July 2, 2018
by Abhilasha "Abby" Dehankar
Goal: Conjugation of oligonucleotides (single-stranded DNA) to quantum dots transferred to aqueous phase using loops-trains-tails method.
Review of progress-to-date:
Note: QD-PC3 displays surface termination of carboxyl groups.
Note: QD-PC3-dye conjugations were performed as control experiments to optimize the carboiimide chemistry before proceeding to QD-PC3-DNA conjugations that would be conducted using the same chemistry.
Note: Factors tested: Different ratios of DNA: quantum dots (1500: 1 and 15:1), DNA conjugation in the presence of a backfiller molecule (amine terminated polyethylene glycol or PEG-amine)
During our last phone, I reported that QD-PC3-DNA conjugation did not work with the carboiimide chemistry that was optimized for QD-PC3-dye conjugation. We tried tweaking a couple of things in the process, but nothing worked. The most probable reason for our failures could be non-specific interactions of DNA with QD-PC3 surface, potentially hindering our current conjugation chemistry. Therefore, for the future we decided to try two different approaches that could decrease the non-specific interactions:
As per the plan, we ordered the new DNA and carboxylic acid/amine terminated PEG for passivating the amine/carboxyl group on the QD surface, respectively. Changing the DNA sequence, changes its molecular weight as well as its size. As mentioned in the previous phone call, we are using size separation columns for purifying the QD-PC3-DNA conjugates from excess/unreacted DNA. A size separation column generally consists of a gel that can separate components in a specific size range depending on the type of the gel and the length of the column. A taller column length can separate with greater efficiency. Our current gel, although capable of separating the new PolyT DNA sequence from QD-PC3-DNA, is extremely inefficient in PolyT separation as a result of the shorter length of our current column. Thus, we had two options, either to use a new gel or a longer column. We decided to go with a more economic, gel option. Unfortunately, my gel purchase got back-ordered twice and has still not arrived limiting my experimental progress for both the approaches!
Meanwhile, me and my undergraduate (Thomas) scoured through the literature to search for alternative conjugation chemistries in case the carboiimide chemistry completely fails us even after trying everything. Thomas suggested that we try “Click” chemistry as our new direction. This chemistry, although relatively new and a non-zero length crosslinking chemistry, has demonstrated better conjugation efficiencies in the literature. Therefore, we have decided to simultaneously work on this chemistry. Even though this chemistry may have a set of its own challenges, we are hopeful that it will have better conjugation efficiency.