DNA Embedding - One step closer II

February 21, 2019

by Kil Ho Lee

DNA embedding – One step closer II

Introduction

In the previous blog post (see the Jan 15th, 2019), we embedded DNA on CdSe/ZnS quantum dot (QD) surface. We performed the gel electrophoresis on DNA embedded QDs. Based on the gel electrophoresis study, we are now confident that the embedding procedure is working well.

In this report, we tested the effect of increasing ZnS thickness for two reasons. First, the thicker ZnS shell can help increasing the fluorescence stability and/or intensity. Second, we hypothesize that the embedding efficiency may increase with more ssDNA (with thiol, or –SH, group) and Zn react to form thicker ZnS shell.  Lastly, we attempted to bind DNA embedded QDs on DNA origami structure to test if Poly-T sequence embedded QDs would hybridize successfully with Poly-A overhang on DNA hinges.

Result and Discussion

Increasing ZnS thickness

In this experiment, we prepared a set of samples as listed in Table 1. After DNA embedding step, we purified the samples via centrifugal filtration.

Table 1. Samples analyzed using fluorescence spectroscopy

No.

MPA exchanged QD

MPA

Zn2+

DNA

NaOH solution (5M)

Water (uL)

(0.3 mg/ml)

(25 mM)

(25 mM)

(100 nM)

Control

(No Heat)

100 ul

9 ul

4.5 ul

100 ul

3 ul

199 uL

S2

100 ul

9 uL

4.5 ul

100 ul

3 uL

199 uL

S3

100 ul

9 uL

9 ul

100 ul

3 uL

194.5 uL

S4

100 uL

9 uL

18 ul

100 uL

3 uL

185.5 uL

As usual, after embedding DNA on QD surface, we measured the fluorescence intensity of each sample and looked for (1) red-shift of QD emission wavelength, and (2) Cy5 fluorescence signal of DNA.

Figure 1. Fluorescence intensity of QDs and Cy5 terminated DNA after the embedding and the purification steps.

We found that QD fluorescence is increased with the addition of more Zn precursor for forming thicker ZnS shell. Comparing the peak emission of control, the red-shift, indicating the formation of thicker shell, is observed for 3 samples embedded with DNA.

We also found that Cy5 signal from DNA is also increasing with the addition of more Zn precursor for forming thicker ZnS shell. Because the amount of DNA was kept constant for all samples, the increase in Cy5 signal suggests the increased number of DNA embedded on QD surface.

However, Cy5 signal from the control sample showed indicated the presence of free DNA even after repeated purification step. In the previous blog post (see Jan 15th, 2019), we tested the centrifugal filtration using water versus PBS buffer and demonstrated that PBS buffer removes free DNA much more effectively compared to water. However, PBS buffer also removed DNA embedded QDs through the centrifugal filter.

Based on what we reported in the previous blog and what we found in this report, further optimization of increasing the ZnS thickness is desired for two reasons. First, as shown in Figure 1, the thicker ZnS shell enhances the fluorescence intensity of QDs and the number of DNA embedded on the QD surface. Second, the thicker ZnS shell would increase the particle size, or the molecular weight, of DNA embedded QDs; therefore, it may not pass through the centrifugal filter when washed with PBS buffer.

We are currently investigating the effect of PBS buffer washing on DNA embedded QDs with thicker ZnS shell.

Hybridization of PolyT-embedded QDs and PolyA-overhang on DNA hinges

In this study, we used DNA embedded QDs and DNA hinges to test the hybridization of PolyT-embedded QDs and PolyA-modified structures.

DNA hinges, provided by Dr. Carlos Castro’s group in mechanical engineering at the Ohio State University, have PolyA sequences (overhangs) at the end of hinge arms. Hence, PolyT-embedded QDs are expected to bind to the specific sites (Figure 2(A)).

In this experiment, we incubated DNA hinges with DNA embedded QDs (1:5 molar ratio) at 50 C for 10 min. Using the transmission electron microscopy (TEM), we looked for QDs bound on DNA hinges.

As shown in Figure 2(B), we could not find QDs bound on DNA hinges.

This particular experiment was performed as a control study to test DNA embedded QDs’ forming composite structures via hybridization of PolyT-PolyA sequences. We are currently adjusting the molar ratio of QDs to hinges to see if we could find an optimal range.

Conclusion and Summary

Based on the ZnS thickness study, we believe we can enhance the fluorescence intensity of DNA embedded QDs and increase the number of DNA embedded on QD surface.

Based on the hinges binding study, we found that the hybridization of PolyT-PolyA strands needs to be better controlled and optimized. Currently, we are studying the effect of molar ratio of QDs to hinges. We hope to use this information for later experiments, including the hybridization of DNA embedded QDs with gold nanoparticles for FRET study.