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I've been looking for methods for immobilizing 5'-Thiol modified dsDNA on gold slides. Most of what I've come across suggests using DTT followed by a desalting step or using TCEP. However, I haven't had any luck reducing and then conjugating the DNA onto the gold slides. I've seen some methods use DTT, and some use TCEP, and some include an incubation step with MCH. It's been difficult finding a complete and satisfactorily detailed method online.

The DNA I am using is a 1 kb long double-stranded molecule with a 5'-Thio modification at one end and a 5'6-FAM modification at the other end. I need to synthesize the strands myself via PCR using two modified primers and a template strand rather than ordering the fully modified 1 kb strand, so my working concentrations are usually fairly low (~70-100 ng/uL after PCR and spin-column purification).

Can anyone provide a detailed method that has worked for them for reducing thiol modified DNA and immobilizing it onto gold surfaces or gold monolayers?

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  • $\begingroup$ The MCH method is described for ssDNA. Would it still work for dsDNA is up in air. $\endgroup$ Apr 3, 2023 at 16:38

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The most of the procedures you have described are for binding single-stranded DNA (ss-DNA) to gold surfaces via thiolate bonds. To my knowledge, I'm not sure (note that I'm not an expert on this field) these methods would work for double-stranded DNA (ds-DNA) without some modifications. However, the authors of Ref.1 have claimed, to their knowledge, the first procedure for binding DNA (supposedly ds-DNA) via thiolate bonds to gold surfaces.

Accordingly, thiol-substituted deoxy-oligonucleotides were used as primers in PCR, which were obtained by esterification of a 6-mercapto-hexanol-group to the 5'-phosphate of oligonucleotides (20-mers) using a $\beta$-cyanoethyl-phosphoramidite C6-thiol-modifier. The fragments were amplified by PCR using either thiol-modified oligonucleotides or, as a control, unmodified oligonucleotides. To avoid oligomerization of the oligonucleotides during the PCR reaction, dithiothreitol (DTT) with final concentration $\pu{2 mM}$ has been used in the polymerization mixture. After PCR, the DNA fragments were extracted with phenol, precipitated with ethanol, and purified by agarose gel electrophoresis. Since residual agarose, even in minute amounts, might be mistakenly interpreted as DNA threads, the thiolated DNA fragments were further purified by chromatography on Elu Tip d according to a standard protocol given in Ref.2. Purified DNA was dissolved in either C Tris-$\ce{HCl}$, $\mathrm{pH}$ 7.5, or $\pu{10 mM}$ ammonium acetate.

Prior to use, a 200-fold excess of DTT should be added to $\pu{0.1 \mu M}$ solution of DNA fragments, and incubated for $30$-$\pu{60 min}$. The template-stripped gold surfaces were prepared as described in Ref.3. Following the stripping with tetrahydrofurane the mica sheet was lifted gently using a slight vacuum suction. The gold-platelets were dried in a desiccator for $\pu{15 min}$ prior to use.

Routinely, a droplet of $\pu{1 \mu L}$ ($0.3$-$\pu{0.5 fmol}$) of DNA in DTT and either $\pu{10 mM}$ ammonium acetate or Tris-$\ce{HCl}$ buffer was transferred onto the template-stripped gold surface, and left for $45$-$\pu{60 min}$ at room temperature at 100% relative humidity; then dried for $10$-$\pu{30 min}$ in a desiccator, and rehydrated in either ammonium acetate or Tris-buffer. This cycle of drying and rehydration - which should not irreversibly denature the DNAs - was found to lead to a fair amount of DNA being anchored to the gold surface. After mounting in the instrument to be used, the fluid cell was flushed with either ammonium acetate or Tris-buffer, which removed non-thiolate-anchored DNA. In principle, thiolated DNA fragments can be anchored to gold directly from the droplet solution without drying; however, the amount bound was then found to be quite small.

Also read Ref.4 for most recent work in the subject.

References:

  1. Martin Hegner, Peter Wagner, and Giorgio Semenza, "Immobilizing DNA on gold via thiol modification for atomic force microscopy imaging in buffer solutions," FEBS Lett. 1993, 336(3), 452-456(DOI: https://doi.org/10.1016/0014-5793(93)80854-N).
  2. Seldon, R.F. and Chory, J. (1993) in: Current Protocols in Molecular Biology (Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. eds.) Greene Publishing Associates and Wiley-Interscience: New York, NY, chap. 2.6, Suppl. 20.
  3. Martin Hegner, Peter Wagner, and Giorgio Semenza, "Ultralarge atomically flat template-stripped Au surfaces for scanning probe microscopy," Surface Sci. 1993, 291(1-2), 39-46 (DOI: https://doi.org/10.1016/0039-6028(93)91474-4).
  4. Janese C. O'Brien, Vivian W. Jones, Marc D. Porter, Curtis L. Mosher, and Eric Henderson, "Immunosensing Platforms Using Spontaneously Adsorbed Antibody Fragments on Gold," Anal. Chem. 2000, 72(4), 703–710 (DOI: https://doi.org/10.1021/ac990581e).
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