Nucleotide Loop Research Articles

A method for the production, purification and liposome reconstitution of cobamide synthase.

Cobamides are essential for the performance of a variety of reactions such methyl transfers, carbon skeleton rearrangements, and eliminations in both prokaryotes and eukaryotes. However, cobamide biosynthesis is limited to a subset of bacteria and archaea. The biosynthesis pathway culminates with the activation and attachment of a lower ligand to the corrin ring; this branch of the pathway is known as nucleotide loop assembly (NLA) pathway. The cobamide synthase (CobS) enzyme is the penultimate step in NLA pathway, and catalyzes the attachment of an α-ribotide to the activated corrin ring. While other NLA enzymes have been well-studied, studies of CobS have proven difficult to date. CobS is an integral membrane protein, and limitations have been largely due to difficulties in protein purification. Here we provide a method to purify CobS, reconstitute protein in proteoliposomes, and assay for its activity.

Design of a Rapid and Reversible Fluorescence Assay to Detect Covid-19 and other Pathogens

We describe a rapid and reusable biophysical method to assay COVID-19. The method uses fluorescent sensors (i.e., molecular beacons) designed to detect a specific RNA sequence from COVID-19 but is general to any RNA of interest. The assay can be used concurrently with an internal control without the need for amplification. Molecular beacons are stem-loop structures in which a ~10 nucleotide loop region has the complementary sequence of a region of the target RNA, and a fluorophore and quencher are placed on the 5’ and 3’ ends of the stem. The energy of hybridization of the loop with its target is designed to be greater than the hybridization energy of the energy of the stem so that when the beacon encounters its target RNA, the structure opens resulting in dequenching of the fluorophore. Here, we designed a beacon to different COVID-19 variants that is completely quenched in its native form and undergoes a 50-fold increase in fluorescence when exposed to nanomolar amounts of synthetic viral oligonucleotide. No changes in intensity are seen when a control RNA (hGAPDH) is added. This increase in fluorescence with beacon opening can be completely reversed upon addition of single stranded DNA complementary to COVID-19 beacon loop region. Beacons can be attached to an inert matrix allowing their use and reuse in concentrated form and can be made from morphilino oligonucleotides that are resistant to RNases. We present an analysis of the parameters that will allow the development of test strips to detect virus in aerosol, body fluids and community waste.

Cobamide remodeling.

Cobamides are a family of structurally-diverse cofactors which includes vitamin B12 and over a dozen natural analogs. Within the nucleotide loop structure, cobamide analogs have variable lower ligands that fall into three categories: benzimidazoles, purines, and phenols. The range of cobamide analogs that can be utilized by an organism is dependent on the specificity of its cobamide-dependent enzymes, and most bacteria are able to utilize multiple analogs but not all. Some bacteria have pathways for cobamide remodeling, a process in which imported cobamides are converted into compatible analogs. Here we discuss cobamide analog diversity and three pathways for cobamide remodeling, mediated by amidohydrolase CbiZ, phosphodiesterase CbiR, and some homologs of cobamide synthase CobS. Remodeling proteins exhibit varying degrees of specificity for cobamide substrates, reflecting different strategies to ensure that imported cobamides can be utilized.

Twice exploration of tRNA+1 frameshifting in an elongation cycle of protein synthesis.

Inducing tRNA +1 frameshifting to read a quadruplet codon has the potential to incorporate a non-natural amino acid into the polypeptide chain. While this strategy is being considered for genome expansion in biotechnology and bioengineering endeavors, a major limitation is a lack of understanding of where the shift occurs in an elongation cycle of protein synthesis. Here, we use the high-efficiency +1-frameshifting SufB2 tRNA, containing an extra nucleotide in the anticodon loop, to address this question. Physical and kinetic measurements of the ribosome reading frame of SufB2 identify twice exploration of +1 frameshifting in one elongation cycle, with the major fraction making the shift during translocation from the aminoacyl-tRNA binding (A) site to the peptidyl-tRNA binding (P) site and the remaining fraction making the shift within the P site upon occupancy of the A site in the +1-frame. We demonstrate that the twice exploration of +1 frameshifting occurs during active protein synthesis and that each exploration is consistent with ribosomal conformational dynamics that permits changes of the reading frame. This work indicates that the ribosome itself is a determinant of changes of the reading frame and reveals a mechanistic parallel of +1 frameshifting with –1 frameshifting.

