Conjugation of Oligonucleotides to Gold Nanoparticles

Description

Oligonucleotide functionalized gold nanoparticles are used in a wide range of applications, with the most common being as probes in bio assays for recognition and detection of other molecules. For example, aptamers can be used to functionalize gold nanoparticle and a depending on the aptamer used, a probe can be generated to bind any molecule of choice.

Oligonucleotides are generally conjugated to gold nanoparticles by utilizing a thiol linker chemically introduced to either the 5'- or 3'-end. Thiols have very strong affinity for gold surfaces and a thiol-modified oligonucleotide (or aptamer) can be conjugated by directly mixing the modified oligo with gold nanoparticles followed by a "salt-aging" procedure that facilitates the binding of the oligo to the gold surface.   This method results in a stable gold nanoparticle oligonucleotide conjugate that retains the ability to bind complementary targets. However, this classic method is time consuming due to the required "salt-aging" step and can take up to two days to complete. Further, the gold nanoparticle size is restricted to smaller gold nanoparticles and cannot successfully generate oligonucleotide gold nanoparticles with diameters of 30nm and above.

In comparison, Cytodiagnostics OligoREADY gold conjugation kits have been optimized for high efficiency one-step conjugation of thiolated oligonucleotides eliminating the time consuming "salt-aging” steps and size restrictions. Simply mix your reduced thiol-modified oligonucleotide with the pre-activated gold nanoparticles supplied in our kits and your conjugate is ready to be used within 2 hours. As in the classic method above, conjugation of the oligonucleotide is achieved by the formation of a strong and stable gold-thiol bond without any additional linkers. A standard protocol for conjugation of a thiol-modified oligonucleotide to our gold nanoparticles is described below.

Conjugation of Oligonucleotides to Gold Nanoparticles - Schematic image

Figure 1. Conjugation of a reduced thiolated oligonucleotide to Cytodiagnostics unique OligoREADY gold nanoparticles.

Features & Benefits

  • No size restrictions. Allows conjugation of oligonucleotides to gold nanoparticles with sizes between 5nm-100nm.
  • Fast and convenient one-step conjugation reaction with no pre-activation requirements or manipulation of the gold nanoparticles. 
  • Does not require time-consuming "salt-aging” procedures. Reaction can be completed in 2 hours or less.
  • Results in the thiol-oligonucleotide conjugated directly to the gold surface without any linkers.

Materials Required

  • OligoREADY Gold Nanoparticles
  • Thiol-modified (5'- or 3'-end) Oligonucleotide 
  • 1M NaCl

Step I: Reduction of thiol-modified oligonucleotides (e.g. trityl-S- S-Oligo) 

  1. Prepare a 0.15 M sodium phosphate buffer, pH 8.5 supplemented with 0.1 M DTT. Note that the pH in this reaction is important for proper reduction of oligonucleotide.
  2. Dissolve lyophilized oligonucleotide to a final concentration of 500 µM in H2O.
  3. Mix 50 µl of dissolved oligonucleotide with 450 µl sodium phosphate buffer.
  4. Incubate 1-2 hours at room temperature to reduce oligonuclotide.
  5. Separate reduced oligonucleotide from trityl-SH and DTT using a NAP 5 column operated in H2O, GE Healthcare.
  6. Final eluate from NAP 5 column will be 1ml in H2O with an approximate concentration of 25 µM.

Note: The exact concentration of final eluate in step 6 above can be measured with UV-VIS spectroscopy by measuring the absorbance at 260nm.

 

Step II: Conjugation of thiolated oligonucleotide to OligoREADY gold nanoparticles

  1. Resuspend one vial of lyophilized OligoREADY gold nanoparticle with 740 µl of H2O.
  2. Transfer into a 1.5 ml microcentrifuge tube.
  3. Add 160 µl of reduced thiolated oligonucleotide at 7.5 µM (0.0075 nmol/µl)* in H2O as prepared above and incubate for at least 1 hour at room temperature.

    *Note:     7.5 µM oligonucleotide is a good starting concentration, but if aggregation or poor sensitivity is observed, the following oligonucleotide concentrations can be attempted for a given particle size range (based on a 30nt oligonucleotide):

     Particle size (nm)

    5

    10-20

    30-100

    [oligonucleotide] (µM)

    5-50

    5-25

    5-15
  4. Add 100 µl of 1M NaCl
  5. Incubate for at least 1 hour at room temperature to allow binding of the oligonucleotide to the gold surface. Longer incubation times may improve surface coverage.
  6. Centrifuge at the appropriate speed for your particular gold nanoparticle size (see table I) for 30 minutes to pellet your oligonucleotide gold conjugate.
  7. Remove supernatant
  8. Resuspend conjugate in 200 µl of storage buffer. The optical density of the particles should be 10 if a 100% recovery has been achieved.
    Common storage buffer: 10 mM sodium phosphate buffer, pH 7.0, 100 mM NaCl and 0.01% (w/v) NaN3.
  9. Measure optical density with a spectrophotometer and adjust concentration as desired.
  10. Store conjugate at +4°C

 

Table I. Recommended gold nanoparticle centrifugation settings. Values in the table below are for a 1 ml gold nanoparticle volume in a standard 1.5 ml microcentrifuge tube using a table-top centrifuge.

 Gold Nanoparticle Diameter Centrifugation Force MWCO Spin Columns
5nm N/A Required
10nm 22,000 x g * Highly recommended
15nm 17,000 x g  Recommended
20nm 10,000 x g Optional
30nm 2,500 x g  Optional
40nm 1,400 x g Optional
50nm 1,100 x g Optional
60nm 900 x g Optional
70nm 700 x g Optional
80nm 600 x g Optional
90nm 500 x g Optional
100nm 400 x g Optional

*For 10nm gold nanoparticles the recovery is estimated to be approximately 50% at this particular speed. For better recovery, 1) use an ultracentrifuge to achieve higher speeds or 2) use 100kDa MWCO Spin Columns (if molecular weight of the conjugated oligonucleotide is <100kDa). While not required for sizes above 10nm, the use of 100kDa MWCO Spin Columns will still improve product recovery. For centrifugation speeds and time for MWCO Spin Columns, please refer to Table II below.

Table II. Recommended centrifugation speeds and time for MWCO Spin Columns.

 Spin Column Size
 		Centrifugation Force Centrifugation Time
0.5 mL 10,000 x g 10 min.
4 mL 1,700 x g 10 min.
15 mL 1,700 x g 10 min.

 

Agarose Gel Analysis of Conjugated OligoREADY Gold Nanoparticles

Figure 2. 0.5% (w/v) agarose gel analysis of 50nm OligoREADY gold nanoparticles before and after conjugation with a thiol-modified oligonucleotide (20 bp). Gel was operated at 100V constant for in 0.5X TBE buffer for 30 minutes.

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