TY - JOUR
T1 - Amplifiable hybridization probes containing a molecular switch
AU - Blok, Herman J.
AU - Kramer, Fred Russell
N1 - Funding Information:
We thank Dr Sanjay Tyagi (Public Health Research Institute) for insightful discussions and Dr Donald Court (National Cancer Institute) for the gift of E. coli RNase III. This work was supported by the National Institutes of Health (Grants HL-43521 and AI-37015) and by the Eindhoven University of Technology.
PY - 1997/6
Y1 - 1997/6
N2 - In order to reduce background signals in Qβ replicase-mediated bioassays, a target-dependent probe amplification strategy has been proposed that utilizes recombinant RNA hybridization probes that contain an inserted molecular switch. A molecular switch is an internal region of the probe that undergoes a conformational change when the probe hybridizes to its target. We investigated whether non-hybridized probes (which cause background signals) could be selectively destroyed by incubating the probe-target hybrids with ribonuclease III, which should cleave the non-hybridized probes and leave the hybridized probes intact. Two problems with this assay design were observed. First, ribonuclease III cleaved probe-target hybrids non-specifically when the target was an RNA, thereby destroying all of the bound probes. And second, the expected conformational change in the molecular switch did not occur when the probes were bound to their targets, apparently because the hairpin stem formed by the molecular switch was too long. Although these results demonstrated that the original assay design could not work, they provided insights that have led to better designs for target-dependent amplification assays. In these assays, the probes will be DNA molecules containing short-stemmed molecular switches. Non-hybridized probes will be selectively destroyed by incubation with a restriction endonuclease.
AB - In order to reduce background signals in Qβ replicase-mediated bioassays, a target-dependent probe amplification strategy has been proposed that utilizes recombinant RNA hybridization probes that contain an inserted molecular switch. A molecular switch is an internal region of the probe that undergoes a conformational change when the probe hybridizes to its target. We investigated whether non-hybridized probes (which cause background signals) could be selectively destroyed by incubating the probe-target hybrids with ribonuclease III, which should cleave the non-hybridized probes and leave the hybridized probes intact. Two problems with this assay design were observed. First, ribonuclease III cleaved probe-target hybrids non-specifically when the target was an RNA, thereby destroying all of the bound probes. And second, the expected conformational change in the molecular switch did not occur when the probes were bound to their targets, apparently because the hairpin stem formed by the molecular switch was too long. Although these results demonstrated that the original assay design could not work, they provided insights that have led to better designs for target-dependent amplification assays. In these assays, the probes will be DNA molecules containing short-stemmed molecular switches. Non-hybridized probes will be selectively destroyed by incubation with a restriction endonuclease.
KW - Qβ replicase
KW - Recombinant MDV-1 RNA
KW - Ribonuclease III recognition site
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U2 - 10.1006/mcpr.1997.0103
DO - 10.1006/mcpr.1997.0103
M3 - Article
C2 - 9232617
AN - SCOPUS:0031171436
SN - 0890-8508
VL - 11
SP - 187
EP - 194
JO - Molecular and Cellular Probes
JF - Molecular and Cellular Probes
IS - 3
ER -