Microarray gene expression analysis free of reverse transcription and dye labeling

A new microarray system has been developed for gene expression analysis using cationic gold nanoparticles with diameters of 250 nm as a target detection reagent. The approach utilizes nonlabeled target molecules hybridizing with complementary probes on the array, followed by incubation in a colloidal gold solution. The hybridization signal results from the precipitation of nanogold particles on the hybridized spots due to the electrostatic attraction of the cationic gold particles and the anionic phosphate groups in the target DNA backbone. In contrast to conventional fluorescent detection, this nanoparticle-based detection system eliminates the target labeling procedure. The visualization of hybridization signals can be accomplished with a flatbed scanner instead of a confocal laser scanner, which greatly simplifies the process and reduces the cost. The sensitivity is estimated to be less than 2 pg of DNA molecules captured on the array surface. The signal from hybridized spots quantitatively represents the amount of captured target DNA and therefore permits quantitative gene expression analysis. Cross-array reproducibility is adequate for detecting twofold or less signal changes across two microarray experiments.     

Homogeneous Fluorescence Assays for RNA Diagnosis by Pyrene-Conjugated 2′- O -Methyloligoribonucleotides

We developed a bispyrene-conjugated 2 ′-O-methyloligoribonucleotide as an RNA-specific RNA-probe. The probe hybridized with the complementary RNA, greatly enhancing fluorescence and discriminating RNA from DNA. The assay was carried out in homogeneous aqueous media without removing the unbound probe from the detection solution. This homogeneous fluorescence assay also discriminated mismatch sequences in the target RNA. These pyrene probes could possess high potential to detect RNA in biological specimens simply.     

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

Adenosine deaminases that act on RNA (ADAR) catalyze adenosine to inosine (A-to-I) editing in double-stranded RNA (dsRNA) substrates. Inosine is read as guanosine by the translation machinery; therefore A-to-I editing events in coding sequences may result in recoding genetic information. Whereas vertebrates have two catalytically active enzymes, namely ADAR1 and ADAR2, Drosophila has a single ADAR protein (dADAR) related to ADAR2. The structural determinants controlling substrate recognition and editing of a specific adenosine within dsRNA substrates are only partially understood. Here, we report the solution structure of the N-terminal dsRNA binding domain (dsRBD) of dADAR and use NMR chemical shift perturbations to identify the protein surface involved in RNA binding. Additionally, we show that Drosophila ADAR edits the R/G site in the mammalian GluR-2 pre-mRNA which is naturally modified by both ADAR1 and ADAR2. We then constructed a model showing how dADAR dsRBD1 binds to the GluR-2 R/G stem-loop. This model revealed that most side chains interacting with the RNA sugar-phosphate backbone need only small displacement to adapt for dsRNA binding and are thus ready to bind to their dsRNA target. It also predicts that dADAR dsRBD1 would bind to dsRNA with less sequence specificity than dsRBDs of ADAR2. Altogether, this study gives new insights into dsRNA substrate recognition by Drosophila ADAR.     

RNA editing in eag potassium channels: Biophysical consequences of editing a conserved S6 residue

RNA editing at four sites in eag, a Drosophila voltage-gated potassium channel, results in the substitution of amino acids into the final protein product that are not encoded by the genome. These sites and the editing alterations introduced are K467R (Site 1, top of the S6 segment), Y548C, N567D and K699R (sites 2–4, within the cytoplasmic C-terminal domain). We mutated these residues individually and expressed the channels in Xenopus oocytes. A fully edited construct (all four sites) has the slowest activation kinetics and a paucity of inactivation, whereas the fully unedited channel exhibits the fastest activation and most dramatic inactivation. Editing Site 1 inhibits steady-state inactivation. Mutating Site 1 to the neutral residues resulted in intermediate inactivation phenotypes and a leftward shift of the peak current-voltage relationship. Activation kinetics display a Cole-Moore shift that is enhanced by RNA editing. Normalized open probability relationships for 467Q, 467R and 467K are superimposable, indicating little effect of the mutations on steady-state activation. 467Q and 467R enhance instantaneous inward rectification, indicating a role of this residue in ion permeation. Intracellular tetrabutylammonium blocks 467K significantly better than 467R. Block by intracellular, but not extracellular, tetraethylammonium interferes with inactivation. The fraction of inactivated current is reduced at higher extracellular Mg⁺² concentrations, and channels edited at Site 1 are more sensitive to changes in extracellular Mg⁺² than unedited channels. These results show that even a minor change in amino acid side-chain chemistry and size can have a dramatic impact on channel biophysics, and that RNA editing is important for fine-tuning the channel’s function.     

