome preparation encapsulating the aqueous solution. The
aqueous solution contains a buffer at a pH between 4 and 11. The pH
of the buffer is chosen such that when the lipids or liposomes are
added to an assay medium, the final pH in a range suitable to
preserve nucleic acids in solution.
[0044] In the reversed-phase evaporation method, the selected
lipids are dissolved in an organic solvent (e.g., chloroform and
ether), as discussed above, and are added to the aqueous solution
and subjected to agitation, sonication and high pressure
homogenization to uniformly disperse the aqueous solution. The
solvent is evaporated from this dispersion to yield a liposome
preparation encapsulating the aqueous solution.
[0045] In the removal of surfactant approach, the selected lipids
dissolved in organic solvent are mixed with a surfactant (e.g.,
cationic surfactant such as cholic acid or deoxycholic acid, and a
non-ionic surfactant such as Triton X-100 and octyl-D-glucoside)
and added to the aqueous solution, which is followed by agitation,
sonication and high pressure homogenization to uniformly disperse
the aqueous solution. The surfactant is then removed by dialysis,
gel filtration and ultrafiltration, which are applied singly or in
combination.
[0046] In the solvent injection, approach, the selected lipids are
dissolved in organic solvent and are added to the aqueous solution,
which has been set for a temperature about 10.degree. C. higher
than the boiling point of the organic solvent. Then, the organic
solvent is evaporated .
[0047] The aqueous solution of the present nucleic acid releasing
compositions also can include, for example, substances other than
lipids that enhance release of nucleic acid depending on the nature
of the sample and the environment in which the nucleic acid is
contained (e.g., the type of cell). Such nucleic acid releasing
substances include, for example, an enzyme(s) to degrade cell
structure, a non-ionic membrane fluidizing compound(s), and/or a
metal chelator(s).
[0048] Enzymes suitable for use with lipid containing aqueous
solution are available from natural sources or produced by
recombinant DNA methods. Such enzymes include, for example,
lysozyme, lipases, and proteinases such as proteinase K, pronase,
trypsin and chymotrypsin. Lysozymes from bovine, chicken, human and
lipases from wheat germ, human, yeast and other sources also are
suitable enzymes to degrade cell structure. These enzymes
preferably are nuclease free to support stability of released
nucleic acids in solution. The aqueous solution containing lipids
and enzymes for releasing nucleic acid can be encapsulated into a
liposome, if desired.
[0049] The enzymes are used at a molar ratio of lipid to enzyme of
between 10,000: 1 and 1:10,000. The optimal ratio of enzyme to
lipid can be readily determined by one skilled in the art. This can
be accomplished by mixing target cells with various lipid:enzyme
ratios, and determining the effectiveness of releasing nucleic acid
in a probe hybridization assay.
[0050] Non-ionic membrane fluidizing compounds, which have been
described in Suciu et al., Mol. Microbiol., 21:181-95 (1996),
Nabekura et al., Pharm Res., 13(7):1069-72 (1996), and Lindow et
al., Cryobiol., 32(3):247-258 (1995), and include aromatic alcohols
such as all phenyl, napthyl, and higher alcohols, also can be used
to release nucleic acid from cells without denaturation of enzymes
or proteins. The hydrocarbon side chains of aromatic alcohols can
be from C.sub.1 to C.sub.50 and longer, preferably between C.sub.1
and C.sub.20. The --OH residue can be at the Cn terminus carbon for
a primary alcohol or any place as in a secondary or tertiary
alcohol. The C--C bonds in Cn chain in addition to single bond can
have unsaturated linkages in the form of double or triple bonds.
The carbon chain also can have secondary and tertiary C-linkages.
Phenethyl alcohol, sec-phenethyl alcohol, benzyl alcohol are
examples of non-ionic membrane fluidizing compounds.
[0051] Non-ionic membrane fluidizing compounds can be included in
the aqueous solutions of the present invention provided they
enhance release of nucleic acids from cells without creating an
enzyme or protein inhibitory environment. Such compounds can be
present in the aqueous solution at a concentration between 0.001%
and 10.0%. The final concentration of non-ionic membrane fluidizing
compound in a sample for releasing nucleic acid is preferably
between about 0.001 and 10% (v/v), more preferably between 0.01%
and 5%, most preferably between 0. 1% and 2%. The ultimate
concentration of the non-ionic membrane fluidizing compound depends
on the nature of the fluidizing compound and the other components
of the nucleic acid releasing composition. One skilled in the art
can readily determine the proper concentration of membrane
fluidizing compound for effective release of nucleic acid from a
particular sample by determining binding of a specific probe to
nucleic acid released by a particular formulation.
[0052] Most non-ionic membrane fluidizing compounds are more
soluble in non-aqueous solvents. In such cases, stock solutions can
be made in a solvent that is less polar than water, for example, in
ethanol or isopropanol.
[0053] The aqueous solution of the nucleic acid releasing
composition also can include metal chelators such as
ethylenediaminetetraacetic acid (EDTA) and
ethyleneguaninetetraacetic acid (EGTA). In addition, the aqueous
solution can be heated to enhance release of the nucleic acid
essentially as described in U.S. Pat. No. 5,837,452 (1988).
[0054] The compositions of the present invention are useful for
releasing nucleic acid in a non-denatured form suitable for
detection of a specific nucleotide sequence. Thus, it is preferred
that the nucleic acid releasing compositions be non-denaturing and
non-inhibitory of enzymes or proteins used in nucleic acid release,
amplification, labeling or detection. This allows the composition
to include a labeled or unlabeled nucleic acid probe or primer or
other reagents useful in detection of a nucleotide sequence without
additional steps to dilute the sample or neutralize denaturing
conditions.
[0055] In some embodiments, the compositions for releasing nucleic
acid also include reagents to label the released nucleic acid for
later detection of formed hybrids. Such reagents for labeling
nucleic acid comprise a binding ligand comprising a chemical moiety
that binds to a nucleic acid and that, when activated by light
(i.e., photochemistry), forms at least one covalent bond therewith,
a label comprising a detectable moiety and optionally, a binding
enhancer comprising a chemical moiety that has a specific affinity
for nucleic acids (U.S. patent application Ser. No.
09/265,127).
[0056] The photochemical method provides more favorable reaction
conditions than the usual chemical coupling method for
biochemically sensitive substances. The DNA binding ligand and
label can first be coupled and then photoreacted with the nucleic
acid, or the nucleic acid can first be photoreacted with the
binding ligand and then coupled to the label.
[0057] DNA-binding ligands useful herein for linking the nucleic
acid component to the label can be any suitable photoreactive form
of known DNA-binding ligand. Particularly preferred DNA-binding
ligands are intercalator compounds such as the furocoumarins, e.g.,
angelicin (isopsoralen) or psoralen or derivatives thereof, which
photochemically react with nucleic acids, e.g.,
4'-aminomethyl-4,5'-dimethylangelicin, 4'-aminomethyl-trioxsalen
(4'-aminomethyl-4,5',8-trimethyl-psoralen), 3-carboxy-5- or
-8-amino- or-hydroxy-psoralen, as well as mono- or bis-azido
aminoalkyl methidium or ethidium compounds.
[0058] Particularly useful photoreactive forms of intercalating
agents are the azidointercalators. Their reactive nitrenes are
readily generated at long wavelength ultraviolet or visible light
and the nitrenes of arylazides prefer insertion reactions over
their rearrangement products (White, et al., Meth. Enzymol., 46:644
(1977)). Representative intercalating agents include azidoacridine,
ethidium monoazide, ethidium diazide, ethidium dimer azide
(Mitchell, et al., J. Am. Chem. Soc., 104:4265 (1982)),
4-azido-7-chloroquinoline, and 2-azidofluorene. A specific nucleic
acid binding azido compound has been described by Forster, et al.,
Nucleic Acid Res., 13:745 (1985). Other useful photoreactable
intercalators are the furocoumarins which form (2+2) cycloadducts
with pyrimidine residues. Alkylating agents also can be used as the
DNA binding ligand, including, for example, bis-chloroethylamines
and epoxides or aziridines, e.g., aflatoxins, polycyclic
hydrocarbon epoxides, mitomycin and norphillin A.
[0059] DNA-binding ligands which do not inhibit nucleic acid
amplification enzymes under amplification reaction conditions
include, for example, 4'-Biotinyl-PEG-4,5'-dimethylangelicin
("BPA"), Angelicin-DAPI-Biotin ("BDA"),
Angelicin-bisbenzimidazole-PEG-azidonitrobenzene ("AZPIMA"),
Angelicin-bisbenzimidazole-PEG-acridine ("APIMA"),
Angelicin-bisbenzimidazole-PEG-biotin ("BPIMA") and compounds
described in U.S. Pat. Nos. 4,950,744 and 5,026,840. In such
compounds, PEG represents any of the known forms of
polyethyleneglycol, including pentaoxaheptadecane.
[0060] Usually, a stock solution of these compounds is prepared
such that an aliquot of the stock solution is added to the reaction
mixture to the desired final concentration. The desired
concentration can be determined by one skilled in the art using
known methods. Such methods include binding studies of the ligand
with nucleic acids in a mock clinical sample. The concentration of
the labeling reagent in the mixture should be between about 0.001
nanomolar and 10.0 millimolar, preferably between about 0.1
micromolar and 100 micromolar, and most preferably between about
0.1 micromolar and 10 micromolar. The DNA-binding ligand will be
present in the aqueous solution of the present invention either as
a mixture or as a component of a liposomal formulation.
[0061] The label, which is linked to the nucleic acid through the
DNA-binding ligand, can be any chemical group or residue having a
detectable physical or chemical property, i.e., labeling can be
conducted by chemical reaction or physical adsorption. The label
includes a functional chemical group to enable it to be chemically
linked to the DNA binding ligand. Such labeling materials have been
well developed in the field of immunoassays and, in general, most
any label useful in such methods can be applied to label DNA as
described herein.
