Research Article: A guide to large-scale RNA sample preparation

Date Published: March 15, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Lorenzo Baronti, Hampus Karlsson, Maja Marušič, Katja Petzold.

http://doi.org/10.1007/s00216-018-0943-8

Abstract

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.

Partial Text

In an ever-growing world of new classes of RNAs, the need to reveal their function and structure is expanding [1]. This need coincides with the advancement in structural biology methods, such as the resolution revolution in cryo-electron microscopy (cryo-EM) [2] or the discovery of invisible RNA states by nuclear magnetic resonance (NMR) methods [3, 4]. Multiple techniques are now available to probe the features of a given RNA, and each one uniquely accesses structural information at different resolution, depending on the question posed. The required sample preparation on a large scale often constitutes the limiting step, as each structural method strongly relies on the quality (and often quantity) of the RNA sample that has to be provided (Fig. 1). Hence first we will give a short overview of the available techniques to determine RNA structure and dynamics.Fig. 1Overview of commonly used sample preparation methods for structural characterization of RNA. The size of RNAs that can be obtained is indicated by gradients for each of the methods ranging from black, for the most suitable, to white, indicating not applicable. White dots indicate which RNA sample preparation method is commonly used and well established for the specific structural biology method, whereas gray dots indicate a less common application or an upcoming new method. Preparation methods that were recently developed and are yet to show their full potential are written in gray above the gradients (Table 1). References for relevant examples are indicated to the right of each structural biology method. Abbreviations: biophys. biophysical, cryo-EM cryo-electron microscopy, EPR electron paramagnetic resonance, FRET fluorescence/Förster resonance energy transfer, NMR nuclear magnetic resonance, nt nucleotide, PCT polymerase chain transcription, PLOR position-selective labeling of RNA, SANS small-angle neutron scattering, SAXS small-angle X-ray scattering, sec. secondary

Sample preparation is currently a bottleneck in the structural characterization of RNA, impeded by RNases and a lack of development for large-scale preparative methods. However, new developments are opening up new avenues that can be combined to answer many new, challenging, and interesting questions in RNA structural biology. Here we reviewed recent progress in well-established production and purification methods that allows preparation of large amounts of RNA commonly needed for structural characterization. Several new exciting methods that are emerging, such as circular RNAs, PLOR, and polymerase chain transcription, have great potential to reduce the amount of work and time needed for RNA sample production. On the other side, obtaining a sample that is pure and homogeneous is usually the most challenging step in structural studies, and new, fast, and innovative methods for sample preparation are currently lacking. However, a combination of typically two different purification methods will most often result in a reliable, easy to work with sample that will save time in the long run.

 

Source:

http://doi.org/10.1007/s00216-018-0943-8

 

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