Single-Molecule FRET-Based Dynamic DNA Sensor

Abstract

Selective and sensitive detection of nucleic acid biomarkers is of great significance in early-stage diagnosis and targeted therapy. Therefore, the development of diagnostic methods capable of detecting diseases at the molecular level in biological fluids is vital to the emerging revolution in the early diagnosis of diseases. However, the vast majority of the currently available ultrasensitive detection strategies involve either target/signal amplification or involve complex designs. Here, using a p53 tumor suppressor gene whose mutation has been implicated in more than 50% of human cancers, we show a background-free ultrasensitive detection of this gene on a simple platform. The sensor exhibits a relatively static mid-FRET state in the absence of a target that can be attributed to the time-averaged fluorescence intensity of fast transitions among multiple states, but it undergoes continuous dynamic switching between a low- and a high-FRET state in the presence of a target, allowing a high-confidence detection. In addition to its simple design, the sensor has a detection limit down to low femtomolar (fM) concentration without the need for target amplification. We also show that this sensor is highly effective in discriminating against single-nucleotide polymorphisms (SNPs). Given the generic hybridization-based detection platform, the sensing strategy developed here can be used to detect a wide range of nucleic acid sequences enabling early diagnosis of diseases and screening genetic disorders.

Keywords: DNA/RNA detection; biomarkers; fluorescence resonance energy transfer (FRET); hybridization sensor; single molecule; single-nucleotide polymorphism (SNP); ultrasensitive sensor.

Figure 1.

Working principle of the sensor. The sensor is composed of synthetic DNA strands, two of which are labeled with either a Cy3 or a Cy5 fluorophore. The DNA construct exhibits a relatively steady FRET efficiency in the absence of a target due to averaging of fast transitions among multiple states. However, binding of the target forms a four-way structure resulting in a dynamic switching between a high (FRET₁) and a low (FRET₂) FRET state. FRET represents FRET efficiency.

Figure 2.

Typical single-molecule traces in the absence of a target. Typical intensity–time (left) and corresponding FRET–time traces (right). Five representative molecules are shown. The molecules exhibit static fluorescence intensities of Cy3 and Cy5 in the absence of a target, and a static FRET level of ~0.5 is observed in the absence of target DNA. All experiments are done at room temperature (23 °C). FRET represents FRET efficiency.

Figure 3.

Detection of a target sequence (p53 tumor suppressor gene) using single-molecule FRET. Typical intensity–time (left) and corresponding FRET–time traces. Five representative molecules are shown. The molecules exhibit dynamic and anticorrelated fluorescence intensities of Cy3 and Cy5. Such dynamic FRET–time traces with FRET levels of ~0.3 and ~0.7 are obtained only in the presence of target DNA. All of the experiments are performed at room temperature (23 °C). FRET represents FRET efficiency. Note that a slightly revised design to improve the sensor stability will be discussed later.

Figure 4.

Determination of the limit of detection (LOD). A calibration curve is obtained by plotting the number of dynamic molecules (≈target-bound molecules) as a function of the target concentration. The inset shows the linear range of the calibration curve with a LOD of 50 fM. Considering our experimental volume of ~100 μL, this LOD is equivalent to 5 attomoles of the DNA target (1 attomoles = 1 × 10⁻¹⁸ moles). The results for both the original and revised designs are shown. The percentage of dynamic molecules is determined from more than 150 single molecules at each concentration. The error bars represent the standard deviation from three groups of independent movie files.

Figure 5.

Specificity of sensors and their compatibility in serum. (A) Specificity test of the sensor using 100 pM target/mutants. While the target is perfectly complementary, mutants have one or two mismatched nucleotides (bolded and underlined). We found that 70% of molecules were dynamic in the presence of 100 pM target, while a negligible fraction of molecules showed dynamic behavior in the presence of 100 pM mutant, thus demonstrating a high specificity of our approach. (B) Sensors behave similarly in 1× Tris HCl and 10% human serum. Notice the zero background in the absence of the target.