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Back when he was a postdoctoral fellow, Professor Niles screened a random library of RNA aptamers to find one that binds to heme – the iron-containing site in hemoglobin. It is known that heme can bind to certain transcription factors and modulate gene expression (see references Zhang and Hach, 1999 and Ogawa, et al., 2001), and RNA aptamers are one potential tool for learning more about signaling networks involving heme. To select heme-binding aptamers, Professor Niles ran a pool of RNAs with 50 randomized base pairs through a heme affinity column. He then amplified the column-selected pool of aptamers and repeated the process several times. An aptamer called "6-5" survived through the 6th round of screening, but ultimately was found not to bind to heme. An aptamer called "8-12" survived through all 8 rounds of screening, and has a heme binding affinity of 220 nM. Both were described in the Niles, et al., 2006 paper referenced below.
Today you will be given two archival plasmids containing the 6-5 and 8-12 sequences, respectively. RNA is not very stable compared to DNA; thus, RNA aptamers are copied into their associated DNA sequences for long-term storage. Ligating the DNA fragment into a plasmid that can be carried in bacteria provides further amplification and storage capabilities. We will make use of these capabilities more extensively in Module 2.
In order to select and amplify just the short DNA fragment that encodes for the aptamer, you will use the polymerase chain reaction, PCR. PCR comprises three main steps: 1) template DNA containing a desired sequence is melted, 2) primers anneal to specific locations on the now melted (i.e., single-stranded) DNA, and 3) the primers are extended by a polymerase to select and create the desired product. Extension occurs at ~70 °C, melting at ~95°C, and annealing at a temperature ~5 °C below the primer melting temperature; thus, the repetition of these steps is called thermal cycling. After each cycle, the newly formed products themselves become templates, causing exponential amplification of the selected sequence. (Note that the early rounds of PCR will not produce the desired product - we will see why in today's pre-lab lecture.)
Once the PCR is running, you will begin to explore some computational tools for RNA analysis. During this module, you will ultimately use three different programs to explore both sequence similarities among RNA candidate aptamers and higher-order structures that arise from the primary sequences. For today, you will look at degrees of sequence similarity among a list of aptamers, some of which bind to heme and some that don't.
Based on the numerous applications of PCR, it may seem that the technique has been around forever. In fact it is only 25 years old. In 1984, Kary Mullis described this technique for amplifying DNA of known or unknown sequence, realizing immediately the significance of his insight.
"Dear Thor!," I exclaimed. I had solved the most annoying problems in DNA chemistry in a single lightening bolt. Abundance and distinction. With two oligonucleotides, DNA polymerase, and the four nucleosidetriphosphates I could make as much of a DNA sequence as I wanted and I could make it on a fragment of a specific size that I could distinguish easily. Somehow, I thought, it had to be an illusion. Otherwise it would change DNA chemistry forever. Otherwise it would make me famous. It was too easy. Someone else would have done it and I would surely have heard of it. We would be doing it all the time. What was I failing to see? "Jennifer, wake up. I've thought of something incredible." --Kary Mullis from his Nobel Lecture, December 8, 1983.
(Copyright © The Nobel Foundation 1993. All rights reserved. This content is excluded from our Creative Commons license. For more information, see http://ocw.mit.edu/fairuse.)
Zhang, L., and A. Hach. "Molecular Mechanism of Heme Signaling in Yeast: the Transcriptional Activator Hap1 Serves as the Key Mediator." Cell Mol Life Sci 56, no. 5-6 (October 30, 1999): 415-26.
Ogawa, K., et al. "Heme Mediates Derepression of Maf Recognition Element Through Direct Binding to Transcription Repressor Bach1." EMBO J 20, no. 11 (January 1, 2001): 2835-43.
Niles, J. C. "Utilizing RNA Aptamers to Probe a Physiologically Important Heme-Regulated Cellular Network." ACS Chem Biol 1, no. 8 (September 19, 2006): 515-24.
You and your partner may work together on the lab practical. (Note: this will not be the case for future quizzes.) You are of course welcome to give different answers should you disagree.
Before starting today's wet lab work, you may want to wipe down your pipettes and your benchtop with 70% ethanol.
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Your understanding of this module will in part be evaluated by the RNA Computational Analysis assignment. Although you will not be prepared to understand the entire assignment today - until you have a better grasp of what SELEX entails - you should be able to get a good start on the first section.
Note that all the sequences in the data file are shown 5' to 3'.