Improved peptidyl linkers for self-assembly of semiconductor quantum dot bioconjugates

Abstract

We demonstrate improved peptide linkers which allow both conjugation to biomolecules such as DNA and self-assembly with luminescent semiconductor quantum dots. A hexahistidine peptidyl sequence was generated by standard solid phase peptide synthesis and modified with the succinimidyl ester of iodoacetamide to yield a thiol-reactive iodoacetyl polyhistidine linker. The reactive peptide was conjugated to dye-labeled thiolated DNA which was utilized as a model target biomolecule. Agarose gel electrophoresis and fl uorescence resonance energy transfer analysis confi rmed that the linker allowed the DNA to self-assemble with quantum dots via metal-affi nity driven coordination. In contrast to previous peptidyl linkers that were based on disulfi de exchange and were thus labile to reduction, the reactive haloacetyl chemistry demonstrated here results in a more stable thioether bond linking the DNA to the peptide which can withstand strongly reducing environments such as the intracellular cytoplasm. As thiol groups occur naturally in proteins, can be engineered into cloned proteins, inserted into nascent peptides or added to DNA during synthesis, the chemistry demonstrated here can provide a simple method for self-assembling a variety of stable quantum dot bioconjugates.

Quantum Dot DNA Bioconjugates: Attachment Chemistry Strongly Influences the Resulting Composite Architecture

“…20 We, and others, have demonstrated that a variety of QDs can be self-assembled with proteins and peptides expressing clearly available (His) n -sequences in a rapid manner (<30 min) due to the high-affinity equilibrium binding constants of this solution interaction ( K d −1 ~1 nM). 20,21,26–29 Importantly, control can be exercised over the ratio or valence of molecules assembled per QD through the molar equivalents utilized. We have previously shown that DNA sequences terminally-modified with (His) n -peptidyl sequences can also self-assemble to QDs in the same manner.…”

“…We have previously shown that DNA sequences terminally-modified with (His) n -peptidyl sequences can also self-assemble to QDs in the same manner. 21,30 Here, we exploit aniline-catalyzed hydrazone ligation chemistry to join the modified backbone DNA to a (His) 6 -peptidyl sequence (Figure 1B). The Dawson lab has shown that this coupling reaction is characterized by enhanced bioconjugation rates of 10 1 – 10 3 M −1 s −1 in mild, aqueous conditions (slightly acidic to neutral pH) and can reach equilibrium ( K eq = 2.3×10 6 M −1 ) in under 30 min using 100 mM aniline catalyst with 10 µM of reactants.…”

Peptide nucleic acid probe for protein affinity purification based on biotin–streptavidin interaction and peptide nucleic acid strand hybridization

“…2), were synthesized from PNA monomers followed by the addition of three 2-(2-aminoethoxy)ethoxy acetic acid (eg1) groups on an Expedite 8909 Nucleic Acid Synthesis System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) using the standard manufacturer's protocols for Fmoc chemistry (2-lmol scale). Iodoacetate was added to the third eg1 group on the linker by reacting the resin-bound PNA strand with 2 mM N-succinimidyl iodoacetate by the method of Berti and coworkers [26]. Biotin was added to the third eg1 group of the linker on another PNA strand with complementary base sequence using 2 mM (+)-biotin Nhydroxysuccinimide ester (Alfa Aesar, Ward Hill, MA, USA) as described by Guerasimova [27].…”

Quantum dots: a powerful tool for understanding the intricacies of nanoparticle-mediated drug delivery

“…Improved NP and biomolecule chemistries allowing for orthogonal attachment of proteins and other (bio) molecules as desired with control over number and orientation need to be pursued [114]. Beyond what is currently utilized, several established and developing chemistries are available to draw from, including self-assembly driven by polyhistidine or other peptidyl sequences [24,115,116], various chemoselective ligations [117,118], and the family of 'click' cycloaddition and related chemistries [119] to name but a Expert Opin. Drug Deliv.…”