TY - JOUR
T1 - Design of Gaussia luciferase-based bioluminescent stem-loop probe for sensitive detection of HIV-1 nucleic acids
AU - Joda, Hamdi
AU - Moutsiopoulou, Angeliki
AU - Stone, Geoffrey
AU - Daunert, Sylvia
AU - Deo, Sapna
N1 - Funding Information:
The work was supported through NIGMS funding (R01GM114321), the National Science Foundation (CHE-1506740), and the State of Florida Department of Health (7ZK01/7ZK11).
Funding Information:
The work was supported through NIGMS funding (R01GM114321), the National Science Foundation (CHE-1506740), and the State of Florida Department of Health (7ZK01/7ZK11). Sylvia Daunert thanks the Miller School of Medicine of the University of Miami for the Lucille P. Markey Chair in Biochemistry and Molecular Biology.
Publisher Copyright:
© 2018 The Royal Society of Chemistry.
PY - 2018/7/21
Y1 - 2018/7/21
N2 - Here we describe the design of a bioluminescent stem-loop probe for the sensitive detection of HIV-1 spliced RNA. In this study, we employed Gaussia luciferase (GLuc), a bioluminescent protein that has several advantages over other bioluminescent proteins, including smaller size, higher bioluminescent intensity, and chemical and thermal stability. GLuc was chemically conjugated to the DABCYL-modified stem-loop probe (SLP) and was purified with a 2-step process to remove unconjugated GLuc and SLP. The binding of the target RNA to the loop region of the SLP results in the open conformation separating the stem part of SLP. GLuc conjugated to the stem acts as a reporter that produces light by a chemical reaction upon adding its substrate, coelenterazine in the presence of the target, while DABCYL serves as a quencher of bioluminescence in the closed conformation of SLP in the absence of the target. The optimized GLuc based-SLP assay resulted in a signal-to-background ratio of 47, which is the highest reported with bioluminescent SLPs and is significantly higher compared to traditional fluorescence-based SLPs that yield low signal to background ratio. Moreover, the assay showed an excellent selectivity against a single and double mismatched nucleic acid target, low detection limit, and ability to detect spiked HIV-1 RNA in human serum matrix.
AB - Here we describe the design of a bioluminescent stem-loop probe for the sensitive detection of HIV-1 spliced RNA. In this study, we employed Gaussia luciferase (GLuc), a bioluminescent protein that has several advantages over other bioluminescent proteins, including smaller size, higher bioluminescent intensity, and chemical and thermal stability. GLuc was chemically conjugated to the DABCYL-modified stem-loop probe (SLP) and was purified with a 2-step process to remove unconjugated GLuc and SLP. The binding of the target RNA to the loop region of the SLP results in the open conformation separating the stem part of SLP. GLuc conjugated to the stem acts as a reporter that produces light by a chemical reaction upon adding its substrate, coelenterazine in the presence of the target, while DABCYL serves as a quencher of bioluminescence in the closed conformation of SLP in the absence of the target. The optimized GLuc based-SLP assay resulted in a signal-to-background ratio of 47, which is the highest reported with bioluminescent SLPs and is significantly higher compared to traditional fluorescence-based SLPs that yield low signal to background ratio. Moreover, the assay showed an excellent selectivity against a single and double mismatched nucleic acid target, low detection limit, and ability to detect spiked HIV-1 RNA in human serum matrix.
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U2 - 10.1039/c8an00047f
DO - 10.1039/c8an00047f
M3 - Article
C2 - 29897056
AN - SCOPUS:85049692509
VL - 143
SP - 3374
EP - 3381
JO - The Analyst
JF - The Analyst
SN - 0003-2654
IS - 14
ER -