RESUMO
The introduction of chemical modifications into long RNA molecules at specific positions for visualization, biophysical investigations, diagnostic and therapeutic applications still remains challenging. In this review, we present recent approaches for covalent internal labeling of long RNAs. Topics included are the assembly of large modified RNAs via enzymatic ligation of short synthetic oligonucleotides and synthetic biology approaches preparing site-specifically modified RNAs via inâ vitro transcription using an expanded genetic alphabet. Moreover, recent approaches to employ deoxyribozymes (DNAzymes) and ribozymes for RNA labeling and RNA methyltransferase based labeling strategies are presented. We discuss the potentials and limits of the individual methods, their applicability for RNAs with several hundred to thousands of nucleotides in length and indicate future directions in the field.
Assuntos
DNA Catalítico/metabolismo , RNA Catalítico/metabolismo , RNA Longo não Codificante/metabolismo , Coloração e Rotulagem , Humanos , RNA Longo não Codificante/análiseRESUMO
We present herein a novel nitroxide spin label-containing RNA triphosphate TPT3NO and its application for site-specific spin-labeling of RNA through inâ 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 inâ 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 185â nucleotide non-coding RNA, the B.â subtilis glmS ribozyme. The approach is then applied to probe for the first time the folding of the 377â nucleotide A-region of the long non-coding RNA Xist, by PELDOR.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Transcrição Gênica , Óxidos de Nitrogênio/química , Conformação de Ácido Nucleico , Marcadores de SpinRESUMO
Recent advances in pulsed electron paramagnetic resonance (EPR) spectroscopy enable studying structure and folding of nucleic acids. An efficient introduction of spin labels at specific positions within the oligonucleotide sequence is a prerequisite. We here present a step-by-step guide to synthesize long RNA oligonucleotides bearing spin labels at specific positions within the sequence. RNA preparation is achieved enzymatically via in vitro transcription using an expanded genetic alphabet. Highly structured, several hundred nucleotides long RNAs with two nitroxide spin labels at specific positions can be prepared by this method.
Assuntos
Oligonucleotídeos , RNA , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Oligonucleotídeos/química , Oligonucleotídeos/genética , RNA/química , RNA/genética , Marcadores de SpinRESUMO
The preparation of highly modified mRNAs and visualization of their cellular distribution are challenging. We report in-cell application of in vitro transcribed mRNA containing natural base modifications and site-specifically introduced artificial nucleotides. Click chemistry on mRNA allows visualization in cells with excellent signal intensities. While non-specific introduction of reporter groups often leads to loss in mRNA functionality, we combined the benefits from site-specificity in the 3'-UTR incorporated unnatural nucleotides with the improved translation efficiency of the natural base modifications Ψ and 5mC. A series of experiments is described to observe, quantify and verify mRNA functionality. This approach represents a new way to visualize mRNA delivery into cells and monitor its spread on a cellular level and translation efficiency. We observed increased protein expression from this twofold chemically modified, artificial mRNA counterbalancing a reduced transfection rate. This synergetic effect can be exploited as a powerful tool for future research on mRNA therapeutics.