Search

It is important to understand the dynamics and higher energy structures of RNA, called excited states, to achieve better understanding of RNA function. R1ρ relaxation dispersion NMR spectroscopy (RD) determines chemical shift differences between the most stable, ground state and the short-lived, low-populated excited states. We describe a procedure for deducing the excited state structure from these chemical shift differences using the mutate-and-chemical-shift-fingerprint (MCSF) method, which requires ~2-6 weeks and moderate understanding of NMR and RNA structure. We recently applied the MCSF methodology to elucidate the excited state of microRNA 34a targeting the SIRT1 mRNA and use this example to demonstrate the analysis. The protocol comprises the following steps: (i) determination of the secondary structure of the excited state from RD chemical shift data, (ii) design of trapped excited state RNA, (iii) validation of the excited state structure by NMR, and (iv) MCSF analysis comparing the chemical shifts of the trapped excited state with the RD-derived chemical shift differences. MCSF enables observation of the short-lived RNA structures, which can be functionally and structurally characterized by entrapment.

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 mRNA1. Here we use R1ρ relaxation-dispersion nuclear magnetic resonance2 and molecular simulations3 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 Ago4,5. 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. Repression of a messenger RNA by a cognate microRNA depends not only on complementary base pairing, but also on the rearrangement of a single base pair, producing a conformation that fits better within the human Ago2 protein.

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 series of five homoisoflavanone analogues have been synthesized from the corresponding 3,5-methoxy phenols via chroman-4-one in three steps. The complete NMR elucidation of these homoisoflavanone analogues is reported. The use of 2D NMR techniques (COSY, NOESY, HSQC and HMBC) proved to be very useful tools in the elucidation of homoisoflavanone analogues. The homoisoflavanone analogues exhibit an AA′BB′ spin pattern in the ring B of the homoisoflavanone. These homoisoflavanone analogues are potential antifungal and anti-inflammatory agents.

Mutating RNA one nucleotide at a time and measuring the impact of this on its chemical reactivity provides a strategy for determining its three-dimensional structure, and from there, hopefully, its function.