Commentary Cellscience Reviews Vol 2 No.2 ISSN 1742-8130

Molecular beacons for intracellular analysis

Colin D. Medley & Weihong Tan

Introduction

Since their development in 1996¹, molecular beacons (MBs) have seen ever-growing use in a wide variety of fields from chemistry to biology to medical sciences^2-4. MBs are fluorescent oligonucleotide based probes that possess excellent specificity and sensitivity. MBs are comprised of two discrete regions comprised of the loop region and the stem region as illustrated in figure 1. Due to their unique hairpin structure, MBs undergo a conformational change upon hybridizing to their targets. The conformational change causes the stem portion of the MB to melt and separate. The melting of the stem causes the fluorophore and quencher on each end of the stem to become spatially separated. When unhybridized the fluorophore and quencher are in close proximity, suppressing the fluorescent signal, after the fluorophore and quencher are separated upon hybridization, the fluorescence signal is restored. This method of signal transduction gives the MB its property of detection without separation. That is, the target can be detected without separating the hybridized probes from the unhybridized probes. This feature of MBs is very significant for detection of mRNA inside of living cells since previous methods of mRNA detection inside of cells required fixed or pretreated cells so that the unbound probes could be removed⁵. Such techniques did not allow analysis on living cells. MBs inherently possess specificity through Watson-Crick base pairing and sensitivity through fluorescence detection that makes them ideal for intracellular mRNA detection. The ability to monitor mRNA expression inside cells both selectively and in real time gives MBs the potential to improve the understanding of many biological issues. It can shed light on many fundamental processes such as the mechanisms and kinetics of mRNA production, transportation and localization of mRNA inside of a cell, and cellular responses to external stimuli.

Figure 1. Diagram of a molecular beacon in a hybridized and unhybridized state.

The use of MBs for intracellular RNA detection and localization requires the design of the MB to be tailored towards mRNA and delivery of the probe into the cell. The major concern in designing MBs for intracellular use is selecting an appropriate target region on the mRNA sequence for the MB. This is especially critical due to the complex secondary structure exhibited by large mRNA sequences. The MB must have access to its complementary sequence or the MB will not hybridize and produce the fluorescent signal for detection. Since current mRNA folding programs do not give a completely reliable secondary structure, a series of MBs is generally selected for each mRNA sequence. The series of probes are tested in vitro with further testing in vivo until a probe is found that can hybridize with mRNA inside a cell.

Delivery of MBs into a living cell has been an area where a lot of effort has been applied, resulting in many very effective options for intracellular delivery. The most common delivery methods include microinjection⁶, electroporation⁷, peptide assisted delivery⁸, and reversible permeabilization⁹. Microinjection has several advantages, and most importantly it allows for the immediate monitoring of the cell for the response of the probe. Secondly, it delivers relatively reproducible amounts of probe into the cell of choice of the experimenter. The disadvantages of using microinjection are related to the technique itself in that it requires additional instruments and expertise while being a very low throughput technique. Electroporation and reversible permeabilization offer a much higher throughput by producing pores in the cell membrane and relying on passive diffusion to deliver probes in the cell media. However the pores can also allow the loss of materials from inside of the cell and there are variations in the amount of probes delivered into each cell. Peptide-assisted delivery allows the probes to pass through the cell membrane with disturbing the cell however it requires the peptide to be conjugated to the probe which can increase the cost and complexity of the probe synthesis. Thus, each method has its advantages and disadvantages and can be chosen based on the needs of the experiment.

In addition to the MB’s ability for separation without detection, MBs have other important characteristics for intracellular experiments. Since MBs are comprised of DNA, they are readily adapted for intracellular experiments since DNA is non-toxic to cells. Another very important characteristic of MBs is that they can be specifically designed for virtually any mRNA sequence. Whilst there are current limitations as to how low an expression level can be detected, in principle a MB can be used to detect any mRNA sequence derived from a cell’s genome. Combined with multitude of delivery methods, their inherent signal transduction method that allows for detection without separation, and the ability to design a MB for any gene, MBs have a wide range of applications for various intracellular studies of biochemical, biological, and medical significance.

Detection of mRNA inside of living cells

In 2001 the real-time hybridization of a MB to mRNA was visualized inside of a single cell¹⁰. In this study, MBs specific for ß-actin and ß-1 andrenergic mRNA were detected in kangaroo rat kidney (PtK2) cells. Figure 1 shows the response of a ß-1 andrenergic mRNA MB inside of a PtK2 cell. The MBs were delivered into the cells by microinjection and then monitored for 18 minutes. In the PtK2 cells, both ß-actin and ß-1 andrenergic mRNA were specifically detected as a negative control MB showed no increase in fluorescence, demonstrating that the fluorescence from the targeted MBs was due to hybridization as opposed to a non-specific interaction. This study demonstrated the real-time mRNA detection capabilities of MBs and showed conclusively that MBs could be very valuable tools for intracellular analysis.

