Biotin Labeled RNA

What is the biotin labeled RNA?

Biotin-labeled RNA denotes RNA molecules that have undergone chemical modification via the inclusion of biotin, a water-soluble vitamin often known as vitamin B7 or H. This diminutive, yet very adaptable, molecule facilitates the effective detection, separation, and manipulation of RNA molecules owing to the strong interaction between biotin and streptavidin (or avidin). Biotin labeling in RNA research has become an essential technique for several molecular biology, biochemical, and diagnostic applications, especially in contexts requiring non-radioactive and highly precise labeling. The advancement of nucleic acid-based therapies and the growing intricacy of RNA research are enhancing the significance of biotin-labeled RNA.

Preparation of native RNA using biotin-PC GMP as an initiator. (Luo Y., et al., 2011)

Preparing biotin-labeled RNA

Incorporation during in vitro transcription: This method entails the integration of biotinylated nucleotides (e.g., biotin-11-UTP) with natural ribonucleotides during RNA synthesis utilizing T7, T3, or SP6 RNA polymerase. This approach facilitates the efficient synthesis of RNA molecules including biotin, uniformly dispersed throughout the transcript, hence allowing for detection or immobilization. This technique can be meticulously adjusted to generate RNA molecules with biotin residues at designated locations or with a certain level of biotinylation, hence augmenting experimental versatility.

Post-transcriptional biotinylation: A different approach is the chemical conjugation of biotin to RNA post-synthesis. Reactive cross-linkers, such sulfo-NHS-biotin, enable the selective attachment of biotin to exposed functional groups on RNA, including primary amines or thiols. This method is advantageous when it is crucial to include biotin at specific sites on the RNA strand, hence assuring minimum interference with the RNA's biological function.

Procedure for biotin labeling of RNA. (Huang F., et al., 2008)

RNA conjugation technology at BOC Sciences

Applications of biotin-Labeled RNA

RNA detection and visualization: Biotin-labeled RNA probes serve as a dependable and extremely sensitive alternative to typical radioactive probes in current molecular methods such as Northern blotting, RNA in situ hybridization, and RNA microarrays. Biotin-tagged RNA may be easily detected using streptavidin-conjugated reporters like horseradish peroxidase (HRP) or fluorescent molecules, resulting in non-toxic, high-throughput RNA detection.

RNA purification and isolation: Biotin-labeled RNA facilitates swift and effective purification from intricate biological specimens. The utilization of streptavidin-coated magnetic beads or affinity columns facilitates the selective isolation of biotin-tagged RNA, which is especially advantageous in RNA pull-down studies. This approach is essential for finding RNA-binding proteins (RBPs) and offers insights into post-transcriptional regulatory networks.

RNA-protein interaction studies: Biotin-labeled RNA is employed in RNA immunoprecipitation (RIP) and cross-linking immunoprecipitation (CLIP) procedures to identify and analyze RNA-protein complexes, clarifying the functions of certain proteins in mRNA splicing, translation, and degradation. Recent research, particularly in RNA biology, has employed biotin-labeled RNA to identify new riboprotein complexes that modulate gene expression across various cellular environments.

Schematic of the strategy for surface immobilization of biotin labeled RNA. (Vincent H A., et al., 2013)

Therapeutic potential in gene therapy: The therapeutic applications of biotin-labeled RNA have broadened to include gene therapy and RNA-based pharmaceuticals. Conjugating biotin-labeled RNA molecules to streptavidin-linked delivery systems enables the targeted distribution of functional RNA, including siRNA or antisense oligonucleotides, to specific cells. The targeted delivery, together with the non-toxic properties of biotin, has created new opportunities for precise gene silencing and modification in disease situations, especially in the treatment of genetic diseases and malignancies.

RNA structural and functional analysis: Biotin-labeled RNA is notably utilized in structural biology for footprinting tests and ribosome profiling. Researchers utilize biotin labeling to delineate RNA secondary structures or investigate conformational changes upon protein interaction, providing a comprehensive insight into RNA folding mechanisms.

Development of RNA-based biosensors and methods: Recent research has concentrated on the development of biosensors and methods that detect specific molecules, such as metabolites or pathogens, by utilizing biotin-labeled RNA to form aptamers or ribozymes. Rapid diagnostics can be achieved by incorporating these RNA-based biosensors into lab-on-a-chip systems.

