U.S. patent application number 11/716181 was filed with the patent office on 2007-08-02 for compositions and methods for the use of fmoc derivatives in dna/rna synthesis.
This patent application is currently assigned to SYNGEN, INC.. Invention is credited to Gabriel Alvarado.
Application Number | 20070179289 11/716181 |
Document ID | / |
Family ID | 32045270 |
Filed Date | 2007-08-02 |
United States Patent
Application |
20070179289 |
Kind Code |
A1 |
Alvarado; Gabriel |
August 2, 2007 |
Compositions and methods for the use of FMOC derivatives in DNA/RNA
synthesis
Abstract
Methods of nucleic acid preparation are described, including
preparation of mRNA, using FMOC derivatives to synthesize
oligonucleotides in addition to applying FMOC protocols to various
therapeutic and diagnostic methods. In some embodiments a single
stranded oligonucleotide is synthesized bound to a polymer support
(such as optic fiber glass filters) using said FMOC
derivatives.
Inventors: |
Alvarado; Gabriel; (San
Mateo, CA) |
Correspondence
Address: |
PERKINS COIE LLP
P.O. BOX 2168
MENLO PARK
CA
94026
US
|
Assignee: |
SYNGEN, INC.
|
Family ID: |
32045270 |
Appl. No.: |
11/716181 |
Filed: |
March 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10670015 |
Sep 24, 2003 |
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11716181 |
Mar 9, 2007 |
|
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60414061 |
Sep 27, 2002 |
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Current U.S.
Class: |
536/25.33 |
Current CPC
Class: |
C12Q 1/6806 20130101;
C07H 21/00 20130101; C07H 21/02 20130101; C12Q 1/6806 20130101;
C12Q 2525/117 20130101 |
Class at
Publication: |
536/025.33 |
International
Class: |
C07H 21/04 20060101
C07H021/04 |
Claims
1. A method for synthesizing and purifying a phosphotriester
oligonucleotide immobilized on a solid support complex, comprising:
a) providing i) a polymer support, and ii) four pools of nucleoside
3'-monomers in solution, said monomers having a nucleoside base and
a phosphoramidate group, wherein at least one of said monomers
contains 9-fluorenylmethoxycarbonyl as an amino protecting group on
the monomer base, and said monomers have an alkyl residue on the
phosphate of the phosphoramidite group; b) sequentially contacting
said polymer support with a solution of monomers from one or more
of said four pools under conditions such that a sequence of
monomers is immobilized on said polymer support to generate an
oligonucleotide/polymer support complex, wherein said complex
comprises at least one monomer containing
9-fluorenylmethoxycarbonyl base protecting group and wherein said
solution, after said contacting, comprises unreacted material; c)
treating said complex under conditions such that said unreacted
material is substantially removed, thereby creating a purified
oligonucleotide/polymer support complex, and d) treating said
purified oligonucleotide/polymer support complex under conditions
whereby said 9-fluorenylmethoxycarbonyl, but not the
phosphoramidate alkyl residue, is released.
2. The method of claim 1, wherein said nucleoside
3'-phosphoramidite monomer containing 9-fluorenylmethoxycarbonyl as
an amino protecting group is a
9-fluorenylmethoxycarbonyl-2'-cytidine 3'-phosphoramidite.
3. The method of claim 1, wherein said nucleoside
3'-phosphoramidite monomer containing 9-fluorenylmethoxycarbonyl as
an amino protecting group is a 9-fluorenyl
methoxycarbonyl-2'-adenosine 3'-phosphoramidite.
4. The method of claim 1, wherein said deoxyribonucleoside
3'-phosphoramidite monomer containing 9-fluorenylmethoxycarbonyl as
an amino protecting group is a
9-fluorenylmethoxycarbonyl-2'-guanosine 3'-phosphoramidite.
Description
[0001] This patent application is a continuation of U.S. patent
application Ser. No. 10/670,015 filed on Sep. 24, 2003, which
claims priority to U.S. Provisional Patent Application No.
60/414,061 filed Sep. 27, 2002, both of which are incorporated
herein in their entirety by reference.
TECHNICAL FIELD
[0002] The present invention relates to compositions and methods
using FMOC derivatives in DNA/RNA synthesis and in particular the
use, in a variety of formats, of immobilized oligonucleotides.
BACKGROUND
[0003] More than twenty years ago the first gene was synthesized by
means of traditional chemical methods. These traditional methods
relied on the use of protecting reagents for those reaction groups
in nucleosides and nucleotides which are sensitive to the
condensing agents required to form the phosphodiester bounds. These
sensitive groups include the amino groups of the bases, the 3'- and
5'-hydroxy groups of the deoxyribose, and the phosphate group
itself. Additionally, these traditional methods of nucleic acid
synthesis relied on supports and linker compounds as an anchor for
the nascent nucleic acid strand.
[0004] In many applications, it is desirable to have the nucleic
acid strand released from the linker support. This cleavage is
usually accomplished at the linker through chemical treatment that
leads to the removal of certain groups and the resultant release of
the nucleic acid from the support. Thus various methods have been
developed to bind the beginning of the nucleic acid strand to the
solid support via a linker that is susceptible to cleavage by
chemical treatment, as well as methods for the efficient release of
undamaged nucleic acid strand from the anchor.
[0005] These traditional approaches, however, are cumbersome and
time consuming in terms of resulting percentage yield of the
desired product (e.g. DNA and/or RNA). What is needed, therefore,
is a more efficient and versatile method for nucleic acid
synthesis.
SUMMARY
[0006] A key step in the solid support synthesis of
oligonucleotides is the protection of exocyclic amino groups of
2'-deoxyadenosine, 2'-deoxycytidine and 2'-deoxyguanosine. The
known N-acyl residues, synthesis by the phosphorotriester,
H-phosphonate and phosphoramidite methods. These groups are
relatively stable under neutral and acidic conditions. However,
their rates of removal under alkaline conditions are dependant upon
the nature of base residues and therefore the full deprotection
period of the resulting oligonucleotide is too long.
[0007] To cure this problem the Applicants used, in some
embodiments, labile 9-fluorenylmethoxycarbonyl group (FMOC) for the
protection of the exocyclic amino functions of deoxynucleoside
bases. In addition, the Applicants incorporated the use of FMOC
under synthesis conditions of the monomeric building blocks and
their corresponding phosphoramidites. That is to say, as
illustrated in FIG. 1, the fully protected FMOC deoxynucleoside
phosphoramidites and their corresponding FMOC derivatized polymer
supports were used in the synthesis of different DNA/RNA
chains.
[0008] In selected embodiments, the Applicants use this DNA/RNA
synthetic motif in novel adaptations of various diagnostic and
therapeutic methods. These methods include, but are not limited to,
the improved synthesis of antisense compounds, high throughput DNA
synthesis, the preparation of labeled nucleic acids, and the
preparation and use of multifuntional columns comprising
oligonucleotides bound to a polymer support.
