Phosphoramidate Ligation of Oligonucleotides in Nanoscale Structures

Abstract

The folding of long DNA strands into designed nanostructures has evolved into an art. Being based on linear chains only, the resulting nanostructures cannot readily be transformed into covalently linked frameworks. Covalently linking strands in the context of folded DNA structures requires a robust method that avoids sterically demanding reagents or enzymes. Here we report chemical ligation of the 3'-amino termini of oligonucleotides and 5'-phosphorylated partner strands in templated reactions that produce phosphoramidate linkages. These reactions produce inter-nucleotide linkages that are isoelectronic and largely isosteric to phosphodiesters. Ligations were performed at three levels of complexity, including the extension of branched DNA hybrids and the ligation of six scaffold strands in a small origami.

Enzyme‐Free Ligation of 5′‐Phosphorylated Oligodeoxynucleotides in a DNA Nanostructure

“…The reagents for chemical ligation are also less expensive than the enyzmes and substrates used in enzymatic protocols. Since the band pattern of phosphodiester products observed here are quite similar to those observed for phosphoramidate ligation, we suspect that local folding is as important as chemical reactivity in the ligation reaction. The phosphodiester products reported here have the advantage of being the natural substrates of polymerases and other enzymes of molecular biology, so that standard procedures can be used to process the products of the long strands obtained by chemical ‘in‐origami’ ligation.…”

“…It consists of a planar DNA origami sheet, in which six 5′‐phosphorylated oligonucleotides, between 97 and 104 nucleotides in length, act as scaffold strands ( 1a – f ), and 19 shorter oligonucleotides, 21 – 45 nucleotides in length, act as staple strands ( 2a – s ). The sequence chosen is that of the main portion of the scaffold dubbed ‘M1.3’, which was also used in our earlier studies on phosphoramidate ligations . The full sequences of all strands are listed in the Supporting Information ( SI ).…”

“…Recently we were able to show that the simultaneous ligation of six 100mer oligonucleotides to form a 600mer can be induced in a preassembled small DNA origami when the 3′‐termini of the strands feature 3′‐amino‐2′,3′‐dideoxynucleosides and the 5′‐termini are phosphorylated . The resulting linkages between the synthetic strands are phosphoramidates, though, not natural phosphodiesters.…”

Capturing and Stabilizing Folded Proteins in Lattices Formed with Branched Oligonucleotide Hybrids

“…The propensity of hybrids to self‐assemble can be tuned through changes in the structure of the organic core . Expanded versions of such hybrids containing triplex‐binding motifs have been developed for capturing cofactors . However, the pore size of the materials formed by the self‐assembling hybrids with short sticky ends are too small to accommodate proteins.…”

Chemical ligation of oligonucleotides using an electrophilic phosphorothioester

“…In general, the chemical ligation between two functional groups requires the presence of condensing reagents. Since the first chemical ligation of ONs was achieved with a water-soluble carbodiimide (12), various condensing reagents, for example 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDCI) (13,14), cyanogen bromide (15), imidazole derivatives (16,17) and 1-hydroxybenzotriazole (HOAt) (18), have been utilized to activate the phosphate (PO) group (19). Additionally, a variety of functional group pairs, for example, a nucleophilic group and an electrophilic group, or alkyne and azide groups, have been examined for use in chemical ligations with various applications in biology and biotechnology (2,20).…”