Product Overview

Azido-hexynyl-pApG is a chemically synthesized capping initiator designed for site-specific modification of the 5′-end of mRNA during in vitro transcription (IVT). Incorporation of this molecule introduces a reactive azide group at the 5′-terminus, enabling precise, bioorthogonal conjugation via click chemistry, including both SPAAC (copper-free) and CuAAC (copper-catalyzed) reactions.

Unlike natural cap analogs that facilitate ribosomal binding and high-efficiency translation, Azido-hexynyl-pApG provides a stable, translationally almost silent capping structure. While it does not achieve the translation levels of a traditional Cap 0 or Cap 1 structure, it still offers enhanced translation efficiency compared to uncapped RNA, while preserving RNA integrity and functionality for downstream applications.

Current Limitation in mRNA Labeling Technologies

Most existing mRNA labeling or functionalization techniques focus on the 3′ end of the molecule, where enzymatic strategies enable site-selective attachment of fluorophores, affinity tags, or targeting ligands. However, this focus limits spatial control and may interfere with poly(A) tail function, stability, or protein interactions.

In contrast, 5′-end modification has historically been underutilized due to the difficulty of introducing functional handles at the initiation site without disrupting cap-dependent translation or structural stability.

Outcome & Advantage of Azido-hexynyl-pApG

Azido-hexynyl-pApG fills this technological gap by enabling:

• Site-specific 5′-end labeling of mRNA during synthesis
• Orthogonal click chemistry compatibility (SPAAC & CuAAC) for post-synthetic modification
• Preservation of structural integrity and partial translation capacity
• Improved experimental flexibility, particularly for RNA imaging, delivery, and bioconjugation studies

This allows researchers to:

• Expand the functional versatility of synthetic mRNAs
• Attach cell-targeting ligands, tracking probes, or therapeutic cargos to the 5′-end — complementing existing 3′-end approaches
• Perform dual-end labeling, supporting deeper insight into RNA transport, localization, and interaction dynamics

Applications

• Fluorescent labeling of mRNA for imaging and tracking
• Targeted delivery by conjugating ligands, peptides, or nanoparticles
• RNA therapeutics and gene replacement strategies where 5′-end modification enhances delivery or selectivity
• Structure–function studies in RNA biology, translation, and RNA–protein interactions

Summary

Azido-hexynyl-pApG offers a powerful and modular solution for expanding the toolkit of RNA chemists and molecular biologists. By enabling bioorthogonal, site-specific 5′-end labeling, it overcomes the limitations of 3′-biased modification strategies and opens new directions in synthetic mRNA design, delivery, and detection.