[0009] In one embodiment, the present invention describes a method
for synthesizing and purifying an oligonucleotide immobilized on a
solid support complex, comprising: a) providing: i) a polymer
support, and ii) four pools of deoxyribonucleoside
3'-phosphoramidite monomers in solution, wherein at least one of
said monomers contains 9-fluorenylmethoxycarbonyl as an amino
protecting group; b) sequentially contacting said polymer support
with a solution of monomers from one or more of said four pools
under conditions such that a sequence of monomers is immobilized on
said polymer support to generate an oligonucleotide/polymer support
complex, wherein said complex comprises at least one monomer
containing 9-fluorenylmethoxycarbonyl and wherein said solution,
after said contacting, comprises unreacted material; c) treating
said complex under conditions such that said unreacted material is
substantially removed, thereby creating a purified
oligonucleotide/polymer support complex, and d) treating said
purified oligonucleotide/polymer support complex under conditions
whereby said 9-fluorenylmethoxycarbonyl is released.
[0010] In one embodiment, the above referenced method using said
deoxyribonucleoside 3'-phosphoramidite monomer containing
9-fluorenylmethoxycarbonyl as an amino protecting group is a
9-fluorenylmethoxycarbonyl-2'-deoxycytidine 3'-phosphoramidite. In
one embodiment, the above referenced method using said
deoxyribonucleoside 3'-phosphoramidite monomer containing
9-fluorenylmethoxycarbonyl as an amino protecting group is a
9-fluorenylmethoxycarbonyl-2'-deoxyadenosine
3'-phosphoramidite.
[0011] In one embodiment, the above referenced method using said
deoxyribonucleoside 3'-phosphoramidite monomer containing
9-fluorenylmethoxycarbonyl as an amino protecting group is a
9-fluorenylmethoxycarbonyl-2'-deoxyguanosine
3'-phosphoramidite.
Definitions
[0012] To facilitate understanding of the invention, a number of
terms are defined below.
[0013] A "solvent" is a liquid substance capable of dissolving or
dispersing one or more other substances. It is not intended that
the present invention be limited by the nature of the solvent
used.
[0014] As used herein, when a solution passes through a polymer
support, it comprises the "flow through." Material that does not
bind, if present, passes with the solution through the polymer
support into the flow through. To eliminate all non-specific
binding, the polymer support is "washed" with one or more wash
solutions which, after passing through the polymer support,
comprise one or more "effluents." "Eluent" is a chemical solution
capable of dissociating material bound to the matrix (if any); this
dissociated material passes through the matrix and comprises an
"eluate."
[0015] The term "gene" refers to a DNA sequence that comprises
control and coding sequences necessary for the production of a
polypeptide or precursor thereof. The polypeptide can be encoded by
a full length coding sequence or by any portion of the coding
sequence so long as the desired enzymatic activity is retained.
[0016] The term "oligonucleotide" as used herein is defined as a
molecule comprised of two or more deoxyribonucleotides or
ribonucleotides, usually more than three (3), and typically more
than ten (10) and up to one hundred (100) or more (although
preferably between twenty and thirty). The exact size will depend
on many factors; which in turn depends on the ultimate function or
use of the oligonucleotide. The oligonucleotide may be generated in
any manner, including chemical synthesis, DNA replication, reverse
transcription, or a combination thereof.
[0017] Because mononucleotides are reacted to make oligonucleotides
in a manner such that the 5' phosphate of one mononucleotide
pentose ring is attached to the 3' oxygen of its neighbor in one
direction via a phosphodiester linkage, an end of an
oligonucleotide is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring and
as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of
a subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5' and 3' ends.
[0018] When two different, non-overlapping oligonucleotides anneal
to different regions of the same linear complementary nucleic acid
sequence, and the 3' end of one oligonucleotide points towards the
5' end of the other, the former may be called the "upstream"
oligonucleotide and the latter the "downstream"
oligonucleotide.
[0019] The term "primer" refers to an oligonucleotide which is
capable of acting as a point of initiation of synthesis when placed
under conditions in which primer extension is initiated. An
oligonucleotide "primer" may occur naturally, as in a purified
restriction digest or may be produced synthetically.
[0020] A primer is selected to be "substantially" complementary to
a strand of specific sequence of the template. A primer must be
sufficiently complementary to hybridize with a template strand for
primer elongation to occur. A primer sequence need not reflect the
exact sequence of the template. For example, a non-complementary
nucleotide fragment may be attached to the 5' end of the primer,
with the remainder of the primer sequence being substantially
complementary to the strand. Non-complementary bases or longer
sequences can be interspersed into the primer, provided that the
primer sequence has sufficient complementarity with the sequence of
the template to hybridize and thereby form a template primer
complex for synthesis of the extension product of the primer.
[0021] "Hybridization" methods involve the annealing of a
complementary sequence to the target nucleic acid (the sequence to
be detected). The ability of two polymers of nucleic acid
containing complementary sequences to find each other and anneal
through base pairing interaction is a well-recognized phenomenon.
The initial observations of the "hybridization" process by Marmur
and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al.,
Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the
refinement of this process into an essential tool of modern
biology. Nonetheless, a number of problems have prevented the wide
scale use of hybridization as a tool in human diagnostics. Among
the more formidable problems are: 1) the inefficiency of
hybridization; 2) the low concentration of specific target
sequences in a mixture of genomic DNA; and 3) the hybridization of
only partially complementary probes and targets.
[0022] With regard to efficiency, it is experimentally observed
that only a fraction of the possible number of probe-target
complexes is formed in a hybridization reaction. This is
particularly true with short oligonucleotide probes (less than 100
bases in length). There are three fundamental causes: a)
hybridization cannot occur because of secondary and tertiary
structure interactions; b) strands of DNA containing the target
sequence have rehybridized (reannealed) to their complementary
strand; and c) some target molecules are prevented from
hybridization when they are used in hybridization formats that
immobilize the target nucleic acids to a solid surface.
[0023] Even where the sequence of a probe is completely
complementary to the sequence of the target, i.e., the target's
primary structure, the target sequence must be made accessible to
the probe via rearrangements of higher-order structure. These
higher-order structural rearrangements may concern either the
secondary structure or tertiary structure of the molecule.
Secondary structure is determined by intramolecular bonding. In the
case of DNA or RNA targets this consists of hybridization within a
single, continuous strand of bases (as opposed to hybridization
between two different strands). Depending on the extent and
position of intramolecular bonding, the probe can be displaced from
the target sequence preventing hybridization.
[0024] Solution hybridization of oligonucleotide probes to
denatured double-stranded DNA is further complicated by the fact
that the longer complementary target strands can renature or
reanneal. Again, hybridized probe is displaced by this process.
This results in a low yield of hybridization (low "coverage")
relative to the starting concentrations of probe and target.
[0025] With regard to low target sequence concentration, the DNA
fragment containing the target sequence is usually in relatively
low abundance in genomic DNA. This presents great technical
difficulties; most conventional methods that use oligonucleotide
probes lack the sensitivity necessary to detect hybridization at
such low levels.
[0026] One attempt at a solution to the target sequence
concentration problem is the amplification of the detection signal.
Most often this entails placing one or more labels on an
oligonucleotide probe. In the case of non-radioactive labels, even
the highest affinity reagents have been found to be unsuitable for
the detection of single copy genes in genomic DNA with
oligonucleotide probes. See Wallace et al., Biochimie 67:755
(1985). In the case of radioactive oligonucleotide probes, only
extremely high specific activities are found to show satisfactory
results. See Studencki and Wallace, DNA 3:1 (1984) and Studencki et
al., Human Genetics 37:42 (1985).
