The RNA chaperone StpA enables fast RNA refolding by destabilization of mutually exclusive base pairs within competing secondary structure elements.

In bacteria RNA gene regulatory elements refold dependent on environmental clues between two or more long-lived conformational states each associated with a distinct regulatory state. The refolding kinetics are strongly temperature-dependent and especially at lower temperatures they reach timescales that are biologically not accessible. To overcome this problem, RNA chaperones have evolved. However, the precise molecular mechanism of how these proteins accelerate RNA refolding reactions remains enigmatic. Here we show how the RNA chaperone StpA of Escherichia coli leads to an acceleration of a bistable RNA's refolding kinetics through the selective destabilization of key base pairing interactions. We find in laser assisted real-time NMR experiments on photocaged bistable RNAs that the RNA chaperone leads to a two-fold increase in refolding rates at low temperatures due to reduced stability of ground state conformations. Further, we can show that upon interaction with StpA, base pairing interactions in the bistable RNA are modulated to favor refolding through the dominant pseudoknotted transition pathway. Our results shed light on the molecular mechanism of the interaction between RNA chaperones and bistable RNAs and are the first step into a functional classification of chaperones dependent on their biophysical mode of operation.

Parallel reaction pathways accelerate folding of a guanine quadruplex.

G-quadruplexes (G4s) are four-stranded, guanine-rich nucleic acid structures that can influence a variety of biological processes such as the transcription and translation of genes and DNA replication. In many cases, a single G4-forming nucleic acid sequence can adopt multiple different folded conformations that interconvert on biologically relevant timescales, entropically stabilizing the folded state. The coexistence of different folded conformations also suggests that there are multiple pathways leading from the unfolded to the folded state ensembles, potentially modulating the folding rate and biological activity. We have developed an experimental method for quantifying the contributions of individual pathways to the folding of conformationally heterogeneous G4s that is based on mutagenesis, thermal hysteresis kinetic experiments and global analysis, and validated our results using photocaged kinetic NMR experiments. We studied the regulatory Pu22 G4 from the c-myc oncogene promoter, which adopts at least four distinct folded isomers. We found that the presence of four parallel pathways leads to a 2.5-fold acceleration in folding; that is, the effective folding rate from the unfolded to folded ensembles is 2.5 times as large as the rate constant for the fastest individual pathway. Since many G4 sequences can adopt many more than four isomers, folding accelerations of more than an order of magnitude are possible via this mechanism.

Solid-Phase-Supported Chemoenzymatic Synthesis of a Light-Activatable tRNA Derivative.

Herein, we present a multi-cycle chemoenzymatic synthesis of modified RNA with simplified solid-phase handling to overcome size limitations of RNA synthesis. It combines the advantages of classical chemical solid-phase synthesis and enzymatic synthesis using magnetic streptavidin beads and biotinylated RNA. Successful introduction of light-controllable RNA nucleotides into the tRNAMet sequence was confirmed by gel electrophoresis and mass spectrometry. The methods tolerate modifications in the RNA phosphodiester backbone and allow introductions of photocaged and photoswitchable nucleotides as well as photocleavable strand breaks and fluorophores.

Unraveling the Kinetics of Spare-Tire DNA G-Quadruplex Folding.

The folding of DNA G-quadruplexes (G4) is essential to regulate expression of oncogenes and involves polymorphic long-lived intermediate states. G4 formation requires four G-tracts, but human gene-promoters often contain multiple G-tracts that act as spare-tires. These additional G-tracts are highly conserved and add multiple layers of functional complexity, as they are crucial to maintain G4 function after oxidative damage. Herein, we unravel the folding dynamics of the G4 sequence containing five G-tracts from cMYC, the major proliferation-driving oncogene. We devise a general method to induce folding at constant experimental conditions using a photochemical trapping strategy. Our data dissect the individual kinetics and thermodynamics of the spare-tire mechanism of cMYC-G4.

Chemo-Enzymatic Synthesis of Position-Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy.

The investigation of non-coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo-enzymatic RNA synthesis containing photoactivatable or 13 C,15 N-labelled nucleosides. All four ribonucleotides containing ortho-nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C-nucleotides and 13 C,15 N-labelled nucleotides were incorporated position-specifically in high yields. We applied this approach for the synthesis of light-inducible 2'dG-sensing riboswitch variants and detected ligand-induced structural reorganization upon irradiation by NMR spectroscopy. This chemo-enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid-phase synthesis.