Efficient preparation of internally modified single-molecule constructs using nicking enzymes

Abstract

Investigations of enzymes involved in DNA metabolism have strongly benefited from the establishment of single molecule techniques. These experiments frequently require elaborate DNA substrates, which carry chemical labels or nucleic acid tertiary structures. Preparing such constructs often represents a technical challenge: long modified DNA molecules are usually produced via multi-step processes, involving low efficiency intermolecular ligations of several fragments. Here, we show how long stretches of DNA (>50 bp) can be modified using nicking enzymes to produce complex DNA constructs. Multiple different chemical and structural modifications can be placed internally along DNA, in a specific and precise manner. Furthermore, the nicks created can be resealed efficiently yielding intact molecules, whose mechanical properties are preserved. Additionally, the same strategy is applied to obtain long single-strand overhangs subsequently used for efficient ligation of ss- to dsDNA molecules. This technique offers promise for a wide range of applications, in particular single-molecule experiments, where frequently multiple internal DNA modifications are required.

Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

“…In this way, DNA oligomer FL905 can be inserted between the two Nt.BbvCI sites according to a strategy described in Fig. 2A (a similar strategy was used to study DNA recombination by Levene and coworks16 and label large DNA fragments1718). Because rx pAB1_FL905 is the only theoretical ligation product, the production yeild should be near 100%.…”

Fork sensing and strand switching control antagonistic activities of RecQ helicases

“…Short DNA hairpins (40, 90 bp) were prepared by annealing synthetic DNA oligomers, with one of them being biotinylated on the 5′-end, followed by ligation48. The long DNA hairpin was prepared by ligating two dsDNA PCR fragments, each consisting of a 488 bp long homologous region followed by a shorter heterologous region, at their homologous ends.…”

Labeling DNA for single-molecule experiments: methods of labeling internal specific sequences on double-stranded DNA

“…This method has been demonstrated to specifically label individual dsDNA molecules with fluorophores such as Alexa 647 and haptens such as biotin, for characterization by optical and force microscopy. 4,16,20,55-57 …”

“…Luzzietti et al developed another variation of nick translation specifically for attaching rotor-beads to internal DNA sequences for magnetic tweezer experiments. 57 …”

“…4,16,20,55-57 While non-specific nicks are common on DNA and could result in non-specific labeling, treating DNA with ligase first can dramatically reduce this non-specific contribution. 4 However, the repertoire of target sequences is limited to those with an associated nicking enzyme.…”