Warning: this page is under heavy construction. The material is being accumulated, before it is refined. You are (very) welcome to participate.
Experimental methods exist for determining, at the level of individual atoms, the various inter-atomic bindings that determine how an RNA molecule folds. However, this type of experiment can take months to perform, for a single RNA sequence. The EteRNA Cloud Lab process is now synthesizing more than 1000 novel RNA molecules per month, and as players/scientists, we expect the results for all 1000 sequences to be available in weeks, if not days. To meet this need, the EteRNA biochemistry team is pioneering the development of high-throughput RNA analysis, and well as high-throughput RNA synthesis. This high-throuhput analysis is under continual development and evolution, and improvements are continally being introduced into the EteRNA cloud labs. EteRNA players who participate in the cloud labs and question the results that get back are actually part of the scientific review process.
At a very high level of abstraction, SHAPE analysis works by mixing a chemical reagent (e.g. 1M7), called the SHAPE probe, into a solution containing many thousands of copies of each distinct RNA molecule being analyzed. The SHAPE probe has the potential for chemically attacking and modifying every base of every RNA in the solution. However, the speed with which the probe modifies the individual bases varies over several orders of magnitude. By carefully controlling the time of the exposure, the SHAPE procedure can limit the reaction so that only a small percentage of all the bases are modified. Using modern technology for processing nucleic acid chains, the number of times any analogous base has been modified can be counted, and the base-probe reaction rate for each base position of each distinct RNA moleculte can be estimated.
This base-probe reaction rate is not a direct measurement of whether the base is or is not bound to a complementary base via one of the three pair bonds recognized in EteRNA puzzles -- CG, AU and GU. It's not even known exactly what details of a base's configuration goes into determning the reaction rate. But the general consensus is that the reaction rate is an indication of how flexible the RNA's backbone is at each base position. This flexibility is highly correlated with (but not wholly determined by) whether or not the base is paired to one of its complements (C with G, G with either C or U, etc). This high correlation is the justification for using the SHAPE data as a measure of whether or not the base is bound, for the purpose of assigning the "score" for the molecule. But the fact that it is not a perfect correlation ensures that there will be an ongoing discussion among the most engaged players and the EteRNA scientists about how the SHAPE data should be presented and interpreted.
This section is a brief guide to the steps taken to produce the SHAPE data for a round of Cloud labs
The first step in the process is to specify the DNA sequence that will be needed to create each RNA sequence that has been selected for synthesis. The DNA sequence is closely related to the RNA sequence it will create, but differs in the following ways
- The sugars in the DNA backbone all have one less oxygen atom that the sugars in the RNA backbone.
- The uracil bases in RNA are replaced by the similar base thymine in DNA
- A 20-base sequence called the "T7 promoter" is added to the 5' end of the DNA. This sequene is crucial for the Transcription step, below.
- "Padding" bases are added to the 3' end of the DNA. Question: what role does fhe padding play?
These DNA specifications are sent to an independent lab that specializes in creating custom DNA molecules. Typically, the Das lab will order about a dozen copies of each DNA molecule.
DNA Amplification - PCR
Teaching about PCR
Transcription (DNA to RNA)
Here's a short video on transcription in the living cell, emphasizing how rapidly it occurs.
This step is described in much detail in the Das Lab paper Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol
Looks like a good figure, covering chemical mod and RT
Reverse transcription (RNA to DNA)
Data collection (Sequencing)
possible reference for this paragraph: http://rnajournal.cshlp.org/content/17/9/1688.full.pdf section "Data processing"
- http://www.chem.unc.edu/rna/pdf-files/2005_em_jacs.pdf the original 2005 scientific paper
- The figure 1 of 'Probing Retroviral and Retrotransposon Genome Structures: The "SHAPE" of Things to Come.' presents a good overview of this method.
- The Wikipedia article about the reverse transcriptase: http://en.wikipedia.org/wiki/Reverse_transcriptase
- Correlating SHAPE signatures with three-dimensional RNA structures
- Massively Parallel RNA Chemical Mapping with a Reduced Bias MAP-seq Protocol
Matthew Seetin, Wipapat Kladwang, J. P. Bida, and Rhiju Das