[0062] Particularly useful labels are enzymatically active groups
such as enzymes (Clin. Chem., 22:1243 (1976)), enzyme substrates
(British Pat. No. 1,548,741), coenzymes (U.S. Pat. Nos. 4,230,797
and 4,238,565) and enzyme inhibitors (U.S. Pat. No. 4,134,792;
fluorescers (Clin. Chem., 25:353 (1979)), and chromophores
including phycobiliproteins; luminescers such as chemiluminescers
and bioluminescers (Clin. Chem., 25:512 (1979) and ibid, 1531);
specifically bindable ligands, i.e., protein binding ligands;
antigens; and residues comprising radioisotopes such as .sup.3H,
.sup.35S, .sup.32P, .sup.125I, and .sup.14C. Such labels are
detected on the basis of their own physical properties (e.g.,
fluorescers, chromophores and radioisotopes) or their reactive or
binding properties (e.g., enzymes, substrates, coenzymes and
inhibitors).
[0063] For example, a cofactor-labeled nucleic acid can be detected
by adding the enzyme for which the label is a cofactor and a
substrate for the enzyme. A hapten or ligand (e.g., biotin) labeled
nucleic acid can be detected by adding an antibody or an antibody
pigment to the hapten or a protein that binds the ligand (e.g.,
avidin), tagged with a detectable molecule. A detectable molecule
has a measurable physical property (e.g, fluorescence or
absorbence) or is participant in an enzyme reaction (e.g., see
above list). For example, one can use an enzyme which acts upon a
substrate to generate a product with a measurable physical
property. Examples of the latter include, but are not limited to,
beta-galactosidase, alkaline phosphatase, papain and peroxidase.
For in situ hybridization studies, the final product of the
substrate is preferably water insoluble. Other labels, e.g., dyes,
will be evident to one having ordinary skill in the art.
[0064] If the label is an enzyme, the labeled DNA is ultimately
placed in a suitable medium to determine the extent of catalysis.
Thus, if the enzyme is a phosphatase, the medium can contain
nitrophenyl phosphate and one can monitor the amount of nitrophenol
generated by observing the color. If the enzyme is a
beta-galactosidase, the medium can contain
o-nitro-phenyl-D-galacto-pyranoside, which also liberates
nitrophenol. The label can be linked to the DNA binding ligand,
e.g., acridine dyes, phenanthridines, phenazines, furocoumarins,
phenothiazines and quinolines, by direct chemical linkage such as
involving covalent bonds, or by indirect linkage such as by the
incorporation of the label in a microcapsule or liposome, which in
turn is linked to the binding ligand.. Methods by which the label
is linked to a DNA binding ligand such as an intercalator compound
are well known in the art and any convenient method can be
used.
[0065] Advantageously, the DNA binding ligand is first combined
with label chemically and thereafter combined with the nucleic acid
component. For example, since biotin carries a carboxyl group, it
can be combined with a furocoumarin by way of amide or ester
formation without interfering with the photochemical reactivity of
the furocoumarin or the biological activity of the biotin.
Aminomethylangelicin, psoralen and phenanthridium derivatives can
similarly be linked to a label, as can phenanthridium halides and
derivatives thereof such as aminopropyl methidium chloride
(Hertzberg et al, J. Amer. Chem. Soc., 104:313 (1982)).
Alternatively, a bifunctional reagent such as dithiobis
succinimidyl propionate or 1,4-butanediol diglycidyl ether can be
used directly to couple the DNA binding ligand to the label where
the reactants have alkyl amino residues, again in a known manner
with regard to solvents, proportions and reaction conditions.
Certain bifunctional reagents, possibly glutaraldehyde may not be
suitable because, while they couple, they may modify nucleic acid
and thus interfere with the assay. Routine precautions can be taken
to prevent such difficulties.
[0066] The particular sequence used in making the labeled nucleic
acid can be varied. Thus, for example, an amino-substituted
psoralen can first be photochemically coupled with a nucleic acid,
the product having pendant amino groups by which it can be coupled
to the label, i.e., labeling is carried out by photochemically
reacting a DNA binding ligand with the nucleic acid in the test
sample. Alternatively, the psoralen can first be coupled to a label
such as an enzyme and then to the nucleic acid.
[0067] Advantageously, the DNA binding ligand can be linked to the
label by a spacer, which includes a chain of up to about 40 atoms,
preferably about 2 to 20 atoms, selected from the group consisting
of carbon, oxygen, nitrogen and sulfur. Such spacer can be the
polyfunctional radical of a member selected from the group
consisting of peptide, hydrocarbon, polyalcohol, polyether,
polyamine, polyimine and carbohydrate, e.g., -glycyl-glycyl-glycyl-
or other oligopeptide, carbonyl dipeptides, and
omega-amino-alkane-carbonyl radical or the like. Sugar,
polyethylene oxide radicals, glyceryl, pentaerythritol, and like
radicals also can serve as spacers. Spacers can be directly linked
to the nucleic acid-binding ligand and/or the label, or the
linkages may include a divalent radical of a coupler such as
dithiobis succinimidyl propionate, 1,4-butanediol diglycidyl ether,
a diisocyanate, carbodiinide, glyoxal, glutaraldehyde, or the
like.
[0068] Nucleic acid labeling reagents including the binding ligand
and label also optionally can include a binding enhancer as
described U.S. application Ser. No. 09/265,127. Covalent or
non-covalent complexes of a binding ligand, a binding enhancer and
a label is referred to herein as a "LAC."
[0069] The nucleic acid binding enhancer ("binding enhancer"),
serves to enhance the affinity of the LAC for nucleic acids above
that exhibited with the binding ligand alone. Accordingly, binding
enhancers tend to have a specific affinity for nucleic acids when
compared to non-nucleic acid sample/reaction constituents. The
binding enhancer can be the same as or different from the binding
ligand. In other words, the binding ligand and the binding enhancer
can each be an intercalator, wherein one of the two is a
monoadduct-forming species, and the other is present to enhance
binding by this monoadduct-forming species. Examples of such "dual
role" binding ligands are described in Chaires, et al., J. Med.
Chem., 40:261-266 (1977). Therein, it has been described that
binding of a bis-intercalating anthracycline antibiotic reached as
high as 10.sup.11 at 20.degree. C. It was also shown that the
affinity of a similar monointercalator is not above 10.sup.7
(Chaires, et al., Biochem., 35:2047-2053 (1996)).
[0070] The binding enhancer also can be a non-intercalating
compound. There are many non-intercalating nucleic acid binding
molecules known in the art. A bis-benzimidazole derivative commonly
known as Hoechst 33258 has shown affinity as high as
3.2.times.10.sup.8 M.sup.-1(Haq, et al., J. Mol. Biol., 271:244-257
(1997)). Other non-intercalating binding enhancers are oligo
pyrroles, phenyl indole derivatives and the like. These molecules
do not bind nucleic acids solely on the basis of positive charge.
Other suitable binding enhancers bind nucleic acids on the basis of
hydrogen bond formation, hydrophobic interaction in the major or
minor groove of the nucleic acid double helix and other non-ionic
interactions that give rise to high affinity reactions with nucleic
acids.
[0071] Not every compound capable of forming an electrostatic bond
with a negatively charged nucleic acid can serve as a binding
enhancer. For example, polycations such as polyamines are generally
not suitable for use in the present invention because of their
inability to specifically bind to nucleic acids in crude samples
and in the presence of amplification reaction components. Such
positively charged compounds can, for example, non-specifically
bind to all anionic macromolecules present in the sample, and not
just to nucleic acids. In addition, the binding enhancer should be
capable of specifically binding to nucleic acids in the presence of
10 to 20 mM magnesium, which is typically required for most
amplification reactions. At this concentration, compounds that bind
to nucleic acids solely on the basis of electrostatic interactions
do not form stable complexes with nucleic acids and thus require a
greater concentration of LAC for efficient labeling.
[0072] As discussed above, the binding ligand for labeling nucleic
acid is either directly or indirectly linked to a label. Such
attachment can be either covalent or ionic, so long as it is stable
under the conditions in which the LAC is employed. Chemical
attachments can be accomplished by any of a variety of well known
methods. For example, if the binding ligand contains or is
derivatised to contain an available carboxyl group and the label
contains or is derivatized to contain an available amino group, the
two can be reacted together to form an ester linkage. By
"available", it is meant that the formation of a linkage will not
interfere with the functioning of the label (i.e., its ability to
be detected or to catalyze a detectable reaction) or the ligand
(i.e., it's ability to bind nucleic acids). Particularly useful
labels are enzymes, enzyme substrates, fluorophore, radioisotopic
compounds, chromophores, magnetically responsive compounds,
antibody epitope-containing compounds, haptens, and the like.
[0073] The binding ligand, binding enhancer, and label or labeling
nucleic acid can also be indirectly attached via a linker. Such
linkers are specifically designed to promote efficient binding of
the binding ligand to the nucleic acids and functioning of the
label attached thereto. This occurs by providing adequate physical
separation between the two components of the LAC to prevent
interference of one by the other. The use of linkers is described
generally in U.S. Pat. No. 4,582,789 and 5,026,840. Certain
compounds can serve the dual role of a binding enhancer and a
linker. For example, linkers can be constructed from positively
charged compounds, such that they enhance binding with negatively
charged nucleic acids. However, in order for the linker to also
serve as a binding enhancer, it is necessary for it to have a
specific affinity for nucleic acids, and not just a structure
specific electrostatic affinity for negatively charged compounds.
The polyalkylamine linkers described in U.S. Pat. No. 5,026,840 are
not optimal as binding enhancers but are suitable as linkers.
[0074] In a preferred embodiment, a bifunctional linker is used
that is capable of reacting with both the nucleic acid binding
moiety and the label to form a chemical bridge therebetween.
However, in an alternate embodiment, a multifunctional linker can
be employed, to which the binding ligand, the binding enhancer and
the label are attached as a "branched" complex. Such complex
formats and chemical reactions for forming these types of complexes
are well known in the art.
