12 research outputs found
Functional contacts of a transfer RNA synthetase with 2′-hydroxyl groups in the RNA minor groove
HPLC Purification of RNA Aptamers up to 59 Nucleotides with Single-Nucleotide Resolution
Lactobacillus gasseri CRISPR-Cas9 characterization In Vitro reveals a flexible mode of protospacer-adjacent motif recognition
Ribozyme targeting of the growth factor pleiotrophin in established tumors: a gene therapy approach
Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances
In this protocol, we describe the facile synthesis of the nitroxide spin-label 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene (TPA) and then its coupling to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. Subsequently, we explain how to perform distance measurements between two such spin-labels on RNA/DNA using the pulsed electron paramagnetic resonance method pulsed electron double resonance (PELDOR). This combination of methods can be used to study global structure elements of oligonucleotides in frozen solution at RNA/DNA amounts of similar to 10 nmol. We especially focus on the Sonogashira cross-coupling step, the advantages of the ACE chemistry together with the appropriate parameters for the RNA synthesizer and on the PELDOR data analysis. This procedure is applicable to RNA/DNA strands of up to similar to 80 bases in length and PELDOR yields reliably spin-spin distances up to similar to 6.5 nm. The synthesis of TPA takes similar to 5 days and spin labeling together with purification similar to 4 days. The PELDOR measurements usually take similar to 16 h and data analysis from an hour up to several days depending on the extent of analysis.</p