[0027] K. B. Mullis et al., U.S. Pat. Nos. 4,683,195 and 4,683,202,
hereby incorporated by reference, describe a method for increasing
the concentration of a segment of a target sequence in a mixture of
genomic DNA without cloning or purification. This process for
amplifying the target sequence (which can be used in conjunction
with the present invention to make target molecules) consists of
introducing a large excess of two oligonucleotide primers to the
DNA mixture containing the desired target sequence, followed by a
precise sequence of thermal cycling in the presence of a DNA
polymerase. The two primers are complementary to their respective
strands of the double stranded target sequence. To effect
amplification, the mixture is denatured and the primers then to
annealed to their complementary sequences within the target
molecule. Following annealing, the primers are extended with a
polymerase so as to form a new pair of complementary strands. The
steps of denaturation, primer annealing, and polymerase extension
can be repeated many times (i.e. denaturation, annealing and
extension constitute one "cycle;" there can be numerous "cycles")
to obtain a high concentration of an amplified segment of the
desired target sequence. The length of the amplified segment of the
desired target sequence is determined by the relative positions of
the primers with respect to each other, and therefore, this length
is a controllable parameter. By virtue of the repeating aspect of
the process, the method is referred to by the inventors as the
"Polymerase Chain Reaction" (hereinafter PCR). Because the desired
amplified segments of the target sequence become the predominant
sequences (in terms of concentration) in the mixture, they are said
to be "PCR amplified".
[0028] With PCR, it is possible to amplify a single copy of a
specific target sequence in genomic DNA to a level detectable by
several different methodologies (e.g. hybridization with a labeled
probe; incorporation of biotinylated primers followed by
avidin-enzyme conjugate detection; incorporation of .sup.32P
labeled deoxynucleotide triphosphates, e.g., dCTP or dATP, into the
amplified segment). In addition to genomic DNA, any oligonucleotide
sequence can be amplified with the appropriate set of primer
molecules. In particular, the amplified segments created by the PCR
process itself are, themselves, efficient templates for subsequent
PCR amplifications.
[0029] The PCR amplification process is known to reach a plateau
concentration of specific target sequences of approximately
10.sup.-8 M. A typical reaction volume is 100 .mu.l, which
corresponds to a yield of 6.times.10.sup.11 double stranded product
molecules.
[0030] With regard to complementarity, it is important for some
diagnostic applications to determine whether the hybridization
represents complete or partial complementarity. For example, where
it is desired to detect simply the presence or absence of pathogen
DNA or RNA (such as from a virus, bacterium, fungi, mycoplasma,
protozoan) it is only important that the hybridization method
ensures hybridization when the relevant sequence is present;
conditions can be selected where both partially complementary
probes and completely complementary probes will hybridize. Other
diagnostic applications, however, may require that the
hybridization method distinguish between partial and complete
complementarity. It may be of interest to detect genetic
polymorphisms. For example, human hemoglobin is composed, in part,
of four polypeptide chains. Two of these chains are identical
chains of 141 amino acids (alpha chains) and two of these chains
are identical chains of 146 amino acids (beta chains). The gene
encoding the beta chain is known to exhibit polymorphism. The
normal allele encodes a beta chain having glutamic acid at the
sixth position. The mutant allele encodes a beta chain having
valine at the sixth position. This difference in amino acids has a
profound (most profound when the individual is homozygous for the
mutant allele) physiological impact known clinically as sickle cell
anemia. It is well known that the genetic basis of the amino acid
change involves a single base difference between the normal allele
DNA sequence and the mutant allele DNA sequence.
[0031] Unless combined with other techniques (such as restriction
enzyme analysis), methods that allow for the same level of
hybridization in the case of both partial as well as complete
complementarity are typically unsuited for such applications; the
probe will hybridize to both the normal and variant target
sequence. Hybridization, regardless of the method used, requires
some degree of complementarity between the sequence being assayed
(the target sequence) and the fragment of DNA used to perform the
test (the probe). (Of course, one can obtain binding without any
complementarity but this binding is nonspecific and to be
avoided.)
[0032] The complement of a nucleic acid sequence as used herein
refers to an oligonucleotide which, when aligned with the nucleic
acid sequence such that the 5' end of one sequence is paired with
the 3' end of the other, is in "antiparallel association." Certain
bases not commonly found in natural nucleic acids may be included
in the nucleic acids of the present invention and include, for
example, inosine and 7-deazaguanine. Complementarity need not be
perfect; stable duplexes may contain mismatched base pairs or
unmatched bases. Those skilled in the art of nucleic acid
technology can determine duplex stability empirically considering a
number of variables including, for example, the length of the
oligonucleotide, base composition and sequence of the
oligonucleotide, ionic strength and incidence of mismatched base
pairs.
[0033] Stability of a nucleic acid duplex is measured by the
melting temperature, or "T.sub.m." The T.sub.m of a particular
nucleic acid duplex under specified conditions is the temperature
at which on average half of the base pairs have disassociated. The
equation for calculating the T.sub.m of nucleic acids is well known
in the art. As indicated by standard references, an estimate of the
T.sub.m value may be calculated by the equation:
T.sub.m=81.5.degree. C.+16.6 log M+0.41(% GC)-0.61(%
form)-.sup.500/L where M is the molarity of monovalent cations, %
GC is the percentage of guanosine and cytosine nucleotides in the
DNA, % form is the percentage of formamide in the hybridization
solution, and L=length of the hybrid in base pairs. [See, e.g.,
Guide to Molecular Cloning Techniques, Ed. S. L. Berger and A. R.
Kimmel, in Methods in Enzymology Vol. 152, 401 (1987)]. Other
references include more sophisticated computations which take
structural as well as sequence characteristics into account for the
calculation of T.sub.m.
[0034] The term "probe" as used herein refers to a labeled
oligonucleotide which forms a duplex structure with a sequence in
another nucleic acid, due to complementarity of at least one
sequence in the probe with a sequence in the other nucleic
acid.
[0035] The term "label" as used herein refers to any atom or
molecule which can be used to provide a detectable (preferably
quantifiable) signal, and which can be attached to a nucleic acid
or protein. Labels may provide signals detectable by fluorescence,
radioactivity, colorimetry, gravimetry, X-ray diffraction or
absorption, magnetism, enzymatic activity, and the like. Such
labels can be added to the oligonucleotides of the present
invention.
[0036] The terms "nucleic acid substrate" and nucleic acid
template" are used herein interchangeably and refer to a nucleic
acid molecule which may comprise single- or double-stranded DNA or
RNA.
[0037] The term "substantially single-stranded" when used in
reference to a nucleic acid substrate means that the substrate
molecule exists primarily as a single strand of nucleic acid in
contrast to a double-stranded substrate which exists as two strands
of nucleic acid which are held together by inter-strand base
pairing interactions.
[0038] The term "polymer support", as used herein, refers to any
substrate or solid support having the chemical composition (or
demonstrating the physical properties of) a polymer. Examples of a
polymer support include (but are not limited to) silica, glass,
polystyrene, or optic fiber glass filters.
