Genetic Code Expansion Facilitates Position‐Selective Labeling of RNA for Biophysical Studies

Abstract

Nature relieso nr eading and synthesizingt he genetic codew ith high fidelity.N ucleic acid building blocks that are orthogonal tot he canonical A-T and G-C base-pairs are therefore uniquely suitable to facilitate position-specific labeling of nucleic acids. Here, we employ the orthogonal kappa-xanthosine-base-pair for in vitro transcription of labeled RNA. We devised an improved synthetic route to obtain the phosphoramidite of the deoxy-version of the kappa nucleoside in solid phase synthesis. From this DNA template, we demonstrate the reliable incorporation of xanthosined uring in vitro transcription. Using NMR spectroscopy,w eshowt hat xanthosine introduces only minor structural changes in an RNA helix. We furthermore synthesized a clickable 7-deaza-xanthosine, which allowst os ite-specifically modify transcribed RNA molecules with fluorophores or other labels.

Strategies for Covalent Labeling of Long RNAs

“…[91] Recently, the kappa (k)-xanthosine (X) base pair of Benner, [93] also forming an artificial hydrogen bonding pattern, was used to site-specifically label the 73 nt long guanine sensing riboswitch RNA from B. subtilis by Hegelein et al. [94] For this, they synthesized the clickable X derivative 7-deaza-xanthosine bearing a terminal alkyne residue which was then site-specifically incorporated into RNA by IVT to enable post-transcriptional labeling with Cy3-azide via click chemistry (Figure 4, left panel). [94] In 2004, based on the 2-oxo(1H)pyridine (y) -2-amino-6(2thienyl)purine (s) base pair system, [95] Hirao and co-workers incorporated a photo-sensitive iodine-modified y derivative into an anti(Raf-1) RNA aptamer at a predefined position during transcription thereby enabling photo-crosslinking of two aptamer molecules.…”

“…[94] For this, they synthesized the clickable X derivative 7-deaza-xanthosine bearing a terminal alkyne residue which was then site-specifically incorporated into RNA by IVT to enable post-transcriptional labeling with Cy3-azide via click chemistry (Figure 4, left panel). [94] In 2004, based on the 2-oxo(1H)pyridine (y) -2-amino-6(2thienyl)purine (s) base pair system, [95] Hirao and co-workers incorporated a photo-sensitive iodine-modified y derivative into an anti(Raf-1) RNA aptamer at a predefined position during transcription thereby enabling photo-crosslinking of two aptamer molecules. [96] They also demonstrated incorporation of bulkier y derivatives, modified with biotin or fluorophores, into RNA molecules opposite s or 2-amino-6-(2-thiazolyl)purine (v) in DNA templates by T7 RNAP (Figure 4, left panel).…”

“…. [94] For this, they synthesized the clickable X derivative 7‐deaza‐xanthosine bearing a terminal alkyne residue which was then site‐specifically incorporated into RNA by IVT to enable post‐transcriptional labeling with Cy3‐azide via click chemistry (Figure 4 , left panel). [94] …”

“…[ 8 , 16 , 82 , 83 , 84 , 85 , 86 , 87 ] Compatibility with diverse click reactions, moreover, allows post‐transcriptional functionalization with virtually any modification of choice. [ 16 , 90 , 94 , 103 , 106 , 107 , 110 ]…”

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.…”