Reassembly of a bioluminescent protein Renilla luciferase directed through DNA hybridization.
Reassembly of split reporter proteins, also referred to as protein complementation, is utilized in the detection of protein-protein or protein-nucleic acid interactions. In this strategy, a reporter protein is fragmented into two inactive polypeptides to which interacting/binding partners are fused. The interaction between fused partners leads to the formation of a reassembled, active reporter. In this Communication, we have presented a proof-of-concept for the detection of a target nucleic acid sequence based on the reassembly of the bioluminescent reporter Renilla luciferase (Rluc), which is driven by DNA hybridization. Although, reassembly of Rluc though protein interactions has been demonstrated by others, the Rluc reassembly through DNA hybridization has not been shown yet, which is the novelty of this work. It is well established that bioluminescence detection offers significant advantages due to the absence of any background signal. In our study, two rationally designed fragments of Rluc were conjugated to complementary oligonucleotide probes. Hybridization of the two probes with fused Rluc fragments resulted in the reassembly of the fragments, generating active Rluc, measurable by the intensity of light given off upon addition of coelenterazine. Our study also shows that the reassembly of Rluc can be inhibited by an oligonucleotide probe that competes to bind to the hybridized probe-Rluc fragment complex, indicating a potential strategy for the quantitative detection of target nucleic acid. We were able to achieve the reassembly of Rluc fused to oligonucleotide probes using femtomole amounts of the probe-fragment protein conjugate. This concentration is approximately 4 orders of magnitude less than that reported using green fluorescent protein (GFP) as the reporter. A DNA-driven Rluc reassembly study performed in a cellular matrix did not show any interference from the matrix.
Cissell
Kyle A
KA
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis, Indiana 46202, USA.
Rahimi
Yasmeen
Y
Shrestha
Suresh
S
Deo
Sapna K
SK
D049410:Luciferases, Renilla / Q000737:chemistry*; D008164:Luminescent Proteins / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000737:chemistry*
D008164:Luminescent Proteins; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D049410:Luciferases, Renilla