* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download MOD ODN - rci.rutgers.edu
Comparative genomic hybridization wikipedia , lookup
Maurice Wilkins wikipedia , lookup
Gene expression wikipedia , lookup
Gel electrophoresis of nucleic acids wikipedia , lookup
Silencer (genetics) wikipedia , lookup
Cell-penetrating peptide wikipedia , lookup
Messenger RNA wikipedia , lookup
Non-coding DNA wikipedia , lookup
Molecular cloning wikipedia , lookup
Epitranscriptome wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Molecular evolution wikipedia , lookup
DNA supercoil wikipedia , lookup
Cre-Lox recombination wikipedia , lookup
Oligonucleotide synthesis wikipedia , lookup
Real-time polymerase chain reaction wikipedia , lookup
Community fingerprinting wikipedia , lookup
Point mutation wikipedia , lookup
Green fluorescent protein wikipedia , lookup
Deoxyribozyme wikipedia , lookup
INTERPLAY OF POLYMER AND NUCLEIC ACID PROPERTIES ON EFFECTIVENESS OF ANTISENSE OLIGONUCLEOTIDES Mark Sumati 2 Sundaram , of Biomedical Engineering, 2Department of Chemical & Biochemical Engineering, Rutgers University, Piscataway, NJ METHODS Antisense oligonucleotides present a potentially powerful means to inhibit the expression of a specific gene that is central to a cellular phenotype, such as an oncogene in tumor growth. The antisense mechanism involved comprises an mRNA-specific oligonucleotide complexed with a polymeric carrier. Our research focuses on a synthetic cationic polymer, polyethylenimine (PEI), as the delivery agent and the green fluorescent (GFP) gene as an easily quantified model target. Fluorescence measurements indicate the level of downregulation of GFP, which in turn is dependent on the mechanism of complex formation and dissociation that we have assessed with biophysical measurements. Our experiments compare PEI of five molecular weights and oligonucleotides with three different backbone structures. The results indicate that PEIoligonucleotide complexes with phosphodiester (PO) backbones dissociate much more rapidly than those with phosphorothioate (PS) backbones. A third “end capped” ODN backbone structure, where only the outer two nucleotides are connected by phosphorothioate linkages, exhibits dissociation behavior similar to that of PO backbone oligonucleotides. Current work focuses on evaluating the effectiveness of end-capped ODN using stably expressing GFP cells. Vortex Incubate for ~15 min at POLYMER PEI – 5MW MOD ODN R.T. Add OLIGREEN to complex 5’…-A-G-G-U-C-A-C-U-U-U-G-C-A-A-C-G-…3’ • • • • • • • • • • • • 3’-C-A-G-T-G-A-A-A-C-G-T-T-5’ Normal Protein Production target mRNA antisense ODN HO SO P O O O O Experiment 2 – COMPLEX DISSOCIATION: Complexes are prepared at a single concentration and oligreen is added. Heparin in introduced last to mediate complex dissociation. Oligreen binds to released ODN. BASE PS ODN 60 25K 70K 40 600K 20 0 0.1 1.0 10.0 100.0 Results 2: 100 90 80 70 60 50 40 30 20 10 0 PEIPEI MW MW 10K 25K 70K 600K • Molecular weight dependency is insignificant in dissociation. • Increasing heparin concentration results in a more than proportionate dissociation response 100 ug/ml HEPARIN CONCENTRATION PEI MW No Protein Production CY-TAGGED ODN IN PEI/ODN COMPLEX V. TIME Antisense Oligonucleotide Experiment 3 - ANTISENSE EXPERIMENT: ODN is tagged with Cy5 dye (red) to measure cellular uptake, while fluorescence level measures downregulation over time. 120 100 PEI MW 80 10K 25K 60 70K 40 600K 20 250 PEI MW NORMALIZED FLUORESCENCE Protein NORMALIZED FLUORESCENCE FLUORESCENCE V. TIME FOR ALL PEI MW/ODN 200 10K 150 