Green fluorescent protein (GFP) and its derivatives revolutionized the study of proteins. Spinach is a recently reported in vitro evolved RNA mimic of GFP, which as genetically encoded fusions, makes possible live-cell, real-time imaging of biological RNAs, without resorting to large RNA-binding protein-GFP fusions. To elucidate the molecular basis of Spinach fluorescence, we have solved its co-crystal structure bound to its cognate exogenous chromophore, revealing that Spinach activates the small molecule by immobilizing it between a base triple, a G-quadruplex, and an unpaired guanine. Mutational and NMR analyses indicate that the G-quadruplex is essential for Spinach fluorescence, is also present in other fluorogenic RNAs, and may represent a general strategy for RNAs to induce fluorescence of chromophores. The structure has guided the design of a miniaturized 'Baby Spinach', and provides the foundation for structure-driven design and tuning of fluorescent RNAs.

Guanine-rich nucleic acid sequences challenge the replication, transcription, and translation machinery by spontaneously folding into G-quadruplexes, the unfolding of which requires forces greater than what most polymerases can exert1,2. Eukaryotic cells host numerous helicases capable of unfolding G-quadruplexes3. The molecular basis for helicase recognition and unfolding of G-quadruplexes remains poorly understood. DHX36 (RHAU, G4R1), a member of the DEAH/RHA family of helicases, binds both DNA and RNA G-quadruplexes with uniquely high affinity4,5,6, is consistently found bound to G-quadruplexes in cells7,8, and is a major source of G-quadruplex unfolding activity in HeLa cell lysates6. DHX36 is a multi-functional helicase implicated in G-quadruplex-mediated transcriptional and post-transcriptional regulation, and is essential for heart development, hematopoiesis, and embryogenesis in mice9,10,11,12. Here, we report the co-crystal structure of bovine DHX36 bound to a DNA with a G-quadruplex and a 3’ single-stranded (ssDNA) segment. We show that the N-terminal DHX36-specific motif folds into a DNA-binding-induced α-helix that together with the OB-fold-like subdomain selectively binds parallel G-quadruplexes. Comparison with our unliganded and ATP-analog-bound DHX36 structures, together with single-molecule FRET analysis, suggests that G-quadruplex binding alone induces rearrangements of the helicase core, which by pulling on its ssDNA tail, drive G-quadruplex unfolding by one residue at a time.

Molecular self-assembly is widely appreciated to result from a delicate balance between several noncovalent interactions and solvation effects. However, current design approaches for achieving self-assembly in water with small, synthetic molecules do not consider all aspects of the hydrophobic effect, in particular the requirement of surface areas greater than 1 nm(2) for an appreciable free energy of hydration. With the concept of a minimum hydrophobic surface area in mind, we designed a system that achieves highly cooperative self-assembly in water. Two weakly interacting low-molecular-weight monomers (cyanuric acid and a modified triaminopyrimidine) are shown to form extremely long supramolecular polymer assemblies that retain water solubility. The complete absence of intermediate assemblies means that the observed equilibrium is between free monomers and supramolecular assemblies. These observations are in excellent agreement with literature values for the free energy of nucleic acid base interactions as well as the calculated free energy penalty for the exposure of hydrophobic structures in water. The results of our study have implications for the design of new self-assembling structures and hydrogel-forming molecules and may provide insights into the origin of the first RNA-like polymers.

The RNA World hypothesis is central to many current theories regarding the origin and early evolution of life. However, the formation of RNA by plausible prebiotic reactions remains problematic. Formidable challenges include glycosidic bond formation between ribose and the canonical nucleobases, as well as the inability of nucleosides to mutually select their pairing partners from a complex mixture of other molecules prior to polymerization. Here we report a one-pot model prebiotic reaction between a pyrimidine nucleobase (2,4,6-triaminopyrimidine, TAP) and ribose, which produces TAP-ribose conjugates in high yield (60-90%). When cyanuric acid (CA), a plausible ancestral nucleobase, is mixed with a crude TAP+ribose reaction mixture, micrometer-length supramolecular, noncovalent assemblies are formed. A major product of the TAP+ribose reaction is a β-ribofuranoside of TAP, which we term TARC. This nucleoside is also shown to efficiently form supramolecular assemblies in water by pairing and stacking with CA. These results provide a proof-of-concept system demonstrating that several challenges associated with the prebiotic emergence of RNA, or pre-RNA polymers, may not be as problematic as widely believed.

