User talk:ElNando888/Blog/Reverse Transcriptase
Re Unbreakable stacks
Once it gets past the bar code, wouldn't the effect of the reverse transcription being blocked be seen in the SHAPE data as a highly reactive base immediately preceding the blocked base? I don't see that in the Try 1 Bracing data.
This would seem logical, but actually, I don't think it would. It's only an intuition, but if Reverse Transcriptase is anything like the other polymerases, then it has a 3D geometry, with an entrance, a channel where the single-stranded nucleotides slide one by one, and only then, it reaches an internal "pocket" called the active site. The corollary is that the stacks have to be broken open some time before the RT adds the complementary DNA base. What length exactly? no clue, but there's a hint that says that it could be as much as 6 bases. And the blocking point may not be at the base of the stack as we see it in the MFE structure. So it's hard to tell...
But where in the SHAPE data for that design do you find any evidence for the interpretation that the reverse transcription gets blocked, once it has gotten by the barcode?
I'm not claiming to have solid evidences. I'm only listing designs with high reactivity error rates and looking at patterns to attempt to discern a possible origin. This said, Rhiju mentioned in the past that this phenomenon of "RT getting stuck" does happen, and here, it seems to me to fit some of the cases.
If you're curious, go check Brourd's lab with the CC mismatches, and sort by GC pair count. You should notice something in the sequences (if you have reactivity errors turned on). My personal impression is that the same cause is present here, but with a little "twist" that would be too long to explain here (I'm thinking of making it the topic of a future blog post)
Oh, and when you get a chance, ask Brourd about his theory concerning SHAPE values exactly 0.