[0075] Compositions comprising an aqueous solution for releasing
nucleic acid of the present invention having the appropriate
combination of nucleic acid releasing, labeling and detecting
reagents to achieve single step processing and detection also are
provided herein. Such compositions require that all the components
of the composition not be denaturing or inhibitory to enzymes or
proteins used in nucleic acid release, amplification, labeling or
detection. All these components when mixed to produce the final
reagent are delivered to the sample in an aqueous solution which
can be water or a buffer solution pH of which is preferably between
3 and 12. More preferably between 5 and 10 such that the released
nucleic acids are not substantially degraded. The particular
reagents to be added and their optimal concentration depends on
various factors including the nature of the sample and the
particular reagents chosen. One skilled in the art can readily
select the proper reagents and determine an optimal concentration
of each without resort to undue experimentation.
[0076] The present invention also provides methods and kits for
using the disclosed compositions in assays for detecting the
presence of a nucleotide sequence in nucleic acid of a sample
containing cells. Such assays are used for diagnosis of infectious
diseases, cancer, human genetic disorders, and others like
histocompatibility (e.g., HLA) typing, forensic and paternity
testing. For example, by contacting and treating the sample with
the above described compositions that contain reagents for
releasing nucleic acid from cells and appropriate labeling reagents
(e.g., LACs), the samples can be used for hybridization diagnosis
without any further processing of the sample. Thus, a urine sample,
for instance, that is suspected of bacterial infections can be
labeled without centrifugation, filtration or dialysis and the
cells in the samples are lysed without any separation step.
[0077] Test samples include body fluids, e.g., urine, blood, semen,
cerebrospinal fluid, pus, amniotic fluid, tears, or semisolid or
fluid discharge, e.g., sputum, saliva, lung aspirate, vaginal or
urethral discharge, stool or solid tissue samples, such as a biopsy
or chorionic villi specimens. Test samples also include samples
collected with swabs from the skin, genitalia, or throat. The
compositions of the invention can be added directly to the sample
or to cells isolated from the sample.
[0078] The assay method can detect the nucleic acid from
essentially any species of organism, including, for example,
Acintobacter, Actinomyces, Aerococcus, Aeromonas, Alclaigenes,
Bacillus, Bacteriodes, Bordetella, Branhamella, Bevibacterium,
Campylobacter, Candida, Capnocytophagia, Chlamydia,
Chromobacterium, Clostridium, Corynebacterium, Cryptococcus,
Deinococcus, Enterococcus, Erysielothrix, Escherichia,
Flavobacterium, Gemella, Gonorrhea, Haemophilus, Klebsiella,
Lactobacillus, Lactococcus, Legionella, Leuconostoc, Listeria,
Micrococcus, Mycobacterium, Neisseria, Nocardia, Oerskovia,
Paracoccus, Pediococcus, Peptostreptococcus, Propionibacterium,
Proteus, Psuedomonas, Rahnella, Rhodococcus, Rhodospirillium,
Staphlococcus, Streptomyces, Streptococcus, Vibrio, and Yersinia.
Also included are viruses such as the hepatitis viruses and human
immunodeficiency viruses (HIV).
[0079] The present methods also can be used to detect nucleic acid
from eukaroytes (protists) in samples from higher organisms, such
as animals or humans. Eukaroytes include algae, protozoa, fungi and
slime molds. The term "algae" refers in general to
chlorophyll-containing protists, descriptions of which are found in
Smith, Cryptogamic Botany, 2nd ed. Vol. 1, Algae and Fungi,
McGraw-Hill, (1955). Eukaryotic sequences according to the present
invention includes all disease sequences. Accordingly, the
detection of genetic diseases, for example, also are embraced by
the present invention.
[0080] Methods of detecting a nucleotide sequence involve
contacting the above described aqueous compositions for releasing
nucleic acid with a sample suspected of containing the nucleotide
sequence of interest. The mixture is incubated for an appropriate
period of time and under conditions suitable for releasing the
nucleic acid from the cells. If release and detection of the
nucleic acid is sought as a single step, the nucleic acid releasing
composition also includes one or more nucleic acid probes or
primers that are complementary to the nucleotide sequence to be
detected and other reagents depending on the detection format to be
used. Such nucleic acid primers or probes can be an oligonucleotide
or, in some cases, a larger nucleic acid molecule.
[0081] If the sample already contains released or isolated nucleic
acid, the incubation period can be between a few seconds to five
min. When the sample contains whole cells, incubation between two
minutes (min) to two hours ("hrs") may be necessary.
[0082] Amplification methods suitable for use with the present
methods include, for example, polymerase chain reaction (PCR),
ligase chain reaction (LCR), transcription mediated amplification
(TMA) reaction, nucleic acid sequence based amplification (NASBA)
reaction, and strand displacement amplification (SDA) reaction.
These methods of amplification are well known in the art.
[0083] PCR can be performed as according to Whelan, et al, J. Clin.
Microbiol., 33(3):556-561 *(1995). Briefly, a PCR reaction mixture
includes two specific primers, dNTP, 0.25 Units (U) of Taq
polymerase, and 1.times.PCR Buffer. For every 25 .mu.l PCR
reaction, a 2 .mu.l sample (e.g., isolated DNA from target
organism) is added and amplified on a thermal cycler. The
amplification cycle includes an initial denaturation, and up to 50
cycles of annealing, strand elongation and strand separation
(denaturation).
[0084] LCR can be performed as according to Moore, et al., J. Clin.
Microbiol., 36(4):1028-1031 (1998). Briefly, an LCR reaction
mixture contains two pair of probes, dNTP, DNA ligase and DNA
polymerase representing about 90 .mu.l, to which is added 100 .mu.l
of isolated nucleic acid from the target organism. Amplification is
performed in a thermal cycler (e.g., LCx.RTM. thermal cycler,
Abbott Labs, North Chicago, Ill.).
[0085] SDA can be performed as according to Walker, et al., Nucleic
Acids Res., 20(7):1691-1696 (1992). Briefly, an SDA reaction
mixture contains four SDA primers, dGTP, dCTP, TTP, dATPS, 150 U of
Hinc II, and 5 U of exonuclease deficient E. coli DNA polymerase I.
The sample mixture is heated 95.degree. C. for 4 min to denature
target DNA prior to addition of the enzymes. After addition of the
two enzymes, amplification is carried out for 120 min. at
37.degree. C. in a total volume of 50 .mu.l. The reaction is
terminated by heating for 2 min at 95.degree. C.
[0086] NASBA can be performed as according to Heim, et al., Nucleic
Acids Res., 26(9):2250-2251 (1998). Briefly, an NASBA reaction
mixture contains two specific primers, dNTP, NTP, 6.4 U of AMV
reverse transcriptase, 0.08 U of Escherichia coli Rnase H, and 32 U
of T7 RNA polymerase. The amplification is carried out for 120 min
at 41.degree. C. in a total volume of 20 .mu.l.
[0087] TMA can be performed as according to Wylie, et al., Journal
of Clinical Microbiology, 36(12):3488-3491 (1998). In TMA, nucleic
acid targets are captured with magnetic beads containing specific
capture primers. The beads with captured targets are washed and
pelleted before adding amplification reagents, which contain
amplification primers, dNTP, NTP, 2500 U of reverse transcriptase
and 2500 U of T7 RNA polymerase. A 100 .mu.l TMA reaction mixture
is placed in a tube, 200 .mu.l oil reagent is added and
amplification is accomplished by incubation at 42.degree. C. in a
waterbath for one hour ("hr").
[0088] A variety of amplification enzymes are well known in the art
and include, for example, DNA polymerase, RNA polymerase, reverse
transcriptase, Q-beta replicase, thermostable DNA and RNA
polymerases. Because these and other amplification reactions are
catalyzed by enzymes, it is important for a single step assay that
the nucleic acid releasing reagents and the detection reagents are
not potential inhibitors of amplification enzymes if the ultimate
detection is to be amplification based.
[0089] Also included in the composition for amplification are
appropriate nucleoside triphosphates, amplification buffer and
certain ions. The concentrations of nucleic acid primers and
enzymes can be selected for specific use. For example, for
polymerase chain reaction, the concentration of the nucleic acid
primer is between 1 picomole and 1 millimole when added to the
sample. The enzyme concentration can vary between about 0.01 U and
100,000U. One skilled in the art can determine the optimal
concentration of enzyme and other reagents by routine
experimentation.
[0090] Detection of the nucleotide sequences also can be performed
directly without amplification by hybridizing the sample nucleic
acid to the nucleic acid probe present in the composition. In this
case, the nucleic acid is contacted and incubated with the labeling
reagents (provided in the nucleic acid release composition or
separately) and the mixture is irradiated at a particular
wavelength for the covalent interaction between the photochemically
reactive DNA binding ligand and the test sample to take place.
After labeling, the material is hybridized under specified
hybridization conditions with a probe specific for the target
nucleic acid.
[0091] Hybridization of the labeled sample nucleic acid or the
labeled nucleic acid probe can be detected in any conventional
hybridization assay format and, in general, in any format suitable
for detecting the hybridized product or aggregate comprising the
labeled nucleic acid. If the sample nucleic acid has been labeled,
it can be used for hybridization in solution and solid-phase
formats, including, in the latter case, formats involving
immobilization of either sample or nucleic acid probe. For example,
preimmobilized nucleic acid probe can be hybridized with labeled
sample nucleic acid. The presence of label associated with the
solid phase indicates hybridization between the probe and the
sample nucleic acid and, thus, detection of the target nucleotide
sequence. Alternatively, unlabeled sample nucleic acid can be
preimmobilized and a labeled probe evaluated for hybridization
thereto.
[0092] Preferable concentration for the probe is between about 0.01
picomole and 10 millimoles, more preferably between about 1
picomole and 1 millimole, and most preferably between about 10
picomole and 10 micromoles. Methods of detecting hybrids on solid
phases are well known in the art and have been extensively
described (e.g., U.S. Pat. Nos. 5,232,831, 4,950,613, 486,539 and
4,563,419).
