An Electrophilic Uridine Building Block for Post-Synthetic RNA Modification as Exemplified for Spin Labeling.

Area of Science:

• Biochemistry
• Organic Chemistry
• Molecular Biology

Background:

• Isothiocyanate (ITC) conjugation is a common method for protein labeling.
• Existing methods involve labeling reagents with reactive amino groups.
• A new approach is needed for efficient and minimally perturbing RNA modification.

Purpose of the Study:

• To synthesize a 5-isothiocyanato methyl-modified uridine building block for RNA.
• To enable facile post-synthetic modification of RNA with primary amines.
• To position unnatural tags in the major groove of RNA with minimal fold perturbation.

Main Methods:

• Developed an "upside-down" ITC labeling approach where RNA carries the electrophilic ITC functionality.
• Utilized solid-phase synthesis for RNA modification.
• Applied the method to a 36-nucleotide (nt) stem loop C from Moloney murine leukemia virus (MMLV) 5'-Leader (SLC^A RNA).
• Performed a paramagnetic relaxation enhancement study using 4-amino-2,2,6,6-tetramethyl-1-piperidine-1-oxyl (TEMPO-NH₂) as a labeling reagent.

Main Results:

• Successfully synthesized the 5-isothiocyanato methyl-modified uridine building block.
• Demonstrated facile post-synthetic modification of RNA with primary amines.
• Showcased minimal perturbation of RNA structure by labeling in the major groove.
• Exemplified the labeling procedure on SLC^A RNA for relaxation enhancement studies.

Conclusions:

• The novel uridine building block and "upside-down" ITC chemistry offer a versatile tool for RNA modification.
• This method allows for precise and minimally disruptive labeling of RNA.
• The technique is suitable for advanced biophysical studies, such as paramagnetic relaxation enhancement, providing insights into RNA structure and function.