Figure 2: A transmission and then the fluorescent images of a PtK2 cell inject with a ß-1 andrenergic mRNA MB at 3-minute intervals for 18 minutes.

Visualization of mRNA distribution and transport

In 2003, Tyagi et al demonstrated that MBs could be used for the visualization of the distribution and transport of mRNA¹¹. In this study an MB for oskar mRNA was investigated in Drosophila melanogaster oocytes. Initially they demonstrated visualizing the distribution of oskar mRNA in the cell. Due to the background exhibited from the MBs, they decided to use a binary MB approach that utilized two MBs that targeted adjacent positions on the mRNA. When both MBs were hybridized to the mRNA sequence a donor and acceptor fluorophore were brought within close proximity allowing FRET to occur and generating a new signal that indicates hybridization of the MBs with the mRNA. In addition to visualizing mRNA distribution, they were also able to track the migration of the mRNA throughout the cell and even into adjacent cells in the oocyte demonstrating the potential of MBs for intracellular tracking. Other studies have imaged MBs targeted against viral mRNA inside their host cells to study the behavior of the mRNA¹². This study investigated both the localization of viral mRNA within the cell and also utilized photobleaching the fluorophore on the MB in order to study the diffusion of the MB mRNA hybrid. These studies demonstrate the wealth of information that can be gained through the visualization of MB hybridization inside a single cell.

In addition to localization and distribution, expression levels of mRNA have also been studied inside of living cells using MBs. Using a two MB FRET approach, the relative expression levels of K-ras and survivin mRNA were determined in human dermal fibroblasts⁹. In this study, human dermal fibroblasts and pancreatic carcinoma cells were used to study the expression levels and the localization of K-ras and survivin mRNA. In order to accomplish this, a two MB FRET approach was used. This encompassed designing two MBs for adjacent target regions. Once both MBs hybridized to the same mRNA, the fluorophores on the MB were brought within a close distance and FRET was allowed to occur. Using this process they demonstrated the localization of the two mRNA sequences in the different cell types and how each different mRNA was localized within different regions inside the cell. The use of the two MBs allowed for much greater specificity, since the signal required two separate hybridization events to be generated.

Using MBs for Gene Expression Analysis

One of the limitations of MBs for intracellular analysis has been the inherent variability in the fluorescent measurements obtained. However, it is difficult to attribute these variations to any one cause such as instrumental sensitivity, experimental variability, variation in gene expression, or simply different amounts of probe being delivered into the cell. In an effort to eliminate experimental and instrumental variations to allow the study the stochasticity of gene expression, a method of ratiometric analysis was developed and applied to cancer cell genomics¹³. In this approach a fluorophore labeled DNA reference probe was injected along with the MBs. The reference probe exhibited a constant level of fluorescence relative to the MB signal that resulted in a ratio value for the MB which was independent of instrumental variations such as the amount of probe delivered into the cell and experimental variations like different cell volumes. This is due to the ratio of the reference probe to the MB being constant, therefore any increase in the ratio was due to the increase of the molecular beacon fluorescence indicating target hybridization. The concept of ratiometric analysis is demonstrated in Figure 3. This figure shows that the reference signal remains constant and that differences between hybridized and unhybridized MBs can be easily distinguished.

Figure 3. Intracellular imaging of single cells using MB probes. A ratiometric approach has been used to minimize experimental variations and enable more reliable data collection. On the top row are the cellular response for ‘closed’ MBs. On the bottom row are the cellular responses for ‘open’ MBs. (a) and (d) are the fluorescence emission images of a reference probe. (b) and (e) correspond to fluorescence emission images of the MB probe response. (c) and (f) are representative ratiometric images of the MB responses by dividing the image from the MB by the image of the reference probe.

Using the ratiometric analysis to normalize fluorescence measurements and to circumvent the sources of variation, the stochasticity of the gene expression could then be studied in detail using cells at basal expression levels, chemical gene stimulation in cells and cells transfected to overexpress a specific gene. Figure 4 shows the stochasticity of the basal gene expression of Manganese Superoxide Dismutase (MnSOD) within single cancer cells. As can be seen in figure 4 the gene of expression of MnSOD varies from cell to cell from very low expression levels to much higher expression levels. Once the ratiometric analysis was developed, the stochasticity of each group of cells was studied for both MnSOD and ß-actin mRNA expression using scatter plots. While the ß-actin mRNA expression was similar in each group of cells, the MnSOD expression for each group of cells had a distinct pattern. Thus the applicability of the ratiometric analysis was demonstrated and shown to compensate for many of the variations encountered within single cell analysis.