Unique anticodon loop conformation with the flipped-out wobble nucleotide in the crystal structure of unbound tRNAVal.

During protein synthesis on ribosome, tRNA recognizes its cognate codon of mRNA through base-pairing with the anticodon. The 5'-end nucleotide of the anticodon is capable of wobble base-pairing, offering a molecular basis for codon degeneracy. The wobble nucleotide is often targeted for post-transcriptional modification, which affects the specificity and fidelity of the decoding process. Flipping-out of a wobble nucleotide in the anticodon loop has been proposed to be necessary for modifying enzymes to access the target nucleotide, which has been captured in selective structures of protein-bound complexes. Meanwhile, all other structures of free or ribosome-bound tRNA display anticodon bases arranged in stacked conformation. We report the X-ray crystal structure of unbound tRNAVal1 to a 2.04 Å resolution showing two different conformational states of wobble uridine in the anticodon loop, one stacked on the neighboring base and the other swiveled out toward solvent. In addition, the structure reveals a rare magnesium ion coordination to the nitrogen atom of a nucleobase, which has been sampled very rarely among known structures of nucleic acids.

The emerging structural complexity of G-quadruplex RNAs.

G-quadruplexes (G4s) are four-stranded nucleic acid structures that arise from the stacking of G-quartets, cyclic arrangements of four guanines engaged in Hoogsteen base-pairing. Until recently, most RNA G4 structures were thought to conform to a sequence pattern in which guanines stacking within the G4 would also be contiguous in sequence (e.g., four successive guanine trinucleotide tracts separated by loop nucleotides). Such a sequence restriction, and the stereochemical constraints inherent to RNA (arising, in particular, from the presence of the 2′-OH), dictate relatively simple RNA G4 structures. Recent crystallographic and solution NMR structure determinations of a number of in vitro selected RNA aptamers have revealed RNA G4 structures of unprecedented complexity. Structures of the Sc1 aptamer that binds an RGG peptide from the Fragile-X mental retardation protein, various fluorescence turn-on aptamers (Corn, Mango, and Spinach), and the spiegelmer that binds the complement protein C5a, in particular, reveal complexity hitherto unsuspected in RNA G4s, including nucleotides in syn conformation, locally inverted strand polarity, and nucleotide quartets that are not all-G. Common to these new structures, the sequences folding into G4s do not conform to the requirement that guanine stacks arise from consecutive (contiguous in sequence) nucleotides. This review highlights how emancipation from this constraint drastically expands the structural possibilities of RNA G-quadruplexes.

Light Mediated Properties of a Thiolato-Derivative of Vitamin B12.

Vitamin B12 derivatives (Cbls = cobalamins) exhibit photolytic properties upon excitation with light. These properties can be modulated by several factors including the nature of the axial ligands. Upon excitation, homolytic cleavage of the organometallic bond to the upper axial ligand takes place in photolabile Cbls. The photosensitive nature of Cbls has made them potential candidates for light-activated drug delivery. The addition of a fluorophore to the nucleotide loop of thiolato Cbls has been shown to shift the region of photohomolysis to within the optical window of tissue (600-900 nm). With this possibility, there is a need to analyze photolytic properties of unique Cbls which contain a Co-S bond. Herein, the photodissociation of one such Cbl, namely, N-acetylcysteinylcobalamin (NACCbl), is analyzed based on density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations. The S0 and S1 potential energy surfaces (PESs), as a function of axial bond lengths, were computed to determine the mechanism of photodissociation. Like other Cbls, the S1 PES contains metal-to-ligand charge transfer (MLCT) and ligand field (LF) regions, but there are some unique differences. Interestingly, the S1 PES of NACCbl contains three distinct minima regions opening several possibilities for the mechanism of radical pair (RP) formation. The mild photoresponsiveness, observed experimentally, can be attributed to the small gap in energy between the S1 and S0 PESs. Compared to other Cbls, the gap shown for NACCbl is neither exactly in line with the alkyl Cbls nor the nonalkyl Cbls.

