Biomod/2013/HKBU/introduction

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BIOMOD 2013 The Rising Power

Contents

Project Title

Quantum Dot-Functionalized DNA Origami Serves as Effective Biomolecular Sensors on a Protein Nanofibril Platform

Project Abstract

DNA origami has enabled manipulations of sophisticated nanoscale constructions; however, the biomedical applications of DNA origami are not yet well explored. Quantum dots (QD) are well-known for their photostability and unique physical properties; they can be visualized individually under fluorescence microscopy. Previous work demonstrates the spontaneous attachment of QD onto self-assembling amyloid fibrils. In this project, self-assembling DNA origami modified with biotins was conjugated to streptavidin-QD625, and then the complex was aligned spontaneously onto biotinylated beta-amyloid fibrils. Each of the fibrils was clearly monitored under both fluorescence microscope and atomic force microscope, illustrating the effective attachment of QD-functionalized DNA origami on a well-defined self-assembling protein fibril. The whole as-prepared complex is ready to be served as biosensor for specific target molecules when a probe is attached to the QD. It is of high potential for development in biocompatible nanomaterials to detect cellular and molecular components, which advance clinically disease diagnosis.

Diagram of the hypothesis

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Step 1. Self-assembly of biotinylated Aβ fibrils. Monomeric Aβ and biotin-conjugated Aβ is mixed in 5:1 ratio.

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Step 2. Applying streptavidin onto the nanofibril platform. Streptavidin will coat the fibril through conjugating with biotin.

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Step 3. Introducing self-assembled biotin-modified DNA origami onto the nanofibril platform. The origami will bind onto streptavidin through the strong biotin-streptavidin interaction.

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Step 4. Applying streptavidin-conjugated quantum dots onto the nanofibril platform. The quantum dot will bind onto the DNA origami through the biotin-streptavidin conjugation.

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Schematic diagram of using Total Internal Reflection Fluorescence Microscopy (TIRFM) for detection of QD-functionalized DNA origami on self-assembled nanofibril platform.

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Schematic diagram of Atomic Force Microscopy (AFM) for detection of QD-functionalized DNA origami on self-assembled nanofibril platform.

Result

Gel electrophoresis of the DNA origami

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Figure 1. Gel electrophoresis of the DNA origami. All self-assembly reactions were performed in 20mM MgCl2 in a 23-hour annealing condition (see methodology). 1)100bp DNA Ladder; 2) - 9)13μM staples: 1μM scaffold; 10)100bp DNA Ladder. As is shown in the image, excess staples were separated from the assembled origami.

Height of nanofibril platform increased after attachment of the particles

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Figure 2. Average height of the biotinylated Abeta fibrils. A: AFM imaging of the fibrils under different treatment; (1) Biotinylated Abeta fibrils control; (2) Quantum dot-conjugated biotinylated Abeta fibrils; (3) Quantum dot-DNA origami-streptavidin-conjugated biotinylated Abeta fibrils. B: Diagram demonstration of the average height after attachment on nanofibril platform. When the platform is coated with streptavidin, DNA origami, and quantum dots, the height significantly increased to around 7.0 nm, compared with the control (3.2 nm). *** P<0.001.

Width of nanofibril platform increased after attachment of the particles

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Figure 3. Average width of the biotinylated Abeta fibrils. A: AFM imaging of the fibrils under different treatment; (1) Biotinylated Abeta fibrils control; (2) Quantum dot-conjugated biotinylated Abeta fibrils; (3) Quantum dot-DNA origami-streptavidin-conjugated biotinylated Abeta fibrils. B: Diagram demonstration of the average height after attachment on nanofibril platform. When the platform is coated with streptavidin, DNA origami, and quantum dots, the width significantly increased to around 53 nm, compared with the control (22 nm). *** P<0.001.