[0093] The nucleic acid probe comprises at least one hybridizable,
e.g., single-stranded, base sequence substantially complementary to
or homologous with the nucleotide sequence to be detected. However,
such base sequence need not be a single continuous polynucleotide
segment, but can comprise two or more individual segments
interrupted by non-homologous sequences. These non-homologous
sequences can be linear or they can be self-complementary and form
hairpin loops. In addition, the homologous region of the probe can
be flanked at the 3'- and 5' termini by non-homologous sequences,
such as those comprising the DNA or RNA or a vector into which the
homologous sequence had been inserted for propagation. In either
instance, the probe as presented as an analytical reagent will
exhibit detectable hybridization at one or more points with sample
nucleic acids of interest. Linear or circular hybridizable, e.g.,
single-stranded polynucleotides can be used as the probe element,
with major or minor portions being duplexed with a complementary
polynucleotide strand or strands, provided that the critical
homologous segment or segments are in single-stranded form and
available for hybridization with sample DNA or RNA. Useful probes
include linear or circular probes wherein the homologous probe
sequence essentially is a single-stranded form (Hu et al., Gene,
17:271 (1982)).
[0094] The nucleic acid probe can be used in any conventional
hybridization technique. As improvements are made and conceptually
new formats are developed, such can be readily applied to the
present probes. Conventional hybridization formats that are
particularly useful include those wherein the sample nucleic acids
or the polynucleotide probe are immobilized on a solid support
(solid-phase hybridization) and those wherein the polynucleotide
species are all in solution (solution hybridization).
[0095] In solid-phase hybridization formats, one of the
polynucleotide species participating in hybridization is fixed in
an appropriate manner in its single-stranded form to a solid
support. Useful solid supports are well known in the art and
include those, for example, which bind nucleic acids either
covalently or non-covalently. Non-covalent binding supports, which
are generally understood to involve hydrophobic bonding include
naturally occurring and synthetic polymeric materials, such as
nitrocellulose, derivatized nylon and fluorinated polyhydrocarbons,
in a variety of forms such as filters, beads or solid sheets.
Covalent binding supports (in the form of filters, beads or solid
sheets, just to mention a few) are also useful and comprise
materials having chemically reactive groups or groups such as
dichlorotriazine, diazobenzyloxymethyl, and the like, which can be
activated for binding to polynucleotides.
[0096] It well known that non-covalent immobilization of an
oligonucleotide to a solid support such as nitrocellulose paper is
generally ineffective for detecting hybridization. Thus, covalent
immobilization is preferred and can be achieved by phosphorylation
of an oligonucleotide by a polynucleotide kinase or by ligation of
the 5'-phosphorylated oligonucleotide to produce
multioligonucleotide molecules capable of immobilization. The
conditions for kinase and ligation reaction have been described
previously (e.g., Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, 1.53 and 5.33 (1989). Thus
oligonucleotide probes specific for genetic defects related to
hemoglobinopathies, such as sickle cell anemia and
alpha-thalassemias can be immobilized on nitrocellulose paper and
contacted with patient sample nucleic acid labeled by the above
described method. The photochemical labeling can be done in a
single step without the need to obtain purified nucleic acid
samples and without affecting the specific hybridizability of the
labeled sample.
[0097] A typical solid-phase hybridization technique begins with
immobilization of sample nucleic acids onto the support in
single-stranded form. This initial step essentially prevents
reannealing of complementary strands from the sample and can be
used for concentrating sample material on the support for enhanced
delectability. The nucleic acid probe is then contacted with the
support and hybridization detected by measurement of the label as
described herein. The solid support provides a convenient means for
separating labeled probe, which has hybridized to the sequence to
be detected, from probe that has not hybridized.
[0098] Another method of interest is the sandwich hybridization
technique wherein one of two mutually exclusive fragments of the
homologous sequence of the probe is immobilized and the other is
labeled. The presence of the polynucleotide sequence of interest
results in dual hybridization to the immobilized and labeled probe
segments (G. Rankim, et al., 21:77-85 (1983)).
[0099] In one embodiment, the immobile phase of the hybridization
system can be a series or matrix of spots of known kinds and/or
dilutions of denatured DNA. This can be prepared by pipetting
appropriate small volumes of native DNA onto a dry nitrocellulose
or nylon sheet, floating the sheet on a sodium hydroxide solution
to denature the DNA, rinsing the sheet in a neutralizing solution,
then baking the sheet to fix the DNA. Before DNA:DNA hybridization,
the sheet is usually treated with a solution that inhibits
nonspecific binding of added DNA during hybridization.
[0100] In solid phase detection systems, unhybridized labeled test
sample can be removed by washing following hybridization. After
washing, the hybrid is detected through the label carried by the
test sample, which is specifically hybridized with a specific
probe.
[0101] The present invention further features kits that
incorporates the components of the invention and makes possible
convenient performance of the invention. Such kit may also include
other materials that would make the invention a part of other
procedures including adaptation to multi-well technologies. The
items comprising the kit may be supplied in separate vials or may
be mixed together, where appropriate.
EXAMPLES
[0102] Materials:
[0103] The synthesis of several new lipids is described in Examples
1-3, other lipids DOPE (Avanti Polar Lipids); DODMECAP, DOMCATOP,
DOMHYTOP, DODMECAP, OBEHYTOP and OBECATOP were prepared as
described in PCT WO 96/40627.
[0104] The lipids and other materials used in the present invention
include the materials described in WO 96/40627 and other
commercially available materials. The synthesis of new compounds
are described below. Unless indicated otherwise, parts are parts by
weight, molecular weight is weight average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1
Synthesis of 3-(2-aminopropyl-1,3-dihexadecyloxypropyl) hexadecyl
ether
[0105] This example describes the synthesis of a lipid compound
useful for releasing nucleic acids from cells according to the
methods and compositions of the invention. A three step procedure
is provided as follows.
[0106] Step 1. Synthesis of 1,3-dihexadecyloxy-2-aminopropane
[0107] A solution of 2-amino-1,3-propanediol (Serinol: Aldrich
Chem. Co., Milwaukee, Wis.; Catalog No. 35,789-8) in
tetrahydrofuran (THF) is added dropwise with vigorous stirring to a
slurry of sodium hydride in THF over a period of 1-2 hrs. The
reaction mixture is stirred for an additional 30 minutes to 1 hr at
room temperature. Hexadecyl tosylate in THF is added dropwise to
the mixture with vigorous stirring over a period of 1-1.5 hrs. The
reaction mixture is stirred at room temperature for 1 hr and worked
up by addition of isopropanol to destroy excess sodium hydride. The
reaction mixture is extracted with chloroform (3.times.100 mL) and
the combined chloroform layers are washed with water (2.times.50
mL), saturated NaCl (1.times.50 mL) and dried (MgSO.sub.4). The
dried chloroform layer is evaporated under vacuum to afford the
product as an off-white solid.
[0108] Step 2. Synthesis of
N-(3-hydroxypropyl)-1,3-dihexadecyloxypropyl-2- -amine
[0109] The compound from Step 1, above, is dissolved in methylene
chloride and added to a solution of 3-bromopropanol in methylene
chloride containing triethyl amine with vigorous stirring. The
reaction mixture is stirred at room temperature for an additional
8-36 hrs. Upon completion of reaction, as shown by thin layer
chromatography (TLC), the reaction mixture is extracted with
methylene chloride. The methylene chloride layer is washed with
dilute hydrochloric acid (3.times.50 mL), water (3.times.100 mL),
saturated NaCl (1.times.75 mL) and dried (MgSO.sub.4). The dried
methylene chloride is evaporated under vacuum to afford the product
as a solid.
[0110] Step 3. Synthesis of
3-(2-aminopropyl-1,3-dihexadecyloxypropyl) hexadecyl ether
[0111] The compound from step 2, above, is dissolved in THF
containing a trace of methylene chloride and is added dropwise with
vigorous stirring to a suspension of sodium hydride in THF over a
period of 45 minutes to 2 hrs. The reaction mixture is stirred for
an additional 1 hr at room temperature. A solution of hexadecyl
bromide (Aldrich Chem. Co., catalog No. 23,445-1) in THF is added
dropwise with vigorous stirring over a period of 2 hrs. The
reaction mixture is stirred for additional 2-4 hrs at room
temperature. The reaction mixture is quenched by adding isopropanol
and the mixture is worked up by extraction with methylene chloride.
The methylene chloride layer is washed in water (3.times.100 mL),
saturated NaCl (1.times.50 mL) and dried (MgSO.sub.4). The dried
organic layer is evaporated under vacuum to afford the product as a
white solid.
Example 2
Synthesis of 3-(2aminopropyl-1-octadecyloxy-3-benzyloxypropyl)
benzyl sulfide.
[0112] This example describes the synthesis of a lipid compound
useful for releasing nucleic acids according to the methods and
compositions of the invention. A three step procedure is provided
as follows.
[0113] Step 1. Synthesis of
1-Octadecyloxy-3-benzyloxy-2-aminopropane
[0114] A solution of 2-amino-1,3-propanediol in THF is added
dropwise with vigorous stirring to a suspension of sodium hydride
over a period of 45 minutes to 2 hrs. The reaction mixture is
stirred at room temperature for an additional hr and sequentially
treated with a solution of one equivalent each of octadecyl bromide
and benzyl bromide, respectively. The reaction mixture is stirred
at room temperature for 4-14 hrs. The reaction mixture is worked up
by extraction with methylene chloride. The methylene chloride layer
is washed with water (3.times.50 mL), saturated NaCl (1.times.50
mL) and dried (MgSO.sub.4). The dried organic layer is evaporated
under vacuum to afford the product as a white solid.
