Conjugation of Oligonucleotides to Gold Nanoparticles

1. Introduction

The conjugation of oligonucleotides to gold nanoparticles combines the unique properties of both components. Gold nanoparticles offer optical, electrical, and catalytic properties, while oligonucleotides provide molecular recognition and programmability. This protocol details a common approach to achieve this conjugation, which is widely used in bioassays, sensing, and nanobiotechnology applications.

2. Materials

Gold Nanoparticles: Citrate-stabilized gold nanoparticles with a desired diameter (e.g., 10¨C50 nm). The choice of size can affect the final application; smaller nanoparticles may be more suitable for some bioassays, while larger ones might be better for certain imaging applications.

Oligonucleotides: Thiol-modified oligonucleotides. The thiol group can be attached to either the 5'- or 3'- end. The sequence of the oligonucleotide depends on the specific application, such as for target recognition in a bioassay.

Buffers and Reagents

Sodium Phosphate Buffer: Prepare a 0.15 M sodium phosphate buffer, pH 8.5, for the reduction of thiol-modified oligonucleotides.

Dithiothreitol (DTT): 0.1 M DTT solution in the sodium phosphate buffer for reducing the disulfide bonds in trityl-S-S-oligo.

Sodium Chloride (NaCl): 1 M NaCl solution for the "salt-aging" step that facilitates the binding of the oligonucleotide to the gold surface.

Storage Buffer: A buffer such as 10 mM sodium phosphate buffer, pH 7.0, containing 100 mM NaCl and 0.01% (w/v) NaN? for storing the final conjugate.

Equipment

Microcentrifuge Tubes: 1.5 ml microcentrifuge tubes for carrying out the reactions.

Centrifuge: Capable of reaching appropriate speeds for pelleting the oligonucleotide-gold conjugate based on the size of the gold nanoparticles (refer to a centrifuge speed-size table for optimal settings).

Spectrophotometer: To measure the optical density of the nanoparticles and the oligonucleotide solutions, which helps in determining concentrations and the success of the conjugation.

Nap 5 Column (GE Healthcare): For separating reduced oligonucleotides from trityl-SH and DTT.

3. Procedure

3.1 Reduction of Thiol-Modified Oligonucleotides

Buffer Preparation: First, make a 0.15 M sodium phosphate buffer with a pH of 8.5. Add 0.1 M DTT to this buffer. The correct pH is crucial for the proper reduction of the oligonucleotide.

Oligonucleotide Dissolution: Take the lyophilized thiol-modified oligonucleotide and dissolve it in water to a final concentration of 500 ¦ÌM.

Mixing for Reduction: Combine 50 ¦Ìl of the dissolved oligonucleotide with 450 ¦Ìl of the prepared sodium phosphate buffer containing DTT.

Incubation: Incubate this mixture at room temperature for 1-2 hours. This incubation period allows the DTT to reduce the disulfide bonds in the trityl-S-S-oligo, activating the thiol group for conjugation.

Separation: Use a Nap 5 column (operated in water) to separate the reduced oligonucleotide from trityl-SH and DTT. The final eluate from the Nap 5 column will be approximately 1 ml in volume with an approximate concentration of 25 ¦ÌM.

Concentration Determination: Measure the exact concentration of the final eluate using UV-vis spectroscopy. Take the absorbance at 260 nm and use the appropriate extinction coefficient for the oligonucleotide to calculate the concentration.

3.2 Conjugation of Thiolated Oligonucleotide to Gold Nanoparticles

Resuspension of Gold Nanoparticles: Take a vial of lyophilized gold nanoparticles and resuspend it in 740 ¦Ìl of water. Transfer this resuspended solution into a 1.5 ml microcentrifuge tube.

Addition of Reduced Thiolated Oligonucleotide: Add 160 ¦Ìl of the reduced thiolated oligonucleotide at a concentration of 7.5 ¦ÌM (0.0075 nmol/¦Ìl) in water to the gold nanoparticle solution. Incubate this mixture at room temperature for at least 1 hour. The 7.5 ¦ÌM oligonucleotide concentration is a good starting point, but if aggregation or poor sensitivity is observed in later applications, adjust the oligonucleotide concentration according to the particle size range (for example, for a 30-nt oligonucleotide, different concentrations can be tried as recommended in relevant literature).

Salt-Aging Step: Add 100 ¦Ìl of 1 M NaCl to the mixture. Incubate the solution at room temperature for at least 1 hour. This "salt-aging" step helps to screen the electrostatic repulsion between the negatively charged gold nanoparticles and the oligonucleotides, facilitating the binding of the oligonucleotide to the gold surface. Longer incubation times may improve the surface coverage of the oligonucleotides on the gold nanoparticles.

Centrifugation: Centrifuge the mixture at the appropriate speed for the specific size of the gold nanoparticles for 30 minutes. This step pellets the oligonucleotide-gold conjugate. Refer to a pre-determined table that correlates gold nanoparticle size with the optimal centrifugation speed.

Removal of Supernatant: Carefully remove the supernatant from the microcentrifuge tube.

Resuspension: Resuspend the conjugate in 200 ¦Ìl of the storage buffer. If 100% recovery has been achieved, the optical density of the particles should be 10.

Measurement and Adjustment: Measure the optical density of the conjugate using a spectrophotometer. Adjust the concentration as desired, either by dilution or further concentration methods.

Storage: Store the conjugate at +4 ¡ãC for future use.

4. Troubleshooting

Aggregation: If the gold nanoparticles aggregate during the conjugation process, it could be due to incorrect oligonucleotide concentration, improper salt-aging conditions, or issues with the gold nanoparticle stability. Try adjusting the oligonucleotide concentration as described above, and ensure that the salt-aging time and temperature are followed precisely. If the gold nanoparticles were not properly stabilized initially, they may be more prone to aggregation. Consider using a different batch of gold nanoparticles or optimizing their stabilization method.

Low Conjugation Efficiency: Low conjugation efficiency may be observed if the reduction of the thiol-modified oligonucleotide was incomplete, or if the binding conditions were not optimal. Check the concentration of the reduced oligonucleotide accurately before conjugation. Also, ensure that the incubation times for both the reduction step and the conjugation steps are sufficient. If the problem persists, consider using a different protocol or modifying the reaction conditions, such as adjusting the pH or the ratio of oligonucleotide to gold nanoparticles.

5. Conclusion

This protocol provides a step-by-step guide to conjugate oligonucleotides to gold nanoparticles. By following these steps carefully, researchers can obtain stable oligonucleotide-gold nanoparticle conjugates that can be used in a variety of applications, such as bioassays for the detection of specific molecules, imaging in biological systems, and the development of novel nanobiotechnological tools. However, it is important to note that the success of the conjugation may vary depending on the quality of the starting materials, the accuracy of the procedures, and the specific requirements of the intended application.