A Simple Quenching Method for Fluorescence Background Reduction and Its Application to the Direct, Quantitative Detection of Specific mRNA

Abstract

New genome sequence information is rapidly increasing the number of nucleic acid (NA) targets of use for characterizing and treating diseases. Detection of these targets by fluorescence-based assays is often limited by fluorescence background from unincorporated or unbound probes that are present in large excess over the target. To solve this problem, energy transfer-based probes have been developed and used to reduce the fluorescence from unbound probes. Although these probes have revolutionized NA target detection, their use requires scrupulous attention to design constraints, extensive probe quality control, and individually optimized experimental conditions. Here, we describe a simpler background reduction approach using singly labeled quencher oligomers to suppress excess unbound probe fluorescence following probe-target hybridization. A second limitation of most fluorescence-based NA target detection and quantification assays is the requirement for enzymatic amplification of target or signal for sensitivity. Amplification steps make quantification of original target copy number problematic because of variations in amplification efficiencies between the sequence targets and the experimental conditions. To avoid amplification, we coupled our quenching approach to a two-color NA assay with correlated, two-color, single-molecule fluorescence detection. We demonstrate a >100-fold background reduction and detection of targets present at concentrations as low as 100 fM using the two-color assay. The application of this technique to the detection and quantification of specific mRNA sequences enabled us to estimate beta-actin copy numbers in cell-derived total RNA without an amplification step.

Single-Molecule Detection and Probe Strategies for Rapid and Ultrasensitive Genomic Detection

“…Ultrasensitive coincidence detection of doubly-covalently-labeled DNA molecules down to 50 fM in the presence of a 1000-fold excess of singlycovalently-labeled DNA molecules was demonstrated. Coincidence detection in combination with two additional quencher-labeled oligonucleotides that quenched the unbound probe fluorescence was also reported [16]. Detections of target DNA sequences at concentrations as low as 100 fM in the presence of a ~ 5000-fold excess of labeled hybridization probes were shown.…”

“…In the last decade, several assay methods for nucleic acid detection based on fluorescence correlation spectroscopy (FCS) have been reported [16,18,38,[40][41][42][43][44][45][46][47][48][49]. The principle of FCS lies in temporal and spatial fluorescence fluctuation due to molecules diffusing in and out of the small detection volume.…”

“…In addition, simpler mathematical evaluation and shorter readout time can also be achieved [45]. Dualcolor FCS [41,46] has been demonstrated for the detection of amplified DNA target sequences in multiplexed polymerase chain reactions (PCR) [47], and mRNA [16,18,48,49]. The typical concentration range of FCS measurements is around 1~100 nM and an average of one to hundreds of fluorescent molecules are under interrogation within the detection volume at any time.…”

“…In the past, SMD techniques have been largely used for the fundamental study in molecular interactions [10][11][12]. Recently, by incorporating new probes or probe strategies, such as fluorescence resonance energy transfer (FRET)-based probes [13][14][15] and dual-color fluorescence coincidence analysis [16][17][18][19][20], SMD has been applied to sequence-specific detection of DNA/RNA targets in solutions.…”

Fluorescence Fluctuation Spectroscopic Approaches to the Study of a Single Molecule Diffusing in Solution and a Live Cell without Systemic Drift or Convection: A Theoretical Study

“…Goodwin and co-workers first measured single-stranded mRNA and DNA at about three femtomolar bulk concentration (3·10 -15 M) by means of two-color fluorescence crosscorrelation spectroscopy (FCCS) [53]. Földes-Papp and coworkers first measured double-stranded, genomic DNA at about thirty femtomolar (30·10 -15 M) bulk allele concentration by means of FCCS [54].…”