Even alignment of the particles was observed along nanofibril platform

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Figure 4. Graphical analysis of the Abeta fibrils coated with the particles (cluster of streptavidin, DNA origami, and QD). As is shown in the result, the height of the particles attached is around the same level (4.79 nm), and the distance varied a little between different particles; overall, a pattern of even alignment of the particles are clearly demonstrated.

Less quantum dots were attached onto the DNA-coated nanofibril platform

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Figure 5. Less quantum dots were attached on the nanofibril platform after the treatment with streptavidin and DNA origami. A: FM imaging of the fibrils with different treatment; (1) Quantum dot-conjugated biotinylated Abeta fibrils; (2) Quantum dot-DNA origami-streptavidin-conjugated biotinylated Abeta fibrils. B: Diagram demonstration of the amount of quantum dots attached on the nanofibril platform under different treatment. A significant lower level of the amount was observed when the platform had been treated with streptavidin and DNA origami, which are about 350 less than the QD-conjugated fibrils. *** P<0.001.

Discussion

Yielding rate of DNA origami

In the experiment, around 4.35 ng/μL of DNA could be extracted out from the agarose gel, which is around 82.65 nM according to the calculation (see Lab Logs of October). However, according to the recipes for self-assembling of DNA origami, the estimated yielding should be around 13 μM, much higher than the exact amount obtained. The low yielding rate may be caused by the inefficiency of the kit used for gel extraction. Moreover, a relatively low level of 260/230 was shown after the gel extraction, which is also thought to be caused by the remaining residue of the chemicals of the kit. Therefore, to raise the concentration of DNA origami in the future, alternative equipment such as Freeze n’ Squeeze column is suggested; to increase the purity of the DNA origami, precipitation step is necessary after the gel extraction.

Efficiency of DNA precipitation

To improve the purity of the DNA origami, DNA precipitation was performed (see methodology). According to the result, the amount of DNA would decrease by 70% to 80%, and the contaminant could be removed by around 95% in general, which reveals a high efficiency of purification but also a high sample loss. The loss in DNA sample may be caused during the removal of ethanol; however, since both AFM imaging and FM imaging showed positive results on the existence of DNA origami, the large amount of loss did not lead to the failure of quantum dot conjugation.

The decreased efficiency of quantum dot alignment

According to the results in fluorescent microscopic study, clear blinking fibril patterns were observed when applying onto the beta-amyloid with streptavidin (STV), DNA origami, and quantum dots (QD) sequentially. Nonspecific binding effects were minimized by blocking the rest space with 1% BSA solution and the washing using PB buffer between each step. Thus, if the QD exist, they are much more likely to connect to the DNA origami rather than exist everywhere, which could be confirmed from the images. However, compared with the fibril with QD added, less QD were found to attach onto the fibril, which is presumably caused by the repulsion effects between streptavidin molecules and DNA molecules. Since DNA origami are negatively charged biomolecules, they tend to be dispersed from each other rather than forming firm aggregates; therefore, less DNA origami can bind onto the streptavidin-coated nanofibril platform, thus leading to less binding of QD. The atomic force microscopic observation provided evidence to support the assumption of repulsion; not only the binding patterns of STV-DNA-QD particles along the fibrils were clearly shown, but also when comparing with the Abeta+QD sample, the STV-DNA-QD particles are much more bulky and dispersed. But still, the quantitative study on the interaction between streptavidin and DNA origami molecules need to be further analyzed in the future to deepen the understanding.

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Hypothesis of Repulsion

Potential Application

Drug delivery

In the next step, we hope to identify a medical substance whose chemical structure can be conjugated onto the small biotinylated DNA origami, and the whole as-prepared molecule can be delivered to target molecules, such as abeta fibrils, therefore demonstrates pharmacological effects as well as advances clinically disease treatment. This requires much more efforts in reading and conducting experiments, but the positive result of QD-functionalized DNA origami at current stage has opened up a promising future for DNA origami in the field of drug delivery.


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