Periodic Reporting for period 1 - miRanDa (microRNA assay system based on self-assembled nanoscale DNA origami arrays)
Okres sprawozdawczy: 2021-06-01 do 2023-05-31
Breast cancer is the most diagnosed cancer type in women worldwide (with ~2.3 million cases) and the primary cause of cancer-related death (0.7 million deaths) annually. Although mammography screening have significantly reduced breast cancer mortality, it has also some limitations due to the location of the cancer or the density of the breast tissue. For example, 25% of cancer in women ages 40-49 are not detectable by a mammogram screening. Moreover, X-ray based screening techniques could cause further problems including DNA damage in the tissues. For this reason, we aimed to develop a DNA origami-based technique for the early detection of cancer biomarkers.
MicroRNAs (miRNAs) are promising biomarkers for diagnostics and prognosis of cancer. These are class of non-coding endogenous small RNAs of 21-25 nucleotides in length and responsible for the regulation of gene expression. Expression levels of miRNA is dysregulated in various cancer types where they can act as either tumor suppressors or oncogenes. In cancers, multiple miRNAs are typically up or downregulated and their combined alterations are specific to individual cancers and their stages. Therefore, specific and sensitive detection and absolute quantification of multiple miRNAs can deliver essential cues to understand disease progression and have growing relevance to early cancer detection and monitoring. However, current miRNA detection techniques such as polymerization chain reaction, next generation sequencing or DNA microarray have important practical limitations and restricted multiplexing capabilities. Moreover, their routine measurement in plasma is time-consuming, costly, and requires dedicated equipment and specialized laboratories. In this project, our objective was to develop novel miRNA detection system from breast cancer cell populations and human plasma samples using bioinspired DNA origami self-assembly tool and state-of-art super resolution imaging technique, DNA-PAINT (point accumulation for imaging in nanoscale topography).
MicroRNAs (miRNAs) are promising biomarkers for diagnostics and prognosis of cancer. These are class of non-coding endogenous small RNAs of 21-25 nucleotides in length and responsible for the regulation of gene expression. Expression levels of miRNA is dysregulated in various cancer types where they can act as either tumor suppressors or oncogenes. In cancers, multiple miRNAs are typically up or downregulated and their combined alterations are specific to individual cancers and their stages. Therefore, specific and sensitive detection and absolute quantification of multiple miRNAs can deliver essential cues to understand disease progression and have growing relevance to early cancer detection and monitoring. However, current miRNA detection techniques such as polymerization chain reaction, next generation sequencing or DNA microarray have important practical limitations and restricted multiplexing capabilities. Moreover, their routine measurement in plasma is time-consuming, costly, and requires dedicated equipment and specialized laboratories. In this project, our objective was to develop novel miRNA detection system from breast cancer cell populations and human plasma samples using bioinspired DNA origami self-assembly tool and state-of-art super resolution imaging technique, DNA-PAINT (point accumulation for imaging in nanoscale topography).
In this project, we developed a DNA origami-based nanosensor providing distance-dependent recognition of miRNAs by applying super-resolution microscopy technique; DNA-PAINT. The sensor is a 8-helix bundle DNA origami structure designed by caDNAno software. It consists of 2 layers of DNA double helices, each layer made of 4 DNA double helices with a dimensions of 306 × 10 x 5 nm. The upper layer is used for decoration of anchor strands to capture miRNAs and the lower layer is used for decoration with biotin labelled strands for immobilization on the glass slide for super-resolution microscopy imaging. The sensor can detect up to 4 miRNAs either separately or in combination based on the relative distance to the boundary markers on the structure using a single imager strand. We managed the ultrasensitive detection of miRNAs, with a limit of detection down to the low femtomolar range (11 fM - 388 fM) without need for amplification. Moreover, our detection system can discriminate single base mutations with low false positive rates. Additionally, we showed that the multiplexing capacity of our nanoarray can be improved by one dimensional polymerization. Using our strategy, we successfully demonstrate the detection of endogenous miRNAs from cell extracts of breast cancer cell lines and plasma from breast cancer patients. The work has been published in a high ranked international journal, Biosensors and Bioelectronics.
In addition to this work, we developed another dynamic DNA origami book biosensor that is precisely decorated with arrays of fluorophores acting as donors and acceptors/quenchers that produce a strong optical readout upon exposure to external stimuli for the single or dual detection of target oligonucleotides and miRNAs. This biosensor allowed the detection of target molecules either through the decrease of Förster resonance energy transfer (FRET) or through an increase in the fluorescence intensity profile owing to a rotation of the constituent top layer of the structure. We demonstrated that the detection of two miRNAs can be achieved simultaneously within 10 min with a limit of detection in the range of 1–10 pM. This work has been published in a high-impact nanotechnology journal, Nanoscale.
The press release about these research findings can be reached by the following link: https://www.unifr.ch/scimed/en/info/news/28437/bioinspired-nanosensors-for-cancer-detection(odnośnik otworzy się w nowym oknie).
A patent application to protect the FRET-based DNA origami book biosensor invention was submitted in October 2022.
The research findings were also presented in national and international conferences including BMT-2022 (Annual conference on Biomedical Engineering), FNANO (Foundations of Nanoscience), NCCR (National Centre of Competence in Research)-Bioinspired Materials and internal University of Fribourg department seminars.
In addition to this work, we developed another dynamic DNA origami book biosensor that is precisely decorated with arrays of fluorophores acting as donors and acceptors/quenchers that produce a strong optical readout upon exposure to external stimuli for the single or dual detection of target oligonucleotides and miRNAs. This biosensor allowed the detection of target molecules either through the decrease of Förster resonance energy transfer (FRET) or through an increase in the fluorescence intensity profile owing to a rotation of the constituent top layer of the structure. We demonstrated that the detection of two miRNAs can be achieved simultaneously within 10 min with a limit of detection in the range of 1–10 pM. This work has been published in a high-impact nanotechnology journal, Nanoscale.
The press release about these research findings can be reached by the following link: https://www.unifr.ch/scimed/en/info/news/28437/bioinspired-nanosensors-for-cancer-detection(odnośnik otworzy się w nowym oknie).
A patent application to protect the FRET-based DNA origami book biosensor invention was submitted in October 2022.
The research findings were also presented in national and international conferences including BMT-2022 (Annual conference on Biomedical Engineering), FNANO (Foundations of Nanoscience), NCCR (National Centre of Competence in Research)-Bioinspired Materials and internal University of Fribourg department seminars.
These nanosensors may open the way towards the new generation of blood-based tests for early cancer detection and monitoring. First, they represent a basis for the development of more sophisticated sensors allowing the simultaneous detection of tens to hundreds of different nucleic acids (multiplexing), or the detection of mutated DNA or large RNA molecules (mRNA). From a technical perspective, the sensor can easily be redesigned to allow detection of other DNA or RNA targets by changing the anchor sequences of the DNA origami sensors. This would allow to easily expand the use of the developed sensor to other cancer types and other disease conditions, in particular cardiovascular, inflammatory, autoimmune, infectious (e.g. SARS-COV-2) and degenerative diseases. Secondly, they open the way to the development of rapid, safe, simple, and low-cost clinically applicable tests for cancer detection and monitoring. Our short-term goal is to develop a diagnostic kit consisting of the nanosensor, reagents, and instructions according to the target oligonucleotide. The long-term goal of the project is to eventually improve cancer diagnostic and patient care by assisting physicians in making informed decisions regarding personalized treatment strategies.