25K 100 70K 600K 50 0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 TIM E (HR) TIM E (HR) O PO ODN O P O O O BASE CHO blank pd1EGFP blank GFP down-regulation ODN release Results 3: • Maximum down-regulation occurs 8 hours after introduction of AS ODN for all MW. • Fluorescence levels return to normal by 30 hours. • 10K MW PEI shows unexpected behavior at 4 hours in both plots. O O 10K PEI POLYMER / ODN RATIO SUGAR BASE SUGAR 1.2K 80 Results 1 • Complex formation increases with polymer concentration. • 1.2KDa MW PEI does not bind as well to the ODN (high fluorescence level) DNA mRNA mRNA DNA PEI MW PEI MW 100 25 ug/ml Down-regulation duration depends on ODN backbone structure and its susceptibility to BASE nuclease degradation. O HO 5’…-A-G-G-U-C-A-C-U-U-U-G-C-A-A-C-G-…3’ PO ODN • Rapidly degraded by cellular nucleases • Transitory effects. 120 Antisense Inhibition RATIONALE O Experiment 1 – COMPLEX FORMATION: Complexes are prepared in vitro with varied polymer concentrations and allowed to form before a DNA binding dye, oligreen (OG), is added to interact with any unbound DNA. High fluorescence levels indicate high levels of unbound DNA. PEI-MOD ODN COMPLEXATION PEI-MOD ODN DISSOCIATION LEVEL @ T=2HR HEPARIN MEDIATED COMPLEX DISSOCIATION - negatively charged heparin binds competitively with the PEI carrier, displacing the ODN. BACKGROUND • Double stranded DNA separates and one DNA strand is copied onto a single mRNA “sense” strand during transcription. The original template strand encodes the antisense sequence. • In antisense therapy a synthetic antisense DNA strand enters the cell and binds to target mRNA of complementary sequence, preventing translation. PEI-ODN complex solution RESULTS % FLUORESCENCE ABSTRACT What is antisense? and Charles M. 1,2 Roth % FLUORESCENCE 1Department 1 Hwang , 5’…*A*G*G*U*C*A*C*U*U*U*G*C*A*A*C*G*…3’ PS ODN ( * bonds) • Less susceptible to nuclease attack • Longer lasting down-regulation, but displays a • Non-specific effects due to its high charge ratio. Binds to non-target molecules. MOD ODN By combining both backbone structures we hope to extend the life of the AS ODN, while decreasing non-specific effects. To the right is a modified (MOD) hybrid ODN: 5’…*A*G-G-U-C-A-C-U-U-U-G-C-A-A-C*G*…3’ MOD ODN MATERIALS GFP F (FL1) GFP F (FL1) GFP F (FL1) Cy5 F (FL4) CONCLUSIONS FUTURE DIRECTIONS DNA backbone structure affects polymer-oligonucleotide complexation and dissociation. Comparing data from each of the end-capped MOD sequence experiments to previous lab experiments on PO and PS sequences shows that the hybrid backbone is influenced by both its PO and PS components. Control experiments necessary to determine if non-specific effects accompany the extended in-cell life of the MOD ODN. Experiments would compare the downregulation of scrambled control MOD ODN with that of anti-GFP MOD ODN sequences. • Overlaying the MOD complex formation plot onto previous PO/PS complexation plots yields a greater match with PO than PS plots. The PEI 1.2K MW does not complex significantly to either PO and MOD oligonucleotides, while complexation occurs in the PS ODN. • Experiments indicate that PEI-MOD complex dissociation following heparin addition is almost complete at 2hrs, compared to 15 minutes for PO and 8 hours for PS sequences. SPECIFIC GOAL: evaluate effectiveness of end-capped MOD ODN sequence in antisense therapy. • Anti-GFP MOD sequence causes maximum down-regulation at 8 hours. ACKNOWLEDGEMENTS We wish to thank Dr. Li Kim Lee and Sandra Viriyayuthakorn for useful images that aided in highlighting key points.