[0115] Step 2. Synthesis of
2-N-(3-mercaptopropyl)-amino-1-octadecyloxy-3--
benzyloxypropane
[0116] A solution of 1-octadecyloxy-3-benzyloxy-2-aminopropane from
step 1, above, and 3-chloro-1-propanethiol (Aldrich, catalog No.
C6,860-1) in methylene chloride containing diisopropylethyl amine
is stirred at room temperature for 8-36 hrs. Upon completion of
reaction, the reaction mixture is extracted with methylene chloride
and washed with dilute acid. The organic layer is washed with water
(3.times.75 mL), saturated NaCl (1.times.75 mL) and dried
(MgSO.sub.4). The dried organic layer is evaporated under vacuum to
yield a white solid product.
[0117] Step 3. Synthesis of
3-(2-aminopropyl-1-octadecyloxy-3-benzyloxypro- pyl) benzyl
sulfide
[0118] A solution of octadecyl bromide in methylene chloride is
added dropwise with vigorous stirring to a solution of the compound
from step 2, above, in methylene chloride containing triethyl amine
over a period of 45 minutes to 2 hrs. The reaction mixture is
stirred for an additional 2-4 hr at room temperature. The reaction
mixture is poured into a mixture of ice-water and the mixture is
worked up by extraction with methylene chloride. The methylene
chloride layer is washed with water (3.times.100 mL), saturated
NaCl (1.times.50 mL) and dried (MgSO.sub.4). The dried organic
layer is evaporated under vacuum to yield a white solid
product.
Example 3
[0119] Synthesis of
bis(3-benzyloxypropyl-1-octadecyloxy-3-benzyloxy-2-pro- pyl
amine)-polyethyleneglycol.
[0120] The following two steps describe the synthesis of a lipid
compound, bis(3-benzyloxypropyl-1-octadecyloxy-3-benzyloxy-2-propyl
amine)-pentaoxaheptadecane. This is one member of the class of
compounds,
bis(3-benzyloxypropyl-1-octadecyloxy-3-benzyloxy-2-propyl
amine)polyethyleneglycol (polyethyleneglycol: "PEG"), which is a
conjugate of a membrane fluidizing compound and a lipid. This
compound can be used instead of a simple lipid compound in
releasing nucleic acid according to the methods and compositions of
the invention.
[0121] Step 1. Synthesis of pentaoxaheptadecane ditosylate
[0122] A solution of p-toluenesufonyl chloride (74 g, 0.39 mol) is
added dropwise to a stirred solution containing hexamethylene
glycol (50 g, 0.18 mol) trimethylamine (40 g, 0.39 mol) in
methylene chloride (400 mL) at 0.degree. C. The reaction mixture is
stirred for 1 hr at room temperature. The mixture is filtered and
the filtrate is concentrated under vacuum in a rotary evaporator.
The residue is suspended in ethylacetate (500 mL) and filtered. The
filtrate concentrated under vacuum to afford yellow oil. The yellow
oil is triturated with hexane and the resulting oil dried under
vacuum to afford 108 g of yellow oil.
[0123] Step 2. Synthesis of
bis(3-benzyloxypropyl-1-octadecyloxy-3-benzylo-
xy-2-propylamine)-PEG
[0124] A solution containing the compound from Example 2, Step 2
and pentaoxaheptadecanoate ditosylate is combined in
dimethylformamide containing diisopropylethyl amine and stirred at
room temperature for 4-48 hrs. Upon completion of reaction as shown
by TLC, the reaction mixture is poured over ice-water. The mixture
is stirred for 1-2 hrs and extracted with methylene chloride. The
organic layer is washed with dilute acid, water (3.times.50 mL),
saturated NaCl (1.times.50 mL) and dried (MgSO.sub.4). The dried
methylene chloride is evaporated under reduced pressure to afford
the product as a white solid.
Example 4
Preparation of Aqueous Solutions Formulated with Lipids for
Releasing Nucleic Acids
[0125] This example describes an aqueous solution containing lipids
for releasing nucleic acid from cells. 80 micromoles of total lipid
(which includes lipid, cholesterol or other sterol, and oleic acid
alone or in combination with titratable amphiphile and sterol in
10:5:2 ratio) is dissolved in chloroform and dried. The dried lipid
is rehydrated with 1 mL of an aqueous solution of the reagents to
be mixed or formulated. Rehydration is performed by vortexing the
mixture overnight at 37.degree. Centigrade ("C."). For liposomal
preparations, the mixture is further processed by freeze thawing
and extruded through polycarbonate filters and further purified by
gel filtration. The formulations can be prepared in presence of a
reversible amplification inhibitor. Such inhibitors are added when
the mixture also contains reagents for an amplification
reaction.
Example 5
Preparation of Aqueous Solutions Formulated with Lipids and
Enzymes
[0126] This example describes a method for preparing aqueous lipid
solutions containing enzymes that are useful for releasing nucleic
acid according to the methods and compositions of the invention.
The following aqueous lipid containing solutions are prepared:
[0127] Reagent A:
[0128] 80 .mu.l dried lipid (Example 4) and 1 mL of 50 mM Sodium
Acetate (pH 6) containing 100,000 units of lysozyme (Sigma Chemical
Co., St. Louis, Mo.).
[0129] Reagent B:
[0130] 80 .mu.l dried lipid (Example 4) and 1 mL of 10 mM Borate
(pH 8) containing 100,000 units of lipase (Sigma Chem. Co.).
[0131] Reagent C:
[0132] 80 .mu.l dried lipid (Example 4) and 1 mL of 10 mM Borate
(pH 8) containing 1 mg proteinase K (Sigma Chem. Co.).
[0133] Reagent D:
[0134] 80 .mu.l dried lipid (Example 4) and 1 mL of 50 mM Sodium
Acetate (pH 6) containing 50,000 units each of lysozyme and
lipase.
[0135] Rehydration is carried out by vortexing the mixture
overnight at 37.degree. C. For liposomes, the mixture is further
processed by freeze thawing and extrusion through polycarbonate
filters (0.1 .mu.M pore). The formulations can be prepared in
presence of a reversible amplification inhibitor. Such inhibitors
are used only when the mixture is prepared for an amplification
reaction.
Example 6
Preparation of Aqueous Solutions Formulated with Lipids and Enzymes
and a Probe
[0136] This example describes the preparation of aqueous lipid
solutions containing enzymes and a probe that are useful for
releasing nucleic acid and hybridizing the nucleic acid to the
probe. The following aqueous lipid containing solutions are
prepared:
[0137] The reagent solution to be mixed or formulated contains an
oligonucleotide probe for subsequent hybridization. The reagents
include:
[0138] Reagent A:
[0139] 80 .mu.l dried lipid (Example 4) and 1 mL of 50 mM Sodium
Acetate (pH 6) containing 100,000 units of lysozyme (Sigma Chem.
Co.) and 1 micromolar of the probe.
[0140] Reagent B:
[0141] 80 .mu.l dried lipid (Example 4) and 1 mL of 10 mM Borate
(pH 8) containing 100,000 units of lipase (Sigma Chem. Co.) and 1
micromolar of the probe.
[0142] Reagent C:
[0143] 80 .mu.l dried lipid (Example 5) and 1 mL of 10 mM Borate
(pH 8) containing 1 mg of proteinase K (Sigma Chem. Co.), 1 mM EDTA
and 1 micromolar of the probe.
[0144] Reagent D:
[0145] 80 .mu.l dried lipid (Example 4) and 1 mL of 50 mM Sodium
Acetate (pH 6) containing 50,000 units each of lysozyme and lipase,
and 1 micromolar of the probe.
[0146] Rehydration is carried out by vortexing the mixture
overnight at 37.degree. C. For liposomes, the mixture is further
processed by freeze thawing and extruded through polycarbonate
filters and further purified by gel filtration. The. formulations
can be prepared in presence of a reversible amplification
inhibitor. Such inhibitors are used only when the mixture is
prepared for an amplification reaction.
Example 7
Preparation of Aqueous Solutions for Releasing and Labeling Nucleic
Acid
[0147] This example describes the preparation of aqueous solutions
containing lipids and other compounds for releasing and labeling
nucleic acid.
[0148] Aqueous solutions containing lipids and formulated with
enzymes and other substances as described in Examples 4, 5 and 6
are combined with a photoreative DNA binding ligand, such as BPA
(Example 17) or spermine-biotin-angelicin (SBA: Albarella et al.,
Nucleic Acids Res., 17:4293-4308 (1989)), BPIMA (Example 18), APIMA
(Example 19), AZPIMA (Example 20) or BDA (Example 21) at a
concentration of about 100 micromolar.
Example 8
Preparation of Aqueous Solutions for Releasing Nucleic Acid and
Amplifying Nucleic Acid
[0149] This example describes the preparation of aqueous solutions
containing lipids for releasing and amplifying nucleic acids.
[0150] In the lipid containing formulations of Examples 4, 5(A),
(B), (C), and 7, additional chemicals for nucleic acid
amplification, which include primers, enzymes and nucleoside
triphosphates are added. Formulations with enzymes are made with
reagents that are free of proteinases and nucleases. The
concentrations of each of the amplification components are adjusted
on the basis of type of procedure to be followed. For a typical
PCR, a five fold higher concentration of materials is used in
formulations so that if one fifth of the mixture is used for
amplification the final amplification concentration is adjusted to
its optimum level.
Example 9
Preparation of an Aqueous Solution for Releasing and Capturing
Nucleic Acid on a Solid Phase
[0151] This example describes the preparation of aqueous solutions
containing lipids for releasing and capturing nucleic acids on a
solid phase.
[0152] Oligo-dT magnetic particles (Novagen, Madison, Wis.) are
used as a solid phase for capturing polyA containing RNA from
cells. 10 .mu.g of the particles are added to any of Reagents A-D
of Examples 5 and 6.
