Dataset Information

Localized fluorescent imaging of multiple proteins on individual extracellular vesicles using rolling circle amplification for cancer diagnosis.

ABSTRACT: Extracellular vesicles (EV) have attracted increasing attention as tumour biomarkers due to their unique biological property. However, conventional methods for EV analysis are mainly based on bulk measurements, which masks the EV-to-EV heterogeneity in tumour diagnosis and classification. Herein, a localized fluorescent imaging method (termed Digital Profiling of Proteins on Individual EV, DPPIE) was developed for analysis of multiple proteins on individual EV. In this assay, an anti-CD9 antibody engineered biochip was used to capture EV from clinical plasma sample. Then the captured EV was specifically recognized by multiple DNA aptamers (CD63/EpCAM/MUC1), followed by rolling circle amplification to generate localized fluorescent signals. By-analyzing the heterogeneity of individual EV, we found that the high-dimensional data collected from each individual EV would provide more precise information than bulk measurement (ELISA) and the percent of CD63/EpCAM/MUC1-triple-positive EV in breast cancer patients was significantly higher than that of healthy donors, and this method can achieve an overall accuracy of 91%. Moreover, using DPPIE, we are able to distinguish the EV between lung adenocarcinoma and lung squamous carcinoma patients. This individual EV heterogeneity analysis strategy provides a new way for digging more information on EV to achieve multi-cancer diagnosis and classification.

SUBMITTER: Zhang J

PROVIDER: S-EPMC7710127 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Localized fluorescent imaging of multiple proteins on individual extracellular vesicles using rolling circle amplification for cancer diagnosis.

Zhang Junli J Shi Jinjin J Zhang Hongling H Zhu Yifan Y Liu Wei W Zhang Kaixiang K Zhang Zhenzhong Z

Journal of extracellular vesicles 20201001 1

Extracellular vesicles (EV) have attracted increasing attention as tumour biomarkers due to their unique biological property. However, conventional methods for EV analysis are mainly based on bulk measurements, which masks the EV-to-EV heterogeneity in tumour diagnosis and classification. Herein, a localized fluorescent imaging method (termed Digital Profiling of Proteins on Individual EV, DPPIE) was developed for analysis of multiple proteins on individual EV. In this assay, an anti-CD9 antibod ...[more]

PMID: 33304477

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Project description:Nucleic acid amplification is a hugely important technology for biology and medicine. While the polymerase chain reaction (PCR) has been highly useful and effective, its reliance on heating and cooling cycles places some constraints on its utility. For example, the heating step of PCR can destroy biological molecules under investigation and heat/cool cycles are not applicable in living systems. Thus, isothermal approaches to DNA and RNA amplification are under widespread study. Perhaps the simplest of these are the rolling circle approaches, including rolling circle amplification (RCA) and rolling circle transcription (RCT). In this strategy, a very small circular oligonucleotide (e.g., 25-100 nucleotides in length) acts as a template for a DNA or an RNA polymerase, producing long repeating product strands that serve as amplified copies of the circle sequence. Here we describe the early developments and studies involving circular oligonucleotides that ultimately led to the burgeoning rolling circle technologies currently under development. This Account starts with our studies on the design of circular oligonucleotides as novel DNA- and RNA-binding motifs. We describe how we developed chemical and biochemical strategies for synthesis of well-defined circular oligonucleotides having defined sequence and open (unpaired) structure, and we outline the unusual ways in which circular DNAs can interact with other nucleic acids. We proceed next to the discovery of DNA and RNA polymerase activity on these very small cyclic DNAs. DNA polymerase "rolling circle" activities were discovered concurrently in our laboratory and that of Andrew Fire. We describe the surprising efficiency of this process even on shockingly small circular DNAs, producing repeating DNAs thousands of nucleotides in length. RNA polymerase activity on circular oligonucleotides was first documented in our group in 1995; especially surprising in this case was the finding that the process occurs efficiently even without promoter sequences in the circle. We describe how one can encode cleavable sites into the product DNAs and RNAs from RCA/RCT, which can then be resolved into large quantities of almost pure oligonucleotides. Our Account then proceeds with a summary describing a broad variety of tools and methods built in many laboratories around the rolling circle concept. Among the important developments are the discovery of highly efficient DNA polymerases for RCA; the invention of exponential ("hyperbranched") RCA amplification made possible by use of a second primer; the development of the "padlock" process for detection of nucleic acids and proteins coupled with RCA; the use of circular oligonucleotides as vectors in cells to encode biologically active RNAs via RCT; and the use of small DNA circles to encode and extend human telomeres. Finally, we finish with some ideas about where the field may go in the future.

Dataset Information

Localized fluorescent imaging of multiple proteins on individual extracellular vesicles using rolling circle amplification for cancer diagnosis.

Publications

Localized fluorescent imaging of multiple proteins on individual extracellular vesicles using rolling circle amplification for cancer diagnosis.

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