[0039] As used herein, the abbreviation "DBU" refers to
1,8-diazabicyclo[5.4.0] undec-7-ene.
[0040] As used herein, the abbreviation "FMOC" refers to a chemical
compound comprising a 9-fluorenylmethoxycarbonyl [also known as
(fluoren-9-yl)methoxycarbonyl] group.
[0041] As used herein, the term "unreacted material" refers to
reagent(s) that are not consumed in a chemical reaction. Examples
of unreacted materials include (but are not limited to)
unincorporated monomers of deoxyribonucleosides after the synthesis
of an oligonucleotide.
[0042] As used herein, the term "purified oligonucleotide/polymer
support complex" refers to a oligonucleotide/polymer support
complex wherein said oligonucleotide is at least 70% pure, more
preferably at least 80% pure, and still more preferably at least
90% pure.
[0043] As used herein the term "substantially removed" refers to a
reaction mixture, after a chemical reaction, wherein unreacted
materials and/or unwanted reaction by products are removed,
thereby, leaving a desired product(s) that is at least 70% pure,
more preferably at least 80% pure, and still more preferably at
least 90% pure As used herein, the words "pool" or "pools" refer to
a contained source of reagent. Example of a pool(s) include (but
are not limited to) a solution of 2'-deoxycytidine
3'-phosphoramidite.
DESCRIPTION OF THE DRAWINGS
[0044] FIG. 1 schematically shows the preparation of FMOC protected
deoxynucleoside phosphoramidite.
[0045] FIG. 2 schematically shows fluorenylmethoxycarbonyl
cleavage.
[0046] FIG. 3 is a schematic of the traditional cleavage and
deprotection "Acyl Strategy" used in the synthesis of
oligonucleotides.
[0047] FIG. 4 is a schematic of the full deprotection "FMOC
Strategy" used in the synthesis of oligonucleotides with the
option, if desired, of cleavage.
[0048] FIG. 5 is a schematic overview of the protocol for the
aminopropylsilylation of silica gel (or controlled pore glass).
DETAILED DESCRIPTION
[0049] A key step in the solid support synthesis of
oligonucleotides is the protection of exocyclic amino groups of
2'-deoxyadenosine, 2'-deoxycytidine and 2'-deoxyguanosine. In
selected embodiments of the present invention, the labile
9-fluorenylmethoxycarbonyl group (FMOC) for the protection of the
exocyclic amino functions of deoxynucleoside bases was used various
protocols in the synthesis of DNA/RNA. These "FMOC based" DNA/RNA
synthetic strategies may then be adapted, in selected embodiments,
to a variety diagnostic and therapeutic methods. These methods
include, but are not limited to, the improved synthesis of
antisense compounds, high throughput DNA synthesis, the preparation
of labeled nucleic acids, and the preparation and use of
multifuntional columns comprising oligonucleotides bound to a solid
support.
Experimental
[0050] The following protocols serve to illustrate certain
preferred embodiments and aspects of the present invention and are
not to be construed as limiting the scope thereof.
[0051] In the experimental disclosure which follows, the following
abbreviations apply: .degree. C. (Centigrade); h [hour(s)]; .mu.g
(micrograms); .mu.mole (micromoles); .mu.l (microliters); mL or ml
(milliliters); mM (milliMolar); r.t. (room temperature).
I. Use Of FMOC Derivatives In DNA/RNA Synthesis
[0052] (Reagents and Reactions)
A. Preparation of 5'-O-DMTR-N.sup.4--FMOC-2'-deoxycytidine
[0053] In one embodiment, 5'-O-DMTR-N.sup.4--FMOC-2'-deoxycytidine
was prepared according to the following protocol. 30 g (37.9 mmol)
of 2'-deoxycytidine hydrochloride were silylated with
trimethylchlorosilane (75 ml) in pyridine (160 ml), followed by
treatment with fluorenylmethoxycarbonyl chloride (33 g, 128 mmol).
After hydrolysis with H.sub.2O (300 ml) and extraction with
AcOEt/H.sub.2O, the N.sup.4--FMOC-2'-deoxycytidine was obtained in
a 97% yield (49.5 g) as a colorless powder.
[0054] For the dimethoxytritylation of the 5'-hydroxy function,
40.8 g (120 mmol) of DMTR-chloride in pyridine (100 ml) and
anhydrous methylene chloride (200 ml) was used. The mixture was
stirred for 30 min at room temperature to yield 74.6 g (90%) of
5'-O-DMTR-N.sup.4--FMOC-2'-deoxycytidine as a white powder. Overall
yield was 87% based on 2'-deoxycytidine hydrochloride. The final
product, as well as all the intermediates, was fully characterized
by HNMR, PNMR, MS and elemental analysis.
B. Preparation of
5'-O-(dimethoxytrityl)-N.sup.6-(9-fluorenylmethoxycarbonyl)-2'-deoxyadeno-
sine and
5'-O-dimethoxytrityl)-N.sup.2-(9-fluorenylmethoxycarbonyl)-2'-deo-
xyguanosine
[0055] In one embodiment, FMOC was used in the synthesis of
5'-O-(dimethoxytrityl)-N.sup.6-(9-fluorenylmethoxy-carbonyl)-2'-deoxyaden-
osine and
5'-O-dimethoxytrityl)-N.sup.2-(9-fluorenylmethoxycarbonyl)-2'-de-
oxyguanosine, and was accompanied by the formation of considerable
amounts of 5'-O-DMTR-N.sup.6-bis-FMOC-2'-da and
5'-ODMTR-N-2-bis-FMOC-2'-dG (respectively 5-7%). All reactions were
carried in one pot and gave excellent yields (e.g. greater than
75%).
C. Preparation of 3'-(2-cyanoethyl)
N,N-diisopropylphosphoramidite)
[0056] For the preparation of 3'-(2-cyanoethyl)
N,N-diisopropylphosphoramidite) of the protected FMOC nucleosides,
diisopropylammonium tetrazolide and tetrazole were used as
activating reagents during the reaction. The resulting residue was
purified by flash silica gel chromatography. All resulting products
were characterized by HNMR, PNMR, MS and elemental analysis.
D. FMOC Cleavage
[0057] For the cleavage of all FMOC protected
5'-O-DMTR-2'-deoxynucleoside-s, a solution of 0.05M DBU in pyridine
was used. The de-protection reaction of the three
mono-FMOC-protected compounds was stopped after five minutes
reaction time with 0.1 M CH.sub.3COOH in pyridine and the resulting
5'-O-DMTR-2'-deoxynucleosides were isolated in 90-98% yields. The
bis-FMOC-protected compounds of the 2'-deoxyguanosine and
2'-deoxyadenosine derivatives were stirred for additional five
minutes with 0.05M DBU in pyridine to afford after neutralization
and isolation the corresponding 5'-DMTR-deoxynucleoside in 90%
yield. While it is not intended that the present invention be
limited by any specific mechanism, kinetic studies shown that the
fluorenylmethoxycarbonyl residue of the corresponding
5'-DMTR-2'-deoxynucleosides were substantially eliminated within
two min. using a 0.05M DBU/CH.sub.3CN or Pyridine (1:1 molar ratio
of 2'-deoxynucleoside/base).
