Cy5 Labeled RNA

What is the Cy5 labeled RNA?

Cy5-labeled RNA is an RNA molecule conjugated with Cy5, a cyanine-derived fluorescent dye. This labeling technique is extensively employed in molecular biology and biochemistry for many applications necessitating the detection, quantification, and imaging of RNA. The Cy5 fluorophore produces red light (~670 nm) when excited, rendering it suitable for fluorescence tests and imaging methods.

Cy5-conjugated poly(A) sequence of mRNA. (Kubota T., et al., 2011)

Preparation of Cy5-Labeled RNA

Cy5 tagging of RNA is often accomplished by including Cy5-labeled nucleotides during RNA synthesis or by employing post-transcriptional chemical conjugation. In the previous method, Cy5-modified nucleotides (e.g., Cy5-UTP or Cy5-CTP) are directly integrated into RNA strands during in vitro transcription, utilizing enzymes like T7 RNA polymerase. This technique guarantees consistent labeling across the RNA strand but requires meticulous regulation to prevent interference with RNA folding or functionality caused by the steric hindrance of the Cy5 dye. In post-synthesis labeling, Cy5 is conjugated to pre-synthesized RNA at designated places, generally via reactive groups on the RNA that establish covalent connections with the Cy5 dye. This approach enhances the accuracy of tagging specific RNA sections, hence reducing potential disruption of its biological function. The difficulty with both methods is preserving the functional integrity of the RNA molecule while achieving sufficient tagging density for successful detection.

Common fluorescent molecules

• Fluorescein isothiocyanate (FITC)
• TAMRA
• FAM
• Alexa Fluor: Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 647
• Cyanine: Cy3, Cy5
• Dylight: Dylight 488, Dylight 550, Dylight 650
• ROX
• BODIPY

RNA conjugation technology at BOC Sciences

Applications of Cy5-Labeled RNA

Fluorescence In Situ Hybridization (FISH): One of the principal uses of Cy5-labeled RNA is in FISH experiments, wherein fluorescent RNA probes are employed to identify particular RNA sequences inside cellular or tissue specimens. The far-red emission of Cy5 provides a notable benefit by diminishing background autofluorescence, therefore enhancing the signal-to-noise ratio and elevating the precision of RNA detection.

RNA localization and dynamics: Utilize Cy5 to label RNA in order to monitor the mobility and spatial distribution of RNA molecules within live cells throughout time. These studies are essential for comprehending the function of RNA in biological processes, including gene expression control and RNA transport. The emergence of super-resolution imaging methods has enabled real-time detection of Cy5-labeled RNA localization with unparalleled accuracy. Without requiring protein translation, Cy5 incorporation allowed for the fluorescence detection of the target mRNA. Research has demonstrated that similarly high levels of fluorescent Cy5 were detected in cells for at least 42 hours after incubation of the Cy5-labeled mCherry-RBD-mRNA with target cells, and fluoresce was still detectable in > 60% of the cells at 68 hours postincubation. Furthermore, cells treated with Cy5-labeled mCherry-RBD-mRNA exhibited a fluorescent (violet) signal, but cells incubated with empty LNPs did not.

Cy5-labeled mCherry-RBD-mRNA. (Tai W., et al., 2022)

RNA-protein interaction studies: Cy5-labeled RNA is extensively utilized in tests, including RNA electrophoretic mobility shift assays (EMSAs) and pull-down procedures, to examine interactions between RNA and certain proteins. The fluorescent label in these investigations enables swift and sensitive detection of RNA-protein complexes, aiding in the discovery of crucial regulators of RNA function in processes including translation, splicing, and destruction.

CRP-RNA complex formation. (Wang M S., et al., 2011)

RNA structural analysis: In structural investigations, Cy5-labeled RNA is frequently utilized in fluorescence resonance energy transfer (FRET) assays. FRET pairings, including Cy5 and Cy3, are employed to assess distances between various sections of an RNA molecule, yielding insights into its folding processes. These research are essential for comprehending the structural foundation of RNA activity and for creating RNA-based therapies.

Cy3 and Cy5 signal from same set of molecules. (Sarkar J., et al., 2019)

Stereoselective modification of small RNA with Cy5. (Shioi R., et al., 2023)

Quantitative Real-Time PCR (qPCR): Cy5-labeled RNA serves as a fluorescent probe in qPCR tests for the detection and quantification of particular RNA sequences with high sensitivity. The fluorescent signal produced by Cy5 during PCR amplification facilitates real-time monitoring of RNA levels, rendering this approach essential in gene expression research and diagnostic testing.

Drug discovery and development: In drug development, Cy5-labeled RNA is utilized in high-throughput screening tests to find drugs that interact with RNA targets. The fluorescence signal from Cy5 serves as an indicator for binding events, enabling the detection of tiny compounds or peptides that influence RNA activity. This use is especially pertinent in the advancement of RNA-targeting therapies, which are attracting interest for their ability to address disorders resulting from aberrant RNA processing or expression.

Advantages of Cy5-Labeled RNA

Cy5-labeled RNA is a favored option for a wide variety of experimental applications because it possesses a number of important benefits that make it an attractive option. One of the most important advantages is that it has a high sensitivity, which enables the detection of low-abundance RNA species. This makes it useful for applications in which the target RNA levels are either restricted or difficult to view. Furthermore, the far-red emission spectrum of Cy5 helps to reduce interference from background autofluorescence in biological materials, which in turn improves the clarity and reliability of measurements that are dependent on fluorescence. Moreover, the potential of Cy5 to do multiplexing is an additional key advantage. Combining Cy5 with other fluorophores, such as Cy3 or FITC, makes it possible to detect various RNA species simultaneously inside the same sample. This is made possible by the fact that Cy5 may be coupled with other fluorophores. When it comes to complicated tests, where it is important to monitor the expression of several RNA molecules, this functionality is especially helpful. In addition, Cy5 provides a non-radioactive alternative to the conventional methods of radiolabeling. This reduces the regulatory and disposal problems that are connected with radioactive materials, in addition to improving the safety of the laboratory and making it easier to handle the materials. In conjunction with this, Cy5 is well-known for its photostability, which enables it to operate for extended periods of time without suffering a major reduction in signal strength.

Disadvantages of Cy5-Labeled RNA

Photobleaching: In prolonged imaging investigations or fluorescence-based tests, the signal from Cy5-labeled RNA may diminish, complicating the proper tracking or visualization of RNA. This constrains the duration and dependability of experiments necessitating extended observation.

Steric hindrance: The Cy5 fluorophore is a comparatively sizable molecule. When bound to RNA, it can induce steric hindrance, hence influencing the inherent interactions between the RNA and its binding partners (e.g., proteins or complementary RNA/DNA sequences). This may disrupt RNA-protein interactions, RNA folding, or RNA hybridization, thereby modifying the typical function and behavior of RNA in biological systems.

Signal sensitivity to environmental conditions: The fluorescence intensity of Cy5 is susceptible to environmental variables including pH, ionic strength, and temperature. Under poor conditions, the fluorescent signal from Cy5-labeled RNA may vary or decrease, resulting in inconsistent outcomes or reduced sensitivity in fluorescence-based tests.

Altered RNA functionality: The conjugation of a Cy5 molecule to RNA may occasionally interfere with the RNA's native structure or function. This is especially troubling if Cy5 is affixed near functionally significant sections, such as ribosomal binding sites or places essential for RNA folding. Altered RNA may fail to interact appropriately with other molecules (including ribosomes, enzymes, or proteins), resulting in erroneous outcomes in functional assays, such as translation studies or RNA-binding protein research.