Incorporation of selectably cleavable sites into oligonucleotide and polynucleotide chains has been described in related U.S. Pat. Nos. 5,118,605 and 4,775,619, the disclosures of which are incorporated by reference herein. Selectably cleavable sites are useful in a number of different types of hybridization assay formats. For example, in one type of assay in which hybridization gives rise to a solid-supported duplex of a labeled probe and sample DNA, a selectably cleavable site contained within the hybrid structure will enable ready separation of the label from the solid support. U.S. Pat. No. 4,775,619 is primarily directed to the use of restriction endonuclease-cleavable sites in such assays, while U.S. Pat. No. 5,118,605 concerns chemically cleavable sites, e.g., disulfide linkages, 1,2-diols, and the like. These chemically clearable sites can be introduced during oligonucleotide synthesis, and are cleavable with particular chemical reagents, e.g., with thiols, periodate, or the like.
The present invention is also directed to selectably cleavable sites. However, the present method involves introduction of sites which are clearable by photolysis as well as sites which are cleavable by other means, e.g., using chemical or enzymatic reagents, e.g., reducing agents. The cleavable sites of the invention are created by incorporation of chemical moieties, preferably photolabile moieties, into oligonucleotide or polynucleotide chains. The novel photolabile moieties are useful in a number of different types of hybridization assay formats, including those described in the above-cited applications, as well as in the amplification nucleic acid hybridization assay described in co-pending, commonly assigned U.S. patent application Ser. No. 252,638, filed 30 Sep. 1988, abandoned in favor of continuation-in-part application Ser. No. 340,031, now U.S. Pat. No. 5,124,246, also incorporated by reference herein.
Another use of the reagents of the invention is, in general terms, the creation of abasic sites within oligonucleotides. By "abasic site" is meant an ether moiety --OR at a position which normally contains a hydroxyl group --OH or a nucleobase. The utility of such derivatization is extensive as will be disclosed in detail hereinbelow.
Still another use of the reagents of the invention is in chemical phosphorylation. In many different aspects of oligonucleotide chemistry, chemical phosphorylation of hydroxyl groups is necessary. For example, in oligonucleotide synthesis, after synthesis and deprotection, the free 5'-hydroxyl group of the oligonucleotide must be phosphorylated for use in most biological processes. Also, phosphorylation of the 3'-hydroxyl functionality is necessary: (1) to prevent extension of the 3' terminus by a polymerase; and (2) in the chemical ligation of DNA, i.e., a 3' phosphate moiety is typically required in the coupling of oligonucleotides using chemical means.
5'-phosphorylation has conventionally been carried out with T4 polynucleotide kinase and ATP, a reaction that is not particularly reliable or efficient. Several methods for chemical 5'-phosphorylation are also known, including those described by Nadeaux et al., Biochemistry 23:6153-6159 (1984), van der Marel et al., Tetrahedron Lett. 22:1463-1466 (1981), Himmelsbach and Pfleiderer, Tetrahedron Letters 23:4793-4796 (1982), Marugg et al., Nucleic Acids Research 12:8639-8651 (1984), and Kondo et al., Nucleic Acids Research Symposium Series 16:161-164 (1985). However, most of these methods involve the use of unstable reagents or require extensive modification of standard deprotection and purification procedures. Similar problems have been found with monofunctional and bifunctional 3'-phosphorylating reagents (see Sonveaux, supra, at 297).
Thus, in addition to utility in providing clearable and/or abasic sites within oligonucleotide or polynucleotide chains, many of the compounds of the present invention are additionally useful as phosphorylating reagents which overcome the limitations of current phosphorylation procedures (and may also be useful in phosphorylation reactions that are used conventional dimethoxytrityl [DMT] purification schemes).