Cobamide remodeling in the freshwater microalga Chlamydomonas reinhardtii.

Microalgae are not able to produce cobamides (Cbas, B12 vitamers) de novo. Hence, the production of catalytically active Cba-containing methionine synthase (MetH), which is present in selected representatives, is dependent on the availability of exogenous B12 vitamers. Preferences in the utilization of exogenous Cbas equipped with either adenine or 5,6-dimethylbenzimidazole as lower base have been reported for some microalgae. Here, we investigated the utilization of norcobamides (NorCbas) for growth by the Cba-dependent Chlamydomonas reinhardtii mutant strain (ΔmetE). The growth yields in the presence of NorCbas were lower in comparison to those achieved with Cbas. NorCbas lack a methyl group in the linker moiety of the nucleotide loop. C. reinhardtii was also tested for the remodeling of NorCbas (e.g. adeninyl-norcobamide) in the presence of different benzimidazoles. Extraction of the NorCbas from C. reinhardtii, their purification, and identification confirmed the exchange of the lower base of the vitamers. However, the linker moiety of the NorCbas nucleotide loop was not exchanged. This observation strongly indicates the presence of an alternative mode of Cba deconstruction in C. reinhardtii that differs from the amidohydrolase (CbiZ)-dependent pathway described in Cba-remodeling bacteria and archaea.

Resonance Raman Optical Activity Spectroscopy in Probing Structural Changes Invisible to Circular Dichroism Spectroscopy: A Study on Truncated Vitamin B12 Derivatives.

This work demonstrates resonance Raman optical activity (RROA) spectra of three truncated vitamin B12 derivatives modified within the nucleotide loop. Since truncated cobalamins possess sufficiently high rotational strength in the range of ROA excitation (532 nm), it was possible to record their spectra in the resonance condition. They showed several distinct spectral features allowing for the distinguishing of studied compounds, in contrast to other methods, i.e., UV-Vis absorption, electronic circular dichroism, and resonance Raman spectroscopy. The improved capacity of the RROA method is based here on the excitation of molecules via more than two electronic states, giving rise to the bisignate RROA spectrum, significantly distinct from a parent Raman spectrum. This observation is an important step in the dissemination of using RROA spectroscopy in studying the complex structure of corrinoids which may prove crucial for a better understanding of their biological role.

Design and Evaluation of AgoshRNAs with 3'-Terminal HDV Ribozymes to Enhance the Silencing Activity.

Since the first application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNA (shRNA) molecules for targeted gene silencing has become a benchmark technology. Plasmid and viral vector systems can be used to express shRNA precursor transcripts that are processed by the cellular RNAi pathway to trigger sequence-specific gene knockdown. Intensive RNAi investigations documented that only a small percentage of computationally predicted target sequences can be used for efficient gene silencing, in part because not all shRNA designs are active. Many factors influence the shRNA activity and guidelines for optimal shRNA design have been proposed. We recently described an alternatively processed shRNA molecule termed AgoshRNA with a ~18base pairs (bp) stem and a 3-5 nucleotides(nt) loop. This molecule is alternatively processed by the Argonaute (Ago) protein into a single guide RNA strand that efficiently induces the RNAi mechanism. The design rules proposed for regular shRNAs do not apply to AgoshRNA molecules and therefore new rules had to be defined. We optimized the AgoshRNA design and managed to create a set of active AgoshRNAs targeted against the human immunodeficiency virus (HIV). In an attempt to enhance the silencing activity of the AgoshRNA molecules, we included the hepatitis delta virus (HDV) ribozyme at the 3' terminus, which generates a uniform 3' end instead of a 3' U-tail of variable length. We evaluated the impact of this 3'-end modification on AgoshRNA processing and its gene silencing activity and we demonstrate that this novel AgoshRNA-HDV design exhibits enhanced antiviral activity.