Example 10
Releasing Nucleic Acids from Gram Negative Bacteria Using Aqueous
Lipid Solutions
[0153] E. Coli strain ATCC 35218 (gram negative) is grown in
culture medium as described by Isenberg, H. D., (Antimicrobial
Susceptibility Testing, ASM press, (1994) pp 5.2.2), to an OD at
600 nm of 1.0. One mL of cell culture is added (in duplicate) to
100 microliters (".mu.l") of reagent (A) or (B) or (C) or (D) of
Example 5. The mixture is incubated at 37.degree. C. for 15 minutes
until the absorbency at 600 nm reads less than 0.1 indicating more
than 90% lysis.
Example 11
Releasing Nucleic Acids from Gram Positive Bacteria Using Aqueous
Lipid Solutions
[0154] Staphylococcus aureus strain ATCC 29213 (gram positive) is
grown as described for E. Coli to an OD at 550 nm of 1.0. One mL of
cell culture is added (in duplicate) to 100 microliters (".mu.l")
of reagent (A) or (B) or (C) or (D) of Example 5. The mixture is
incubated at 60.degree. C. for 15 minutes until the absorbency at
550 nm reads less than 0.1 indicating more than 90% lysis.
Example 12
Releasing Nucleic Acids from a Clinical Sample Containing Chlamydia
Trachomitis
[0155] Cervical swabs samples are collected in transport medium
(Manual of Clinical microbiology, 5.sup.TH Ed., ASM press (1991),
p1238) lacking any detergent. 0.2 mL of Reagent A described in
Example 5 is added to the medium containing the swab. The mixture
is incubated at 37.degree. C. for 15 min. 100 .mu.l of the mixture
is then subjected to Gen-Probe's commercial PACE 2 assay format
(Gen-Probe, Inc., San Diego, Calif.) according to the
manufacturer's instructions. The results support efficient release
of RNA as judged by the hybridization assay.
Example 13
Releasing, Labeling and Detecting Nucleic acid from a Clinical
Sample Suspected of Chlamydial Infection
[0156] Cervical swabs samples are collected in transport medium
lacking any detergent as described in Example 12. 0.2 mL of the
aqueous solution containing BPA described in Example 7 is added to
the medium containing the swab. The mixture is incubated at
37.degree. C. for 60 minutes. During the incubation step, the
sample is exposed to light of 340.+-.30 nm using a
transilluminator. After illumination, the labeled sample is
hybridized with the PACE 2 probe (Gen-Probe, Inc., San Diego,
Calif.), immobilized to nitrocellulose paper. The presence of the
label on the nitrocellulose indicates hybridized nucleic acids and
demonstrates the effectiveness of the simultaneous lysis and
labeling of the released nucleic acids.
Example 14
Releasing Nucleic Acids from a Clinical Sample Infected with
Mycobacterium Tuberculosis
[0157] A sputum sample from a tuberculosis positive individual is
processed by treatment with N-Acetyl-L-cysteine-NaOH to generate a
sediment as described in the Manual of Clinical microbiology,
5.sup.th Ed., ASM press (1991), p307-309. 100 .mu.l of the sediment
is added to 10 .mu.l of reagent (D) in Example 5 and 90 .mu.l of
Tris buffer pH 7.4.+-.0.2. A control extraction sample is prepared
containing 100 .mu.l of the sediment and 100 .mu.l of the Tris
buffer. The mixtures are incubated at 60.degree. C. for 1 hr and
then heated at 90.degree. C. for 15 minutes. The control sample is
lysed by sonication. The samples are then tested by PCR as
described in Christian et al., J. Clin. Microbiol. 33(3):556-561
(1995). The results indicate efficient lysis of both samples.
Example 15
Releasing, Labeling and Detecting Nucleic Acid from a Urine Sample
with an Aqueous Lipid Solution
[0158] This example demonstrates releasing, labeling and detecting
nucleic acids from a urine sample with Reagent A of Example 7
(Reagent A contains and BPA as the labeling agent). Urine is
processed by centrifugation as described in Dattagupta, et al.,
Analytical Biochemistry, 177:85-89 (1989), and resuspended in 50 mM
sodium acetate buffer pH 6. 0.9 mL of the suspension is added to
0.1 nL of Reagent A and the mixture is incubated at 37.degree. C.
for 2 hrs. The step of photoactivation and detection of the labeled
product is performed as described by Dattagupta, et al. supra
(1989). Briefly, after nucleic acid is released (or during
incubation), the mixture is exposed to light (365.+-.30 nm) for 60
minutes to conjugate the BPA to the nucleic acid. The labeled
nucleic acid is then hybridized to a specific probe.
Example 16
Releasing and labeling Nucleic Acid from a Clinical Serum Sample
Suspected of Containing Hepatitis B virus.
[0159] This example demonstrates releasing and labeling nucleic
acid from a serum sample with an aqueous solution comprising BPA
prepared as described in Example 7 (based on any of Regents A-D
from Examples 5 or 6) is added to 50%v/v. 100 .mu.l of the serum
sample is added to 100 .mu.l of the aqueous solution and the
mixture heated at 60.degree. C. for 10 minutes. The step of
photoactivation and detection of the labeled product is performed
as described by Dattagupta, et al., Analytical Biochemistry,
177:85-89 (1989). Briefly, after nucleic acid is released (or
simultaneously with incubation), the mixture is exposed to light
(365.+-.30 nm) for 60 minutes to conjugate the BPA to the nucleic
acid. The labeled nucleic acid is then hybridized to immobilized
genomic hepatitis B DNA and detected as described in Dattagupta et
al., supra (1989).
Example 17
Preparation of 4'-Biotinyl-pentaoxaheptadecane
-4,5'-dimethylangelicin (BPA)
[0160] This example describes the preparation of the photoreactive
nucleic acid binding ligand, BPA.
[0161] The synthesis of BPA is carried out in the following five
steps.
[0162] Step 1: Preparation of
3,6,9,12,15-pentaoxaheptadecane-1,17-diol ditosylate
[0163] A solution of 73.91 g of p-toluenesulfonyl chloride (0.389
mol) in 400 mL of methylene chloride is added dropwise with
stirring over a 2.5 hrs period to 400 mL of methylene chloride
containing 50 g of hexaethylene glycol (0.177 mol) and 64 mL of
triethylamine (39.36 g, 0.389 mol) at 0.degree. C. The mixture is
stirred for one hr at 0.degree. C. and heated to ambient
temperature for 44 hrs. The mixture is filtered and the filtrate
concentrated in vacuo. The resulting residue is suspended in 500 mL
of ethyl acetate and filtered. The filtrate is concentrated in
vacuo to a yellow oil which was triturated eight times with 250 mL
portions of warm hexane to remove unreacted p-toluenesulfonyl
chloride. The resulting oil is then concentrated under high vacuum
to yield 108.12 g of a yellow oil (quantitative yield).
[0164] Analysis:
[0165] Calculated for C.sub.26H.sub.38O.sub.11S.sub.2: Calc.=C,
52.87; H, 6.48. found: C, 52.56; H, 6.39.
[0166] PMR ("proton magnetic resonance"): (60 MHz,
CDCl.sub.3).delta.: 2.45 (s, 6H); 3.4-3.8 (m, 20H); 4.2 (m, 4H);
7.8 (AB quartet, J=8Hz, 8H).
[0167] IR ("infrared"): (neat) cm.sup.-1: 2870, 1610, 1360, 1185,
1105, 1020 930, 830, 785, 670.
[0168] Step 2: Preparation of 1,17-Diphthalimido
3,6,9,12,15-pentaoxahepta- decane
[0169] A stirred suspension containing 108 g of
3,6,9,12,15-pentaoxaheptad- ecane-1,17-diol ditosylate (0.183 mol),
74-57 g of potassium phthalimide (0.403 mol), and 700 mL of
dimethylacetamide is heated at 160-170.degree. C. for 2 hrs and
then cooled to room temperature. The precipitate is filtered and
washed with water and acetone to yield 53.05 g of product as a
white powder which was dried at 55.degree. C. (0.1 mm); melting
point: 124-126.degree. C.
[0170] A second crop of product is obtained from the
dimethylacetamide filtrate by evaporation in vacuo and the
resulting precipitate is successively washed ethyl acetate, water,
and acetone. A resulting white powder is dried at 55.degree. C.
(0.1 mm of vacuum) to yield an additional 9.7 g of product; melting
point 124.5-126.5.degree. C. The combined yield of product is 62.82
g (68% yield).
[0171] Analysis:
[0172] First crop, calculated for
C.sub.28H.sub.32N.sub.2O.sub.91/2H.sub.2- O; Calc.=C, 61.19; H,
6.05; N, 5.09. found: C, 61.08; H. 6.15; N, 5.05.
[0173] Second crop calculated for C.sub.28H.sub.32N.sub.2O.sub.9:
Calc.=C, 62.21; H, 5.97; N, 5.18. found: C, 61.78; H, 6.15; N,
5.13.
[0174] Second Crop PMR: (60 MHz, DMSO-d.sub.6).delta.: 3.5 (s, 8H);
3.6 (s, 8H); 3.8 (bt, J=3Hz, 8H): 8.1 (s, 8H).
[0175] Second Crop IR: (KBr) cm.sup.-1: 2890, 1785, 1730, 1400,
1100, 735.
[0176] Step 3: Preparation of
1,17-Diamino-3,6,9,12,15-Pentaoxaheptadecane
[0177] The synthesis generally followed the method of Kern et al.,
Makrol. Chem., 180, 2539 (1979). A solution containing 60 g of
1,17-diphthalimido-3,6,9,12,15-pentaoxaheptadecane (0.118 mol),
14.8 g of hydrazine hydrate (0.296 mol), and 500 mL of ethanol is
heated with mechanical stirring in a 100.degree. C. oil bath for
three hrs. The mixture is cooled and filtered. A resultant filter
cake is washed four times with 300 mL portions of ethanol. The
combined filtrates are concentrated to yield 32.35 g of a yellow
opaque glassy oil by evaporative distillation at 150-200.degree. C.
(0.01 mm of vacuum). The result is 22.82 g of a light yellow oil
(69% yield). b.p. 175-177.degree. C. (0.07 mm).
