A novel view of gel-shifts: analysis of RNA-protein complexes using a two-color fluorescence dye procedure

The electrophoretic mobility shift assay (EMSA) is a common technique to identify and analyze RNA-protein interactions, using the altered electrophoretic mobility of RNA and/or protein upon forming an RNA-protein complex. Traditional techniques of visualization of the EMSA results include either prelabeling of RNA before complex formation or specific RNA- or protein-staining after electrophoresis. Recently, two-color fluorescent staining (TCFS) methods were developed, in which the nucleic acid is stained first and scanned; subsequently, the protein is stained and scanned. In the current study, we developed a TCFS system, in which RNA and protein are stained with SYBR Green I and with SYPRO Red, respectively. The gel is subsequently scanned in two channels in a laser scanner to detect both simultaneously. Furthermore, we show that tetramethylrhodamine (TAMRA)-labeled proteins can subsequently be monitored in multicomponent RNA-protein complexes. This novel two-color fluorescence staining is simple, sensitive, and significantly faster than other comparable procedures and allows the independent quantitative determination of both free or complexed nucleic acids and proteins. The interactions between 23S rRNA and ribosomal protein L11 and the ribosomal protein complex L10/L12(4) were used to demonstrate the advantages of this method.     

Ultrahighly Sensitive Homogeneous Detection of DNA and MicroRNA by Using Single‐Silver‐Nanoparticle Counting

DNA and RNA analysis is of high importance for clinical diagnoses, forensic analysis, and basic studies in the biological and biomedical fields. In this paper, we report the ultrahighly sensitive homogeneous detection of DNA and microRNA by using a novel single‐silver‐nanoparticle counting (SSNPC) technique. The principle of SSNPC is based on the photon‐burst counting of single silver nanoparticles (Ag NPs) in a highly focused laser beam (about 0.5 fL detection volume) due to Brownian motion and the strong resonance Rayleigh scattering of single Ag NPs. We first investigated the performance of the SSNPC system and then developed an ultrasensitive homogeneous detection method for DNA and microRNA based on this single‐nanoparticle technique. Sandwich nucleic acid hybridization models were utilized in the assays. In the hybridization process, when two Ag‐NP–oligonucleotide conjugates were mixed in a sample containing DNA (or microRNA) targets, the binding of the targets caused the Ag NPs to form dimers (or oligomers), which led to a reduction in the photon‐burst counts. The SSNPC method was used to measure the change in the photon‐burst counts. The relationship between the change of the photon‐burst counts and the target concentration showed a good linearity. This method was used for the assay of sequence‐specific DNA fragments and microRNAs. The detection limits were at about the 1 fM level, which is 2–5 orders of magnitude more sensitive than current homogeneous methods.     

Target‐Directed Azide‐Alkyne Cycloaddition for Assembling HIV‐1 TAR RNA Binding Ligands

The highly conserved HIV‐1 transactivation response element (TAR) binds to the trans‐activator protein Tat and facilitates viral replication in its latent state. The inhibition of Tat–TAR interactions by selectively targeting TAR RNA has been used as a strategy to develop potent antiviral agents. Therefore, HIV‐1 TAR RNA represents a paradigmatic system for therapeutic intervention. Herein, we have employed biotin‐tagged TAR RNA to assemble its own ligands from a pool of reactive azide and alkyne building blocks. To identify the binding sites and selectivity of the ligands, the in situ cycloaddition has been further performed using control nucleotide (TAR DNA and TAR RNA without bulge) templates. The hit triazole‐linked thiazole peptidomimetic products have been isolated from the biotin‐tagged target templates using streptavidin beads. The major triazole lead generated by the TAR RNA presumably binds in the bulge region, shows specificity for TAR RNA over TAR DNA, and inhibits Tat–TAR interactions.     

Nucleobase‐Modified PNA Suppresses Translation by Forming a Triple Helix with a Hairpin Structure in mRNA In Vitro and in Cells

Compounds that bind specifically to double‐stranded regions of RNA have potential as regulators of structure‐based RNA function; however, sequence‐selective recognition of double‐stranded RNA is challenging. The modification of peptide nucleic acid (PNA) with unnatural nucleobases enables the formation of PNA–RNA triplexes. Herein, we demonstrate that a 9‐mer PNA forms a sequence‐specific PNA–RNA triplex with a dissociation constant of less than 1 nm at physiological pH. The triplex formed within the 5′ untranslated region of an mRNA reduces the protein expression levels both in vitro and in cells. A single triplet mismatch destabilizes the complex, and in this case, no translation suppression is observed. The triplex‐forming PNAs are unique and potent compounds that hold promise as inhibitors of cellular functions that are controlled by double‐stranded RNAs, such as RNA interference, RNA editing, and RNA localization mediated by protein–RNA interactions.     