Disruption of evolutionarily correlated tRNA elements impairs accurate decoding

Bacterial transfer RNAs (tRNAs) contain evolutionarily conserved sequences and modifications that ensure uniform binding to the ribosome and optimal translational accuracy despite differences in their aminoacyl attachments and anticodon nucleotide sequences. In the tRNA anticodon stem-loop, the anticodon sequence is correlated with a base pair in the anticodon loop (nucleotides 32 and 38) to tune the binding of each tRNA to the decoding center in the ribosome. Disruption of this correlation renders the ribosome unable to distinguish correct from incorrect tRNAs. The molecular basis for how these two tRNA features combine to ensure accurate decoding is unclear. Here, we solved structures of the bacterial ribosome containing either wild-type [Formula: see text] or [Formula: see text] containing a reversed 32-38 pair on cognate and near-cognate codons. Structures of wild-type [Formula: see text] bound to the ribosome reveal 23S ribosomal RNA (rRNA) nucleotide A1913 positional changes that are dependent on whether the codon-anticodon interaction is cognate or near cognate. Further, the 32-38 pair is destabilized in the context of a near-cognate codon-anticodon pair. Reversal of the pairing in [Formula: see text] ablates A1913 movement regardless of whether the interaction is cognate or near cognate. These results demonstrate that disrupting 32-38 and anticodon sequences alters interactions with the ribosome that directly contribute to misreading.

Cobamides.

Vitamin B12 is the only known essential human micronutrient made exclusively by prokaryotes. Kennedy and Taga introduce us to the world of cobamides-those cobalt-containing compounds, like B12, that appear to be the proprietary domain of our microbial partners.

Specificity of cobamide remodeling, uptake and utilization in Vibrio cholerae.

Cobamides are a group of compounds including vitamin B12 that can vary at the lower base position of the nucleotide loop. They are synthesized de novo by only a subset of prokaryotes, but some organisms encode partial biosynthesis pathways for converting one variant to another (remodeling) or completing biosynthesis from an intermediate (corrinoid salvaging). Here, we explore the cobamide specificity in Vibrio cholerae through examination of three natural variants representing major cobamide groups: commercially available cobalamin, and isolated pseudocobalamin and p-cresolylcobamide. We show that BtuB, the outer membrane corrinoid transporter, mediates the uptake of all three variants and the intermediate cobinamide. Our previous work suggested that V. cholerae could convert pseudocobalamin produced by cyanobacteria into cobalamin. In this work, cobamide specificity in V. cholerae is demonstrated by remodeling of pseudocobalamin and salvaging of cobinamide to produce cobalamin. Cobamide remodeling in V. cholerae is distinct from the canonical pathway requiring amidohydrolase CbiZ, and heterologous expression of V. cholerae CobS was sufficient for remodeling. Furthermore, function of V. cholerae cobamide-dependent methionine synthase MetH was robustly supported by cobalamin and p-cresolylcobamide, but not pseudocobalamin. Notably, the inability of V. cholerae to produce and utilize pseudocobalamin contrasts with enteric bacteria like Salmonella.

Ultrastable Bimolecular G-Quadruplexes Programmed DNA Nanoassemblies for Reconfigurable Biomimetic DNAzymes.

