The ability to image specific RNAs in living cells in real time can provide essential information on RNA synthesis, processing, transport, and localization, as well as on the dynamics of RNA expression and localization in response to external stimuli; it will offer unprecedented opportunities for advancement in molecular biology, disease pathophysiology, drug discovery, and medical diagnostics. Over the last decade or so, there is increasing evidence to suggest that RNA molecules have a wide range of functions in living cells, from physically conveying and interpreting genetic information, to essential catalytic roles, to providing structural support for molecular machines, and to gene silencing. These functions are realized through control of the expression level and stability, both temporally and spatially, of specific RNAs in a cell. Therefore, determining the dynamics and localization of RNA molecules in living cells will significantly impact on molecular biology and medicine.
Many in vitro
methods have been developed to provide a relative (mostly semi-quantitative) measure of gene expression level within a cell population using purified DNA or RNA obtained from cell lysate. These methods include PCR [1
], Northern hybridization (or Northern blotting) [2
], expressed sequence tag (EST) [3
], serial analysis of gene expression (SAGE) [4
], differential display [5
], and DNA microarrays [6
]. These technologies, combined with the rapidly increasing availability of genomic data for numerous biological entities, present exciting possibilities for understanding human health and disease. For example, pathogenic and carcinogenic sequences are increasingly being used as clinical markers for diseased states. However, using in vitro
methods to detect and identify foreign or mutated nucleic acids is often difficult in a clinical setting due to the low abundance of diseased cells in blood, sputum, and stool samples. Further, these methods cannot reveal the spatial and temporal variation of RNA within a single cell.
Labeled linear oligonucleotide (ODN) probes have been used to study intracellular mRNA via in situ
hybridization (ISH) [7
] in which cells are fixed and permeabilized to increase the probe delivery efficiency. Unbound probes are removed by washing to reduce background and achieve specificity [8
]. To enhance the signal level, multiple probes targeting the same mRNA can be used [7
]. However, fixation agents and other supporting chemicals can have considerable effect on signal level [9
] and possibly on the integrity of certain organelles such as mitochondria. Thus, fixation of cells, by either cross-linking or denaturing agents, and the use of proteases in ISH assays may prevent from obtaining an accurate description of intracellular mRNA localization. It is also difficult to obtain a dynamic picture of gene expression in cells using ISH methods.
Of particular interest is the fluorescence imaging of specific messenger RNAs (mRNAs), both their expression level and subcellular localization, in living cells. As shown schematically in , for eukaryotic cells a pre-mRNA molecule is synthesized in cell nucleus. After processing (including splicing and polyadenylation) the mature mRNAs are transported from cell nucleus to cytoplasm, and often localized at specific sites. The mRNAs are then being translated by ribosome to make specific proteins, and degraded by Rnases after a certain amount of time. The limited lifetime of mRNA enables a cell to alter protein synthesis rapidly in response to its changing needs. During the entire life cycle of an mRNA, it is always complexed with RNA-binding proteins to form a ribonucleoprotein (RNP). This has significant implications to the live-cell imaging of mRNAs (as discussed later).
In order to detect RNA molecules in living cells with high specificity, sensitivity and signal-to-background ratio, especially for low abundance genes and clinical samples containing a small number of diseased cells, the probes need to recognize RNA targets with high specificity, convert target recognition directly into a measurable signal, and differentiate between true and false-positive signals. It is important for the probes to quantify low gene expression levels with high accuracy, and have fast kinetics in tracking alterations in gene expression in real time. For detecting genetic alterations such as mutations, insertions and deletions, the ability to recognize single nucleotide polymorphisms (SNPs) is essential. To achieve this optimal performance, it is necessary to have a good understanding of the structure-function relationship of the probes, probe stability, and RNA target accessibility in living cells. It is also necessary to achieve efficient cellular delivery of probes with minimal probe degradation.
In the remaining sections, we review commonly used fluorescent probes for RNA detection, discuss the critical issues in living cell RNA detection, including probe design, target accessibility, cellular delivery of probes, and detection sensitivity, specificity and signal-to-background ratio. Emphasis is placed on the design and application of molecular beacons, although some of the issues are common to other oligonucleotide probes.