Structure of a Protein–RNA Complex by Solid‐State NMR Spectroscopy

Solid‐state NMR (ssNMR) is applicable to high molecular‐weight (MW) protein assemblies in a non‐amorphous precipitate. The technique yields atomic resolution structural information on both soluble and insoluble particles without limitations of MW or requirement of crystals. Herein, we propose and demonstrate an approach that yields the structure of protein–RNA complexes (RNP) solely from ssNMR data. Instead of using low‐sensitivity magnetization transfer steps between heteronuclei of the protein and the RNA, we measure paramagnetic relaxation enhancement effects elicited on the RNA by a paramagnetic tag coupled to the protein. We demonstrate that this data, together with chemical‐shift‐perturbation data, yields an accurate structure of an RNP complex, starting from the bound structures of its components. The possibility of characterizing protein–RNA interactions by ssNMR may enable applications to large RNP complexes, whose structures are not accessible by other methods.     

The Synthesis of Methylated,Phosphorylated, and Phosphonated 3′‐Aminoacyl‐tRNASec Mimics

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA^Sec) in all domains of life. The multistep pathway involves O‐phosphoseryl‐tRNA:selenocysteinyl‐tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl–tRNA^Sec intermediate is converted into selenocysteinyl‐tRNA^Sec. The SepSecS architecture and the mode of tRNA^Sec recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA^Sec substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl‐derived tRNA^Sec mimics that contain a hydrolysis‐resistant ribose 3′‐amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site‐specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)‐2‐amino‐4‐phosphonobutyric acid–oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three‐strand enzymatic ligation protocol to obtain the corresponding full‐length tRNA^Sec derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS‐tRNA interactions. The novel tRNA^Sec mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X‐ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA‐dependent enzymes.     

Polymer‐Templated Perylene‐Probe Noncovalent Self‐Assembly: A New Strategy for Label‐Free Ultrasensitive Fluorescence Turn‐On Biosensing

A new label‐free fluorescence turn‐on strategy for highly sensitive biosensing has been developed. A negatively charged perylene probe was synthesized. Polycations could induce aggregation of the perylene probe through noncovalent interactions and the fluorescence of the probe’s monomer was efficiently quenched. Upon addition of a single‐stranded nucleic acid, competitive binding of the negatively charged nucleic acid (a polyanion) to the cationic polymer resulted in the release of a monomer and thus a turn‐on fluorescence signal was detected. Without the use of any amplification techniques, a detection limit of 2 pM DNA was obtained. Based on these results, an assay strategy for the highly sensitive detection of alkaline phosphatase (ALP) activity has been demonstrated. λ Exonuclease (λ exo) could degrade 5′‐phosphorylated single‐stranded DNA. However, when the DNA sample was treated with ALP, the phosphate functional group was removed by ALP and it could no longer be degraded by λ exo. Binding of the DNA to the perylene probe–polycation complex resulted in a turn‐on fluorescence signal, which could be used for sensing of ALP. The method is highly sensitive, a limit of detection as low as 0.02 mU mL^?1 ALP was obtained. Our method is simple, convenient, highly sensitive, and inexpensive.     

Crystal‐Structure‐Guided Design of Self‐Assembling RNA Nanotriangles

RNA nanotechnology uses RNA structural motifs to build nanosized architectures that assemble through selective base‐pair interactions. Herein, we report the crystal‐structure‐guided design of highly stable RNA nanotriangles that self‐assemble cooperatively from short oligonucleotides. The crystal structure of an 81 nucleotide nanotriangle determined at 2.6 Å resolution reveals the so‐far smallest circularly closed nanoobject made entirely of double‐stranded RNA. The assembly of the nanotriangle architecture involved RNA corner motifs that were derived from ligand‐responsive RNA switches, which offer the opportunity to control self‐assembly and dissociation.     

