Unidirectional cloning by cleaving heterogeneous sites with a single sandwiched zinc finger nuclease.

We previously developed a novel type of zinc finger nucleases (ZFNs), sandwiched ZFNs that can discriminate DNA substrates from cleavage products and thus cleave DNA much more efficiently than conventional ZFNs as well as perform with multiple turnovers like restriction endonucleases. In the present study, we used the sandwiched ZFN to unidirectionally clone exogenous genes into target vectors by cleaving heterogeneous sites that contained heterogeneous spacer DNAs between two zinc-finger protein binding sites with a single sandwiched ZFN. We demonstrated that the sandwiched ZFN cleaved a 40-fold excess of both insert and vector plasmids within 1h and confirmed by sequencing that the resulting recombinants harbored the inserted DNA fragment in the desired orientation.

Modular blue fluorescent RNA sensors for label-free detection of target molecules.

A blue fluorescent RNA was applied to modular blue fluorescent RNA sensors, wherein a "recognition" event of the target molecule by the aptamer domain is transmitted through a "communication" module, to a "signaling" fluorescent RNA domain, which "lights up".

Sandwiched zinc-finger nucleases harboring a single-chain FokI dimer as a DNA-cleavage domain.

Zinc-finger nucleases (ZFNs) are a powerful tool for manipulation of genomic DNA. Recently, we reported a new ZFN composed of one artificial zinc-finger protein (AZP) and a single-chain FokI dimer (scFokI) that refines ZFN technology. While AZP-scFokI cleaved DNA specifically around the AZP-target site, several nucleotide positions were cleaved due to the mobility of the scFokI domain.