Dataset Information

Applying DNA rolling circle amplification in fluorescence imaging of cell surface glycans labeled by a metabolic method.

ABSTRACT: Glycans on the cell surfaces are essential for cellular communication. Metabolically labeling glycans can introduce unnatural sugars into cellular glycans, which can facilitate further labeling. We report herein imaging cell surface glycosylation by using click chemistry and DNA rolling circle amplification (RCA) to improve detection sensitivity. Through the RCA amplification, the image resolution of a cell was significantly improved and much fewer unnatural sugars were used than required previously. The advantage of this method is that it avoids introducing too much unnatural sugar, which can interfere with normal, physiological cell function. Simultaneously, the enhanced fluorescence intensity conveniently facilitates the detection of cells' own biosynthetic glycans by simply using a microplate reader. The results indicate that the metabolically labelling ability is different for different carbohydrates and different cells. Next, the RCA technique was adopted in a fluorescence resonance energy transfer (FRET)-based methodology that facilitated the glycan imaging of specific proteins on the cell surface. This method is broadly applicable to imaging the glycosylation of cellular proteins. Our results highlight the applications of RCA in metabolic glycan labeling, which can be used to monitor the glycosylation status on cells, and study the means by which glycosylation regulates cell function.

SUBMITTER: Zhang X

PROVIDER: S-EPMC6024553 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Applying DNA rolling circle amplification in fluorescence imaging of cell surface glycans labeled by a metabolic method.

Zhang Xiaoru X Li Ruijuan R Chen Yuanyuan Y Zhang Shusheng S Wang Wenshuang W Li Fuchuan F

Chemical science 20160614 9

Glycans on the cell surfaces are essential for cellular communication. Metabolically labeling glycans can introduce unnatural sugars into cellular glycans, which can facilitate further labeling. We report herein imaging cell surface glycosylation by using click chemistry and DNA rolling circle amplification (RCA) to improve detection sensitivity. Through the RCA amplification, the image resolution of a cell was significantly improved and much fewer unnatural sugars were used than required previo ...[more]

PMID: 30034758

Similar Datasets

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

Applying DNA rolling circle amplification in fluorescence imaging of cell surface glycans labeled by a metabolic method.

Publications

Applying DNA rolling circle amplification in fluorescence imaging of cell surface glycans labeled by a metabolic method.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets