Strategies for Covalent Labeling of Long RNAs

Abstract

The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site-specifically modified RNAs via in vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field.

Azide‐Modified Nucleosides as Versatile Tools for Bioorthogonal Labeling and Functionalization

“…Over the years however, multiple chemical and enzymatic approaches have been developed for sequence and site specific incorporation of azide-modified building blocks. [10,68,69] One such procedure has been described by Rao et al in 2012. [70] It involves the synthesis of an azide-modified UTP derivative, which could be selectively transferred into an RNA by in vitro transcription of a DNA template with T7 RNA polymerase.…”

“…Over the years many such methods, which rely on the selective and site specific introduction of modifications or reactive handles into the ONs of interest have been developed. [9][10][11] Out of this wide and versatile toolbox the copper catalyzed azide alkyne cycloaddition (CuAAC) or the copper free strain-promoted azide alkyne cycloaddition (SPAAC) are of particular usefulness. These so called click reactions are often used for the introduction of affinity tags like biotin, [12] fluorophores, [13] or the formation of bioconjugates.…”

“…It is therefore not surprising that there is an immense demand in the scientific community for methods which allow the identification, tracking and specific isolation of various ONs in vitro and in cells without interference or cross reactions with other biomolecules. Over the years many such methods, which rely on the selective and site specific introduction of modifications or reactive handles into the ONs of interest have been developed [9–11] . Out of this wide and versatile toolbox the copper catalyzed azide alkyne cycloaddition (CuAAC) or the copper free strain‐promoted azide alkyne cycloaddition (SPAAC) are of particular usefulness.…”

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

“…Long RNAs such as mRNAs with several hundred to several thousand nucleotides in length are generally synthesized via in vitro transcription and site-specific introduction of fluorescent reporter groups into such RNA sequences is challenging. 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 .…”

Solid‐Phase‐Supported Chemoenzymatic Synthesis of a Light‐Activatable tRNA Derivative

“…Thus, position-specific labeling is difficult. [19][20][21][22] Chemoenzymatic approaches can be a solution: we reported earlier that a combination of two different T4 RNA ligases and modified 5',3'-bisphosphates enabled us to synthesize a 392mer RNA modified at one specific internal position. [23] This approach does not use the harsh conditions of chemical solid-phase synthesis and thus allows the introduction of more delicate nucleotide modifications.…”