1. Field of the Invention
The invention relates to the chemical synthesis of oligonucleotides and to materials and processes that are useful in such synthesis.
2. Summary of the Related Art
Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive and efficient methods for synthesizing oligonucleotides.
The synthesis of oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs pp. 165-189 (S. Agrawal, Ed., Humana Press, 1993).; Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., 1991); and Uhlmann and Peyman, supra. Agrawal and Iyer, Curr. Op. in Biotech. 6: 12 (1995); and Anti-sense Research and Applications (Crooke and Lebleu; Eds., CRC Press, Boca Raton, 1993). Early synthetic approaches included phosphodiester and phosphotriester chemistries. Khorana et al., J. Molec. Biol. 72: 209 (1972) discloses phosphodiester chemistry for oligonucleotide synthesis. Reese, Tetrahedron Lett. 34: 3143-3179 (1978), discloses phosphotriester chemistry for synthesis of oligonucleotides and polynucleotides. These early approaches have largely given way to the more efficient phosphoramidite and H-phosphonate approaches to synthesis. Beaucage and Caruthers, Tetrahedron Lett. 22: 1859-1862 (1981), discloses the use of deoxynucleoside phosphoramidites in polynucleotide synthesis. Agrawal and Zamecnik, U.S. Pat. No. 5,149,798 (1992), discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
Both of these modern approaches have been used to synthesize oligonucleotides having a variety of modified internucleotide linkages. Agrawal and Goodchild, Tetrahedron Lett. 28: 3539-3542 (1987), teaches synthesis of oligonucleotide methylphosphonates using phosphoramidite chemistry. Connolly et al., Biochemistry 23: 3443 (1984), discloses synthesis of oligonucleotide phosphorothioates using phosphoramidite chemistry. Jager el al., Biochemistry 27: 7237 (1988), discloses synthesis of oligonucleotide phosphoramidates using phosphoramidite chemistry. Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988), discloses synthesis of oligonucleotide phosphoramidates and phosphorothioates using H-phosphonate chemistry.
Solid phase synthesis of oligonucleotides by any of the known approaches ordinarily involves the same generalized protocol. Briefly, this approach comprises anchoring the 3xe2x80x2-most nucleoside to a solid support functionalized with amino and/or hydroxyl moieties and subsequently adding the additional nucleosides in step-wise fashion. Desired internucleoside linkages are formed between the 3xe2x80x2 functional group (e.g., phosphoramidite group) of the incoming nucleoside and the 5xe2x80x2 hydroxyl group of the 5xe2x80x2-most nucleoside of the nascent, support-bound oligonucleotide.
Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 umol to 1 mmol and higher). See Padmapriya et al., Antisense Res. Dev. 4: 185 (1994). Several modifications of the standard phosphoramidite methods have already been reported to facilitate the synthesis and isolation of oligonucleotides. See e.g., Padmapriya et al., supra; Ravikumar et al., Tetrahedron 50: 9255 (1994); Theisen et al., Nucleosides and Nucleotides 12: 43 (1994); and Iyer et al., Nucleosides and Nucleotides 14: 1349 (1995) (Kuijpers et al., Nucl. Acids Res. 18: 5197 (1990); and Reddy et al., Tetrahedron Lett. 35: 4311 (1994).
One limitation in solid phase synthesis resides in the nature of the solid phase support upon which the oligonucleotide is synthesized. A variety of solid support materials have been described for solid phase oligonucleotide synthesis, the most prevalent of which is controlled-pore glass (CPG). (See, e.g., Pon, Methods in Molec. Biol. 20: 465 (1993)). Unfortunately, CPG suffers certain limitations that prevent it from being an ideal support material. See e.g., Ron et al., Biotechniques 6: 768 (1988); McCollum et al., Nucleosides and Nucleotides 6: 821 (1987); Bardella et al., Tetrahedron Lett. 31: 6231-6234 (1990) For example, CPG is unstable under the standard ammonium hydroxide procedure that is used to deprotect the oligonucleotide and to cleave it from the solid support. In addition, oligonucleotide synthesis using CPG as the solid support results in rather high levels of n-1 contaminant in the synthesis product.