2D aggregation and selective desorption of nanoparticle probes: a new method to probe DNA mismatches and damages

A 2D colorimetric DNA sensor is reported based on the 2D aggregation of oligonucleotide-modified gold nanoparticle probes resulting from the molecular hybridization between these latest and their complementary single stranded DNA targets. To increase their mobility the nanoparticles are adsorbed on a fluid lipid bilayer, itself supported on a substrate. The hybridization between the target and the mobile nanoparticle probes creates links between the nanoparticles resulting in the formation of nanoparticle aggregates in the plane of the substrate. This aggregation is detected using a new method based on the selective desorption of non-aggregated nanoparticles. The addition of dextran sulfate induces the substitution of non-aggregated gold nanoparticles while aggregated ones are stable on the substrate. We show that this detection method is highly specific and allows the detection of DNA mismatches and damages.     

A Comprehensive Analysis of Northern versus Liquid Hybridization Assays for mRNAs,Small RNAs,and miRNAs Using a Non-Radiolabeled Approach

Northern blotting (NB), a gold standard for RNA detection, has lost its charm due to its hands-on nature, need for good quality RNA, and radioactivity. With the emergence of the field of microRNAs (miRNAs), the necessity for sensitive and quantitative NBs has again emerged. Here, we developed highly sensitive yet non-radiolabeled, fast, economical NB, and liquid hybridization (LH) assays without radioactivity or specialized reagents like locked nucleic acid (LNA)- or digoxigenin-labeled probes for mRNAs/small RNAs, especially miRNAs using biotinylated probes. An improvised means of hybridizing oligo probes along with efficient transfer, cross-linking, and signal enhancement techniques was employed. Important caveats of each assay were elaborated upon, especially issues related to probe biotinylation, use of exonuclease, and bioimagers not reported earlier. We demonstrate that, while the NBs were sensitive for mRNAs and small RNAs, our LH protocol could efficiently detect these and miRNAs using less than 10–100 times the total amount of RNA, a sensitivity comparable to radiolabeled probes. Compared to NBs, LH was a faster, more sensitive, and specific approach for mRNA/small RNA/miRNA detection. A comparison of present work with six seminal studies is presented along with detailed protocols for easy reproducibility. Overall, our study provides effective platforms to study large and small RNAs in a sensitive, efficient, and cost-effective manner.     

Evaluating conformational changes in protein structures binding RNA

Many protein-RNA recognition events are known to exhibit conformational changes from qualitative observations of individual complexes. However, a quantitative estimation of conformational changes is required if protein-RNA docking and template-based methods for RNA binding site prediction are to be developed. This study presents the first quantitative evaluation of conformational changes that occur when proteins bind RNA. The analysis of twelve RNA-binding proteins in the bound and unbound states using error-scaled difference distance matrices is presented. The binding site residues are mapped to each structure, and the conformational changes that affect these residues are evaluated. Of the twelve proteins four exhibit greater movements in nonbinding site residues, and a further four show the greatest movements in binding site residues. The remaining four proteins display no significant conformational change. When interface residues are found to be in conformationally variable regions of the protein they are typically seen to move less than 2 A between the bound and unbound conformations. The current data indicate that conformational changes in the binding site residues of RNA binding proteins may not be as significant as previously suggested, but a larger data set is required before wider conclusions may be drawn. The implications of the observed conformational changes for protein function prediction are discussed.     

Isoenergetic penta- and hexanucleotide microarray probing and chemical mapping provide a secondary structure model for an RNA element orchestrating R2 retrotransposon protein function

LNA (locked nucleic acids, i.e. oligonucleotides with a methyl bridge between the 2′ oxygen and 4′ carbon of ribose) and 2,6-diaminopurine were incorporated into 2′-O-methyl RNA pentamer and hexamer probes to make a microarray that binds unpaired RNA approximately isoenergetically. That is, binding is roughly independent of target sequence if target is unfolded. The isoenergetic binding and short probe length simplify interpretation of binding to a structured RNA to provide insight into target RNA secondary structure. Microarray binding and chemical mapping were used to probe the secondary structure of a 323 nt segment of the 5′ coding region of the R2 retrotransposon from Bombyx mori (R2Bm 5′ RNA). This R2Bm 5′ RNA orchestrates functioning of the R2 protein responsible for cleaving the second strand of DNA during insertion of the R2 sequence into the genome. The experimental results were used as constraints in a free energy minimization algorithm to provide an initial model for the secondary structure of the R2Bm 5′ RNA.     