E. FMOC Deprotection of 2'-dG
[0058] In one embodiment, the de-protection reaction of the 2'-dG
compound did not take place due to the insolubility of the starting
material. In this case, 0.05 M DBU/pyridine was used. All starting
2'-deoxynucleosides were dissolved within ten seconds if a higher
concentration of DBU was used (e.g. 0.5 M DBU/CH.sub.3CN and a 1.20
molar ratio of deoxynucleoside to DBU). Ten seconds was sufficient
to cleave of the FMOC group via elimination. On HPLC, only two
peaks were detected, one for the de-blocked product and the second
for the eliminated compound. In embodiments using piperidine, a
slow addition of piperidine to the dibenzofulvene was observed. See
FIG. 2.
II. Assembly of Oligonucleotides
[0059] The synthesis of oligonucleotides was carried out using the
Ecosyn D-300 or the Ecosyn D-200 DNA/RNA synthesizers (Eppendorf
Netheler Hinz GmbH, Hamburg, Germany).
[0060] A. Reagents
[0061] In some embodiments, DMTr-dA, dC, dG and T synthesis columns
with a loading of 0.2 .mu.M, DMTr-dA, dC, dG and
T-.beta.-cyanoethyl phosphoamidites, BDTD-sulfurization reagent,
and all reagents for DNA synthesis and modifications on the 5'-end
were purchased from Eppendorf North America (Madison, Wis., USA).
dA, dC, dG and T synthesis columns containing
nucleoside-derivatized synthesis support (500-1000 A pore size) and
a loading of 20-30 .mu.mole/g corresponding to 0.2 .mu.mole
nucleoside/column were purchased from Eppendorf North America
(Madison, Wis., USA). .beta.-cyanoethyl phosphoramidites (dA, dC,
dG, T) were products of Eppendorf North America in prepacked
bottles and anhydrous acetonitrile is added before use to yield 0.1
M solutions. Before the BDTD bottle (Eppendorf North America) was
attached to the port of the synthesizer, anhydrous acetonitrile is
added via a dry syringe to yield a 0.1M solution. Activator
solution (97% acetonitrile, 3% tetrazole), CAP A (80%
tetrahydrofuran, 10% acetic anhydride and 10% pyridine) and CAP B
(90% tetrahydrofuran, 10% 1-methyl-limidazole) solutions, 3%
trichloroacetic acid in dichloromethane and oxidation reagent (80%
tetrahydrofuran, 2% iodine, 5% water, 13% pyridine) were received
in prepacked bottles and used as delivered from Eppendorf North
America.
[0062] B. Automatic Oligodeoxynucleotide Synthesis
[0063] In some embodiments, oligonucleotides were synthesized
according to the following cycle (set out in Tables 1 and 2) with
repeating subroutines, whereby argon and reagent pass the column
from top or bottom connection with the optosensor on or off.
TABLE-US-00001 TABLE 1 Column washing subroutine Time Step [sec]
Source Destination Optosensor 1 1 ACN Waste OFF 2 1 GAS Waste OFF 3
0 ACN CB ON 4 2 ACN CB OFF 5 4 GAS CB OFF 6 0 ACN CB ON 7 0.5 GAS
CT OFF 8 0.5 GAS CB OFF 9 4 GAS CT OFF 10 0 ACN CB ON 11 3 GAS CB
OFF 12 4 GAS CT OFF CB: column bottom, CT: column top, ACN:
acetonitrile
[0064] TABLE-US-00002 TABLE 2 Coupling subroutine Desti- Opto- Time
Step[sec] Source nation Mixed sensor Delay[sec] 1 4 GAS CT OFF 2
0.2 TET Waste OFF 3 0 TET CB NUC ON 10 4 1 NUC CB TET OFF 10 5 0.1
GAS CB OFF 10 6 0.1 GAS CB OFF 10 7 5 GAS CB OFF 8 1 GAS Waste OFF
TET: tetrazole; NUC: phosphoramidite; CB: column bottom; CT: column
top
[0065] In some embodiment, for the synthesis of thiooligos, the
subroutine sequence was modified in comparison to that used for the
chain elongation of unmodified oligos as follows: Deprotection,
washing, coupling, sulfurization, capping.
[0066] Irrespective of the routine selected for oligonucleotide
synthesis, the end procedure was as follows: (a) Cleavage of the
FMOC-protection groups with 0.1 M DBU in CH.sub.3CN for 5-10 min.
(b) Cleavage of the de-blocked oligonucleotide (if required) from
the polymer support with concentrated NH.sub.3 solution for 30 min
at room temperature. The quality of the synthesized
oligonucleotides was evaluated by PAGE, HPLC, CE and the biological
activity may be proven by the synthesis a gene (or portion
thereof).
[0067] It should also be noted that the polymer supports are
important in the methods, described by the present invention, for
the direct immobilization of oligonucleotides. Moreover, the
present invention incorporates an improved process for the
derivatization of different types of silica gel and controlled pore
glass. Specifically, this improved process both keeps the silica
particles intact and also reduces the consumption of reagents.
III. Functionalization of Polymer Supports on Gas Phase: Silica
Gels and Controlled Pore Glasses
[0068] A. Surface Activation
[0069] 3 grams of silica gel, controlled pore glass (native), or
polystyrene based supports were suspended in concentrated
hydrochloric acid solution (30 ml. 25.degree. C., 30 min). The
activated material was recovered by filtration and washed with
distilled water (200 ml), methanol (40 ml) and dried in vacuo.
[0070] B. Gas-Phase Aminopropylsilylation
[0071] Activated silica gel or controlled pore glass (2 g) was
placed in a 20.times.150 mm pyrex tube containing 3-aminopropyl
triethoxysilane (3 ml). A hose was fitted onto the end of the tube
and the vessel and contents were sealed under vacuum using a flame.
The tube was incubated behind a safety shield (140.degree. C., 12
h) and the reaction was terminated by returning to ambient
temperature. Silica or controlled pore glass was recovered by
cautiously cracking open the vessel. Each derivative was washed
with distilled water (200 ml), ethanol (40 ml) and ether (20 ml)
and dried in vacuo.
[0072] C. Gas-Phase Succinylation
[0073] Aminopropylsilylated silica gel or controlled pore glass (2
g) was sealed in vacuo in a pyrex tube containing succinic
anhydride (200 mg). A hose was fitted onto the end of the tube and
the vessel and contents were sealed under vacuum using a flame. The
reaction was incubated overnight (140.degree. C., 16 h) and
terminated by returning to ambient temperature and cracking open
the vessel. The material was washed and dried as described
above.
[0074] D. Conventional Aminopropylsilylation and Succinilation
[0075] Silica gel or controlled pore glass starting material was
aminopropylsilylated, in refluxing toluene and succinilated in
water, according to the following protocol (as set out in the
schematic overview projected in FIG. 5).
