A distinctive feature found in eukaryotic mRNA is the presence of a cap structure at the 5' terminus, which is an important modification and specifically interacts with numerous cellular proteins to involve in pre-mRNA splicing, RNA export, translation initiation and RNA turnover. With access to advanced instruments, fit-for-purpose laboratories, and professional knowledge, Creative Biolabs offers a comprehensive range of mRNA services, including but not limited to mRNA synthesis, modification, delivery, stability test, and mRNA-based cell reprogramming. Amongst, mRNA 5' fluorescent cap is an available and delicate service item in our mRNA modification portfolio.
Almost all eukaryotic mRNAs have a cap at the 5' terminus, which is a unique residue composed of a 7-methylguanosine moiety connected to the mRNA via a triphosphate linkage, m7GpppGpNp(Np)n. This specific methylated modification plays important roles in all stages of mRNA metabolism: synthesis, splicing, nucleocytosolic transport, intracellular localization, translation, and turnover.
Synthetic analogs of the mRNA cap have been proven to be valuable tools in the study of cap-associated processes without interfering with their biological functions, such as radioactive, fluorescent, and other spectroscopic labels. Fluorescence is a potent technique to obtain visualized information about the behavior and distribution of molecules within biological systems, usually aided by the fluorescence microscopy or Förster resonance energy transfer (FRET). Fluorescence mRNA also proves invaluable for mRNA dynamics, interactions and other biophysical studies. The most commonly used fluorophores in mRNA capping are low molecular weight groups that can not interfere with eIF4E binding interactions, such as anthraniloyl (Ant), N-methylanthraniloyl (Mant) and trinitrophenyl (TNP). These dyes are environmentally sensitive fluorophores that emit a stronger signal upon binding to a target mRNA. Nucleotides labeled with these tags can closely mimic naturally occurring nucleotides in their interactions with molecular targets.
Creative Biolabs offers a variety of strategies for the covalent linkage of fluorophores to RNA, mostly focusing on cotranscriptional or posttranscriptional enzymatic labeling approaches.
In vitro transcription (IVT) RNA is subjected to a dedicated enzymatic capping reaction. The capping enzymes are produced recombinantly in E. coli and developed from capping apparatuses of different eukaryotic organisms or DNA viruses (such as vaccinia virus). Usually, this method offers the advantage of the capability of 100% capping on a large quantity of IVT.
Fig.1 Schematic representation of post-transcriptional capping. (Muttach, 2017)
In this way, cap analogs are added directly to the IVT at the 5' end by RNA polymerases with relaxed substrate specificity (e.g. T3, T7 or SP6 RNA polymerases). Therefore, this route allows for a single-step workflow and permits more flexibility to incorporate non-canonical cap structures (besides m7GpppG) because RNA polymerases also accept several modified or alternative cap analogs.
Fig.2 Schematic representation of co-transcriptional capping with different cap analogs. (Muttach, 2017)
Creative Biolabs provides affordable custom mRNA services to help researchers tailor synthesis and introduce modifications to produce transcripts that are optimal for downstream applications. If you are interested in our services, please feel free to contact us.
Reference
