Term
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Definition
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Term
| What is the maximum amount of base pairs in an oligonucleotide? |
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Definition
| Normally 15-30 bases, but can reach to 100 bp |
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Term
True or False?
Most drug molecules have low molecular weight ~500da |
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Definition
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Term
| What is the typical molecular weight of siRNA |
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Definition
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Term
| What makes nucleic acid highly soluble in h2o? |
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Definition
| Its high phosphate (charged) backbone |
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Term
| How are olgionucleotides typically synthesized? |
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Definition
| with a olgionucleotide synthesizing machine, similar to chemical drugs |
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Term
| Antisense olgionucleotide sequence is complementary to the _______ |
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Definition
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Term
| While most drugs target protein, targeting rna that makes protein requires ______ between DNA -> RNA -> proteiin |
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Definition
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Term
| When antisense inhibits gene expression by blocking protein synthesis, oligonucleotides ________ with mrna |
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Definition
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Term
| what are the 2 ways of increasing production of a protein? |
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Definition
1) use an enzyme to degrade the protein, and make more
2) inhibit the suppressor gene |
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Term
True or false?
siRNA and shRNA are usually from inside the body, (does not need to be administered) |
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Definition
| False, from outside, must be administered |
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Term
| in regard to antigene, the hoogstein or reverse hoogsteen allow what to form? |
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Definition
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Term
| What is the advantage of antisense oglionucleotides being specific to one unique mRNA sequence? |
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Definition
| so you can go in and hybridize with it and knock it down |
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Term
| what are the two different mechanisims of inhibition in antisense oligonucleotides hybridizing to mRNA? |
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Definition
1) destruction of mRNA by ribonucleases
2) ribosome movement blocked |
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Term
| How is protein synthesis inhibited by antisense oligo? |
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Definition
| ribonucleases recognize and cleave RNA:DNA duplex |
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Term
| During inhibition of protein synthesis by antisense oligo, the ribonuclease will destroy _____ but leave the _____ |
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Definition
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Term
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Definition
| endogenous enzymes that degrade RNA |
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Term
| What is the role of RNase H? |
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Definition
| ribonuclease that recognizes and cleaves mRNA in an RNA/DNA duplex. Also cleaves RNA in an RNA/RNA duplex |
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Term
| How is protein synthesis inhibited by antisense oligos? (2) |
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Definition
| an antisense oligo prevents ribosome from proceeding along mRNA during translation |
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Term
| What are some advantages of antisense oligos over traditional drugs? (late 1970s) |
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Definition
tradition drugs intervene after a disease causing protein is formed or target enzymes whereas...
antisense therapeutics block mRNA translation and intervene before a disease causing protein is formed |
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Term
| What is the advantage of the 50-60 bonds in antisense oligos that give them an advantage over trad drugs? |
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Definition
| 50-60 bonds == more specific and less side effects |
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Term
| What are the top 3 considerations in designing antisense oligos? |
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Definition
1) gene target
2) length of oligo
3) portion of mRNA to target |
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Term
| Why is cancer a good target for antisense oligo gene targeting? |
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Definition
| often a vast difference in gene expression between cancer and normal cells. |
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Term
| What are the quantitative differences between cancer cells and normal cells in terms of gene expression? |
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Definition
| in a cancer cell, gene is expressed at an abnormally high level where a normal cell would not express at all |
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Term
| Define chromosomal tranlocation |
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Definition
transposition of two segments of different chromosomes; results from abnormal breakage and refusion of the segments
new sequence not found in normal cells |
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Term
| when considering the optimal length of antisense oligos, why is the rna pool of cellular RNA smaller? |
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Definition
| because of the introns being removed by splicing |
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Term
| What are two factors to considering in practical targeting off mRNA? |
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Definition
1 - shape of mRNA
2 - proteins that protect the mRNA |
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Term
| What is a problem with oligos that are too long? |
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Definition
| can bind to itself or have cross interactions with different mRNA |
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Term
| what is a problem with oligos that are too short? |
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Definition
| lose specificity and cant hybridize |
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Term
| What are some limitations of antisense oligos? |
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Definition
1) poor stability
2) poor cellular uptake
3) presence of "non-antisense effects" |
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Term
| what are the only oligos that do not need to use uptake into cells and why? |
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Definition
aptamers
b/c - these target proteins which may be outside the cells |
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Term
| what are the only oligos that do not need to use uptake into cells and why? |
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Definition
aptamers
b/c - these target proteins which may be outside the cells |
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Term
| Antisense contains what bonds that make it degrade quickly and what is the half life in serum? |
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Definition
contains - phosphodiester bonds
1 hour half life in serum |
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Term
| Linkage adds what to increase half life of oligos? |
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Definition
| backbone mods add S or Se to the Oxygen (phosphorothioate) to increase half life where exonuclease are designed to elevate phosphodiester bonds |
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Term
| In medicinal chem of antisense oligos, what bond is most susceptible to hydrolysis? |
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Definition
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Term
| what are the 2 ways to break the phosphate bond? |
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Definition
1) exonuclease - start chewing from 5' or 3' end
2) endonuclease starts in the middle |
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Term
true or false?
