Methods of observing interacting cells without disturbing them; imaging gene expression noninvasively using nucleic acid aptamers; IMAGEtags

Project

To understand how cells communicate, the most desirable condition is to monitor them while in their natural state surrounded by other cells as in the tissue. To accomplish this, reporters are needed with signals that can be read from living cells. Although protein reporters have been used for many years, they have the major disadvantage that they provide a signal that is delayed many hours after the gene is activated. RNA aptamers can fold to their active forms inside cells and are a good option as components of reporters that give an immediate signal of transcription. We have used RNA aptamers to develop reporters of gene expression that we call IMAGEtags (Intracellular Multiaptamer GEnetic tags), which are strings of aptamers that can be used to tag any RNA so that its transcription can be observed. The principle by which IMAGEtags work is that, when the aptamers are transcribed as part of the tagged RNA, they bind to their target molecule, which is a small chemical linked with a fluorophore. Two chemicals are used, each being linked to a different fluorophore. The fluorophores can interact in FRET and thereby signal the presence of the tagged RNA. In this project, the Nilsen-Hamilton group has collaborated with the Kraus group.

IMAGEtags are more sensitive than other RNA reporters that have been developed to detect RNA in living cells so they can be used to monitor mRNA transcription as well as trancription of the more abundant ribosomal RNAs. The FRET fluorescence can be detected in living cells and gives an immediate signal that the RNA is being transcribed. The immediate signal from the IMAGEtags enables real-time monitoring of gene expression. We will use the IMAGEtags to monitor interactions in 3D cell culture systems in which the cell community is perturbed with defined external signals such as happens on inflammation or with drug treatments and the interactions between cancer cells and their normal cell neighbors.

We have also started to develop the means to apply IMAGEtags and other aptamer sensors understand the interaction between microbial communities. These communities are important in the soil (part of the rhizosphere) and in our own bodies (the microbiomes of the gut and of the skin). Microbes cooperate to form metabolically integrated communities in which they communicate by chemical signals. It is these intercellular communications that we aim to interrogate to advance our understanding of how microbes optimize their opportunities for survival in a variety of environments.

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