In vivo proximity labeling strategies for identifying protein partners of an RNA of interest. (Weissinger R., et al., 2021)

Advantages of biotin labelled RNA

High sensitivity and specificity: A primary benefit of biotin-labeled RNA is its exceptional sensitivity in detection experiments. The robust and highly specific binding relationship between biotin and streptavidin (or avidin) facilitates the efficient and reliable capture, separation, or detection of biotinylated RNA. This relationship is especially beneficial in techniques such as RNA pull-down experiments, Northern blotting, and in situ hybridization, where precise detection is crucial.

Versatility in multiple applications: Biotin-labeled RNA is applicable in several domains, such as RNA purification, RNA-protein interaction investigations, RNA structure research, and medicinal delivery mechanisms. This adaptability arises from the simplicity of biotin conjugation to RNA, facilitating the creation of highly flexible experimental methods across several fields of research. The label may be inserted at several locations throughout the RNA strand, providing researchers with control over the extent of labeling and functional versatility.

Non-radioactive labeling: In contrast to conventional labeling techniques employing radioisotopes, biotin labeling offers a non-toxic and safer option. The application of biotin mitigates the risks and intricacies linked to the management of radioactive substances while offering comparable or superior sensitivity in detection techniques. Biotin labeling-RNA has become immensely advantageous for diagnostic and research facilities, especially in high-throughput screening settings.

Compatibility: Biotin-labeled RNA is suitable for several detection technologies, including streptavidin-conjugated enzymes [e.g., horseradish peroxidase (HRP)], fluorescent markers, or magnetic beads. This facilitates the effective detection, purification, or visualization of RNA and provides flexibility in selecting the most appropriate detection method according to individual research requirements, ranging from high-resolution imaging to quantitative tests. Due to its non-radioactive characteristics and detectability through various techniques, biotin-labeled RNA is suitable for high-throughput screening tests. This is especially beneficial in drug development and functional genomics research, where extensive screening of RNA-protein interactions or RNA structure-function connections is required.

Facilitates RNA isolation and purification: Employing biotin-labeled RNA alongside streptavidin-coated magnetic beads or affinity columns offers a quick and effective technique for the isolation of individual RNA molecules from complex biological mixtures. This has demonstrated significant utility in RNA pull-down tests and the investigation of RNA-binding proteins (RBPs), where accuracy and purity are essential. The robust biotin-streptavidin binding guarantees negligible loss of the RNA sample during purification.

How to use the biotin labeled RNA on RNA-protein interaction studies?

RNA Pull-Down Assays: Biotin-conjugated RNA is treated with cellular lysates or nuclear extracts. RNA-binding proteins in the lysate interact with biotin-labeled RNA. The RNA-protein complexes are subsequently isolated utilizing streptavidin-coated magnetic beads or agarose beads, which adhere to the biotin moiety on the RNA. The bound proteins are subsequently eluted, separated by SDS-PAGE, and identified via mass spectrometry or Western blotting. This technique offers a focused strategy to isolate specific RNA-protein interactions, enabling researchers to identify the proteins that directly associate with a given RNA sequence or structure.

CLIP (Crosslinking and Immunoprecipitation) Assays: In certain instances, biotin-labeled RNA may be delivered into cells, subsequently undergoing crosslinking (usually UV-induced) to solidify RNA-protein connections. Following crosslinking, the RNA-protein complexes are isolated utilizing streptavidin-coated beads. The isolated proteins and corresponding RNA fragments are identified using sequencing or mass spectrometry. This approach elucidates protein binding within a more natural setting, demonstrating the RNA sequences that interact with proteins in living cells.

Mapping RNA-Binding sites: Biotin-labeled RNA is utilized with RNase digesting methods, including RNA footprinting. The RNA is associated with proteins, and the portions shielded by these bound proteins are resistant to nuclease digestion, signifying the protein-binding site. The safeguarded fragments can then be isolated utilizing streptavidin-coated beads and examined via sequencing. This approach effectively identifies precise binding sites on RNA, offering structural insights into RNA-protein interactions.