Difference between revisions of "20.109(S10):Aptamer binding assay (Day7)"
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==Introduction== | ==Introduction== | ||
− | + | 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 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? | |
− | + | 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 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== |
− | + | ===Part 1: Purify, quantify, and prepare RNA=== | |
− | + | Repeat the [[20.109%28S10%29:Purify_RNA_and_run_affinity_column_%28Day4%29 | Day 4]] protocol, parts 1 through 3. That is, briefly: | |
− | # | + | #Digest the RNA, then purify on a Micro Bio-Spin column. |
− | # | + | #Quantify the RNA by spectrophotometry. |
− | #* | + | #*If you have less than 1.4 nmol of either "post" sample, let one of the teaching faculty know. |
+ | #Dilute each sample to 8 μM in the selection buffer (SB). | ||
+ | #*Note that to dilute your post-column sample, you will have to make an educated assumption about the ratio of 6-5 to 8-12, because they do not have the same molecular weight. What do you expect to have happened on the column? | ||
+ | #Finally, denature not only your "post" samples, but also your four "pre" samples, at 70 °C and then let them cool for at least 10 minutes. | ||
+ | #*If you are missing some "pre" samples due to low RNA yields, let the teaching faculty know; we have extra 6-5 and 8-12 to give you. | ||
− | ===Part | + | ===Part 2: Binding assay=== |
− | #For each sample in the table below, add 175 | + | #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?'' |
− | #Now add 175 | + | #*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>. |
− | #Incubate for five minutes at room temperature. | + | #For each sample in the table below, add 175 |