Methods for decreasing the expression of specifically targeted genes

#It is well known that nucleic acids, i.e., deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) are essential building blocks in living cells. These compounds are high molecular weight polymers which are made up of many "nucleotide" units, each such nucleotide unit being composed of a base (a purine or a pyrimidine), a sugar (which is either ribose or deoxyribose) and a molecule of phosphoric acid. DNA contains deoxyribose as the sugar moiety and the bases adenine, guanine, cytosine, and thymine (which may be represented as A, G, C and T, respectively). RNA contains ribose instead of deoxyribose and uracil (U) in place of thymine.

#The nucleotide units in DNA and RNA are assembled in definite linear sequences which determine specific biological functions. In the normal mammalian cell, DNA replicates itself, and also serves as the template for the synthesis of RNA molecules whose nucleotide sequences carry the information encoded by the DNA. RNA molecules serve several different functions within the cell. Messenger RNA (mRNA) directs protein synthesis.

#According to the well known Watson-Crick model, DNA molecules consist of two polynucleotide strands coiled about a common axis. The resulting double helix is held together by hydrogen bonds between complementary base pairs in each strand. Hydrogen bonds are formed only between adenine (A) and thymine (T) and between guanine (G) and cytosine (C). Hence within the double helix, adenine (A) and thymine (T) are viewed as complementary bases which bind to each other as A-T. The same is true for guanine (G) and cytosine (C), as G-C.

#FIG. 1 is a schematic illustration to show how the expression of a selected gene is blocked in accordance with the process of this invention by hybridization of an oligonucleotide to its complementary sequence within the mRNA and cleavage of the mRNA by the enzyme RNaseH at the site of the RNA-DNA double helix.

#FIG. 2 illustrates the degradation of a single stranded DNA molecule by the action of a nuclease that removes sequentially one nucleotide at a time from the 3'- to the 5'-end of the chain.

#FIG. 3 is a drawing of an autoradiogram demonstrating that the exclusive pathway of degradation of DNA fragments in human blood is by exonucleolytic cleavage from the 3'- to the 5'-end of the DNA chain. The oligonucleotide utilized in this experiment, MA-15, has the sequence: 5'-GAGCACCATGGTTTC. In lanes 1-5, the oligonucleotide was radiolabeled at the 5'-end of the molecule with .sup.32 P. In lanes 6-10, the .sup.32 P-label is at the 3'-terminal internucleotide link. The labeled oligonucleotide was incubated in human blood at 37.degree. C. Aliquots were removed at the times shown and analyzed by polyacrylamide gel electrophoresis. The labeled bands within the gel were visualized by autoradiography.

#The invention comprises the method, means and composition which together enable the use of oligonucleotides that are modified at the 3'-terminal phosphodiester linkage, and are thereby rendered resistant to degradation within cells and body fluids, to selectively block the expression of a particular gene. The method involves the hybridization of the modified oligonucleotide to the corresponding mRNA to form a substrate fully capable of being recognized by the enzyme RNaseH, followed by the cleavage of the mRNA at the site of the RNA-DNA double helix such that the expression of the targeted gene is blocked. The invention further details the use of DNA and RNA molecules modified at the 3'-terminal phosphodiester linkage as nucleic acid probes for diagnostic applications.

#In the broadest terms, the objectives of the present invention are accomplished by modifying oligonucleotides at just the 3'-terminal phosphodiester linkage. When this is accomplished it has been found that the oligonucleotide is markedly resistant to degradation by nucleases within cells and within blood. Equally important, such modified oligonucleotides hybridize normally with complementary nucleic acid sequences and do form substrates with mRNAs that are recognized and cleaved by RNaseH. As a result of the cleave of the mRNA, expression of the corresponding gene is selectively blocked as is desired in various therapeutic applications.

#Recent advances in molecular biology employing recombinant DNA techniques have led to new diagnostic and therapeutic strategies. In DNA diagnostics, a DNA or RNA probe is used to detect the presence of a complementary nucleic acid (DNA or RNA) sequence. In this process the probe hybridizes to its complementary sequence if it is present within the sample. The target sequence may be a DNA or RNA molecule of a bacteria or virus, or an altered gene leading to a genetic abnormality such as sickle cell disease. Frequently, the sample to be analyzed is immobilized on a solid support such as nitrocellulose or a nylon membrane. The probe carries a label, e.g., a radioactive, fluorescent, or enzyme marker to permit its detection. The field of DNA diagnostics is currently under very active investigation, having applications in the diagnosis of infectious diseases, cancer, and genetic disorders. However, there are as yet no products in widespread clinical use. Existing methods, although extremely useful as research tools, lack adequate sensitivity for many practical applications, and are often too complicated to be carried out routinely and reliably in a clinical laboratory.