The 5′ and 3′ termini of RNA play important roles in many cellular processes. Using Förster resonance energy transfer (FRET), we show that mRNAs and lncRNAs have an intrinsic propensity to fold in the absence of proteins into structures in which the 5′ end and 3′ end are ≤7 nm apart irrespective of mRNA length. Computational estimates suggest that the inherent proximity of the ends is a universal property of most mRNA and lncRNA sequences. Only guanosine-depleted RNA sequences with low sequence complexity are unstructured and exhibit end-to-end distances expected for the random coil conformation of RNA. While the biological implications remain to be explored, short end-to-end distances could facilitate the binding of protein factors that regulate translation initiation by bridging mRNA 5′ and 3′ ends. Furthermore, our studies provide the basis for measuring, computing and manipulating end-to-end distances and secondary structure in RNA in research and biotechnology.

Nucleic acids can be designed to be nano-machines, pharmaceuticals, or probes. RNA secondary structures can form the basis of self-assembling nanostructures. There are only four natural RNA bases, therefore it can be difficult to design sequences that fold to a single, specified structure because many other structures are often possible for a given sequence. One approach taken by state-of-the-art sequence design methods is to select sequences that fold to the specified structure using stochastic, iterative refinement. The goal of this work is to accelerate design. Many existing iterative methods select and refine sequences one base pair and one unpaired nucleotide at a time. Here, the hypothesis that sequences can be preselected in order to accelerate design was tested. To this aim, a database was built of helix sequences that demonstrate thermodynamic features found in natural sequences and that also have little tendency to cross-hybridize. Additionally, a database was assembled of RNA loop sequences with low helix-formation propensity and little tendency to cross-hybridize with either the helices or other loops. These databases of preselected sequences accelerate the selection of sequences that fold with minimal ensemble defect by replacing some of the trial and error of current refinement approaches. When using the database of preselected sequences as compared to randomly chosen sequences, sequences for natural structures are designed 36 times faster, and random structures are designed six times faster. The sequences selected with the aid of the database have similar ensemble defect as those sequences selected at random. The sequence database is part of RNAstructure package at http://rna.urmc.rochester.edu/RNAstructure.html.

A number of protein factors regulate protein synthesis by bridging mRNA ends or untranslated regions (UTRs). Using experimental and computational approaches, we show that mRNAs from various organisms, including humans, have an intrinsic propensity to fold into structures in which the 5' end and 3' end are ≤ 7 nm apart irrespective of mRNA length. Computational estimates performed for ~22,000 human transcripts indicate that the inherent proximity of the ends is a universal property of most, if not all, mRNA sequences. Only specific RNA sequences, which have low sequence complexity and are devoid of guanosines, are unstructured and exhibit end-to-end distances expected for the random coil conformation of RNA. Our results suggest that the intrinsic proximity of mRNA ends may facilitate binding of translation factors that bridge mRNA 5' and 3' UTRs. Furthermore, our studies provide the basis for measuring, computing and manipulating end-to-end distances and secondary structure in mRNAs in research and biotechnology. INRODUCTIONRegulation of mRNA translation in eukaryotes involves protein-mediated interactions between mRNA ends. Translation initiation requires the recruitment of the small ribosomal subunit to the 5' end of the mRNA 1 . The formation of the initiation complex is stimulated by the interaction between the 5' mRNA cap-binding protein eIF4E and the 3' end poly(A) tail binding protein PABP, which is mediated through their binding to different parts of the translational factor eIF4G 2,3 . The eIF4E•eIF4G•PABP complex is thought to enhance translation initiation by circularizing the mRNA and forming the "closed-loop" structure 4-6 . The mechanism by which the mRNA closed loop enhances proteins synthesis is not well understood.Remarkably, translation initiation of many eukaryotic mRNAs is also regulated by sequences in their 3' UTRs and controlled by the formation of protein bridges between the 5' and 3' UTRs. For example, the 3' UTR regulatory sequences recruit protein complexes (e.g. CPEB•Maskin, Bruno•Cup, or GAIT complex), which inhibit translation by interacting with either eIF4E or eIF4E•eIF4G bound to the 5' end of mRNA 7 . The pervasiveness of protein bridges between mRNA UTRs in the evolution of translation regulation is puzzling because of the significant entropic cost expected for protein-mediated mRNA circularization 8 .The entropic penalty for the formation of protein bridges between mRNA ends may be partially mitigated by mRNA compaction through intramolecular basepairing interactions.Recent theoretical analyses suggested that the 5' and 3' ends of long (1,000-10,000 nucleotidelong) RNAs are always brought in the proximity of few nanometers of each other regardless of RNA length and sequence because of the intrinsic propensity of RNA to form widespread intramolecular basepairing interactions 8-10 . One study predicted that the 5' to 3' end distance in RNAs is 3 nm, on average 8 . These theoretical predictions were tested by single-molecule Förster resonance energy transfer (smFRET) measurements o...

Design of RNA sequences that adopt functional folds establishes principles of RNA folding and applications in biotechnology. Inverse folding for RNAs, which allows computational design of sequences that adopt specific structures, can be utilized for unveiling RNA functions and developing genetic tools in synthetic biology. Although many algorithms for inverse RNA folding have been developed, the pseudoknot, which plays a key role in folding of ribozymes and riboswitches, is not addressed in most algorithms. For the few algorithms that attempt to predict pseudoknot-containing ribozymes, self-cleavage activity has not been tested. Herein, we design double-pseudoknot HDV ribozymes using an inverse RNA folding algorithm and test their kinetic mechanisms experimentally. More than 90% of the positively designed ribozymes possess self-cleaving activity, whereas more than 70% of negative control ribozymes, which are predicted to fold to the necessary structure but with low fidelity, do not possess it. Kinetic and mutation analyses reveal that these RNAs cleave site-specifically and with the same mechanism as the WT ribozyme. Most ribozymes react just 50- to 80-fold slower than the WT ribozyme, and this rate can be improved to near WT by modification of a junction. Thus, fast-cleaving functional ribozymes with multiple pseudoknots can be designed computationally.

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