[0076] Silica gel was surface activated by refluxing with HCl,
thereby providing the active SiOH groups on the surface of the
silica gel. This activated gel (see, S-1 in FIG. 4) is derivatized
by refluxing with .gamma.-aminopropyltriethoxysilane in toluene to
provide the functionalized resin (see, S-2 in FIG. 4). The amino
group may be derivatized in a number of ways, including a standard
procedure with succinic anhydride to give the activated resin (see,
S-3 in FIG. 4). The final step in preparing the resin for use in
the automated procedure requires condensing a protected nucleoside
to S-3, with dicyclohexylcarbodiimide (DCC) used as the condensing
agent in pyridine. When diethylaminopyridine is used as catalyst,
the resulting resin (see, S-4 in FIG. 4) contains approximately 0.1
millimole of nucleoside per gram of resin. On removal of the
dimethoxytrityl group (DMT) from the nucleoside with mild acid, the
resin S-5 that is obtained is ready for chain extension. 10
milligrams of the resin (see, S-5 in FIG. 4) was packed into a
synthesis column which snaps into the synthesizer.
[0077] Once the resin (see, S-5 in FIG. 4) has been attached to the
instrument, the chain may be synthesized.
IV. Methods Designed to Capture Nucleic Acid Sequences
[0078] In selected embodiments, the present invention contemplates
a single stranded oligonucleotide bound to a polymer support. Such
oligonucleotide is complementary to a specific sequence on a target
nucleic acid that (in a preferred embodiment) is longer than the
oligonucleotide immobilized on the solid support. This design
allows for the selective hybridization of the target nucleic acid
to the oligonucleotide on the support.
[0079] The present methods of directly immobilizing
oligonucleotides, of a specific sequence, are particularly useful
for the isolation of a single stranded nucleic acid sequence or to
detect the presence of a particular nucleic acid present in a
relatively low concentration. The use of the FMOC-protected
phosphoramidites allows the direct synthesis of any oligonucleotide
and, after de-protection under organic conditions, to keep the
oligonucleotide attached to the polymer support (controlled pore
glass [CPG], polystyrene, tentagel, silica gel, membranes, etc.)
Given the highly labile character of FMOC, the final de-protection
may be carried out under organic conditions (e.g. 10% piperidine in
dichloromethane or dimethyl formamide or 1.0 M DBU in
acetonitrile). The derivatization of the polymer support for the
direct immobilization of oligonucleotides was completed via LCMA
according to the following protocol.
A. Derivatives of LCAMA-CPG and of
5'-O-(2-Dansylethoxycarbonyl)-2'--deoxy-3'
O-succinylnucleosides
[0080] To a mixture of 200 mg of LCAMA-CPG [e.g.
(long-chain-alkyl)methylamine controlled-pore glass)], 45 .mu.mol
of Thy (29 mg), Cyt.sup.npeoc (37 mg), Ade.sup.npeoc (38 mg), or
Guan.sup.Pe/.sub.npeoc (46 mg), and 30 mg (91.4 .mu.mol) of TOTU, 3
ml of abd. MeCN and 19 .mu.l (180 .mu.mol) of N-methylmorpholine
were added. After a short ultrasonic treatment (30 s) and 2 h
standing in the dark at r.t., the CPG material was collected in a
glass suction filter and washed with MeOH, DMF, MeOH, and
Et.sub.2O.
V. Preparation of Immobilized Oligonucleotides to Capture RNA
[0081] The m-RNA of higher cells is usually polyadenylated to a
various degree at its 3' end (known as the Poly A-tail or track).
The present invention contemplates isolating m-RNA by hybridization
(binding/basepairing) to the immobilized oligonucleotides
synthesized as described above. In this manner, m-RNA (PolyA+ RNA)
hybridizes with its complementary sequence or with any specific
sequence linked to a solid support (such as silica gel). When using
an oligonucleotide attached to a polymer support, the target m-RNA
becomes immobilized to the support and can then be separated by
removing the liquid phase containing all the non bound nucleic
acids from the hybridization mixture. After all of the remaining
liquid has been removed, the polyA+ RNA or any specific RNA can be
eluted from the oligo-dT20/30 or specific m-RNA probe support. This
allows the physical separation of polyA+ RNa from
non-polyadelynated, non-binding RNA.
VI. Oligonucleotide Synthesis, Purification and Phosphorylation
[0082] Oligonucleotides were synthesized using the FMOC derivative
cyanoethyl phosphoramidites as described above. For all the
oligonucleotides the average stepwise yield based on tritanol
release was higher than 99.4%. The de-protection was performed with
a solution of 0.1 M DBU in acetonitrile. Cleavage of the
oligonucleotide was performed in the presence of ammonium hydroxide
for 30 min. at room temperature. Synthesis was performed in a 150
nmol scale on CPG-1000.
[0083] The crude oligonucleotides were purified by polyacrylamide
gel electrophoresis. The oligonucleotides, ranging in length from 9
to 84 nt and sharing 9 to 15 complementary overlapping bases,
contained several repeat sequences such as CGGC (6 times), GCGGC (3
times), TCTGCGGCG (2 times) and GCGCCCCGC (2 times).
Oligonucleotides phosphorylation was effected using bacteriophage
T4 polynucleotide kinase (Gibco, Gaithersburg, Md., USA). Annealing
and ligation was carried out with Ampligase TM DNA ligase
(Epicentre Technologies, Inc. Madison, Wis., USA).
[0084] Cloning of the ligated sequences: For direct cloning, the
assembled blocks from the previous reaction were ligated to SmaI
and SstI digested vector pGEM4Z using T4 DNA ligase at 16.degree.
C. overnight.
VII. Antisense Applications
[0085] In some embodiments, the present invention contemplates the
targeting of therapeutic gene products [including, but not limited
to, Human Insulin-like Growth Factor I (IGF-I)]. For example, in
some embodiments, the present invention contemplates compositions
comprising oligomeric antisense compounds, particularly
oligonucleotides (for example, those identified in the drug
screens), for use in modulating the function of nucleic acid
molecules encoding IGF-I, ultimately modulating the amount of IGF-I
expressed. This is accomplished by providing antisense compounds
that specifically hybridize with one or more nucleic acids encoding
IGF-I.
[0086] The specific hybridization of an oligomeric compound with
its target nucleic acid interferes with the normal function of the
nucleic acid. This modulation of function of a target nucleic acid
by compounds that specifically hybridize to it is generally
referred to as "antisense." The functions of DNA to be interfered
with include replication and transcription. The functions of RNA to
be interfered with include all vital functions such as, for
example, translocation of the RNA to the site of protein
translation, translation of protein from the RNA, splicing of the
RNA to yield one or more mRNA species, and catalytic activity that
may be engaged in or facilitated by the RNA. The overall effect of
such interference with target nucleic acid function is modulation
of the expression of IGF-1.
[0087] In the context of the present invention, "modulation" means
either an increase (stimulation) or a decrease (inhibition) in the
expression of a gene. It is preferred to target specific nucleic
acids for antisense. "Targeting" an antisense compound to a
particular nucleic acid, in the context of the present invention,
is a multistep process. The process usually begins with the
identification of a nucleic acid sequence whose function is to be
modulated. This may be, for example, a cellular gene (or mRNA
transcribed from the gene) whose expression is associated with a
particular disorder or disease state, or a nucleic acid molecule
from an infectious agent. The targeting process also includes
determination of a site or sites within this gene for the antisense
interaction to occur such that the desired effect, e.g., detection
or modulation of expression of the protein, will result.