ADLOC: An Aptamer‐Displacement Assay Based on Luminescent Oxygen Channeling

Functional nucleic acids, such as aptamers and allosteric ribozymes, can sense their ligands specifically, thereby undergoing structural alterations that can be converted into a detectable signal. The direct coupling of molecular recognition to signal generation enables the production of versatile reporters that can be applied as molecular probes for various purposes, including high‐throughput screening. Here we describe an unprecedented type of a nucleic acid‐based sensor system and show that it is amenable to high‐throughput screening (HTS) applications. The approach detects the displacement of an aptamer from its bound protein partner by means of luminescent oxygen channeling. In a proof‐of‐principle study we demonstrate that the format is feasible for efficient identification of small drug‐like molecules that bind to a protein target, in this case to the Sec7 domain of cytohesin. We extended the approach to a new cytohesin‐specific single chain DNA aptamer, C10.41, which exhibits a similar binding behavior to cytohesins but has the advantage of being more stable and easier to synthesize and to modify than the RNA‐aptamer M69. The results obtained with both aptamers indicate the general suitability of the aptamer‐displacement assay based on luminescent oxygen channelling (ADLOC) for HTS. We also analyzed the potential for false positive hits and identified from a library of 18 000 drug‐like small molecules two compounds as strong singlet‐oxygen quenchers. With full automation and the use of commercially available plate readers, we estimate that the ADLOC‐based assay described here could be used to screen at least 100 000 compounds per day.     

Second‐Generation Fluorescent Quadracyclic Adenine Analogues: Environment‐Responsive Probes with Enhanced Brightness

Fluorescent base analogues comprise a group of increasingly important molecules for the investigation of nucleic acid structure, dynamics, and interactions with other molecules. Herein, we report on the quantum chemical calculation aided design, synthesis, and characterization of four new putative quadracyclic adenine analogues. The compounds were efficiently synthesized from a common intermediate through a two‐step pathway with the Suzuki–Miyaura coupling as the key step. Two of the compounds, qAN1 and qAN4, display brightnesses (εΦ_F) of 1700 and 2300, respectively, in water and behave as wavelength‐ratiometric pH probes under acidic conditions. The other two, qAN2 and qAN3, display lower brightnesses but exhibit polarity‐sensitive dual‐band emissions that could prove useful to investigate DNA structural changes induced by DNA–protein or –drug interactions. The four qANs are very promising microenvironment‐sensitive fluorescent adenine analogues that display considerable brightness for such compounds.     

High‐Resolution Crystal Structure of a Silver(I)–RNA Hybrid Duplex Containing Watson–Crick‐like CSilver(I)C Metallo‐Base Pairs

Metallo‐base pairs have been extensively studied for applications in nucleic acid‐based nanodevices and genetic code expansion. Metallo‐base pairs composed of natural nucleobases are attractive because nanodevices containing natural metallo‐base pairs can be easily prepared from commercially available sources. Previously, we have reported a crystal structure of a DNA duplex containing T? Hg^II? T base pairs. Herein, we have determined a high‐resolution crystal structure of the second natural metallo‐base pair between pyrimidine bases C? Ag^I? C formed in an RNA duplex. One Ag^I occupies the center between two cytosines and forms a C? Ag^I? C base pair through N3? Ag^I? N3 linear coordination. The C? Ag^I? C base pair formation does not disturb the standard A‐form conformation of RNA. Since the C? Ag^I? C base pair is structurally similar to the canonical Watson–Crick base pairs, it can be a useful building block for structure‐based design and fabrication of nucleic acid‐based nanodevices.     

Geometric similarity between protein–RNA interfaces

A new method is described to measure the geometric similarity between protein–RNA interfaces quantitatively. The method is based on a procedure that dissects the interface geometry in terms of the spatial relationships between individual amino acid nucleotide pairs. Using this technique, we performed an all‐on‐all comparison of 586 protein–RNA interfaces deposited in the current Protein Data Bank, as the result, an interface–interface similarity score matrix was obtained. Based upon this matrix, hierarchical clustering was carried out which yielded a complete clustering tree for the 586 protein–RNA interfaces. By investigating the organizing behavior of the clustering tree and the SCOP classification of protein partners in complexes, a geometrically nonredundant, diverse data set (representative data set) consisting of 45 distinct protein–RNA interfaces was extracted for the purpose of studying protein–RNA interactions, RNA regulations, and drug design. We classified protein–RNA interfaces into three types. In type I, the families and interface structural classes of the protein partners, as well as the interface geometries are all similar. In type II, the interface geometries and the interface structural classes are similar, whereas the protein families are different. In type III, only the interface geometries are similar but the protein families and the interface structural classes are distinct. Furthermore, we also show two new RNA recognition themes derived from the representative data set. © 2009 Wiley Periodicals, Inc. J Comput Chem 2009     