Finding a human telomere DNA–RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: A protective structure for telomere end

Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA–RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA–RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA–RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA–RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA–RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.     

Improving the prediction of RNA secondary structure by detecting and assessing conserved stems

The prediction of RNA secondary structure can be facilitated by incorporating with comparative analysis of homologous sequences. However, most of existing comparative methods are vulnerable to alignment errors and thus are of low accuracy in practical application. Here we improve the prediction of RNA secondary structure by detecting and assessing conserved stems shared by all sequences in the alignment. Our method can be summarized by: 1) we detect possible stems in single RNA sequence using the so-called position matrix with which some possibly paired positions can be uncovered; 2) we detect conserved stems across multiple RNA sequences by multiplying the position matrices; 3) we assess the conserved stems using the Signal-to-Noise; 4) we compute the optimized secondary structure by incorporating the so-called reliable conserved stems with predictions by RNAalifold program. We tested our method on data sets of RNA alignments with known secondary structures. The accuracy, measured as sensitivity and specificity, of our method is greater than predictions by RNAalifold.     

The highest affinity binding site of small protein B on transfer messenger RNA is outside the tRNA domain

Eubacterial ribosomes stalled on defective mRNAs are released through a mechanism referred to as trans-translation, depending on the coordinated actions of small protein B (SmpB) and transfer messenger RNA (tmRNA). A series of tmRNA variants with deletions in each structural domain were produced. Their structures were monitored by enzymatic and chemical probes in vitro, in the presence and absence of SmpB. Dissociation constants between these RNAs and SmpB from Aquifex aeolicus were derived by surface plasmon resonance (SPR) combined with filter binding assays. Three independent experimental evidences, including filter binding assays, SPR, and concentration titrations of the RNA–protein reactivity changes toward structural probes, indicate that the binding site that has the highest affinity for the protein is located outside the tRNA domain, upstream of the internal tag. The minimal tmRNA fragment that contains this high affinity site for SmpB, and also contains another site of lower affinity, includes the tag reading frame and three downstream pseudoknots that form a ring structure in solution.     

Rapid and quantitative quality control of microarrays using cationic nanoparticles

The fabrication quality of microarrays significantly influences the accuracy and reproducibility of microarray experiments. In this report, we present a simple and fast quality control (QC) method for spotted oligonucleotide and cDNA microarrays. It employs a nonspecific electrostatic interaction of colloidal gold nanoparticles with the chemical groups of DNA molecules and other biomolecules immobilized on the microarray surface that bear positive or negative charges. An inexpensive flatbed scanner is used to visualize and quantify the binding of cationic gold particles to the anionic DNA probes on the microarray surface. An image analysis software was designed to assess the various parameters of the array spots including spot intensity, shape and array homogeneity, calculate the overall array quality score, and save the detailed array quality report in an Excel file. The gold staining technique is fast and sensitive. It can be completed in 10 min and detect less than 1% of the probe amount commonly recommended for microarrays. Compared to the current microarray QC method that utilizes the hybridization of probes with short random sequence oligonucleotides labeled with fluorophore, our gold staining method requires less time for the analysis, reduces the reagent cost, and eliminates the need for the expensive laser scanner. Biotechnol. Bioeng. 2009; 102: 960–964. © 2008 Wiley Periodicals, Inc.     

银染增强的纳米金标记探针对微量核酸的检测

本研究利用银染增强的纳米金技术建立了一种简单快速的核酸定量方法.该方法基于纳米金与烷巯基修饰的寡核苷酸分子共价键合作用,将纳米微粒报告基团标记在与靶核酸一端序列互补的寡核苷酸上,同时生物素化修饰另一端互补序列.靶核酸与两段寡核苷酸探针杂交后,借亲和素固定在酶标板孔内,通过纳米金催化的银染放大效应产生高灵敏的识别信号,适时记录其吸光度值从而实现核酸分子的定量.该检测方法检测单链核酸分子的灵敏度达0.1 fM,双链分子为10 fM.