An oligonucleotide is a short polymer consisting of a linear sequence of four nucleotides in a define order. The nucleotide subunits are joined by phosphodiester linkages joining the 3'-hydroxyl moiety of one nucleotided to the 5'-hydroxyl moiety of the next nucleotide. An example of an oligonucleotide is 5'&gt;ApCpGpTpApTpGpGpC&lt;3'. The letters A, C, G, and T refer to the nature of the purine or pyrimidine base coupled at the 1'-position of deoxyribose: A, adenine; C, cytosine; G, guanine; and T, thymine. "p" represents the phosphodiester bond. The chemical structure of a section of an oligonucleotide is shown in Structure 1. ##STR1##
Synthetic oligonucleotides are powerful tools in modern molecular biology and recombinant DNA work. There are numerous applications for these molecules, including a) as probes for the isolation of specific genes based on the protein sequence of the gene product, b) to direct the in vitro mutagenesis of a desired gene, c) as primers for DNA synthesis on a single-stranded template, d) as steps in the total synthesis of genes, and many more, reviewed in Wm. R. Bahl et al, Prog. Nucl. Acid Res. Mol. Biol. 21, 101, (1978).
A very considerable amount of effort has therefore been devoted to the development of efficient chemical methods for the synthesis of such oligonucleotides. A brief review of these methods as they have developed to the present is found in Crockett, G. C., Aldrichimica Acta 16(3), 47-55 (1983), and "Oligonucleotide Synthesis: A Practical Approach", ed. Gait, M.J., IRL Press, Oxford, England (1984). The best methodology currently available utilizes the phosphoramidite derivatives of the nucleosides in combination with a solid phase synthetic procedure, Matteucci, M.D. and Caruthers, M.H. J. Am. Chem. Soc. 103, 3185, (1981); and Beaucage, S.L., and Caruthers, M.H., Tet. Lett. 22(20), 1858-1862 (1981). In this chemistry, the 3'-nucleside of the sequence to be synthesized is attached to a solid support via base-labile linker arm. Subsequent nucleosides are attached sequentially to the previous nucleoside to generate a linear polymer of defined sequence extending off of the solid support. The general structure of a deoxyribonucleoside phosphoramidite is shown in Structure 2: ##STR2## and the chemical steps used in each cycle of oligonucleotide synthesis are shown in Structure 3: ##STR3## Oligonucleotides of length up to 40 bases may be made on a routine basis in this manner, and molecules as long as 106 bases have been made. Machines that employ this chemistry are now commercially available.
There are many reasons to want a method for covalently attaching other chemical species to synthetic oligonucleotides. Fluorescent dyes attached to the oligonucleotides permit one to eliminate radioisotopes from the research, diagnostic, and clinical procedures in which they are used, and improve shelf-life and availability. As described in the assignees co-pending application for a DNA sequencing machine (Ser. No. 570,973, filed Jan. 16, 1984, now abandoned) the synthesis of fluorescent-labeled oligonucleotides permits the automation of the DNA sequencing process. The development of appropriate techniques and instrumentation for the detection and use of fluorescent-labeled oligonucleotides allows the automation of other currently laborious laboratory and clinical techniques. The attachment of DNA cleavage chemicals such as those disclosed by Schultz et al, J. Am. Chem. Soc. 104, 6861 (1982); and Hertzberg, R.P., and Dervan, P.B., J. Am. Chem. Soc. 104, 313 (1982) permits the construction of synthetic restriction enzymes, whose specificity is directed by the oligonucleotide sequence.
