A new reporter design based on DNA origami nanostructures for quantification of short oligonucleotides using microbeads

Youngeun Choi,Carsten Schmidt,Ilko Bald,Stefan Rödiger,Philip Tinnefeld

doi:10.1038/s41598-019-41136-x

Abstract

The DNA origami technique has great potential for the development of brighter and more sensitive reporters for fluorescence based detection schemes such as a microbead-based assay in diagnostic applications. The nanostructures can be programmed to include multiple dye molecules to enhance the measured signal as well as multiple probe strands to increase the binding strength of the target oligonucleotide to these nanostructures. Here we present a proof-of-concept study to quantify short oligonucleotides by developing a novel DNA origami based reporter system, combined with planar microbead assays. Analysis of the assays using the VideoScan digital imaging platform showed DNA origami to be a more suitable reporter candidate for quantification of the target oligonucleotides at lower concentrations than a conventional reporter that consists of one dye molecule attached to a single stranded DNA. Efforts have been made to conduct multiplexed analysis of different targets as well as to enhance fluorescence signals obtained from the reporters. We therefore believe that the quantification of short oligonucleotides that exist in low copy numbers is achieved in a better way with the DNA origami nanostructures as reporters.

Highlights

Development of nucleic acid detection systems has increased rapidly over the past few years, with the discovery of potential biomarkers for early diagnosis of diseases such as cancer or type II diabetes[1]
We present a reporter design based on DNA origami nanostructures to detect short oligonucleotides in a microbead-based assay
Probe strands are positioned on a different trapezoid of the triangular DNA origami nanostructure than where the dye labels are positioned, as well as on the opposite plane of the triangle as to the dye labelling strands

Summary

Introduction

Development of nucleic acid detection systems has increased rapidly over the past few years, with the discovery of potential biomarkers for early diagnosis of diseases such as cancer or type II diabetes[1]. The ability to place molecules with spatial control makes these DNA nanostructures a good platform to develop reporters with multiple chromophores for signal enhancement as well as binding sites for targets Another aspect to consider when profiling bioanalytes at low concentration is the detection method. Microbead-based assays provide many advantages compared to conventional analytical techniques such as smaller sample consumption, high degree of multiplexing, and high throughput capability[16] They have been used extensively in bioanalysis including single nucleotide polymorphisms[17], standard PCR products[18], miRNA19, pathogens[20], as well as reactions such as in vitro cloning of DNA21, aptamer generation[22], and gene expression analysis[23]. Each particle can be thought of as a spot on a microarray, where reactions and detections can be conducted independently, with each population of the microbead assigned to one analyte only[25]

Methods

Results

Conclusion