Difference between revisions of "20.109(S10):Aptamer binding assay (Day7)"
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Between the first-round of journal club talks, and the paper that we all discussed on day three of the module, you have begun to learn a lot about the usefulness of RNA aptamers and the SELEX process. | Between the first-round of journal club talks, and the paper that we all discussed on day three of the module, you have begun to learn a lot about the usefulness of RNA aptamers and the SELEX process. | ||
− | One particularly compelling type of aptamer is that which not only binds to a target, but in doing so reproducibly effects a particular function. Riboswitches consisting of an aptamer domain and an expression platform are common in nature, particularly in bacteria. The aptamer domain often recognizes a small molecule metabolite. Due to a resulting conformational change in the expression platform, transcription of a gene (or translation of its associated protein) may subsequently be turned on or off. For example, target binding may alter premature transcript termination via terminator/anti-terminator pairing. Engineers can mimic | + | One particularly compelling type of aptamer is that which not only binds to a target, but in doing so reproducibly effects a particular function. Riboswitches consisting of an aptamer domain and an expression platform are common in nature, particularly in bacteria. The aptamer domain often recognizes a small molecule metabolite. Due to a resulting conformational change in the expression platform, transcription of a gene (or translation of its associated protein) may subsequently be turned on or off. For example, target binding may alter premature transcript termination via terminator/anti-terminator pairing. Engineers can mimic nature s designs to create riboswitches with arbitrary desired functions. Ribozymes, or RNA with cleaving activity, may be incorporated in engineered riboswitches for additional functions. |
The specificity and affinity exhibited by aptamers is well-suited to several therapeutic uses. You have now seen examples of aptamers acting as drugs, drug antidotes, and potentially as targeted drug carriers that hone in on disease sites. You can start to appreciate the trade-offs inherent in experimental design for human health applications. For example, is the greater cost and labor of ''in vivo'' experiments offset by increased likelihood that the aptamer actually works in the required environment? | The specificity and affinity exhibited by aptamers is well-suited to several therapeutic uses. You have now seen examples of aptamers acting as drugs, drug antidotes, and potentially as targeted drug carriers that hone in on disease sites. You can start to appreciate the trade-offs inherent in experimental design for human health applications. For example, is the greater cost and labor of ''in vivo'' experiments offset by increased likelihood that the aptamer actually works in the required environment? | ||
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While RNA aptamers theoretically present infinite engineering possibilities, in reality researchers are limited by time and resources. Thus, improvements to SELEX efficiency are also an important research topic. Use of microfluidics and magnetic beads, and dual selections, are two examples of modified SELEX you saw in the journal club papers. Some modifications to SELEX are particular to specific applications, such as preparing aptamers to complex targets (e.g., tumors). | While RNA aptamers theoretically present infinite engineering possibilities, in reality researchers are limited by time and resources. Thus, improvements to SELEX efficiency are also an important research topic. Use of microfluidics and magnetic beads, and dual selections, are two examples of modified SELEX you saw in the journal club papers. Some modifications to SELEX are particular to specific applications, such as preparing aptamers to complex targets (e.g., tumors). | ||
− | In this module, we have investigated SELEX efficiency at the column selection step, using known RNA sequences (one that binds to heme and one that | + | In this module, we have investigated SELEX efficiency at the column selection step, using known RNA sequences (one that binds to heme and one that doesn t) as a model system. We varied stringency in two ways: number of washes, and amount of target aptamer present. After today s experiment, you should be able to discover some trends in SELEX efficiency. Consider how applicable your results are to an arbitrary SELEX system, and what further experiments or you might suggest doing in the future. |
==Protocols== | ==Protocols== | ||
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#Retrieve some 6 μM heme from the teaching faculty. ''Why might you use 6 μM instead of 8 μM, if we want 1:1 molar RNA:heme?'' | #Retrieve some 6 μM heme from the teaching faculty. ''Why might you use 6 μM instead of 8 μM, if we want 1:1 molar RNA:heme?'' | ||
#*A 1M stock solution of heme was originally prepared in DMSO, then diluted in multiple steps to 6 μM. Note that the stock solution is prepared by dabbing a little (solid) hemin into DMSO, and then testing the concentration on a spectrophotometer. The extinction coefficient of heme at 405 nm is 180 mM<sup>-1</sup>cm<sup>-1</sup>. | #*A 1M stock solution of heme was originally prepared in DMSO, then diluted in multiple steps to 6 μM. Note that the stock solution is prepared by dabbing a little (solid) hemin into DMSO, and then testing the concentration on a spectrophotometer. The extinction coefficient of heme at 405 nm is 180 mM<sup>-1</sup>cm<sup>-1</sup>. | ||
− | #For each sample in the table below, add 175 | + | #For each sample in the table below, add 175 |