Developing RNA aptamers as single cell biosensors for mammalian cells

OVERVIEW: Cells are the basic building blocks of all organisms. The growth of organisms is controlled by cell proliferation, and in mammals cells are know to have a limited proliferative lifespan (the limit is about 50 cell division cycles) before they irreversibly withdraw for proliferation into a cell state of senescence. Senescent cells retain the ability to perform essential tissue function, but at this stage of cell development are much less effective in performing key functions than younger cells. Hence as organisms age tissues gradually acquire a higher content of senescent cells and reduced functionality. The process of cell ageing is complex and because of this overall biological ageing can differ from one person to the next. This arises because of cell-to-cell heterogeneity (variability) in biological processes. Notably, as cells age the heterogeneity in their metabolic processes also increases and this results in declining efficiency. In simple terms, this declining efficiency defines the process of ageing.

SPECIFIC FOCUS OF PROJECT: This project plans to develop tools to explore how the efficiency of a fundamental biological process - DNA synthesis - varies from one cell to the next. Eventually these tools will allow us to make clear statements about changes in metabolic heterogeneity as cells age. Our aim is to develop small molecule biosensors that will allow us to define with a high level of accuracy the concentration of the DNA precursors within individual cells. The DNA replication precursors are called deoxyribonuceoside triphosphates (dNTPs) and 4 are required during synthesis. Complex pathways regulate the concentration of these precursors in cells, and as cells age the efficiency with which this pools are replenished declines. Imbalance in the pools in ageing cells is known to contribute to genetic changes in DNA and also accelerates changes that drive cell senescence. We propose that by understanding how the pools of precursors change from cell-to-cell, within cell populations, we will be able to develop new thinking about the metabolic heterogeneity linked to cell ageing and begin to understand how/if heterogeneity contributes to differences in the rate that individuals age. This could provide an index of biological ageing, which when combined with lifestyle and environmental factors might provide more reliable predictions of an individuals likely lifespan.

PROJECT PLAN: Accurate measurement of small molecules within individual human cells is extremely difficult and to date synthetic precursors such as dNTP have never been measured in single cells - normal many millions of cells are extracted and precursors in this cell population measured. Recently, a group in US published an elegant approach for measuring small molecules in single cells based on a synthetic RNA molecule called an aptamer. Aptamers are synthesised in the lab and complex mixtures of aptamers can be generated and screened for their ability to interact specifically with a small molecule target. Hence, using a selected dNTPs as targets, specific and selective aptamers can be generated that will interact only with the designated dNTP in the cell. Hence, a single aptamer molecule is introduced into the human cells and when it interacts with the target dNTP a conformational change in the aptamer allows binding of a molecule that emits light. The amount of light emitted is directly related to the concentration of the precursor, based on the biophysical properties of the binding of the target. Our project will develop for the first time the aptamer-based biosensor system in mammalian cells and use this as a reporter for DNA synthesis precursor pools, which we can alter experimentally within cells. Changes in the pools during DNA replication result in DNA damage and this will be monitored for genetic defects by tracking cells through the cell cycle to see how defects in synthesis correlate with subsequent defects in cell division.

Somatic mammalian cells have limited proliferative potential, entering a state of replicative senescence after ~100 cell divisions. As cells age, increasing metabolic stress correlates with a functional decline and gradual accumulation of macromolecular damage. This process is typified by changes in the concentration of reactive oxygen species as a result of changes in mitochondrial efficacy and this type of endogenous metabolic stress results in, for example, protein misfolding and damage to DNA. As a consequence of the unpredictable and stochastic nature of metabolic decline individual cells age at different rates and accumulate unpredictable patterns of age-related defects.

In order to explore the process of metabolic decline during ageing at a single cell level, and so develop detailed insights into the cellular heterogeneity associated with metabolic decline, we propose to develop single cell biosensors that will allow quantification of dNTP pool concentrations in single cells. dNTP metabolism is regulated during different cell cycle phase and different states of growth arrest to ensure that sufficient concentrations of precursor are available to support S phase that occupies a strictly controlled 9-10h of the cell cycle. If pool size is severely perturbed replication is blocked. Drug-induced reduction of the pools leads to alteration in the replication fork rate, increased origin density and often correlates with defects in completion of replication, with associated mitotic defects, which result in cell death or aneuploidy. In addition, metabolic imbalance in dNTP pools leads to increased DNA mutation, altered telomere homeostatis and a range of disease phenotypes.

In collaboration with Professor Samie Jaffrey, who developed the original single cell biosensor system for bacteria, and AptaSol, a commercial Aptamer-based company, we will develop biosensor for analysis of replciation precursor pools in single human cells

In the last 15 years, the applicants have performed world-class research by adopting a multidisciplinary approach to understanding fundamental biological system, namely how cells maintain the fidelity of of their genomes during DNA replication and segregation. These fundamental processes are clearly of huge relevance during development, ageing and in response to cellular stress, processes that are essential for all forms of life. To do this we use functional genomics, focusing on key genes and probing their function in cell based assays. Importantly, interpreting the enormous data sets describing genomes, transcriptomes, proteomes and metabalomes requires a detailed functional annotation of all the individual components; our rigorous approach to characterising individual components in the context of cell signalling, cell proliferation and ageing will continue to make invaluable contributions to the understanding of biological systems. Importantly, in order to remain internationally competitive, we need to innovate and set up new approaches by developing novel tools and technology to generate the single cell information that is needed to fully understand biological systems. A central element of this proposal therefore is to set up new approaches which will not only benefit our science but will also enable the efforts of others. Developing versatile and adaptable biosensors as general tools for understanding the heterogeneity of small molecules in mammalian cells will provide huge benefit in the analysis of molecules which at present can only be analyses in cell populations - and hence neglects cell-to-cell variability.

Though our proposed study will focus on tools development it is also addressing another key strategic remit of BBSRC, that of healthy ageing and well being. In addition, the ability to develop quantitative information on single cells is an essential requirement to underpin aspects of systems modelling that lie at the heart of the BBSRC's interests in computational and systems biology.

By contributing to the state-of-the-art experimental program within two ambitious and vibrant research teams in Manchester this proposed study with provide ongoing opportunities for training world class researchers in the non-clinical life sciences and as such is directly inline with the aspirations described in the BBSRC's 10 Year Vision and Strategic Plan.