Conformational Flexibility and Dynamics of the Internal Loop and Helical Regions of the Kink-Turn Motif in the Glycine Riboswitch by Site-Directed Spin-Labeling.

Site-directed spin-labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy provides a means for a solution state description of site-specific dynamics and flexibility of large RNAs, facilitating our understanding of the effects of environmental conditions such as ligands and ions on RNA structure and dynamics. Here, the utility and capability of EPR line shape analysis and distance measurements to monitor and describe site-specific changes in the conformational dynamics of internal loop nucleobases as well as helix-helix interactions of the kink-turn motif in the Vibrio cholerae (VC) glycine riboswitch that occur upon sequential K(+)-, Mg(2+)-, and glycine-induced folding were explored. Spin-labels were incorporated into the 232-nucleotide sequence via splinted ligation strategies.

Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced.

Site-directed spin-labeling strategies and electron paramagnetic resonance spectroscopy for large riboswitches.

Genetic regulation effected by RNA riboswitches is governed by ligand-induced structural reorganization with modulation of RNA conformation and dynamics. Characterization of the conformational states of riboswitches in the presence or absence of salts and ligands is important for understanding how interconversion of riboswitch RNA folding states influences function. The methodology of site-directed spin labeling (SDSL) coupled with electron paramagnetic resonance (EPR) spectroscopy is suitable for such studies, wherein site-specific incorporation of a nitroxide radical spin probe allows for local dynamics and conformational changes to be investigated.

DNA-rescuable allosteric inhibition of aptamer II ligand affinity by aptamer I element in the shortened Vibrio cholerae glycine riboswitch.

Glycine riboswitches contain two aptamers and turn on the expression of downstream genes in bacteria. Although full-length glycine riboswitches were shown to exhibit no glycine-binding cooperativity, the truncated glycine riboswitches were confirmed to bind two glycine molecules cooperatively. Thorough understanding of the ligand-binding cooperativity may shed light on the molecular basis of the cooperativity and help design novel intricate biosensing genetic circuits for application in synthetic biology.

Characterizing the dynamics of the leader-linker interaction in the glycine riboswitch with site-directed spin labeling.

Site-directed spin labeling with continuous wave electron paramagnetic resonance (EPR) spectroscopy was utilized to characterize dynamic features of the kink-turn motif formed through a leader-linker interaction in the Vibrio cholerae glycine riboswitch. Efficient incorporation of spin-labels into select sites within the phosphate backbone of the leader-linker region proceeded via splinted ligation of chemically synthesized spin-labeled oligonucleotides to in vitro transcribed larger RNA fragments. The resultant nitroxide EPR line shapes have spectral characteristics consistent with a kink-turn motif and reveal differential backbone dynamics that are modulated by the presence of magnesium, potassium, and glycine.

Specific RNA-binding antibodies with a four-amino-acid code.

Numerous large non-coding RNAs are rapidly being discovered, and many of them have been shown to play vital roles in gene expression, gene regulation, and human diseases. Given their often structured nature, specific recognition with an antibody fragment becomes feasible and may help define the structure and function of these non-coding RNAs. As demonstrated for protein antigens, specific antibodies may aid in RNA crystal structure elucidation or the development of diagnostic tools and therapeutic drugs targeting disease-causing RNAs.

An energetically beneficial leader-linker interaction abolishes ligand-binding cooperativity in glycine riboswitches.

Comprised of two aptamers connected by a short nucleotide linker, the glycine riboswitch was the first example of naturally occurring RNA elements reported to bind small organic molecules cooperatively. Earlier works have shown binding of glycine to the second aptamer allows tertiary interactions to be made between the two aptamers, which facilitates binding of a separate glycine molecule to the first aptamer, leading to glycine-binding cooperativity. Prompted by a distinctive protection pattern in the linker region of a minimal glycine riboswitch construct, we have identified a highly conserved (>90%) leader-linker duplex involving leader nucleotides upstream of the previously reported consensus glycine riboswitch sequences.

Improvement of the crystallizability and expression of an RNA crystallization chaperone.

Crystallizing RNA has been an imperative and challenging task in the world of RNA research. Assistive methods such as chaperone-assisted RNA crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by forming crystal contacts and providing initial phasing information. Despite the early successes, the crystallization of large RNA-Fab complex remains a challenge in practice.

Synthetic antibodies for specific recognition and crystallization of structured RNA.

Antibodies that bind protein antigens are indispensable in biochemical research and modern medicine. However, knowledge of RNA-binding antibodies and their application in the ever-growing RNA field is lacking. Here we have developed a robust approach using a synthetic phage-display library to select specific antigen-binding fragments (Fabs) targeting a large functional RNA.

Reactions of phosphate and phosphorothiolate diesters with nucleophiles: comparison of transition state structures.

A series of methyl aryl phosphorothiolate esters (SP) were synthesized and their reactions with pyridine derivatives were compared to those for methyl aryl phosphate esters (OP). Results show that SP esters react with pyridine nucleophiles via a concerted S(N)2(P) mechanism. Brønsted analysis suggests that reactions of both SP and OP esters proceed via transition states with dissociative character.

Synthesis of 2'-C-difluoromethylribonucleosides and their enzymatic incorporation into oligonucleotides.

[Chemical reaction: See text] Nucleosides bearing a branched ribose have significant promise as therapeutic agents and biotechnological and biochemical tools. Here we describe synthetic entry into a new subclass of these analogues, 2'-C-beta-difluoromethylribonucleosides. We constructed the glycosylating agent 4 in three steps from 1,3,5-tri-O-benzoyl-alpha-D-ribofuranose 1.

New strategies for exploring RNA's 2'-OH expose the importance of solvent during group II intron catalysis.

The 2'-hydroxyl group contributes inextricably to the functional behavior of many RNA molecules, fulfilling numerous essential chemical roles. To assess how hydroxyl groups impart functional behavior to RNA, we developed a series of experimental strategies using an array of nucleoside analogs. These strategies provide the means to investigate whether a hydroxyl group influences function directly (via hydrogen bonding or metal ion coordination), indirectly (via space-filling capacity, inductive effects, and sugar conformation), or through interactions with solvent.