The relatively low stability and polymorphism of bimolecular G-quadruplexes (bi-G4s) are big difficulties that are faced in employing them to guide DNA assembly, as they are usually subject to a transformation into more stable tetramolecular or G-wire structures favored by K+ or Mg2+. Although bi-G4s benefit by additional duplex handles, a challenge remains in tailoring their intrinsic properties to resolve the above difficulties. Toward this challenge, here we engineer several ultrastable bi-G4s via replacing their nucleotide loops with special mini-hairpins, which consist of a GAA loop and a short GC-paired stem. Such a structural alteration favors the formation of G:C:G:C tetrads in the head-to-head folding topologies of bi-G4s and improves their thermal stability, with an increase in the melting temperature by up to 25 °C. It dramatically reduces their structural conversion into G-wires, verified by atomic force microscopy. These features enable the utilization of two well-chosen bi-G4s to shape a DNA nanotriangle into the desired framework nucleic acid (FNA) architectures such as "bowknot" and "butterfly" that are reversibly switched by the bi-G4s. On this basis, we further build a reconfigurable DNAzyme device to mimic the activation of human telomerase that is modulated by the G4 dimerization. Our designed ultrastable bi-G4s will offer a promising tool for dynamically manipulating intracellular DNA nanoassemblies with endogenous K+ and exploring the relationship between dimerization and function in some physiological processes.

Stabilization of DNA Loop Structures by Large Cations.

The DNA-binding properties of large cations differ from those of metal ions due to steric exclusion from base-paired regions. In this study, the thermal stability of DNA secondary structures, including duplexes, internal loops, bulge loops, hairpin loops, dangling ends, and G-quadruplexes, was investigated in the presence of cations of different sizes. Large cations, such as tetrabutylammonium and tetrapentylammonium ions, reduced the stability of fully matched duplexes but increased the stability of duplexes with a long loop. The cations also increased the stability of G-quadruplexes with a long loop, and the degree of stabilization was greater for low-stability G-quadruplexes. Analysis of the salt concentration dependence indicates that large cations bind to the loop nucleotides, leading to counteracting the destabilization effect on base pairing. It is likely that binding occurs when loop nucleotides are sufficiently flexible to allow for greater accessibility for large cations. These results provide insight into nucleic acid interactions with large cationic molecules and suggest a potential method for stabilizing noncanonical DNA structures under intracellular conditions.

Structural and functional analysis of an l‐serine O‐phosphate decarboxylase involved in norcobamide biosynthesis

Structural diversity of natural cobamides (Cbas, B12 vitamers) is limited to the nucleotide loop. The loop is connected to the cobalt-containing corrin ring via an (R)-1-aminopropan-2-ol O-2-phosphate (AP-P) linker moiety. AP-P is produced by the l-threonine O-3-phosphate (l-Thr-P) decarboxylase CobD. Here, the CobD homolog SMUL_1544 of the organohalide-respiring epsilonproteobacterium Sulfurospirillummultivorans was characterized as a decarboxylase that produces ethanolamine O-phosphate (EA-P) from l-serine O-phosphate (l-Ser-P). EA-P is assumed to serve as precursor of the linker moiety of norcobamides that function as cofactors in the respiratory reductive dehalogenase. SMUL_1544 (SmCobD) is a pyridoxal-5'-phosphate (PLP)-containing enzyme. The structural analysis of the SmCobD apoprotein combined with the characterization of truncated mutant proteins uncovered a role of the SmCobD N-terminus in efficient l-Ser-P conversion.

From general base to general acid catalysis in a sodium-specific DNAzyme by a guanine-to-adenine mutation.

Recently, a few Na+-specific RNA-cleaving DNAzymes were reported, where nucleobases are likely to play critical roles in catalysis. The NaA43 and NaH1 DNAzymes share the same 16-nt Na+-binding motif, but differ in one or two nucleotides in a small catalytic loop. Nevertheless, they display an opposite pH-dependency, implicating distinct catalytic mechanisms. In this work, rational mutation studies locate a catalytic adenine residue, A22, in NaH1, while previous studies found a guanine (G23) to be important for the catalysis of NaA43. Mutation with pKa-perturbed analogs, such as 2-aminopurine (∼3.8), 2,6-diaminopurine (∼5.6) and hypoxanthine (∼8.7) affected the overall reaction rate. Therefore, we propose that the N1 position of G23 (pKa ∼6.6) in NaA43 functions as a general base, while that of A22 (pKa ∼6.3) in NaH1 as a general acid. Further experiments with base analogs and a phosphorothioate-modified substrate suggest that the exocyclic amine in A22 and both of the non-bridging oxygens at the scissile phosphate are important for catalysis for NaH1. This is an interesting example where single point mutations can change the mechanism of cleavage from general base to general acid, and it can also explain this Na+-dependent DNAzyme scaffold being sensitive to a broad range of metal ions and molecules.

