Enzymatic ligation of large biomolecules to DNA.

The ability to synthesize, characterize, and manipulate DNA forms the foundation of a range of advanced disciplines including genomics, molecular biology, and biomolecular engineering. In particular for the latter field, DNA has proven useful as a structural or functional component in nanoscale self-assembled structures, antisense therapeutics, microarray diagnostics, and biosensors. Such applications frequently require DNA to be modified and conjugated to other macromolecules, including proteins, polymers, or fatty acids, in order to equip the system with properties required for a particular application. However, conjugation of DNA to large molecular components using classical chemistries often suffers from suboptimal yields. Here, we report the use of terminal deoxynucleotidyl transferase (TdT) for direct enzymatic ligation of native DNA to nucleotide triphosphates coupled to proteins and other large macromolecules. We demonstrate facile synthesis routes for a range of NTP-activated macromolecules and subsequent ligation to the 3' hydroxyl group of oligodeoxynucleotides using TdT. The reaction is highly specific and proceeds rapidly and essentially to completion at micromolar concentrations. As a proof of principle, parallelly labeled oligonucleotides were used to produce nanopatterned DNA origami structures, demonstrating rapid and versatile incorporation of non-DNA components into DNA nanoarchitectures.

Synthesis of dopamine and serotonin derivatives for immobilization on a solid support.

The two important neurotransmitters dopamine and serotonin are synthesized with short PEG tethers and immobilized on a magnetic solid support. The tether is attached to the aromatic moiety of the neurotransmitters to conserve their original functional groups. This approach causes minimal alteration of the original structure with the aim of optimizing the immobilized neurotransmitters for aptamer selection by SELEX. For the dopamine derivative, the tether is attached to the aromatic core of a dopamine precursor by the Sonogashira reaction. For serotonin, a link to the indole core is introduced by a Claisen rearrangement from the allylated phenol moiety of serotonin. The tethers are azide-functionalized, which enables coupling to alkyne-modified magnetic beads. The coupling to the magnetic beads is quantified by UV spectroscopy using Fmoc-monitoring of the immobilized dopamine and serotonin derivatives.