Thursday, 15 January 2009 00:00

A Yoctoliter-Scale DNA Reactor for Small-Molecule Evolution

Margit Haahr Hansen, Peter Blakskjær, Lars Kolster Petersen, Tara Heitner Hansen, Jonas Westergaard Højfeldt, Kurt Vesterager Gothelf, and Nils Jakob Vest Hansen

The center of DNA three-way junctions, constituting a yoctoliter (10-24 L) volume, is applied as an efficient reactor to create DNA-encoded libraries of chemical products. Amino acids and short peptides are linked to oligonucleotides via cleavable and noncleavable linkers. The oligonucleotide sequences contain two universal assembling domains at the center and a distal codon sequence specific for the attached building block. Stepwise self-assembly and chemical reactions of these conjugates in a combinatorial fashion create a library of pentapeptides in DNA three-way junctions in a single reaction vessel. We demonstrate the formation of an evenly distributed library of 100 peptides. Each library member contains a short synthetic peptide attached to a unique genetic code creating the necessary “genotype-phenotype” linkage essential to the process of in vitro molecular evolution. Selective enrichment of the [Leu]-enkephalin peptide from an original frequency of 1 in 10 million in a model library to a final frequency of 1.7% in only two rounds of affinity selection is described and demonstrates successful molecular evolution for a non-natural system

CDNA highlights

March 2, 2010

Single-molecule chemical reactions on DNA origami

Researchers at the Danish National Research Foundation’s Centre for DNA Nanotechnology (CDNA) at iNANO demonstrate that it is possible to control chemical reactions on DNA nanostructures and generate images of reactions of individual molecules. The results were published on 28 February in Nature Nanotechnology. The article is available here

CDNA news

elena February 12, 2010

Electrochemical assay for attomole detection of DNA

The project was carried out in collaboration between CDNA and DTI. We combined a magnetic bead sandwich hybridization capture assay used for pre-concentration and bioseparation of target DNA, with a lipase-based amplification and electrochemical readout system. The signal amplification in the DNA assay is based on the catalytic activity of a lipase enzyme, and this “electrochemical blotting” concept is principally new. The use of this hydrolytic enzyme allowed for close to few-molecule detection of lipase-labeled DNAs at the electrode surface, due to accumulation of the catalysis product (ester bond cleavage and removal of the redox label from the zone of electrochemical reaction). The developed electrochemical lipase- and magnetic beads-based sandwich hybridization assay represents a fundamentally new electrochemical approach for sensitive DNA detection.

ChemComm link