Synthesis of branched nucleic acids

Details Synthesis of branched nucleic acids Synthesis of branched nucleic acids

1997-01-03

2001-01-30

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S022100

Reexamination Certificate

active

06180777

ABSTRACT:

The invention relates generally to polymer synthesis, and more particularly, to the synthesis of branched polymers and multiply connected macromolecular structures, such as macrocycles.
BACKGROUND
Polynucleotide detection and analysis is becoming increasingly important in many research, medical, and industrial fields, e.g. Caskey, Science 236: 1223-1228 (1987); Landegren et al, Science, 242: 229-237 (1988); and Arnheim et al, Ann. Rev. Biochem., 61: 131-156 (1992). Several techniques have been developed which exploit the specific hybridization of a probe nucleic acid to a complementary target nucleic acid for detection of the target. Generally, the most powerful of such techniques involve some form of target sequence and/or signal amplification, e.g. polymerase chain reaction, Arnheim et al (cited above); ligation-based amplification, e.g. Barany, PCR Methods and Applications 1: 5-16 (1991); strand-displacement amplification, Walker et al, Proc. Natl. Acad. Sci., 89: 392-396 (1992); branched probe signal amplification, Wang et al, U.S. Pat. No. 4,925,785, Urdea et al, U.S. Pat. No. 5,124,246, Hudson et al, J. Am. Chem. Soc., 115: 2119-2124 (1993); and the like.
In regard to the latter category of techniques, synthesis of the branched probe structures by current methods is typically difficult, results in low yields, and provides only limited and indirect means for monitoring yield and product quality during and after synthesis. Presently, most branched polymer approaches to polynucleotide detection do not lend themselves to the production of practical commercial assays.
SUMMARY OF THE INVENTION
The invention provides methods and compositions relating to the production and use of branched polymers, and other branched and multiply connected macromolecular structures, such as macrocycles. Preferably, branched polymers and multiply connected macromolecular structures of the invention comprise at least two branches and/or macrocycles: at least one branch or macrocycle is a target binding moiety capable of specifically binding to a target molecule of interest and one or more branches or macrocycles are signal generation moieties capable of directly or indirectly generating a detectable signal. Preferably, the branched polymers and macrocycles of the invention comprise at least one oligonucleotide moiety as a target binding moiety. In accordance with the method of the invention branched polymers and other macromolecular structures are assembled from components having phosphorothioate or phosphorodithioate groups and having haloacyl- or haloalkylamino groups. The phosphorothioate or phosphorodithioate groups react rapidly and efficiently with haloacyl- or haloalkylamino groups when brought into contact to form thio- or dithiophosphorylacyl or thio- or dithiophosphorylalkylamino bridges which complete the assembly of the desired structure. The method of the invention permits thorough purification and isolation of the components prior to the final assembly step. Incomplete branched polymers and/or other incomplete structures are readily separated from the desired product by standard means. An important aspect of the invention is that the thio- or dithiophosphorylacyl- or thio- or dithiophosphorylalkylamino bridges are readily and selectively cleaved by oxidation, which allows for convenient post synthesis confirmation of structure. The method of the invention further permits the synthesis of several novel branched polymer configurations.


REFERENCES:
patent: 5424413 (1995-06-01), Hogan et al.
patent: 5681943 (1997-10-01), Letsinger et al.

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