Design of Gaussia luciferase-based bioluminescent stem-loop probe for sensitive detection of HIV-1 nucleic acids

Abstract

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.

Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid**

“…The general concept was introduced by Deo and her associates assembled a (Renilla) luciferase-hairpin oligonucleotide-(3'-dabcyl) conjugate for micro-RNA detection, [12] and subsequently created a (Gaussia) luciferasehairpin oligonucleotide-(3'-dabcyl) conjugate for HIV detection. [11] The Deo sensors were prepared by spontaneous coupling of hairpin-forming oligos onto reactive residues of those luciferase enzymes. Spontaneous coupling is relatively inexpensive, but the technique produces heterogeneity in enzyme: oligonucleotide stoichiometry (1 : 1, 1 : 2, 1 : 3 etc.)…”

“…In enzymatic beacons or E-beacons the fluorophore of a molecular beacon is replaced by the compact, ATP-independent bioluminescent enzyme, nanoluciferase (Nluc). [10][11][12][13] This substitution provides an internal, amplifiable light source. Nluc with the engineered substrate furimazine produces light that is sufficiently bright for measurement using a portable luminometer.…”

Shining Light on the Secreted Luciferases of Marine Copepods: Current Knowledge and Applications

“…GLuc was chemically conjugated to the DABCYL‐modified nucleotide stem‐loop probe complementary to HIV‐1 RNA. DABCYL serves as a BRET quencher of bioluminescence in the closed conformation of SLP in the absence of the target .…”

Single-Step FRET-Based Detection of Femtomoles DNA

“…For example, an enzymatic amplification requires a multi-step process and use of enzyme, which is costly and sensitive to reaction conditions such as temperature, pH, and enzyme inhibitors [8,18]. Other emerging examples demonstrating a sensitive detection of DNA down to low picomolar and high attomolar concentrations in an amplification-free format involve complex systems such as quantum dots, nanoparticles, cationic polymers, or microarrays [19,20,21,22,23,24,25,26,27,28,29]. In this regard, single-molecule FRET (smFRET) with the ability to measure many single molecules simultaneously offers several advantages over these complex systems and the bulk fluorescence assays [30].…”