Liquid‐Phase RNA Synthesis by Using Alkyl‐Chain‐Soluble Support

Recent progress in the RNA therapeutics has increased demand for the synthesis of large quantities of oligoribonucleotides. The assembly of RNA oligomers relies mainly on solid‐phase approaches. These allow rapid product purification and the ability to drive a target reaction to completion through the use of excess reagents. Despite the known advantages of solid‐phase synthesis, some issues in the process remain to be addressed, such as low and limited scale, reagent accessibility, and the use of a very large excess of reagents. Herein, we report a highly efficient and practical method of liquid‐phase synthesis of RNA oligomers by using alkyl‐chain‐soluble support. We demonstrate the utility of the liquid‐phase method through 21‐mer RNA synthesis on a gram scale.     

A nonradioactive electrophoretic mobility shift assay for measurement of pregnane X receptor binding activity to CYP3A4 response element

The electrophoretic mobility shift assay (EMSA) is a method for the study of specific DNA–protein interactions in vitro. The pregnane X receptor (PRX) is a key xenobiotic sensor that regulates the expression of drug‐metabolizing enzymes and many other genes. Radiolabeled ³²P‐DNA‐probes had been used in studies of PXR‐DNA interactions. There is an increasing need for nonradioactive assays, due to the health, safety and environmental issues. In the current study, we present a protocol for the nonradioactive electrophoretic mobility shift assay, allowing studying interactions between human PXR with promoter DNA sequences.     

A Benzophenone‐Based Photocaging Strategy for the N7 Position of Guanosine

Selective modification of nucleobases with photolabile caging groups enables the study and control of processes and interactions of nucleic acids. Numerous positions on nucleobases have been targeted, but all involve formal substitution of a hydrogen atom with a photocaging group. Nature, however, also uses ring‐nitrogen methylation, such as m⁷G and m¹A, to change the electronic structure and properties of RNA and control biomolecular interactions essential for translation and turnover. We report that aryl ketones such as benzophenone and α‐hydroxyalkyl ketone are photolabile caging groups if installed at the N7 position of guanosine or the N1 position of adenosine. Common photocaging groups derived from the ortho‐nitrobenzyl moiety were not suitable. Both chemical and enzymatic methods for site‐specific modification of N7G in nucleosides, dinucleotides, and RNA were developed, thereby opening the door to studying the molecular interactions of m⁷G and m¹A with spatiotemporal control.     

Profiling of Ribose Methylations in RNA by High‐Throughput Sequencing

Ribose methylations are the most abundant chemical modifications of ribosomal RNA and are critical for ribosome assembly and fidelity of translation. Many aspects of ribose methylations have been difficult to study due to lack of efficient mapping methods. Here, we present a sequencing‐based method (RiboMeth‐seq) and its application to yeast ribosomes, presently the best‐studied eukaryotic model system. We demonstrate detection of the known as well as new modifications, reveal partial modifications and unexpected communication between modification events, and determine the order of modification at several sites during ribosome biogenesis. Surprisingly, the method also provides information on a subset of other modifications. Hence, RiboMeth‐seq enables a detailed evaluation of the importance of RNA modifications in the cells most sophisticated molecular machine. RiboMeth‐seq can be adapted to other RNA classes, for example, mRNA, to reveal new biology involving RNA modifications.     

A Carbon‐Dot‐Based Fluorescent Nanosensor for Simple Visualization of Bacterial Nucleic Acids

A simple and facile method for sensing of nucleic acids is in great need for disease biomarker detection and diagnosis. Herein, a fluorescent nanosensor utilizing carbon dot nanoparticles is introduced that form visible precipitates in the presence of target DNA. Carbon dot nanoparticles are fabricated by microwave pyrolysis of polyethylenimine, which emits strong photoluminescence and can form precipitates when added to target DNA oligonucleotides. The precipitates can be easily visualized by UV illumination, and data can be acquired as images using a smartphone, which are analyzed for quantification. This carbon‐dot‐based assay allowed fluorescent sensing of target oligonucleotides with various sizes and visualization even with minimal amount of DNA (≈100 pmol). Finally, the assay can be applied as a nanosensor platform for detecting bacterial DNA for the antibiotic‐resistance gene KPC‐2 from Klebsiella pneumoniae . This method provides a simple technique for detecting molecular targets, showing wide applicability for diagnostics on the bedside or point‐of‐care testing.