Abstract
RNA molecules play different roles in coding, decoding and gene expression regulation. Such roles are often associated to the RNA secondary or tertiary structures. The folding dynamics lead to multiple secondary structures of long RNA molecules, since an RNA molecule might fold into multiple distinct native states. Despite an ensemble of different structures, it has been theoretically proposed that the separation between the 5′ and 3′ ends of long single-stranded RNA molecules (ssRNA) remains constant, independent of their base content and length. Here, we present the first experimental measurements of the end-to-end separation in long ssRNA molecules. To determine this separation, we use single molecule Fluorescence Resonance Energy Transfer of fluorescently end-labeled ssRNA molecules ranging from 500 to 5500 nucleotides in length, obtained from two viruses and a fungus. We found that the end-to-end separation is indeed short, within 5–9 nm. It is remarkable that the separation of the ends of all RNA molecules studied remains small and similar, despite the origin, length and differences in their secondary structure. This implies that the ssRNA molecules are ‘effectively circularized’ something that might be a general feature of RNAs, and could result in fine-tuning for translation and gene expression regulation.
Highlights
Ribonucleic acids (RNAs) are a large family of biomolecules present in all forms of life
Most functional RNA molecules exhibit a secondary structure that is highly conserved across the large evolutionary distance from bacteria to mammals, e.g. the tRNAs [6,7]
To determine the end-to-end distance we performed single molecule Fluorescence Resonance Energy Transfer (smFRET) measurements using the 11 end-labeled mRNA molecules described in Table 1. smFRET experiments were carried out with freely diffusing mRNA molecules at 27◦C either under magnesium-free conditions using TE buffer or in the presence of 5 mM magnesium using TM buffer (Figure 2 and Supplementary Figure S2a and b)
Summary
Ribonucleic acids (RNAs) are a large family of biomolecules present in all forms of life. Calculations of the minimum free energy secondary structures of single-stranded RNA (ssRNA) molecules indicate that the percentage of paired nucleotides (nts) (f) and the average duplex length (k) approach a constant value as the number of nts increases [8,9,10]. This constancy for f and k has been verified for a wide range of viral and yeast ssRNA sequences [11] by application of both the mFOLD [12] and the RNA Vienna algorithms [13].
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