Practical Aspects of Sample Preparation and Setup of 1H R1&#961; Relaxation Dispersion Experiments of RNA

RNA is a highly flexible biomolecule, wherein changes in structures play crucial roles in the functions that RNA molecules execute as cellular messengers ...

Karolinska Institutet

RNA is becoming more important as an increasing number of functions, both regulatory and enzymatic, are being discovered on a daily basis. As the RNA boom has just begun, most techniques are still in development and changes occur frequently. To understand RNA functions, revealing the structure of RNA is of utmost importance, which requires sample preparation. We review the latest methods to produce and purify a variation of RNA molecules for different purposes with the main focus on structural biology and biophysics. We present a guide aimed at identifying the most suitable method for your RNA and your biological question and highlighting the advantages of different methods. Graphical abstract In this review we present different methods for large-scale production and purification of RNAs for structural and biophysical studies.

A guide to large-scale RNA sample preparation.

An ever-increasing number of functional RNAs require a mechanistic understanding. RNA function relies on changes in its structure, so-called dynamics. To reveal dynamic processes and higher energy structures, new NMR methods have been developed to elucidate these dynamics in RNA with atomic resolution. In this Review, we provide an introduction to dynamics novices and an overview of methods that access most dynamic timescales, from picoseconds to hours. Examples are provided as well as insight into theory, data acquisition and analysis for these different methods. Using this broad spectrum of methodology, unprecedented detail and invisible structures have been obtained and are reviewed here. RNA, though often more complicated and therefore neglected, also provides a great system to study structural changes, as these RNA structural changes are more easily defined-Lego like-than in proteins, hence the numerous revelations of RNA excited states.

RNA Dynamics by NMR Spectroscopy.

We present a 2D replica exchange protocol incorporating secondary structure information to dramatically improve 3D RNA folding using molecular dynamics simulations. We show that incorporating base-pairing restraints into all-atom, explicit solvent simulations enables the accurate recapitulation of the global tertiary fold for 4 representative RNAs ranging in length from 24 to 68 nt. This method can potentially utilize base-pairing information from a wide variety of experimental inputs to predict complex RNA tertiary folds including pseudoknots, multi-loop junctions, and non-canonical interactions.

A two-dimensional replica-exchange molecular dynamics method for simulating RNA folding using sparse experimental restraints.

There is an increasing demand for efficient and robust production of short RNA molecules in both pharmaceutics and research. A standard method is  transcription by T7 RNA polymerase. This method is sequence-dependent on efficiency and is limited to products longer than ~12 nucleotides. Additionally, the native initiation sequence is required to achieve high yields, putting a strain on sequence variability. Deviations from this sequence can lead to side products, requiring laborious purification, further decreasing yield. We here present transcribing tandem repeats of the target RNA sequence followed by site-specific cleavage to obtain RNA in high purity and yield. This approach makes use of a plasmid DNA template and RNase H-directed cleavage of the transcript. The method is simpler and faster than previous protocols, as it can be performed as one pot synthesis and provides at the same time higher yields of RNA.

One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts.

Recent findings in genome-wide transcriptomics revealed that RNAs are involved in almost every biological process, across all domains of life. The characterization of native RNAs of unknown function and structure is particularly challenging due to their typical low abundance in the cell and the inherent sensitivity toward ubiquitous RNA degrading enzymes. Therefore, robust in vitro synthesis and extensive work-up methods are often needed to obtain samples amenable for biochemical, biophysical, and structural studies. Here, we present a protocol that combines the most recent advances in T7 in vitro transcription methodology with reverse phase ion pairing and ion exchange HPLC purification of RNAs for the production of yield-optimized large-scale samples. The method is easy to follow, robust and suitable for users with little or no experience within the field of biochemistry or chromatography. The complete execution of this method, for example, for production of isotopically labeled NMR samples, can be performed in less than a week.

A robust and versatile method for production and purification of large-scale RNA samples for structural biology.

MicroRNAs (miRNAs) regulate the levels of translation of messenger RNAs (mRNAs). At present, the major parameter that can explain the selection of the target mRNA and the efficiency of translation repression is the base pairing between the 'seed' region of the miRNA and its counterpart mRNA. Here we use R relaxation-dispersion nuclear magnetic resonance and molecular simulations to reveal a dynamic switch-based on the rearrangement of a single base pair in the miRNA-mRNA duplex-that elongates a weak five-base-pair seed to a complete seven-base-pair seed. This switch also causes coaxial stacking of the seed and supplementary helix fitting into human Argonaute 2 protein (Ago2), reminiscent of an active state in prokaryotic Ago. Stabilizing this transient state leads to enhanced repression of the target mRNA in cells, revealing the importance of this miRNA-mRNA structure. Our observations tie together previous findings regarding the stepwise miRNA targeting process from an initial 'screening' state to an 'active' state, and unveil the role of the RNA duplex beyond the seed in Ago2.

Katja Petzold

Department of Medical Biochemistry and Biophysics

Karolinska Institutet

Practical Aspects of Sample Preparation and Setup of ¹H R_1ρ Relaxation Dispersion Experiments of RNA

Katja Petzold

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Medical Biochemistry and Biophysics

Karolinska Institutet

Practical Aspects of Sample Preparation and Setup of 1H R1ρ Relaxation Dispersion Experiments of RNA

Department of Medical Biochemistry and Biophysics

Practical Aspects of Sample Preparation and Setup of ¹H R_1ρ Relaxation Dispersion Experiments of RNA