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

Abstract

Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of lightcontrollable RNA nucleotides into the tRNA Met sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

“…While this method holds great promise for NMR applications, low yields of bis-phosphorylation (6–22%) and ligation (9–49%) reactions are a major drawback . More recent efforts by Schwalbe and co-workers to improve this technology include the addition of magnetic streptavidin beads as a solid-support and 5′-biotinylated RNA …”

“…As described in Section and shown in Scheme , the chemo-enzymatic labeling method developed by Dayie and co-workers ,, permits the synthesis of a versatile assortment of rNTPs with atom-specific isotope labels (Table ). While there are other enzymatic methods to generate both atom-specific (e.g., the Gilles-Schramm-Williamson ,− or Serriani methods shown in Schemes and , respectively) and position-specific (e.g., PLOR and the Schwalbe method , ) labels, no other technique offers the versatility and simplicity that is afforded by the Dayie method. Our one-pot chemo-enzymatic approach can produce isotope-labeled purine and pyrimidine rNTPs in a few days and with high yield (75–95%) (Table ).…”

“…While the enzymatic production of RNA with isotope-labeled rNTPs ,,− is the most widely used approach to obtain labeled RNA, an attractive alternative is to use isotope-labeled amidites and solid-phase synthesis. Like PLOR introduced by Wang and co-workers and the chemo-enzymatic approach developed by Schwalbe and co-workers, , the amidite method offers the advantage of position-specific RNA labeling. However, even though amidite labeling is currently the most effective and widely used method for position-specific labeling, its utility for NMR studies is limited to RNAs ≈ 60 nt.…”

Trendbericht Organische Chemie 2022

“…143) Heckel und Schwalbe stellten chemoenzymatisch komplexe RNAs her, insbesondere tRNAs, mit lichtschaltbaren, chemisch modifizierten Nukleotidbausteinen. 144) Bei chemisch modifizierten Oligonukleotiden stellten Micura und…”

Blue light-activatable DNA for remote control of cell-free logic gates and synthetic cells

“…Longer wavelength photocages would be preferred, however, as visible light shows reduced damage to biomolecules 42,43 and tissue penetration is increased with longer wavelengths. 44,45 Longer wavelength photocages for use in biological systems have been reported, 46 and there are also several reports showing sequential activation of 2 photocages [47][48][49] . However, orthogonal activation of 2 or more photocages has few reported examples [50][51][52][53][54] , mostly due to chromophores having overlapping absorbance bands and orthogonal release thus being difficult.…”