The unwinding of nucleic acid secondary structures within cells is crucial to maintain genomic integrity and prevent abortive transcription and translation initiation. DHX36, also known as RHAU or G4R1, is a DEAH-box ATP-dependent helicase highly specific for DNA and RNA G-quadruplexes (G4s). A fundamental mechanistic understanding of the interaction between helicases and their G4 substrates is important to elucidate G4 biology and pave the way toward G4-targeted therapies. Here we analyze how the thermodynamic stability of G4 substrates affects binding and unwinding by DHX36. We modulated the stability of the G4 substrates by varying the sequence and the number of G-tetrads and by using small, G4-stabilizing molecules. We found an inverse correlation between the thermodynamic stability of the G4 substrates and rates of unwinding by DHX36. In stark contrast, the ATPase activity of the helicase was largely independent of substrate stability pointing toward a decoupling mechanism akin to what has been observed for many double-stranded DEAD-box RNA helicases. Our study provides the first evidence that DHX36 uses a local, non-processive mechanism to unwind G4 substrates, reminiscent of that of eukaryotic initiation factor 4A (eIF4A) on double-stranded substrates.

The RNA world hypothesis proposes that nucleic acids were once responsible for both information storage and chemical catalysis, before the advent of coded protein synthesis. However, it is difficult to imagine how nucleic acid polymers first appeared, as the abiotic chemical formation of long nucleic acid polymers from mononucleotides or short oligonucleotides remains elusive, and barriers to achieving this goal are substantial. One specific obstacle to abiotic nucleic acid polymerization is strand cyclization. Chemically activated short oligonucleotides cyclize efficiently, which severely impairs polymer growth. We show that intercalation, which stabilizes and rigidifies nucleic acid duplexes, almost totally eliminates strand cyclization, allowing for chemical ligation of tetranucleotides into duplex polymers of up to 100 base pairs in length. In contrast, when these reactions are performed in the absence of intercalators, almost exclusively cyclic tetra-and octanucleotides are produced. Intercalator-free polymerization is not observed, even at tetranucleotide concentrations >10; 000-fold greater than those at which intercalators enable polymerization. We also demonstrate that intercalation-mediated polymerization is most favored if the size of the intercalator matches that of the base pair; intercalators that bind to Watson-Crick base pairs promote the polymerization of oligonucleotides that form these base pairs. Additionally, we demonstrate that intercalationmediated polymerization is possible with an alternative, nonWatson-Crick-paired duplex that selectively binds a complementary intercalator. These results support the hypothesis that intercalators (acting as 'molecular midwives') could have facilitated the polymerization of the first nucleic acids and possibly helped select the first base pairs, even if only trace amounts of suitable oligomers were available.base pair selection | origin of life | RNA world | polymerization | molecular evolution O ver the past two decades, significant evidence has been presented in support of the RNA world hypothesis, which proposes that RNA polymers predated coded proteins in early life (1, 2). Current support for this hypothesis includes the fact that contemporary life still uses RNA as an informational polymer and in chemical catalysis (3). The ability of RNA to catalyze reactions is exemplified by natural and artificial ribozymes that promote a wide variety of chemical reactions (4) as well as the observation that the catalytic core of the ribosome is comprised of RNA (5). Despite the attractiveness of the RNA world as a hypothetical stage of early life, it remains unclear how RNA [or a predecessor of RNA (6-11)] would initially have been synthesized without the aid of protein enzyme catalysis.Several distinct proposals have been presented for the abiotic origin of the first RNA polymers (10,(12)(13)(14)(15)(16)(17). Perhaps the most notable is that of Ferris and coworkers, in which mineral surfaces are used to locally concentrate and promote the polymerization of liga...

Protein structural vibrations impact biology by steering the structure to functional intermediate states; enhancing tunneling events; and optimizing energy transfer. Strong water absorption and a broad continuous vibrational density of states have prevented optical identification of these vibrations. Recently spectroscopic signatures that change with functional state were measured using anisotropic terahertz microscopy. The technique however has complex sample positioning requirements and long measurement times, limiting access for the biomolecular community. Here we demonstrate that a simplified system increases spectroscopic structure to dynamically fingerprint biomacromolecules with a factor of 6 reduction in data acquisition time. Using this technique, polarization varying anisotropy terahertz microscopy, we show sensitivity to inhibitor binding and unique vibrational spectra for several proteins and an RNA G-quadruplex. The technique’s sensitivity to anisotropic absorbance and birefringence provides rapid assessment of macromolecular dynamics that impact biology.

Semagenesis enriches our understanding of the mechanisms available for small-molecule underground information exchange among plants. Critical differences in this process, as used by parasitic plants, are beginning to emerge and point towards new strategies for managing parasitic angiosperms in agricultural settings.