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| | <h2>RNAi</h2> | | <h2>RNAi</h2> |
| - | Fortunately, several well-characterized biological pathways potentially fill this role. Collectively, they fall into a class of mechanisms named "RNAi" (standing for "RNA Interference"), and they work by destroying the mRNA transcripts of particular genes before ribosomal binding and translation into protein. They achieve specificity through the integration of short RNA segments into the RNA Induced Silencing Complex (RISC), the group of proteins responsible for RNAi. The RISC will preferentially degrade sequences with complementarity to integrated short RNA segments. By selecting RISC targeting sequences with complementarity to multiple homologous allergens, we hope to achieve a degree of specificity that will prevent the expression of allergens and their homologues while permitting plant survival and growth.
| + | RNAi (RNA interference) can be used to cleave mRNA sequences in order to prevent translation of specific proteins. Target mRNA sequences that are complementary to short RNA segments, which are recognized by the cellular machinery, are cleaved. By introducing short RNA sequences complimentary to the sequences of the various allergens that we would like to target, we hope to knockdown the expression of these allergens and their homologues. |
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| | <h2>hpRNA</h2> | | <h2>hpRNA</h2> |
methods
As proteins provoke the majority of allergic reactions, we hope to diminish the allergenic properties of our plants by suppressing the expression of proteins that aren't necessary for the plant's survival but have been found to cause allergic responses in humans. This problem is complicated by the fact that proteins with allergenic properties may have several slightly different (but similar enough to remain allergens) homologues spread throughout the plant's genome. Subject to the constraint of the plant's survival, we would like to reduce or eliminate the expression of these homologues as well.
RNAi
RNAi (RNA interference) can be used to cleave mRNA sequences in order to prevent translation of specific proteins. Target mRNA sequences that are complementary to short RNA segments, which are recognized by the cellular machinery, are cleaved. By introducing short RNA sequences complimentary to the sequences of the various allergens that we would like to target, we hope to knockdown the expression of these allergens and their homologues.
hpRNA
With RNAi, the problem of creating a hypoallergenic plant reduces to the problem of integrating short RNA strands into the RISC, each with complementarity to RNA which ribosomes would otherwise transcribe into allergenic proteins. One mechanism of flagging RNA for RISC-incorporation is to place the RISC-targeting sequence (~300bp), an intron-specific sequence (200-, and the reverse complement of the RISC-targeting sequence (~300bp) under a promoter (in nature these constructs could also be found in an intron). Upon transcription, this construct will form a hairpin: the targeting sequence and its reverse complement will anneal to each other while the intron-specific sequence will form the hairpin's loop. This structure is called a hpRNA, short for "hairpin RNA." The RISC will then process and incorporate part of one of the legs of the hairpin (targeting sequences) with which it will search for and destroy complementary RNA sequences.
amiRNA
The process of hairpin RNA (hpRNA) incorporation involves customizing a small (~21bp) portion of one of the hairpin legs which will actually be used for RISC specificity. Since the usual processing of hpRNA isn't entirely deterministic we can profitably bypass part of the processing, effectively jumping in to the hpRNA pipeline at a later stage that gives us more control over the sequence which the RISC will use for specificity.
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