To overcome these problems, various attempts have been made to develop polymer supports to replace CPG. See e.g., Gao et al., Tetrahedron Lett. 32: 5477-5479 (1991); The Gene Assembler(trademark), A Fully Automated DNA Synthesizer, Pharmacia Fine Chemicals, Uppsala, Sweden (1986). The use of organic supports in this context has been explored. Reddy et al., Tetrahedron Lett. 35: 5771-5774 (1994) discloses an organic support based on native Fractogel (xe2x80x9cToyopearlxe2x80x9d, TosoHaas, Philadelphia, Pa.). Fractogel, however, has inherent limitations as a support for oligonucleotide synthesis, due to its low density when packed in acetonitrile and its limited pore volume per unit bed volume. Although it would be desirable to replace CPG with a support that lacks its limitations, none of the polymer supports developed to date have provided the efficiency that CPG provides.
There is, therefore, a need for polymer supports for oligonucleotide synthesis that provide the efficiency of CPG, but that do not suffer from the instability or n-1 contamination problems inherent in CPG.
The invention provides passivated organic polymer supports, processes for their preparation and processes for their use in oligonucleotide synthesis that allow for highly efficient solid phase synthesis of oligonucleotides. The efficiency of synthesis provided when using the organic polymer supports according to the invention is at least as good as that provided by controlled pore glass (CPG). Unlike CPG, the organic polymer supports according to the invention are highly stable under standard ammonium hydroxide conditions used to deprotect the oligonucleotides and to cleave them from the solid support. In addition, solid phase oligonucleotide synthesis using the organic polymer supports according to the invention results in greatly reduced production of n-1 contaminant oligonucleotide.
In a first aspect, the invention provides a passivated organic polymer support for solid phase synthesis of oligonucleotides. The passivated organic polymer support according to the invention comprises a plurality of microscopic particles. Each particle has amino and/or hydroxyl groups covalently bound to the particle. Each particle further has nucleosides covalently bound to some of the amino and/or hydroxyl groups. At least some of the amino and/or hydroxyl groups that are not covalently bound to nucleosides are covalently bound to hydrophobic passivating groups.
In a second aspect, the invention provides a process for passivating an organic polymer support for oligonucleotide synthesis. The process according to the invention comprises introducing hydrophobic passivating groups at the site of free amino and/or hydroxyl groups that are covalently bound to the particles that comprise the organic polymer support. Organic polymer supports for oligonucleotide synthesis have amino and/or hydroxyl groups covalently bound to the particles that comprise the support. Some of the amino and/or hydroxyl groups are covalently bound to nucleosides, while others remain as free amino and/or hydroxyl groups. The presence of these amino and/or hydroxyl groups lends a hydrophilic character to the particles. In the process according to the invention, the hydrophilic character of the particles is reduced by covalently attaching hydrophobic passivating groups to the amino and/or hydroxyl groups. Passivation of the particles in this manner results in greatly improved efficiency of oligonucleotide synthesis.
In a third aspect, the invention provides an improved process for solid phase oligonucleotide synthesis. In this improved process according to the invention, the improvement comprises carrying out solid phase synthesis on the passivated organic polymer support according to the invention. This process of oligonucleotide synthesis according to the invention produces oligonucleotides at least as efficiently as processes utilizing CPG, but with greatly reduced contamination by n-1 and without chemical breakdown of the solid support.
The organic polymer supports and process for their use according to the invention are useful for synthesizing oligonucleotides on a scale ranging from small laboratory scale to large commercial scale. Thus, the organic polymer supports and process for their use according to the invention can be used to supply oligonucleotides for research purposes, for diagnostic purposes and for therapeutic purposes using the antisense approach.