[0178] Analysis:
[0179] For C.sub.12H.sub.12N.sub.2O.sub.51/2H.sub.2O: Calc.=C,
49.80, H, 10.10; N, 9.68. found: C, 50.36; H, 9.58; N, 9.38.
[0180] PMR: (60 MHz, CDCl.sub.3).delta.: 1.77 (s, 4H, NH.sub.2);
2.85 (t, J=5Hz, 4H); 3.53 (t, J=5Hz, 4H); 3.67 (m, 16H).
[0181] (CHCl.sub.3) cm.sup.-1: 3640, 3360, 2860, 1640, 1585, 1460,
1350, 1250, 1100, 945, 920, 870.
[0182] Step 4: Preparation of
1-Amino-17-N-(Biotinylamido)-3,6,9,12,15-pen- taoxaheptadecane
[0183] A solution containing 7.2 g of
1,17-diamino-3,6,9,12,15-pentaoxahep- tadecane (25 mmol) in 75 mL
of dimethylformamide ("DMF") under an argon atmosphere is treated
with 3.41 g of N-succinimidyl biotin (10 mmol) added in portions
over 1.0 hour. The resulting solution is stirred for four hrs at
ambient temperature. A sample of the solution run on TLC
(SiO.sub.2; solvent: 70:10.1 CHCL.sub.3--CH.sub.3OH-conc.
NH.sub.4OH) and visualized by dimethylaminocinnamaldehyde spray
reagent to determine conversion to a new product (Rf=0.18). The
solution is divided in half and each half absorbed onto SiO.sub.2
and purified by flash column chromatography on 500 g of
SiO.sub.2-60 (230-400 mesh) using a 70:10.1
CHCl.sub.3--CH.sub.3OH-conc. NH.sub.4OH solvent mixture. Fractions
containing the product are pooled and concentrated to a yield 2.42
g of a gelatinous, waxy solid. The product is precipitated as a
solid from isopropanol-ether, washed with hexane, and dried at
55.degree. C. (0.1 mm) to result in 1.761 g of a white powder (35%
yield).
[0184] Analysis:
[0185] Calculated for C.sub.22H.sub.42N.sub.24O.sub.7S.3/2H.sub.2O:
Calc.=C, 49.51; H, 8.50; N. 10.49. found: C, 49.59; H, 8.13; N,
10.39.
[0186] PMR: (90 MHz, DMSO-d.sub.6).delta.: 1.1-1.7 (m, 6H); 2.05
(t, J=7Hz, 2H); 2.62 (t, J=4Hz, 1H); 2.74 (t, J=4Hz, 1H); 3.0-3.4
(m, 14H). 3.50 (s, 14H); 4.14 (m, 1H); 4.30 (m, 1H); 6.35 (d,
J=4Hz, 1H); 7.80 (m, 1H).
[0187] CMR: (22.5 MHz, DMSO-d.sub.6).delta.: 25.2, 28.0, 28.2,
35.1, 40.6, 55.3, 59.2, 61.1, 69.6, 69.8, 71,2, 162.7, 172.1.
[0188] IR: (KBr) cm.sup.-1: 2900, 2850, 1690, 1640, 1580, 1540,
1450, 1100.
[0189] Mass Spectrum (FAB) m/e: 507.3 (M+1, 56% )
[0190] Step 5: Preparation of 4'-Biotinyl-pentaoxaheptadecane
-4,5'-dimethylangelicin (BPA)
[0191] The synthesis generally followed the method of Albarella, J.
P., et al., Nucl. Acids Res., 17:4293 (1989). A solution of 203 mg
of 1-amino-17-N(biotinylamido)-3,6,9,12,15-pentaoxaheptadecane (0.4
mmol) in 1 mL of DMF under an argon atmosphere is treated with 78
of N,N-carbonyldimidazole (0.48 mmol). The resulting mixture is
stirred for four hrs and then treated with 55 mg of
4'-aminomethyl-4,5'-dimethylingel- icin hydrochloride (0.2 mmol),
140 .mu.l of diisopropylethylamine, and 100 .mu.l of DMF. The
resulting mixture is stirred overnight at 50.degree. C. and then
evaporated onto SiO.sub.2 in vacuo and the resultant solid is
purified by chromatography on 60 g of SiO.sub.2 (230-400 mesh), and
eluted with 1.5 liters of 7% CHCl.sub.3--CH.sub.3OH, followed by 1
liter of 10% CHCl.sub.3--CH.sub.3OH. Fractions containing the
product are pooled and concentrated to yield 72 mg of a glassy
solid (47% yield).
[0192] Analysis:
[0193] PMR: (90 MHz, DMSO-d.sub.6):.delta.. 1.1-1.8 (m, 6H); 2.04
(bt, J=7Hz, 2H); 2.5 (s, 6H); 2.56 (m, 1H); 2.74 (bd, J=4Hz, 1H);
2.8-3.4 (m, 14H); 3.40 (m, 14H); 4.14 (m, 1H); 4.25 (m, 1H); 4.40
(bd, J=6Hz, 2H); 6.5 (m, 1H); 6.35 (s, 1H); 7.02 (s, 1H); 7.45 (d,
J=8Hz, 1H); 7.62 (d, J=8Hz, 1H); 7.80 (m, 1H).
[0194] CMR: (22.5 MHz, DMSO-d.sub.6).delta.: 11.9, 18.9, 25.3, 28.2
28.3, 33.4, 35.2, 55.4, 59.2, 61.0, 69.2, 69.6, 69.8, 70.0, 89.0,
107.8, 112.0, 113.1, 114.3, 120.6, 121.6, 153.6, 154.4, 155.6.
157.9, 159.5, 162.7, 172.1.
Example 18
Synthesis of angelicin bisbenzimidazole-pentaoxaheptadecane-biotin
("BPIMA")
[0195] This example describes the preparation of BPIMA, a LAC
comprising a photoreactive binding ligand, binding enhancer and a
label. The label is biotin and the enhancer moiety is
bisbenzimidazole.
[0196] The synthesis of BPIMA is carried out in the following eight
steps.
[0197] Step 1: Synthesis of dihexadecyl-3-bromo-propanediol
[0198] In a 210 mL round bottomed flask equipped with a magnetic
stir bar, 2 g of dihexadecylglycerol (Sigma Chem.Co.) is dissolved
into 120 mL of toluene. To this solution is added 3.54 g (10.7
mmoles) of carbon tetrabromide and 2.80 g (10.7 mmoles) of
tripenylphosphine and the reaction mixture is stirred overnight for
18-20 hrs at room temperature. A resulting yellow suspension is
filtered and the filtrate concentrated on a rotary evaporator to
afford a white solid residue. This residue is dissolved in toluene,
washed once with saturated sodium chloride, dried over anhydrous
magnesium sulfate and concentrated under vacuum on a rotary
evaporator to afford 2.5 g of crude product as a white powder. This
crude product is purified further by flask column chromatography on
a silica gel 60 (E. Merck, Germany) column by sequential elution
with 100 mL each of hexane, 14 ethyl acetate in hexane, 21 ethyl
acetate in hexane and, fmally, 31 ethyl acetate in hexane.
Fractions (8 mL) are collected and screened by TLC (silica gel;
solvent: 5:1 ethyl acetate--hexane) and those fractions that
contain pure product are pooled. The pooled fractions are
concentrated under vacuum on a rotary evaporator to afford a
quantitative yield of 1,2-0-dihexadecyl-3-bromo-1,2 propanediol as
a white powder.
[0199] Step 2: Synthesis of Bisbenzimidazole Succinate Ester
[0200] A solution of bisbenzimidazole (6 g; 0.01 mol)
dicyclohexylcarbodiimide (0.05 mol) and Succinic acid (0.01 mol) in
100 mL chloroform is stirred overnight for 18-24 hrs. During this
time, a white precipitate is formed. The precipitate is filtered
and washed with chloroform (2.times.50 mL). The chloroform washes
are combined and concentrated under vacuum in a rotary evaporator
and the residue purified by flash column chromatography. The
fractions containing the product are combined and concentrated
under vacuum in a rotary evaporate to afford bisbenzimide
succinnate ester (80%) as a white solid.
[0201] Step 3: Synthesis of pentaoxaheptadecane ditosylate
[0202] A solution of p-toluenesufonyl chloride (74 g; 0.39 mol) is
added dropwise to a stirred solution containing hexamethylene
glycol (50 g; 0.18 mol) trimethylamine (40 g; 0.39 mol) in
methylene chloride (400 mL) at 0.degree. C. The reaction mixture is
then stirred for 1 hr at room temperature. The mixture is filtered
and the filtrate concentrated under vacuum in a rotary evaporator.
The residue is suspended in ethylacetate (500 mL) and filtered. The
filtrate is concentrated under vacuum to afford yellow oil. The
yellow oil is triturated with hexane and the resulting oil vacuum
dried to afford 108 g of yellow oil.
[0203] Step 4: Synthesis of Diphthalimido pentaoxaheptadecane
ditosylate.
[0204] A suspension of ditosylate (Step 3; 108 g), potassium
phthalimide (75 g) in dimethylacetamide (700 mL) is heated at
165.degree. C. for 2 hrs with vigorous stirring. The reaction
mixture is then cooled to room temperature and the precipitate
filtered. The precipitate is washed with water and acetone to
afford 53 g of the desired product as a white solid.
[0205] Step 5: Synthesis of Diaminopentaoxaheptadecane (PEG)
[0206] A solution of diphthalimide (Step 4: 60 g), hydrazine
hydrate (15 g) and ethanol (500 mL) is heated at 100.degree. C.
with stirring for 3 hrs. The reaction mixture is cooled to room
temperature and filtered. The solid is washed with cold ethanol.
The combined filtrate is concentrated under vacuum in a rotary
evaporator to afford 33 g of yellow oil.
