EPR Distance Measurements on Long Non‐coding RNAs Empowered by Genetic Alphabet Expansion Transcription

Abstract

We present herein a novel nitroxide spin label‐containing RNA triphosphate TPT3NO and its application for site‐specific spin‐labeling of RNA through in vitro transcription using an expanded genetic alphabet. Our strategy allows the facile preparation of spin‐labeled RNAs with sizes ranging from short RNA oligonucleotides to large, complex RNA molecules with over 370 nucleotides by standard in vitro transcription. As a proof of concept, inter‐spin distance distributions are measured by pulsed electron paramagnetic resonance (EPR) spectroscopy in short self‐complementary RNA sequences and in a well‐studied 185 nucleotide non‐coding RNA, the B. subtilis glmS ribozyme. The approach is then applied to probe for the first time the folding of the 377 nucleotide A‐region of the long non‐coding RNA Xist, by PELDOR.

Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions

“…This preserves the co-transcriptional folding of the target RNA, and is advantageous for RNA structure-function relationship studies. We note that the rTPT3 CO labeling scheme is compatible with denaturing methods, as either rTPT3/rTPT3 CO incorporated RNAs 41,65 or nitroxide-labeled RNAs 20,66 have been puried with denaturing gels.…”

“…During our manuscript preparation, a similar UBP-based spin labeling approach for RNAs was reported by Domnick and co-workers. 41 In that report, Domnick and co-workers synthesize a nitroxide-modied triphosphate ribonucleotide TPT3 NO that is incorporated into RNAs during in vitro transcription. The direct use of TPT3 NO in in vitro transcription incurs potential risks of damaging the nitroxide, for example, exposure to reducing agents during in vitro transcription, and may account for the slightly lower labeling efficiency reported by Domnick and co-workers 41 (see Table S5 †).…”

“…41 In that report, Domnick and co-workers synthesize a nitroxide-modied triphosphate ribonucleotide TPT3 NO that is incorporated into RNAs during in vitro transcription. The direct use of TPT3 NO in in vitro transcription incurs potential risks of damaging the nitroxide, for example, exposure to reducing agents during in vitro transcription, and may account for the slightly lower labeling efficiency reported by Domnick and co-workers 41 (see Table S5 †). In addition, constrained by the requirement that the RNA polymerase must accept the nitroxide-modied unnatural base (i.e., TPT3 NO ) as a substrate, a relatively long and exible linker is utilized to connect the nitroxide to TPT3.…”

“…39,40 Very recently, a nitroxide-modied triphosphate TPT3 (TPT3 No ) was developed to achieve site-specic spin labeling of RNAs through in vitro transcription. 41 Here, we present the synthesis of alkyne-derivatized TPT3 (rTPT3 CO TP) ( Fig. 1) and demonstrate its application in posttranscriptional site-directed spin labeling of large RNAs.…”

Cognate base‐pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase

“…The successful development of replicable unnatural base pairs (UBPs) with high fidelity has founded genetic alphabet expansion technologies. [1][2][3] Practical applications in vitro and in vivo have rapidly advanced in the wide areas of novel quantitative PCR methods, [4,5] high-affinity DNA aptamer generation, [6,7] RNA labeling, [8,9] and the creation of semi-synthetic organisms for protein synthesis involving unnatural amino acids. [10,11] Basic DNA recombination technology involving UBPs expedites UBP-applications.…”