[0088] Within the context of the present invention, a preferred
intragenic site is the region encompassing the translation
initiation or termination codon of the open reading frame (ORF) of
the gene. Since the translation initiation codon is typically
5'-AUG (in transcribed mRNA molecules; 5'-ATG in the corresponding
DNA molecule), the translation initiation codon is also referred to
as the "AUG codon," the "start codon" or the "AUG start codon." A
minority of genes have a translation initiation codon having the
RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and
5'-CUG have been shown to function in vivo. Thus, the terms
"translation initiation codon" and "start codon" can encompass many
codon sequences, even though the initiator amino acid in each
instance is typically methionine (in eukaryotes) or
formylmethionine (in prokaryotes). Eukaryotic and prokaryotic genes
may have two or more alternative start codons, any one of which may
be preferentially utilized for translation initiation in a
particular cell type or tissue, or under a particular set of
conditions. In the context of the present invention, "start codon"
and "translation initiation codon" refer to the codon or codons
that are used in vivo to initiate translation of an mRNA molecule
transcribed from a gene encoding a tumor antigen of the present
invention, regardless of the sequence(s) of such codons.
[0089] Translation termination codon (or "stop codon") of a gene
may have one of three sequences (i.e., 5'-UAA, 5'-UAG and 5'-UGA;
the corresponding DNA sequences are 5'-TAA, 5'-TAG and 5'-TGA,
respectively). The terms "start codon region" and "translation
initiation codon region" refer to a portion of such an mRNA or gene
that encompasses from about 25 to about 50 contiguous nucleotides
in either direction (i.e., 5' or 3') from a translation initiation
codon. Similarly, the terms "stop codon region" and "translation
termination codon region" refer to a portion of such an mRNA or
gene that encompasses from about 25 to about 50 contiguous
nucleotides in either direction (i.e., 5' or 3') from a translation
termination codon.
[0090] The open reading frame (ORF) or "coding region," which
refers to the region between the translation initiation codon and
the translation termination codon, is also a region that may be
targeted effectively. Other target regions include the 5'
untranslated region (5' UTR), referring to the portion of an mRNA
in the 5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA or corresponding nucleotides on the
gene, and the 3' untranslated region (3' UTR), referring to the
portion of an mRNA in the 3' direction from the translation
termination codon, and thus including nucleotides between the
translation termination codon and 3' end of an mRNA or
corresponding nucleotides on the gene. The 5' cap of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap. The
cap region may also be a preferred target region.
[0091] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
that are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence. mRNA
splice sites (i.e., intron-exon junctions) may also be preferred
target regions, and are particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular mRNA splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also preferred targets. It has also been found that
introns can also be effective, and therefore preferred, target
regions for antisense compounds targeted, for example, to DNA or
pre-mRNA.
[0092] Once one or more target sites have been identified,
oligonucleotides are chosen that are sufficiently complementary to
the target (i.e., hybridize sufficiently well and with sufficient
specificity) to give the desired effect. For example, in preferred
embodiments of the present invention, antisense oligonucleotides
are targeted to or near the start codon.
[0093] In the context of this invention, "hybridization," with
respect to antisense compositions and methods, means hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleoside or nucleotide
bases. For example, adenine and thymine are complementary
nucleobases that pair through the formation of hydrogen bonds. It
is understood that the sequence of an antisense compound need not
be 100% complementary to that of its target nucleic acid to be
specifically hybridizable. An antisense compound is specifically
hybridizable when binding of the compound to the target DNA or RNA
molecule interferes with the normal function of the target DNA or
RNA to cause a loss of utility, and there is a sufficient degree of
complementarity to avoid non-specific binding of the antisense
compound to non-target sequences under conditions in which specific
binding is desired (i.e., under physiological conditions in the
case of in vivo assays or therapeutic treatment, and in the case of
in vitro assays, under conditions in which the assays are
performed).
[0094] Antisense compounds are commonly used as research reagents
and diagnostics. For example, antisense oligonucleotides, which are
able to inhibit gene expression with specificity, can be used to
elucidate the function of particular genes. Antisense compounds are
also used, for example, to distinguish between functions of various
members of a biological pathway.
[0095] The specificity and sensitivity of antisense is also applied
for therapeutic uses. For example, antisense oligonucleotides have
been employed as therapeutic moieties in the treatment of disease
states in animals and man. Antisense oligonucleotides have been
safely and effectively administered to humans and numerous clinical
trials are presently underway. It is thus established that
oligonucleotides are useful therapeutic modalities that can be
configured to be useful in treatment regimes for treatment of
cells, tissues, and animals, especially humans.
[0096] While antisense oligonucleotides are a preferred form of
antisense compound, the present invention comprehends other
oligomeric antisense compounds, including but not limited to
oligonucleotide mimetics.
[0097] Specific examples of preferred antisense compounds useful
with the present invention include oligonucleotides containing
modified backbones or non-natural internucleoside linkages. As
defined in this specification, oligonucleotides having modified
backbones include those that retain a phosphorus atom in the
backbone and those that do not have a phosphorus atom in the
backbone. For the purposes of this specification, modified
oligonucleotides that do not have a phosphorus atom in their
internucleoside backbone can also be considered to be
oligonucleosides.
[0098] The present invention contemplates that modified
oligonucleotide backbones include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates and chiral-phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked
analogues of these, and those having inverted polarity wherein the
adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or
2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are
also included.
[0099] It is also contemplated that modified oligonucleotide
backbones (that do not include a phosphorus atom therein have
backbones that are formed by short chain alkyl or cycloalkyl
internucleoside linkages) mixed heteroatom and alkyl or cycloalkyl
internucleoside linkages, or one or more short chain heteroatomic
or heterocyclic internucleoside linkages. These include those
having morpholino linkages (formed in part from the sugar portion
of a nucleoside); siloxane backbones; sulfide, sulfoxide and
sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH.sub.2
component parts.
[0100] Specifically, the present invention contemplates the use of
FMOC-protected phosphoramidites for the synthesis of antisense
compounds corresponding to the "RO--P--O" backbone of DNA wherein
(in preferred embodiments R is selected from the group consisting
of methyl, ethyl, akly, and aryl group. While it is not intended
the present invention be limited by any specific mechanism, the
FMOC derivatives described in the present invention facilitate
deprotection under organic conditions.
[0101] That is to say, standard protocols for deprotection, i.e.
using ammonium hydroxide, are characterized by cleavage followed by
deprotection. Specifically, the deprotection of the protecting
groups on the phosphorous and the exocyclic amine (using these
standard protocols) occur at the same time. That is to say, using
the traditional "Acyl Strategy" there is no deprotection without
cleavage. See, for example, the "Acyl Strategy" schematic presented
in FIG. 3. In contrast, using the FMOC protected phosphoramidites
described in the present invention, the deprotection selectively
occurs first on the exocyclic amino groups; leaving intact the
protection on the phosphorous. It should be noted, however, the use
of DBU in CH.sub.3CN cleaves both FMOC and the cyanoethyl group
from the phosphate (of the phosphoramidite). That is to say, in
embodiments using DBU, an active oligonucleotide is produced when
the FMOC and the cyanoethyl group are cleaved, by DBU, from the
phosphate of the phosphoramidite. See, for example, the "FMOC
Strategy" presented in FIG. 4.
