Chemoenzymatic Preparation of Functional Click‐Labeled Messenger RNA

Abstract

Synthetic mRNAs are promising candidates for a new class of transformative drugs that provide genetic information for patients’ cells to develop their own cure. One key advancement to develop so‐called druggable mRNAs was the preparation of chemically modified mRNAs, by replacing standard bases with modified bases, such as uridine with pseudouridine, which can ameliorate the immunogenic profile and translation efficiency of the mRNA. Thus the introduction of modified nucleobases was the foundation for the clinical use of such mRNAs. Herein we describe modular and simple methods to chemoenzymatically modify mRNA. Alkyne‐ and/or azide‐modified nucleotides are enzymatically incorporated into mRNA and subsequently conjugated to fluorescent dyes using click chemistry. This allows visualization of the labeled mRNA inside cells. mRNA coding for the enhanced green fluorescent protein (eGFP) was chosen as a model system and the successful expression of eGFP demonstrated that our modified mRNA is accepted by the translation machinery.

Development of a Universal RNA Dual-Terminal Labeling Method for Sensing RNA-Ligand Interactions

“…RNA single-labeling strategies have been successfully developed during the past few years [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] . In general, chemo-enzymatic approaches are used to directly install various labels (such as alkyne or azide) into different ncRNAs for the second-step fast click modifications of various functional groups 17,18 . In addition, direct chemical reaction of RNA at 3'-terminus can be achieved based on periodate chemistry 25 .…”

“…RNA 5'-terminus can be chemically labeled by several methods [14][15][16][17][18][19][20][21][22] . For example, direct 5'-functionalization with diazo reagents was achieved for short RNAs 20 , while the use toward long RNAs needs to be further validated.…”

Stealth fluorescence labeling for live microscopy imaging of mRNA delivery

“…23 Click-chemistry approaches have also been used to introduce fluorophores into the coding sequence of an mRNA post-transcription. 24 Despite progress in these areas, external, molecular dyes, such as the cyanines Cy3 and Cy5 remain the most common labeling choice. 25,26 Design and implementation of smaller and uncharged fluorophores for intrinsic native-like labeling of RNAs, compatible with both transcriptional and translational machineries, will therefore represent a major step forward.…”

Stronger together for in-cell translation: natural and unnatural base modified mRNA

“…16 Random introduction of functionalized nucleoside triphosphates during IVT in the coding region of the mRNA influences its expression pattern. 17 Post-transcriptional fluorescent mRNA functionalization applying copper-catalyzed azide-alkyne click reactions (CuAAC) allows mRNA visualization in cells 17 but is not suitable for live cell applications due to cell toxicity 18 and further limited by promoting RNA hydrolysis 19 . Expanding the approach to strainpromoted azide-alkyne click reactions (SPAAC) 17,20 enables click functionalization inside cells 17,20 but was so far only applied in protocols modifying an mRNA's poly(A) tail either at a single terminal position 17 or multiple times at random positions within the poly(A) sequence 20 .…”