Single-Step FRET-Based Detection of Femtomoles DNA.

Sapkota Sapkota,Dhakal Dhakal,Donkoh-Moore Donkoh-Moore,Megalathan Megalathan,Kaur Kaur

doi:10.3390/s19163495

Abstract

Sensitive detection of nucleic acids and identification of single nucleotide polymorphism (SNP) is crucial in diagnosis of genetic diseases. Many strategies have been developed for detection and analysis of DNA, including fluorescence, electrical, optical, and mechanical methods. Recent advances in fluorescence resonance energy transfer (FRET)-based sensing have provided a new avenue for sensitive and quantitative detection of various types of biomolecules in simple, rapid, and recyclable platforms. Here, we report single-step FRET-based DNA sensors designed to work via a toehold-mediated strand displacement (TMSD) process, leading to a distinct change in the FRET efficiency upon target binding. Using single-molecule FRET (smFRET), we show that these sensors can be regenerated in situ, and they allow detection of femtomoles DNA without the need for target amplification while still using a dramatically small sample size (fewer than three orders of magnitude compared to the typical sample size of bulk fluorescence). In addition, these single-molecule sensors exhibit a dynamic range of approximately two orders of magnitude. Using one of the sensors, we demonstrate that the single-base mismatch sequence can be discriminated from a fully matched DNA target, showing a high specificity of the method. These sensors with simple and recyclable design, sensitive detection of DNA, and the ability to discriminate single-base mismatch sequences may find applications in quantitative analysis of nucleic acid biomarkers.

Highlights

Over the years, a myriad of sensors have been developed for the specific detection of nucleic acids spanning a wide array of fields such as fundamental research, clinical diagnosis, and biotechnology [1,2,3].With the advent of DNA nanotechnology, fluorescence resonance energy transfer (FRET)-based sensors have especially surged in popularity for their ability to detect a variety of targets including nucleic acids, proteins, and small molecules
The sensors were assembled in their open conformation in the presence of a probe oligonucleotide that was partially complementary to the Cy3-labeled strand (Figure 1a), which allowed the sensor molecules to stay in their open conformation, resulting in a low FRET efficiency
The bulk EFRET analysis of sensor-I showed that the sensor assumed a closed conformation which was not able to open (Figure 2), suggesting that the 20 bp stem was too long to be invaded by the probe

Summary

Introduction

With the advent of DNA nanotechnology, fluorescence resonance energy transfer (FRET)-based sensors have especially surged in popularity for their ability to detect a variety of targets including nucleic acids, proteins, and small molecules Due to their simple and highly predictable design, ability to be regenerated through the toehold-mediated strand displacement (TMSD) process [4,5,6], and specificity towards pre-defined targets, DNA-based sensors have found applications in fluorescent hybridization assays [2,7,8], aptamer-based assays [7,9,10], electrochemical assays [3,11], surface enhanced Raman spectroscopy [12,13], and surface plasmon resonance analysis of biomolecules [14,15], just to name a few. 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

Methods

Results

Conclusion