[0207] Step 6: Synthesis of
1-Amino-17-N-(Biotinylamido)-pentaoxaheptadeca- ne
[0208] A solution of diaminopentaoxaheptadecane (Step 5: 7 g) in
dimethyformamide is mixed with 3.4 g of N-succinimidylbiotin and
then stirred at room temperature for 4 hrs. The product is purified
by flash column chromatography on a silica gel 60 column. The
fractions containing the product are pooled and concentrated under
vacuum in a rotary evaporator to afford 2.5 g of a waxy solid. The
waxy solid is recrystallized from isopropanol/ether mixture-to
afford 1.8 g of white powder.
[0209] Step 7: Synthesis of bisbenzimidazole-PEG-biotin
[0210] A solution of biotinylamido pentaoxaheptadecane (Step 6; 3
g), bisbenzimidazole succinate ester (Step 2; 2 g) and
dicydclohexylcarbodimide (5 g) in chloroform (200 mL) is stirred at
room temperature for 20-24 hrs. The white precipitate formed is
filtered and the precipitate washed with chloroform. The chloroform
washes are combined and concentrated under vacuum in a rotary
evaporator and the residue purified by flash column chromatography.
The fractions containing the product are combined and concentrated
under vacuum in a rotary evaporator to afford
bisbenzamide-PEG-biotin as an off-white solid (1.5 g).
[0211] Step 8: Synthesis of Angelicin
bisbenzimidazole-PEG-biotin
[0212] To a solution of bisbenzimidazole-PEG-biotin (Step 7; 0.4
mmol) in dimethylformamide is added N,N-carbonyldiimidazole (0.5
mmol). The resulting mixture is stirred for 3-5 hrs and is then
treated with aminomethylangelicin (0.2 mmol), diispropylethylamine
(150 mL) and dimethylformamide (100 mL). The reaction mixture is
stirred overnight at 50-55.degree. C. The mixture is evaporated
under vacuum in a rotary evaporator and the residue is loaded onto
a column of silica gel and eluted sequentially with 7% methanol in
chloroform and 10% methanol in chloroform. The fractions containing
the product are pooled and concentrated to afford (0.2 mmol) BPIMA
as a glassy solid.
Example 19
Synthesis of Angelicin
bisbenzimidazole-pentaoxaheptadecane-acridine ("APIMA")
[0213] This example describes the preparation of APIMA, a LAC
comprising a photoreactive binding ligand, binding enhancer and
label. The label is a chemiluminescent acridinium ester.
[0214] The following six steps describes the synthesis of
APIMA.
[0215] Step 1: Synthesis of acrdinecarbonylchloride
[0216] A solution of acridine carboxyl acid (Aldrich Chem. Co.) and
thionyl chloride is stirred at room temperature for 20-24 hrs.
Excess thionyl chloride is removed under vacuum in a rotary
evaporator. The residue is treated with toluene and evaporated to
remove traces of thionyl chloride.
[0217] Step 2: Synthesis of acridine-4-hydroxypropionic acid
succinimide ester
[0218] A solution of acridine carbonyl chloride(Step 1: 2.3 g) in
dry pyridine (35 mL) is treated with hydroxyphenolpropionic acid
N-hydroxysuccinimide ester (2.5 g) at room temperature for 8-24
hrs. The resulting triethylaminehydochloride is filtered and the
solution is concentrated under vacuum in a rotary evaporator to
afford the succinimide ester as an off white solid.
[0219] Step 3: Synthesis of methyl fluorosulfonate succinimido
acridine
[0220] A solution of succinimide ester (Step 2; 2 g) and methyl
fluorosulfonate (3 mL) in dry chloroform is stirred for 8-24 hrs at
room temperature. The resulting solid is filtered and the solution
concentrated under vacuum in a rotary evaporator to afford 1.5 g of
product as a yellow solid.
[0221] Step 4: Synthesis of
1-Amino-17-N(acridnylamido)-pentaoxaheptadecan- e
[0222] A solution of diaminopentaoxaheptadecane (a "PEG": step 5,
Example 18) in dimethylformamide (75 mL) is treated with acridine
NHS ester (step 3).
[0223] The resulting solution is stirred at room temperature for 4
hrs. The solvent is removed under vacuum in a rotary evaporator and
the residue is triturated with hexane to afford the compound as a
pale yellow solid.
[0224] Step 5: Synthesis of bisbenzimidazole-PEG-acridine
[0225] A solution of acridinylamido pentaoxaheptadecane (step 4),
bisbenzimidazole succinic acid half ester (step 2, Example 18) and
dicyclohexylcarbodimide in chloroform is stirred at room
temperature for 18-24 hrs. A white precipitate is filtered and the
precipitate washed with chloroform. The combined chloroform washes
are concentrated under vacuum in a rotary evaporation to afford the
product as an off white solid.
[0226] Step 6: Synthesis of angelicin
bisbenzimidazole-PEG-acridine
[0227] N,N-carbonyldiimidazole is added to a solution of
bisbenzimidazole-PEG-acridine (step 5) in dimethylformamide. The
resulting mixture is stirred for 3-8 hrs and then treated with
aminomethyldimethylangelicin, diisopropylethylamine and
dimethylformamide. The mixture is stirred overnight at
50-55.degree. C. and evaporated under vacuum in a rotary
evaporator. The residue is purified by flash column chromatography
on a column of silica gel. Sequential elution with 7% methanol in
chloroform and 10% methanol in chloroform affords fractions
containing the product. The fractions are pooled and concentrated
to yield APIMA as a solid.
Example 20
Synthesis of
angelicin-bisbenzimidazole-pentaoxaheptadecane-azidonitrobenz- ene
("AZPIMA")
[0228] This example describes the preparation of a AZPIMA, a LAC
comprising a photoreactive binding ligand and a binding enhancer,
both of which are intercalating moieties.
[0229] The following two steps describes the synthesis of
AZPIMA.
[0230] Step 1: Synthesis of
bisbenzimidazole-PEG-azidonitrobenzene
[0231] A solution of diaminopentaoxaheptadecane (a "PEG": Step 5,
Example 18) and sulfoSANPH.RTM. (Pierce Chemicals, Rockford, Ill.)
is stirred at room temperature overnight. The solution is
concentrated under vacuum in a rotary evaporator and the residue is
dissolved in DMF. The solution is treated with bisbenzamide
succinate ester (step 2, Example 18) and stirred overnight.
Following completion of reaction as determined by TLC, the solution
is concentrated to afford an off white crystalline solid.
[0232] Step 2: Synthesis of angelicin
bisbenzimidazole-PEG-azidonitrobenze- ne
[0233] A solution of bisbenzimidazole-PEG-azidonitrobenzene (Step
1, above) and N,N-carbonyldiimidazole in dimethylformamide is
stirred for 4-14 hrs at room temperature. The resulting mixture is
treated with aminomethyldimethylangelicin, diisopropylethylamine
and the resulting mixture is stirred overnight at 50-55.degree. C.
Following completion of reaction, the reaction mixture is
concentrated in a rotary evaporator. The residue is purified by
flash column chromatography on a column of silica gel. The column
is eluted with a mixture of chloroform/methanol and the fractions
containing APIMA are pooled and concentrated to afford APIMA as a
solid.
Example 21
Synthesis of Angelicin-4',6'-diamidino-2-phenylindole-Biotin
("BDA")
[0234] This example describes the preparation of BDA, as LAC
comprising a photoreactive binding ligand, binding enhancer and a
label.
[0235] The following two steps describes the synthesis of BDA.
[0236] Step 1: Synthesis of 1-4',6'-diamidino-2-phenylindole
17-pentaoxaheptadecane tosylate (4',6'-diamidino-2-phenylindole:
"DAPI").
[0237] A solution of pentaoxaheptadecane ditosylate (Step 3,
Example 18) and DAPI (Aldrich Chem. Co., Cat.No 21,708-5) in
dimethylsulfoxide is stirred at room temperature for 8-24 hrs. Upon
completion of the reaction, as shown by TLC, the mixture is
evaporated under vacuum in a rotary evaporator and the residue
loaded onto a column of silica gel and eluted with a solution of
0-50% methanol in chloroform. The fractions containing the product
are pooled and concentrated under vacuum in a rotary evaporator to
afford the product as an off-white solid.
[0238] Step 2: Synthesis of Angelicin-DAPI
[0239] A solution of 1-DAPI-17-pentaoxaheptadecane tosylate (step
1) and aminomethyldimethylangelicin in dinthylformamide is stirred
at 25-60.degree. C. for 8-48 hrs. Upon completion of the reaction,
as shown by TLC, the reaction mixture is evaporated under vacuum in
a rotary evaporator and the residue is loaded onto a column of
silica gel and eluted with a solution of 0-30% methanol in
chloroform containing a trace of ammonia. The fractions containing
the product are pooled and concentrated to afford the product as a
pale yellow solid. The crude product is recrystallized from a
mixture of dimethylformamide and hexane.
[0240] Step 3: Synthesis of Angelicin-DAPI-Biotin
[0241] A solution of angelicin-DAPI (Step 2) and biotin-NHS ester
(Sigma Chem. Co., Cat. No. 1759) in DMF is stirred at 25-70.degree.
C. for 8-72 hrs. Upon completion of the reaction, the reaction
mixture is evaporated under vacuum in a rotary evaporator and the
residue is treated with petroleum ether. The solid is collected by
filtration and washed with petroleum ether (3.times.50 mL). The
crude solid is recrystallized to afford BDA as a white solid.
[0242] The examples set forth above are provided to give those of
ordinary skill in the art with a complete disclosure and
description of how to make and use the preferred embodiments of the
compositions, and are not intended to limit the scope of what the
inventors regard as their invention. Modifications of the
above-described modes for carrying out the invention that are
obvious to persons of skill in the art are intended to be within
the scope of the following claims. All publications, patents, and
patent applications cited in this specification are incorporated
herein by reference as if each such publication, patent or patent
application were specifically and individually indicated to be
incorporated herein by reference.
* * * * *