The ligation reaction that I keep referring to requires three pieces of DNA. They get fused together all in one shot, that is slightly complicated. The most crucial of which is the adapter duplex, because without it the anchor and the unzipping DNA would not attach and the reaction would yield nothing. And because of how important the adapter is, this has been the source of my troubles for the past 4 years. But before I go into that, let me tell you about the duplex.
It’s called an adapter duplex because it is actually two single stranded pieces of DNA. We call them the top and bottom strand. They are short DNA sequences manufactured from biotech companies. In the past we’ve used Alpha DNA, but I’m thinking of trying someone new. How short are the strands? The bottom strand has about 35 bases and the top is only a few bases longer. Compare that to the anchor sequence which is either 1100 bp (base pairs) or 4400 bp or the unzipping sequence which can be as long or as short as we want (but typically around 3000bp for calibration sequences).
Once our single stranded sequences arrive via mail (we call these short sequence oligonucleotides, or oligos for short), we need to bind the top and bottom strands together in a process called annealing. Most molecular biological reactions involve some kind of enzyme to help the reaction, but annealing is quite a natural process. DNA naturally wants the bases to bind to complementary bases (A-T, G-C) and even in single stranded form, the DNA will self anneal, that is bind to itself. So to get our top and bottom strands to stick together we just put them together in the same tube, heat it up to near boiling temperatures, and slowly bring the temp down so that the top and bottom strands find each other and bind. Once it’s cooled, the adapter duplex is formed and will stay that way unless heated to very high temperatures (near boiling).
There are three key features of the adapter duplex: (1) a biotin molecule, (2) a gap in the DNA backbone, and (3) two non-palindromic overhangs. The overhangs are designed to bind with a very specific sequence. One side can only bind with the overhang I mentioned in the anchor DNA, the other side can only bind with the overhang contained in the unzipping DNA. Right now that particular sequence is very specific to cutting plasmid pBR322 with the enzyme SapI (and any other plasmids that share similar properties).
The biotin is necessary for unzipping. The biotin has a high affinity for streptavidin which coats the microspheres we use for optical tweezing. Typically the biotin in our bottom adapter strand is near the start, but not at the start of the sequence. In more recent iterations, we moved it to the 5′ end completely or added a poly-A overhang with several biotin there. The reason for this is because we’ve been having issues actually unzipping, which I’ll explain in another post. The hope was that by moving the biotin we would get better tethering efficiency and better unzipping. We ended up not getting unzipping results and the tethering efficiency studies were inconclusive.
The bottom strand has both the biotin and the gap (key feature 2), which actually plays a role in the unzipping. Since the tweezers will pull on this side, the gap was designed to aid in the unzipping. Basically the gap was the weakest point in the complete DNA chain and since the microsphere is so close to it the DNA would begin to unzip from this location. The gap is actually a missing phosphorus (the yellow in the image to the right), which prevents the anchor and the bottom adapter strand from connecting to each other. In later iterations we completely removed the first base to make the gap wider, and the poly-A tail I mentioned was also used to prevent there from being any attachment.
Ultimately I never got unzipping to work. Oddly enough, I ran experiments that verified the ligation reaction worked, but could never get the completed structure to unzip. That’s what this new set of experiments is going to attempt. But before I get to that, I need to tell you about the unzipping DNA portion!