[0102] In some embodiments it may be desirable to remove (using
piperidine instead of DBU) only the FMOC group from the exocylic
amine of the phosphoramidite, thereby, leaving the alkyl residue on
the phosphate of the phosphoramidite such that the phosphate is
protected by the cyanoethyl group in this particular embodiment.
These embodiments are especially suited to evaluating the antisense
properties of a given oligonucleotide such that the phosphate
groups of said oligonucleotide are protected, only, with said
cyanoethyl group or any other alkyl residue.
[0103] One skilled in the relevant art knows well how to generate
oligonucleotides containing the above-described modifications. The
present invention is not limited to the antisense oligonucleotides
described above. Any suitable modification or substitution may be
utilized.
[0104] Furthermore, it is not necessary for all positions in a
given compound to be uniformly modified, and in fact more than one
of the aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also contemplates antisense compounds that
are chimeric compounds. "Chimeric" antisense compounds or
"chimeras," in the context of the present invention, are antisense
compounds, particularly oligonucleotides, which contain two or more
chemically distinct regions, each made up of at least one monomer
unit, i.e., a nucleotide in the case of an oligonucleotide
compound. These oligonucleotides typically contain at least one
region wherein the oligonucleotide is modified so as to confer upon
the oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids.
[0105] By way of example, RNaseH is a cellular endonuclease that
cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,
therefore, results in cleavage of the RNA target, thereby greatly
enhancing the efficiency of oligonucleotide inhibition of gene
expression. Consequently, comparable results could be obtained with
shorter oligonucleotides when chimeric oligonucleotides are used,
compared to phosphorothioate deoxyoligonucleotides hybridizing to
the same target region. Cleavage of the RNA target can be routinely
detected by gel electrophoresis and, if necessary, associated
nucleic acid hybridization techniques known in the art.
[0106] The chimeric antisense compounds contemplated by the present
invention may be formed as composite structures of two or more
oligonucleotides, modified oligonucleotides, oligonucleosides
and/or oligonucleotide mimetics. The present invention also
contemplates pharmaceutical compositions and formulations
comprising the antisense compounds of the present invention.
VIII. High Throughput DNA Synthesis
[0107] The present invention contemplates the use of FMOC
derivatives in the high throughput synthesis of DNA. It is not
intended that the present invention be limited to any specific
format for high throughput synthesis. In one embodiment, a standard
96-well plate is contemplated as the solid support to which
oligonucleotides are synthesized using the FMOC protocols
elaborated above. The adaptation of these FMOC protocols to
oligonucleotide synthesis in a 96 well plate offers advantages over
standard conditions which rely on the use of ammonium hydroxide
concentrated at 55.degree. C. for 16 h and the use of methyl amine:
ammonium hydroxide (9:1 v/v) at 95.degree. C. for one hour.
[0108] The use, in these standard methods, of ammonium hydroxide
and a mixture of methyl amine and ammonium hydroxide (both highly
volatile reagents) promotes leakage between reaction well and is
therefore problematic (especially in 96-well format). Specifically,
this leakage problem can cause the incomplete deprotected of
oligonucleotides. The FMOC chemistry described in the instant
application, in contrast, facilitates the complete deprotection of
oligonucleotides at room temperature (with an organic reagent: 0.1
M DBU/Acetonitrile) substantially without leakage.
[0109] Moreover, this FMOC synthetic motif allows for the
production of multifuntional columns. That is to say, if
oligonucleotides are immobilized on polymer resins suitable for
incorporation into flow through columns these immobilized
oligonucleotides (synthesized using the FMOC chemistries described
in the instant application) may be washed, and thereby purified,
while still attached to the resins comprising the column. In this
way a multifuctional column is created such that the same column
may be used for the synthesis and purification of a given
oligonucleotide. In one embodiment of the present invention these
substantially purified, but immobilized, oligonucleotides are
cleaved from their solid support by eluting the column with
ammonium hydroxide.
[0110] In other embodiments, FMOC protected phosphoramidites
eliminate the erosion, once again caused by the use of ammonium
hydroxide in the final deprotection, observed in traditional
microbiochip manufacturing wherein oligonucleotides are immobilized
on a silicon "chip".
IX. Preparation Of Labeled Nucleic Acids
[0111] FMOC phosphoramidites may be incorporated with various
labels, thereby, creating a "reporter molecule". These labels
include, but are not limited to: .sup.32P, .sup.33P, .sup.35S,
enzymes, or fluorescent molecules (e.g., fluorescent dyes). Once
again, the incorporation of detectable labels with FMOC
phosphoramidites (described in the instant application) confers
decided advantages over traditional labeling techniques.
Specifically, theses traditional labeling techniques use sodium
hydroxide as a methanolic solution for deprotection in a reaction
that occurs at room temperature for 18 h. Under these relatively
"harsh" conditions, the cleavage of the label, from the
oligonucleotide, is impossible to avoid. These traditional labeling
techniques typically provide recovery of labeled product in the
range of 20-30%. Using the relatively "milder" conditions
characterized by the chemistries of the present invention, almost
100% of the synthesized labeled oligonucleotide may be
recovered.
[0112] In other embodiments, it is contemplated that FMOC
phosphoramidites may be incorporated into fluorescence in situ
hybridization (e.g. "FISH"), a technique in which detectably
labeled DNA probes (which can be prepared, for example, from cDNA
sequences or genomic sequences contained in cosmids or bacterial
artificial chromosomes (BACs)) are hybridized to cytogenetic or
histological specimens. Such specimens include, but are not limited
to, metaphase chromosome spreads and interphase nuclei prepared
from tissue or blood specimens, and formaldehyde-fixed,
paraffin-embedded tissue sections. Fluorescent labels can be
directly incorporated into the probe, or can be applied as
antibody-label conjugates which bind to affinity labels (for
example, biotin or digoxigenin) incorporated into the probe, either
directly, or as an antibody "sandwich" (i.e. a primary and a
secondary antibody). The fluorescent dyes include, but are not
limited to rhodamine, texas red, FITC (fluorescein isothiocyanate)
and TRITC (tetramethyl rhodamine isothiocyanate). The fluorescent
labels are detected using a fluorescence microscope equipped with a
mercury or xenon lamp (as an illumination source) and appropriate
filters for excitation and emission. The pattern of fluorescence
can be used to assess copy number of the locus recognized by the
probe, or, in cases where two or more (differentially) labeled
probes are used, to assess the relative positions of the probes
(for example to detect chromosomal rearrangements, such as
translocations and inversions).
[0113] From the above, it should be evident that the present
invention provides compositions and methods that allow for
efficient (i.e., high yield) synthesis of DNA/RNA using FMOC
derivatives. The present invention provides compositions and
methods which provide high quality, high volume preparations of
DNA/RNA. This recovered DNA/RNA, in turn, can be used in a variety
of diagnostic and therapeutic methods.
Sequence CWU 1
1
4 1 24 DNA Artificial Sequence Synthetic 1 cggccggccg gccggccggc
cggc 24 2 15 DNA Artificial Sequence Synthetic 2 gcggcgcggc gcggc
15 3 18 DNA Artificial Sequence Synthetic 3 tctgcggcgt ctgcggcg 18
4 18 DNA Artificial Sequence Synthetic 4 gcgccccgcg cgccccgc 18
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