Figure 4. Ratiometric images of MDA-MB-231 cells at 10 minutes after the injection of MB-2. R values indicate the average ratiometric value of the cytoplasm after analysis.

Another recent publication also utilized ratiometric analysis as a means of normalizing the MB signal in order to compare gene expression profiles in different groups of cells¹⁴. In this publication multiple genes were measured simultaneously using ratiometric analysis. In these experiments, ß-actin and MnSOD mRNA were localized inside human breast cancer cells. In addition to MBs for ß-actin and MnSOD, a control MB was also injected that had no complement within the cell. The MBs were delivered using microinjection and imaged using confocal microscopy. The confocal microscope was able to resolve the three MB fluorophores in addition to the reference probe fluorophore. A sample cell in figure 5 shows the fluorescent images of the MBs and the reference probe. It is important to note in this figure that the reference probe fluorescence stays constant over the duration of the monitoring period which is essential to ratiometric analysis. Another noteworthy feature is that the control MB shows a very low level of fluorescence with no increase in signal during the monitoring period. This reinforces the conviction that the increase of the fluorescence seen in the ß-actin MB channel is due to a specific hybridization event, for if the signal increase was due to a non-specific interaction the control MB would also be affected. In order to demonstrate the potential of the technique, the chemical induction of gene expression was also studied. This was accomplished by incubating one group of cells with Lipopolysaccharide (LPS). LPS was previously shown to increase the expression of MnSOD mRNA. Twenty cells of each type were injected with the mixture of MBs and the reference probe and the gene expression profile of each cell was determined. The results from this study after ratiometric analysis illustrated that the control MB signal remains at a low level for both groups of cells. Similarly, levels of ß-actin expression remained very similar for both groups of cells. However, MnSOD mRNA expression increased dramatically in response to LPS as may be seen by the shift in the MnSOD expression in the histogram. While the overall trends are consistent, this study also revealed much variability in gene expression between cells. This is consistent with the aforementioned study which also demonstrated a high degree of stochasticity in levels of gene expression between cells. Moreover, these results were consistent with the growing realization that an ‘average’ cell, as defined by cell population analysis, might not exist^15,16.

Figure 5 Time elapsed fluorescent images of each MB inside of a single MDA-MB-231 cell. A) the image of the ß-actin MB (green), B) the image of the control MB (red), C) the image of the MnSOD MB (blue) , and D) the image of the reference probe (orange).

Problems with MBs inside the cell

While MBs have shown a great deal of promise, some limitations currently exist. One of the main limitations is a susceptibility of the MB to endogenous nucleases and single strand binding proteins. Both of these can induce a false positive response signal from the MB, and thus greatly limit the viability of the MB inside of a cell. MBs are degraded in human breast cancer cells regardless of their sequence within approximately 30 minutes. In addition, MB’s also exhibit a background fluorescence even in the closed state which limits the threshold of detection of the MB target sequence within the cell. Currently, many groups are working to combat these problems and have achieved some successes. These technological advancements include the use of modified DNA backbones to imbue the MB with resistance to nucleases, for example incorporating 2’-O-methyl bases¹⁷, locked nucleic acid bases¹⁸, novel superquenching moieties to achieve lower fluorescence backgrounds¹⁹, or amplifying polymer to increase signal²⁰. Combing nanoparticles and nanosensors with immobilized surface MB’s will lead to nanosized MB sensors for intracellular measurements in living cells^21-24. The continuing improvement of MB design and synthesis further extends its potential as an intracellular probe.

Conclusions

Thus far MBs have shown great potential as probes for intracellular analysis. The combination of their specificity and their ability to detect targets without separation makes them uniquely suited to intracellular applications. MBs enable the reporting of mRNA within single living cells, a feat that is presently not possible with other forms of mRNA detection and analysis. As these previous studies have shown, MB’s have a variety of uses including detection of mRNA, visualizing intracellular mRNA transport and distribution, monitoring changes in levels of gene expression, and even elucidating biological trends. Further research into delivery methods and design of MBs has made intracellular experiments much easier to perform, expanding this field of research. In the future, MBs will continue to prove effective probes for many purposes, since in theory, an MB can be designed for any mRNA sequence. Current advances in MB technology and imaging techniques continue to push the limits of MBs in terms of both sensitivity and reliability. Lower background fluorescence signals, more intense target signals, and a greater intracellular viability extend the potential range of uses for MBs in intracellular analysis, making the goals of detecting a low mRNA copy number and intracellular quantification attainable possibilities. Whilst using MB’s for intracellular analysis is still a nascent field, the unique properties of MBs ensure their continued application in biological and medical measurements at the cellular level.

Acknowledgement

This work was supported by NSF NIRT and NIH grants.

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