Our goal was to develop a bifunctional peptide linker that could allow easy attachment of DNA oligonucleotides at one end while the other is modified with a polyhistidine tag to facilitate self-assembly of the full peptide-DNA complex onto QDs via metal-histidine interactions.
We have developed a conjugation strategy based on metal-affinity-driven
interactions between CdSe-ZnS core-shell QDs and proteins or peptides appended with polyhistidine (Hisn) tags. … The His tag drives self assembly by interacting directly with the metallic surface …
- (DNA) DNA conjugates of target DNA are thiolated (sulf-hydride group attached):
- (linker) The synthesis of the peptide module was performed by standard solid-phase peptide synthesis (SPPS) on Rink amide resin to create the desired His6-Cys sequence.
The crude peptide was precipitated, and the final bifunctional reactive linker His6-Cys(Ac)-S-S-Py was obtained through a direct disulfide exchange reaction.
(the initial reactive chemistry for His6 attachment to thiolated-DNA is one of the fastest and most common linkage chemistries used in bioconjugation)
- (tag) Functionalization of free 5‘-thiol DNA with the peptide linker (4) to yield the His6-tailed oligo (5) was rapid and straightforward …
and the mixture was allowed to react anywhere from 1 h to overnight
- (Quantum Dot) The His tag drives self assembly by interacting directly with the metallic surface [of the Quantum Dot]
The QD-peptide-DNA conjugates were further characterized by atomic force microscopy (AFM) imaging, where association of oligos with a central QD was observed only for samples made of QDs mixed with His6-peptide-DNA …
We also demonstrate the potential utility of this His-reactive-peptide modification of DNA by assembling and testing a QD-DNA molecular beacon that specifically detected the presence of its complementary sequence.
The potential of this reactive linker was demonstrated by self-assembling several QD-DNA conjugates as well as a QD-MB construct able to discriminate between different sequences of DNA. A variety of other applications, such as highly luminescent multilabeled hybridization probes, are possible using this construct. Preforming MB sensors with different color QDs and then mixing them may allow “multiplexing”. Beyond nanoparticle-MBs, this selfassembly technique may be applicable to attaching biomolecules to a variety of other similarly prepared surfaces.
And now for the shortened version. Above, you will see a very stylized expression of the process of self-assembly. Again, we will do this by steps:
- Obtain the His6 tags (Polyhistidine-tag)
- Obtain Cy5 dye (a fluorescent/fluorophore nano particle)
- Obtain the Thiolated DNA
- Label the Thiolated DNA with Cy5 dye, the dye becoming a quenched fluorophore;
- Obtain the Quantum Dots
- Combine where:
- The Thiolated DNA, which has an affinity for the one end of the His6 tag, becomes attached;
- The His6 tag, which has an affinity for the DHLA covering of the Quantum Dot, becomes attached;
- (not a production step, but found in the illustration prior to the addition of the complementary DNA) Excite the batch with high energy light as seen in the center of the graphic where:
- the proximity of the Cy5 particle in the His6–Thiolated DNA and the Quantum Dot allows the molecular beacon to absorb energy through FRET (Förster resonance energy transfer) and emit a color shifted light in fluorescence;
- (not a production step, but, rather, the set-up for a test of the marker beacon) Obtain and add Complementary DNA to the process
- the molecular beacon (the His6–Thiolated DNA–Cy5) unzips, and takes on the Complementary DNA;
- (not a production step, but, rather, a test of the marker beacon) The right side of the graphic shows the action the new bioconjugated Quantum Dot undergoes to stimulation by (a now second) high energy light to emit a color shifted light in fluorescence.