The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5'-azide and 3'-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.

A generic strategy to expand the analytical scope of electrical nanopore sensing is presented. We specifically and electrically detect the activity of a diagnostically relevant hydrolytic enzyme and remove the analytically harmful interference from the biochemically complex sample matrix of blood serum. Our strategy is demonstrated at the example of the renin protease which is involved in regulation of blood pressure. The analysis scheme exploits a new approach to reduce sample complexity while generating a specific read-out signal. Within a single spin-column (i), the protease cleaves a resin-tethered peptide substrate (ii) which is affinity-purified using the same multifunctional resin to remove interfering blood serum components, followed by (iii) detecting the peptide via electrical nanopore recordings. Our approach is beneficial in several ways. First, by eliminating serum components, we overcome limitations of nanopore sensing when challenging samples lead to membrane instability and a poor signal-to-noise ratio. Second, the label-free sensing avoids drawbacks of currently used radiolabel-immunoassays for renin. Finally, the strategy of simultaneous generation and purification of a signal peptide within a multifunctional resin can very likely be expanded to other hydrolytic enzymes dissolved in any analyte matrix and exploited for analytical read-out methods other than nanopore sensing.

We describe the synthesis of 2'-deoxyuridine-5'-triphosphate derivatives bearing linkers of varying length, bulk and flexibility, at position 5 of the pyrimidine base. Nucleotide analogues with terminal functional groups are of interest due to their application potential for the functional labelling of DNA strands. In the course of the synthesis of the nucleotide analogues, the methodology for the Yoshikawa phosphorylation procedure was optimised, resulting in an approach which reduces the amount of side-products and is compatible with labile functional groups attached to the base. The effect of linker composition on the enzymatic incorporation into DNA was systematically investigated using two different DNA polymerases. Deep Vent(R) exo(-) from the B-polymerase family accepted most nucleotide analogues as substrates, while Taq from the A-family was slightly less proficient. Both polymerases had difficulties incorporating 5-(3-amino-prop-1-ynyl)-2'-deoxyuridine triphosphate. A molecular model of the active site of the polymerase was used to rationalise why this nucleotide was not accepted as a substrate.