Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-03-13
2001-07-31
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091100, C435S091200, C536S022100, C536S023100, C536S024300, C536S024310, C536S024320, C536S024330, C536S024500, C536S025300, C536S025310, C536S025320
Reexamination Certificate
active
06268132
ABSTRACT:
BACKGROUND OF THE INVENTION
A. Field of the Invention
The present invention relates to fluorescent structural analogs of the non-fluorescent nucleosides commonly found in DNA and RNA, methods of their derivatization and subsequent use in the synthesis of fluorescent oligonucleotides, and to their new and useful applications both as fluorescent monomers and in fluorescent oligonucleotides having prescribed sequences. Additionally, it relates to applications in which fluorescent structural analogs are substituted for specific non-fluorescent nucleosides in prescribed DNA or RNA sequences and to methods of using fluorescent oligonucleotides as hybridization reagents and probes for diagnostic and therapeutic purposes and as diagnostic and therapeutic research tools.
B. General Description of the Art
The six commonly occurring N-nucleosides which predominate in the composition of DNA and RNA from all sources have the structures shown in
FIG. 1
wherein R
6
is H for inosine and NH
2
for guanosine, R
9
is H for uridine and CH
3
for thymidine. Furthermore, R
12
, R
14
═OH for ribonucleotides, R
12
═OH, R
14
═H for 2′-deoxy nucleotides, R
12
═H, R
14
═OH for 3′-deoxy nucleotides, and R
12
, R
14
═H in dideoxy nucleotides.
The six commonly occurring nucleotides do not absorb light at wavelengths >290 nm and are effectively non-fluorescent under physiological conditions. Derivatives of the commonly occurring N-nucleotides for a variety of synthetic, diagnostic, and therapeutic purposes are common, including substitutions on both the heterocyclic base and the furanose ring. These substitutions can be made at the loci shown in
FIG. 2
in which R
4
is a reactive group derivatizible with a detectable label (NH
2
, SH,═O, and which can include an optional linking moiety including, but not limited to, an amide, thioether, or disulfide linkage or a combination thereof with additional variable reactive groups, R
1
through R
3
, e.g., R
1
—(CH
2
)
x
—R
2
, or R
1
—R
2
—(CH
2
)
x
—R
3
—, where x is an integer in the range of 1 and 25 inclusive; and R
1
, R
2
, and R
3
can be H, OH, alkyl, acyl, amide, thioether, or disulfide); R
5
is H or part of an etheno linkage with R
4
; R
6
is H, NH
2
, SH, or ═O; R
9
is hydrogen, methyl, bromine, fluorine, or iodine, or an alkyl or aromatic substituent, or an optional linking moiety including an amide, thioether, or disulfide linkage or a combination thereof such as R
1
—(CH
2
)
x
—R
2
, or R
1
—R
2
—(CH
2
)
3
—R
3
—, where x is an integer in the range of 1 and 25 inclusive; R
10
is hydrogen, or an acid-sensitive base stable blocking group, or a phosphorous derivative, R
11
═R
12
═H; R
12
is hydrogen, OH, or a phosphorous derivative; R
14
is H, OH, or OR
3
where R
3
is a protecting group or additional fluorophore. The letters N and C in the N-nucleosides and C-nucleosides designate the atom at which the glycosidic covalent bond connects the sugar and the heterocyclic base. In the cases of the commonly occurring nucleosides, the bases are either adenine, guanine, cytosine, inosine, uracil, or thymine. The bases are attached to a furanose sugar, a general structure of which is shown in FIG.
3
. The sugar substituents for the fluorescent analogs share the same numbering system for all R groups, but the numbering system for some of the heterocycle analogs may differ.
I. Known Methods of Labeling Nucleotides
Nucleotide sequences are commonly utilized in a variety of applications including diagnostic and therapeutic probes which hybridize target DNA and RNA and amplification of target sequences. It is often necessary, or useful, to label nucleotide sequences.
