15-Nucleic Acid Technology-updated.ppt

NEW CHAPTER Recombinant DNA based Applications (Nucleic acid Technology) 1 * Learning objectives - Define oligonucleotides: structural and physico-chemical properties. - Learn about the approaches applied: 1) Anti-sense technology. 2) Triplex technology. 3) Aptamers. 4) Ribozymes. 2 * Backgrou...

NEW CHAPTER Recombinant DNA based Applications (Nucleic acid Technology) 1 * Learning objectives - Define oligonucleotides: structural and physico-chemical properties. - Learn about the approaches applied: 1) Anti-sense technology. 2) Triplex technology. 3) Aptamers. 4) Ribozymes. 2 * Background: • Nucleic acid technology include all the techniques based on oligonucleotides and their analogs (isolated and synthesized) and which are used in Pharmacy and Medicine. • Nucleic acid technology involves: 1) triplex (DNA level) and antisense technology (RNA level), 2) aptamer technology (protein level) and 3) study of ribozymes. • Nucleic acid technology can be used for diagnostic purposes (microarray); to study diseases and genetic disorders (transgenesis); and treatment of certain disrorders (gene therapy). 3 Oligonucleotides * Biochemistry: 1)Oligonucleotides (or “oligos” as they are sometimes called) are short polymeric segments of deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), generally 100 or fewer bases long. 2)Oligodeoxyribonucleotides (ODNs) contain (T A C G) nucleotides. In oligoribonucleotides, the pyrimidine base uracil (U) is substituted for T and hydrogen bonds with A. 4 * Nomenclature: • The nomenclature for oligonucleotides follows a consistent pattern. For a monomer, dimer, trimer up to a decamer, the names would be mononucleotide, dinucleotide, trinucleotide, etc. Beyond that, the name of an oligonucleotide is given by its length as a number followed by “ -mer”. Thus a 21-base containing oligonucleotide would be a 21-mer (twenty one-mer). 5 * Physicochemical Properties: - Oligodeoxyribonucleotides and oligoribonucleotides are highly susceptible to rapid degradation by structurally nonspecific intracellular nucleases - The phosphodiester linkage possesses a negative charge preventing passive diffusion through cellular and nuclear membranes. - Natural oligonucleotides have been shown to accumulate in cells by receptormediated endocytosis. 6 → Enhance Drug Properties: - Replacing the negatively charged oxygen (i.e. phosphate group) on the phosphodiester linkages with more nuclease resistant and lipophilic groups. This results in a series of modified oligonucleotides with improved physiochemical properties potentially more useful to enhance bioavailability and stability (Slide 8). - A peptide nucleic acid (PNA) is a more recent variation of the phosphatesugar backbone of oligonucleotides, aminoethyl glycine (gly-NH-CH2-CH2-gly) units serve as the backbone of the polymer, which has improved physiochemical characteristics. 7 Alkyl phosphonate Phosphorothioate Formacetal Phosphoramidate 8 Triplex Technology • An antigene nucleic acid approach. It is triple helix (triplex) technology in which short oligodeoxyribonucleotides of 15—27 nucleotides in length can bind sequence-specifically to complementary segments of duplex DNA. 9 10 Anti-sense Technology • Antisense oligonucleotide interruption of the flow of genetic information may occur at the mRNA level in the cytoplasm or by interacting with the mRNA precursor in the nucleus. Antisense RNA would be oligoribonucleotides that are complementary for the mRNA sequence that is targeted. Antisense DNA would be single stranded oligodeoxyribonucleotides that are complementary to mRNA. • Mechanism can be: 1) masking the ribosome binding site, 2) cross-linking the oligonucleotide to the target mRNA, 3) induce ribonuclease H (RNase H) action on RNA-RNA / DNA-RNA duplex where the RNA part is digested. 11 Anti-sense Technology 12 Therapeutic application of anti-sence approach • Fomivirsen (Vitravene), the first antisense biotech drug to be approved and marketed, is a 21-mer phosphorothioate oligodeoxynucleotide. This nucleic acid agent is indicated for the treatment of human cytomegalovirus (CMV) induced retinitis in AIDS patients. 13 5'-GCG TTT GCT CTT CTT CTT GCG-3' 14 Aptamers • Aptamers are single-stranded or doublestranded sequences of oligonucleotides that bind to proteins (rather than nucleic acids) or other small molecules. Aptamers can be synthetic or natural. • Aptamers can be classified as: 1) DNA or RNA aptamers. They consist of (usually short) strands of oligonucleotides (slide 16). 2) Peptide aptamers. They consist of a short variable peptide domain, attached at both ends to a protein scaffold. * Example: • Pegaptanib sodium injection (brand name Macugen) is an aptamer, acts as antiangiogenic medicine for the treatment of neovascular age-related macular degeneration (AMD). • Pegaptanib works as an antagonist to VEGF, which when injected into the eye blocks the actions of VEGF. This then reduces the growth of the blood vessels located within the eye and works to control the leakage and swelling. 16 Ribozymes • Ribozymes are catalytic RNAs that cleave covalent bonds, generally degrading a target RNA. • Natural ribozymes: the functional part of the ribosome, the molecular machine that translates RNA into proteins, is fundamentally a ribozyme, composed of RNA tertiary structural motifs that are often coordinated to metal ions such as Mg2+ as cofactors. 17 Learning outcomes • Short oligonucleotide fragments are designed to target different stages of DNA replication and gene expression. • There are many approaches that Short oligonucleotides can follow to achieve therapeutic outcome. Among these approaches is the antisense technology which has clinical applications. 18