changes of the heterocycle (bases) does not effect the activity of the nucleases, changes will cause change in affinity of oligo and target |
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Definition
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Term
| How can you increase the binding affinity for the phosphotiate in modification of oligos? |
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Definition
| increase the length of the oligos or change sequences (use CG instead of AT) or chemical modification of the base to increase affinity |
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Term
| What happens when you modify oligos? |
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Definition
1) stability is increased (usually)
2) enzymes
3) adverse effects - changing O -> S makes oligo have different activity |
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Term
| What makes it hard for the phosphorothiate in an oliog to enter the cell? |
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Definition
| charged backbone (negatively charged macromolecule) will not enter cell by passive diffusion |
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Term
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Definition
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Term
| The nucleus membrane is fairly leaky, thus _____ and ____ serve as a barrier for oligos? |
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Definition
| cell membrane and endosome |
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Term
| Once in the endosome, what are the steps that occur to oligos? |
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Definition
1) get degraded
2) fuse back into membrane
3) unknown mechanism that let the oligo out and have effect on cell |
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Term
| ASOs are formulated with _______ ________ to enhance cellular uptake |
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Definition
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Term
| how do cationic surfactants help cellular intake? |
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Definition
| when placed on backbone, its liophilic and increase absorptive pinocytosis |
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Term
| How are the most effective liposomes charged - and why is this charge most effective? |
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Definition
positive -
because our cell surface has a lot of negatively charged protein, etc increases absorptive pinocytosis |
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Term
| Mipomersen is a first in class apo-B synthesis inhibitor used to treat hyperlipidemiea by interefering with what? |
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Definition
| normal cholesterol pathway and how its made |
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Term
| What are some non-antisense action of oligos? |
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Definition
immune stimulation is an undesirable side-effect of certain ASOs and dependent upon 1) presence of unmethylated CpG dinucleotides within ASO
2) phosphorothioate backbone |
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Term
| what is the role of DNa methylation in vertebrate cells? |
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Definition
methylation of CpG islannds frequently occur in the promoterss of many genes
high methylation = low transcription |
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Term
| what does dna methylation have to do with ASOs? |
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Definition
presence of unmethylated CpG dinucleotides within ASOs causes immunostimulation
mechanism used to design ASOs containing CpG dinucleotides that trigger immune sys to destroy cancer cells |
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Term
| how is siRNa different from oligos? |
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Definition
| siRNA starts out double stranded |
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Term
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Definition
| RNA interference - describing interference of RNA as natural mechanism and as a scientific tool |
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Term
| How are siRNAs introduced? |
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Definition
| usually through virus and can come from nucleus but has no mismatch |
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Term
| Describe the steps in RISC - RNA induced silencing complex |
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Definition
| siRNA binds to RISC, Risc separates the DS, leaving one within the complex, the RNA within the complex targets the mRNA and degrades it |
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Term
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Definition
| short dsRNA molecules with 21 nucleotides strands of RNA in staggered duplex, 19 nucleotides double stranded with 2 base overhangs |
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Term
| siRNA are created with the help of what enzyme? |
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Definition
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Term
| what is the main difficulty in designing miRNA? |
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Definition
| we do not know exactly how to design the loop where there is a mismatch |
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Term
| what is added to siRNA to target ligands? |
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Definition
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Term
True or False?
miRNA are endogenous factors that control translation? |
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Definition
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Term
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Definition
an RNA sequence
can hybridize and catalytically cleave
the guandine of the hairpin loop serves as a nucleophile |
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