A. Labeling of oligonucleotide probes with radioisotopes. Hybridization of specific DNA or RNA sequences typically involves annealing oligonucleotides of lengths which range from as little as 5 bases to more than 10,000 bases (10 kb). The majority of oligonucleotide probes currently in research use are radioactively labeled; however, because of (a) the short half lives of the isotopes in common usage, (b) the safety requirements, and (c) the costs of handling and disposal of radioactive probes, convenient and sensitive non-isotopic methods of detection are required for hybridization diagnostic methods to achieve widespread acceptance and application.
B. Non-isotopic methods of labeling oligonucleotide probes. In general, all of the non-isotopic methods of detecting hybridization probes that are currently available depend on some type of derivatization of the nucleotides to allow for detection, whether through antibody binding, or enzymatic processing, or through the fluorescence or chemiluminescence of an attached “reporter” molecule. In most cases, oligonucleotides have been derivatized to incorporate single or multiple molecules of the same reporter group, generally at specific cyclic or exocyclic positions. Techniques for attaching reporter groups have largely relied upon (a) functionalization of 5′ or 3′ termini of either the monomeric nucleosides or the oligonucleotide strands by numerous chemical reactions using deprotected oligonucleotides in aqueous or largely aqueous media (see Cardullo et al. [1988
] PNAS
85:8790-8794); (b) synthesizing modified nucleosides containing (i) protected reactive groups, such as NH
2
, SH, CHO, or COOH, (ii) activatable monofunctional linkers, such as NHS esters, aldehydes, or hydrazides, or (iii) affinity binding groups, such as biotin, attached to either the heterocyclic base or the furanose moiety. Modifications have been made on intact oligonucleotides or to monomeric nucleosides which have subsequently been incorporated into oligonucleotides during chemical synthesis via terminal transferase or “nick translation” (see, e.g., Brumbaugh et al. [1988
] PNAS
85:5610-5614; Sproat, B. S., A. I. Lamond, B. Beijer,P. Neuner,P. Ryder [1989
] Nucl. Acids Res
. 17:3371-3386; Allen, D. J., P. L. Darke, S. J. Benkovic [1989
] Biochemistry
28:4601-4607); (c) use of suitably protected chemical moieties, which can be coupled at the 5′ terminus of protected oligonucleotides during chemical synthesis, e.g., 5′-aminohexyl-3′-O-phosphoramidite (Haralambidis, J., L. Duncan, G. W. Tregar [1990
] Nucl. Acids Res
. 18:493-499); and, (d) addition of functional groups on the sugar moiety or in the phosphodiester backbone of the polymer (see Conway, N. E., J. Fidanza, L. W. McLaughlin [1989
] Nucl. Acids Res. Symposium Series
21:43-44; Agrawal, S., P. C. Zamecnik [1990
] Nucl. Acids Res
. 18:5419-5423).
At the simplest, non-nucleoside linkers and labels have been attached to the 3′ or 5′ end of existing oligonucleotides by either enzymatic or chemical methods. Modification of nucleoside residues internal to the sequence of a DNA or RNA strand has proven to be a difficult procedure, since the reaction conditions must be mild enough to leave the RNA or DNA oligomers intact and still yield reaction products which can participate in normal Watson-Crick base pairing and stacking interactions (see FIG.
4
).
C. Derivatizations of the heterocyclic base (B). Numerous methods for both cyclic and exocyclic derivatization of the N-nucleoside base have been described, including the following:
(1) Hapten labeling. DNA probes have been amino modified and subsequently derivatized to carry a hapten such as 2,4-dinitrophenol (DNP) to which enzyme-conjugated anti-hapten antibodies bind which subsequently can be processed using a colorimetric substrate as a label (Keller et al. [1988
] Analytical Biochemistry
170:441-450).
(2) Amino- and thiol-derivatized oligonucleotides. Takeda and Ikeda ([1984
] Nucl. Acids Research Symposium Series
15:101-104) used phosphotriester derivatives of putresceinyl thymidine for the preparation of amino-derived oligomers. Ruth and colleagues have described methods for synthesizing a deoxyuridine analog with a primary amine “linker arm” 12 carbons in length at C
5
Chromagen, Inc.
Riley Jezia
Saliwanchik Lloyd & Saliwanchik
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