Conjugation of N‐(3‐(9‐Ethynyl‐6H‐indolo[2,3‐b]quinoxalin‐6‐yl)propyl)‐2,2,2‐trifluoroacetamide Intercalator to a Triplex Forming Oligonucleotide, a Three‐Way Junction, and a G‐Quadruplex

A new intercalating nucleic acid monomer Z comprising an 3‐(9‐((4‐oxyphenyl)ethynyl)‐6H‐indolo[2,3‐b]quinoxalin‐6‐yl)propan‐1‐amine moiety was synthesized. When Z was inserted into triplex forming oligonucleotides, high thermal stability was observed for its corresponding Hoogsteen‐type triplexes. Three‐way junction (TWJ) was studied by targeting a DNA strand to the foot of a DNA or an RNA hairpin. When Z was inserted into the DNA strand, this resulted in the highest increase of thermal melting ever reported for a TWJ modified by insertion of an intercalator into the junction site. Experiments with mismatches confirmed formation of the TWJ. Improvements in stability of a G‐quadruplex were achieved by insertion of the monomer Z by replacement of one of the nucleotides in the TGT loop. The first steps in the synthesis of the monomer Z were condensation of 5‐iodoisatin with o‐phenylene diamine under reflux in acetic acid and subsequent alkylation reaction with 2‐(3‐bromopropyl)isoindoline‐1,3‐dione followed by suitable reduction led to 3‐(9‐Iodo‐6H‐indolo[2,3‐b]quinoxalin‐6‐yl)propan‐1‐amine which in turn was treated with ethyl trifluoroacetate with formation of 2,2,2‐trifluoro‐N‐(3‐(9‐iodo‐6H‐indolo[2,3‐b]quinoxalin‐6‐yl)propyl)acetamide. This compound was reacted by Sonogashira coupling conditions to give the diol which was converted into the DMT‐protected phosphoramidite, which in turn was used to incorporate the monomer Z into oligonucleotides.

A structure-activity relationship of a thrombin-binding aptamer containing LNA in novel sites.

In this report, structural characterization, aptamer stability and thrombin of a new modified thrombin-ligand complex binding aptamer (TBA) containing anti-guanine bases and a loop position locked nucleic acid (LNA) are presented. NMR, circular dichroic spectroscopy and molecular modeling were used to characterize the three-dimensional structure of two G-quadruplexes. LNA-modification of the anti-guanosines yields G-quadruplexes that show affinity and inhibitory activity toward thrombin, whereas LNA-modification of a thymine nucleotide in the TGT loop increases the thermal stability of TBA. As assessed by denatured PAGE electrophoresis, all modified aptamers display an increase in environmental stability. The prothrombin time assay and fibrinogen assay showed that the aptamers still had good inhibitory activity, and 15 of them had the longest PT time. Therefore, the LNA modification is well suited to improve the physicochemical and biological properties of the native thrombin-binding aptamer.

Truncated vitamin B12 derivative with the phosphate group retained

Cobyric acid and cobinamide are valuable building blocks for the synthesis of artificial cobalamins modified at the nucleotide loop. However, truncated vitamin B[Formula: see text] derivatives are devoid of the phosphate group. We have found that 2-iodoxy benzoic acid-mediated phosphorolysis leads to the cleavage of only one of the phosphodiester bond giving a vitamin B[Formula: see text] analogue with the phosphate moiety preserved. Subsequent alkylation with an organic halide establishes its role as a precursor for the synthesis of vitamin B[Formula: see text] mimics modified at the nucleotide loop.