There are several reports in the literature of the derivitization of DNA. A modified nucleoside triphosphate has been developed wherein a bictin group is conjugated to an aliphatic amino group at the 5-position of uracil, Langer et al., Proc. Nat. Acad. Sci. U.S.A. 78, 6633-6637 (1981). This nucleotide derivative is effectively incorporated into double stranded DNA in a process referred to as "nick translation." Once in DNA it may be bound by anti-biotin antibody which can then be used for detection by fluorescence or enzymatic methods. The DNA which has had biotin-conjugated nucleosides incorporated therein by the method of Langer et al is fragmented into smaller single and double stranded pieces which are heterogeneous with respect to the sequence of nucleoside subunits and variable in molecular weight. Draper and Gold, Biochemistry 19, 1774-1781 (1980), reported the introduction of aliphatic amino groups by a bisulfite catalyzed transamination reaction, and their subsequent reaction with a fluorescent tag. In Draper and Gold the amino group is attached directly to a pyrimidine base. The amino group so positioned inhibits hydrogen bonding and for this reason, these materials are not useful in hybridization and the like. Also, this method does not permit amino groups to be inserted selectively at a desired position. Chu et al, Nucleic Acids Res. 11(18), 6513-6529 (1983), have reported a method for attaching an amine to the terminal 5'-phosphate of oligonucleotides or nucleic acids. This method involves a number of sequential reaction and purification steps which are laborious to perform and difficult to scale up. It also is restricted to the introduction of a single amino group at the 5'-terminus of the oligonucleotide. Subsequent to the filing of the original patent application of which the present case is a Continuation-In-Part, Takeda and Ikeda, Nucl. Acids Res. Symp. Series 15, 101-104 (1984) have reported the synthesis and use of phosphotriester derivatives of putrescinyl thymidine for the preparation of amino-derivatized oligonucleotides. These materials differ from those reported herein in that the amino containing moiety is attached to the base moiety and not to the sugar moiety of the oligonucleotides, and also in that the DNA synthetic chemistry used was phosphotriester and not phosphoramidite.
The present invention presents a general method for the introduction of one or more free aliphatic amino groups into synthetic oligonucleotides. These groups may be selectively inserted at any desired position in the oligonucleotide. They are readily and specifically reacted with a variety of amino reactive functionalities, and thereby permit the covalent attachment of a wide variety of chemical species in a position specific manner. This is illustrated by the preparation of a number of fluorescent oligonucleotide derivatives. The materials prepared in this fashion are effective in DNA hybridization methods, as illustrated by their use as primers in DNA sequence analysis, and also by a study of their melting behaviour in DNA duplex formation.
According to the present invention, aliphatic amino groups are introduced into an oligonucleotide by first synthesizing a 3'-0-phosphoramidite derivative of a nucleoside analogue containing a protected aliphatic amino group attached to the sugar moiety of the nucleoside. This phosphoramidite is then reacted with the oligonucleotide being synthesized on a solid support. If the amino protecting group is base-labile, the process of oligonucleotide cleavage from the solid phase and deprotection of the base moieties and aliphatic amino group yields the amino-derivatized oligonucleotide. If the amino protecting group is acid-labile, it may be removed by treatment with anhydrous or aqueous acid prior to cleavage of the oligonucleotide from the support and deprotection of the base moieties, or it may be retained during cleavage and deprotection to simplify and improve the chromatographic purification of the oligonucleotide, and then removed subsequently by treatment with aqueous acid, yielding the amino-derivatized oligonucleotide in either case.
More specifically, the present invention concerns modified deoxynucleoside phosphoramidites in which an aliphatic amino group, which has been suitably protected, is attached to the sugar moiety of the nucleoside. The chemical structure of a typical nucleoside is shown in Structure 4. ##STR4## It is characterized by a heterocyclic pyrimidine or purine base (B) linked by a carbon-nitrogen bond to the furanose (sugar) ring of ribose (R=R'=R"=OH) or deoxyribose (R=R'=OH; R"=H). The numbering of the sugar carbon atoms is 1' to 5' as indicated in the figure; thus, the base is connected to C-1' of the sugar. An aliphatic amino group may be attached in principle to any of the five ring carbons. It also comprises the respective phosphoramidite derivatives which are synthesized by reacting an appropriate phosphine with the free 3'-hydroxyl group of the suitably protected amino nucleosides.
USPTO Backgrounds
USPTO Backgrounds