It Clicked!

July 31, 2018

by Abhilasha "Abby" Dehankar

Goal: To conjugate oligonucleotides (single-stranded DNA) to quantum dots transferred to aqueous phase using the loops-trains-tails method.  The conjugated QD-DNA particles are intended for use as part of a fluorescence control system for STORM imaging.

 

Review of progress-to-date:

1. We successfully transferred organic, NN-labs quantum dots (QDs) to the aqueous phase by ligand exchange with phytochelatin-3 (PC3), using a process known as the ‘loops-train-tails’ method.

Note: The resulting QD-PC3 has surface carboxyl groups (–COOH) amine (NH2) groups available for conjugation to other molecules. For example, -COOH conjugate to NH2 groups.

2. We successfully conjugated QD-PC3-COOH to amine-modified (-NH2) dye molecules using “carbodiimide” chemistry. This formed an amide (-CONH2-) bond between the carboxyl groups (-COOH) on the QD surface and the amine (-NH2) group of dye molecules.

Note: QD-PC3-dye conjugations were performed as control experiments to optimize the carboiimide chemistry before proceeding to QD-PC3 conjugation to DNA using the same approach.

3. We were unsuccessful making QD-PC3-oligo conjugations using carboiimide chemistry.

Note: We tested several different DNA sequences, such as random, mixed base pairs and PolyT sequences. We also tried partial modification of the QD-PC3 surface with Polyethylglycol (PEG) molecules to minimize non-specific interactions. None of these approaches or modifications were successful.

4. Because carbodiimide chemistry worked for small dye molecules but not for larger DNA molecules, we decided to move in a different direction, testing the newer “Click Chemistry” for DNA conjugation to QD-PC3.
Note: Click Chemistry uses strain-induced cycloaddition chemistry on alkynes to bind them to azides. Click Chemistry is also referred to as a bio-orthogonal chemistry because it does not interfere with native biochemical reactions in a living system.

 

Monthly Update:

Last month, we discussed our efforts to minimize non-specific interactions between oligonucleotides and the QD surface - and thus to increase DNA conjugation - through investigating various DNA and QD surface modifications. We had hypothesized that these might enhance conjugation by improving the chances for specific amide bond formation between –COOH group on QD surface and the amine group at DNA termination. But, because the modified PolyT DNA molecules are longer than the mixed-base-pair DNA we had previously studied, we also had to develop a new purification procedure. At the time of our last meeting, we were still waiting the new purification material.

Simultaneously, we began preparations to evaluate Click Chemistry as an alternative for DNA conjugation. In this post, I will discuss the results of the QD/DNA modification approaches (unsuccessful) followed by updates on the Click Chemistry (successful).

 

DNA modifications (unsuccessful)

The first modification that we attempted was to see if we could improve conjugation efficiency by using a DNA sequence consisting of a single, repeated base pair of thymine (T, i.e., polyT) that is known to perform better in conjugation because this nucleotide has shown the lowest affinity towards nanoparticle surface charges.  We also used our newly developed column purification process to obtain the final product. However, in our trials, the PolyT DNA also failed to conjugate, and of PolyT DNA completely separated from the QDs in the purification column.

 

QD modifications (unsuccessful)

We attempted to modify the QD surface groups (-COOH and –NH2) with PEG to reduce the number of charges on QD surface available for non-specific interaction with the DNA. This approach involved two steps:

1. Partial passivation of QD surface groups with PEG molecules using standardized carboiimide chemistry.

2. Conjugation of modified QDs from step 1 to DNA using standard carboiimide chemistry.

 

Since the QD-PC3 surface has two different charge groups: amine (-NH2) and carboxyl (-COOH), the experimental design involved three sub-approaches for step 1:

1. Partial passivation of only amine groups on QD-PC3 surface

2. Partial passivation of only the carboxyl groups on QD-PC3 surface

3. Partial passivation of both amine as well as carboxyl groups on QD-PC3 surface

These experiments were performed at high salt concentrations to stabilize the inherent negative charge of DNA that comes from its backbone. Unfortunately, all three approaches failed: DNA did not conjugate to the QD surface.

These results indicate fundamental issues with either the DNA integrity or the carbodiimide chemistry. We could perform an assay to test for DNA integrity and the required presence of an active amine group. As to the carbodiimide chemistry, we note that it is has already proven successful for small dye molecules., so we have a successful control. To mirror the larger molecules at play, and to rule out any QD surface issues, we could perform conjugation of oligonucleotides to commercially-available carboxyl-terminated polystyrene beads.

 

Click Chemistry (successful!)

Simultaneously, we began experiments with the alternative, Click Chemistry, route (Figure 1). Click Chemistry consists of two processes:

Step 1: QD-PC3-COOH groups are conjugated to dibenzocyclooctyne (DBCO) using carboiimide chemistry (Note: this is the same chemistry as used previously with success, but only for conjugating small molecules.

Step 2: A strain-promoted alkyne-azide cyloaddition click reaction occurs between DBCO-terminated QDs and azide-DNA to form QD-DNA conjugates. (Note: this requires us ro purchase DNA with pre-existing azide terminations).

 Click Chemistry
Figure 1: Click Chemistry

 

We performed the first step using our standard carboiimide chemistry, originally designed to conjugate amine-terminated dye molecules to QDs. Then, the DBCO-terminated QDs were allowed to react with azide DNA for over 7 hours. The final product was purified using size-exclusion chromatography, similar to the methods used in our earlier carbodiimide chemistry work.

To evaluate our conjugation results, the azide-DNA was pre-modified with a dye molecule for detection through fluorescence spectroscopy. If the dye molecules were present, we would observe a fluorescence peak corresponding to ~ 675 nm, as well as a ~ 610 nm peak corresponding to the QDs. This would indicate that DNA was successfully conjugated.

This trial was successful! As seen in Fig 2, both the QD peak (610 nm) and the dye peak (670 nm) are present in the experimental sample (black line). The control shows fluorescence peak for the quantum dots only (610 nm) (gray line).

 Fluorescence spectra for QD_DNA conjugations through click chemistry
Figure 2: Fluorescence spectra for QD_DNA conjugations through click chemistry

 

To confirm these positive preliminary results, we will test these DNA-modified QDs to see if they conjugate to DNA origami hinges with a complementary sequence.  Future experiments would then involve optimization of the Click Chemistry pathway for DNA conjugation.