Nucleic Acids Research

Brandt, Ole; Feldner, Julia; Stephan, Achim; Schröder, Markus; Schnölzer, Martina; Arlinghaus, Heinrich F.; Hoheisel, Jörg D.; Jacob, Anette

doi: 10.1093/nar/gng120pmid: 14500847

Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full‐length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time‐of‐flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter‐bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full‐length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI‐TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol‐modified PNAs on maleimide surfaces.

Flavell, Andrew J.; Bolshakov, Viacheslav N.; Booth, Allan; Jing, Runchun; Russell, Joanne; Ellis, T. H. Noel; Isaac, Peter

doi: 10.1093/nar/gng113pmid: 14500843

A microarray‐based method has been developed for scoring thousands of DNAs for a co‐dominant molecular marker on a glass slide. The approach was developed to detect insertional polymorphism of transposons and works well with single nucleotide polymorphism (SNP) markers. Biotin‐ terminated allele‐specific PCR products are spotted unpurified onto streptavidin‐coated glass slides and visualised by hybridisation of fluorescent detector oligonucleotides to tags attached to the allele‐ specific PCR primers. Two tagged primer oligonucleotides are used per locus and each tag is detected by hybridisation to a concatameric DNA probe labelled with multiple fluorochromes.

Yonezawa, Masato; Doi, Nobuhide; Kawahashi, Yuko; Higashinakagawa, Toru; Yanagawa, Hiroshi

doi: 10.1093/nar/gng119pmid: 14500846

We describe the use of a DNA display system for in vitro selection of peptide ligands from a large library of peptides displayed on their encoding DNAs. The method permits completely in vitro construction of a DNA‐tagged peptide library by using a wheat germ in vitro transcription/translation system compartmentalized in water‐in‐oil emulsions. Starting with a library of 109–1010 random decapeptides, 21 different peptide ligands were isolated for monoclonal antibody anti‐FLAG M2. DNA display selected more diverse peptides with a DYKXXD consensus motif than previously reported phage display systems. Binding and recovery rates of three peptides were significantly higher than those of the original FLAG peptide, implying that these peptides would be superior to the FLAG peptide for purification of tagged proteins. The simplicity of DNA display enables two selection rounds per day to be conducted. Further, DNA display can overcome the limitations of previous display technologies by avoiding the use of bacterial cells and RNA tags. Thus, DNA display is expected to be useful for rapid screening of a wide variety of peptide ligands for corresponding receptors.

Vernis, Laurence; Piskur, Jure; Diffley, John F. X.

doi: 10.1093/nar/gng121pmid: 14500848

The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations. Using this novel approach, we show that depletion of a single deoxyribonucleoside causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase, Rad53. We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse–chase labelling.

King, Oliver D.; Roth, Frederick P.

doi: 10.1093/nar/gng117pmid: 14500844

We introduce a non‐parametric representation of transcription factor binding sites which can model arbitrary dependencies between positions. As two parameters are varied, this representation smoothly interpolates between the empirical distribution of binding sites and the standard position‐specific scoring matrix (PSSM). In a test of generalization to unseen binding sites using 10‐fold cross‐validation on known binding sites for 95 TRANSFAC transcription factors, this representation outperforms PSSMs on between 65 and 89 of the 95 transcription factors, depending on the choice of the two adjustable parameters. We also discuss how the non‐ parametric representation may be incorporated into frameworks for finding binding sites given only a collection of unaligned promoter regions.

Rudi, Knut; Holck, Askild L.

doi: 10.1093/nar/gng118pmid: 14500845

Quencher extension (QEXT) is a novel single step closed tube real‐time method to quantify SNPs using reporters and quenchers in combination with primer extension. A probe with a 5′‐reporter dye is single base extended with a dideoxy nucleotide containing a quencher dye if the target SNP allele is present. The extension is recorded from the quenching (reduced fluorescence) of the reporter dye. This avoids the influence of the unincorporated dye‐labeled nucleotides, resulting in high accuracy and a high signal‐to‐noise ratio. The relative amount of a specific SNP allele is determined from the nucleotide incorporation rate in a thermo‐cycling reaction. We tested the QEXT assay using five SNPs in the Listeria monocytogenesinlA gene as a model system. The presence of the target SNP alleles was determined with high statistical confidence (P < 0.0005). The quantitative detection limits were between 0 and 5% for the targeted SNP alleles on a background of other SNP alleles (P < 0.05). The QEXT method is directly adaptable to current real‐time PCR equipment and is thus suited for high throughput and a wide application range.

Jasinska, Anna; Michlewski, Gracjan; de Mezer, Mateusz; Sobczak, Krzysztof; Kozlowski, Piotr; Napierala, Marek; Krzyzosiak, Wlodzimierz J.

doi: 10.1093/nar/gkg767pmid: 14500808

Among the goals of RNA structural and functional genomics is determining structures and establishing the functions of a rich repertoire of simple sequence repeats in transcripts. These repeats are present in transcripts from their ‘birth’ in the nucleus to their ‘death’ in cytoplasm and have the potential of being involved in many steps of RNA regulation. The knowledge of their structural features and functional roles will also shed more light on the postulated mechanisms of RNA pathogenesis in a growing list of neurological diseases caused by simple sequence repeat expansions. Here, we discuss several different lines of research to support the hypothesis that the mechanism of RNA pathogenesis may be a more common phenomenon triggered or modulated also by abundant long normal repeats. We propose structures of the repeat regions in transcripts of genes involved in Triplet Repeat Expansion Diseases. We have classified the polymorphic repeat alleles of these genes according to their ability to form hairpin structures in transcripts, and describe the distribution of different structural forms of the repeats in the human population. We have also reported the results of a systematic survey of the human transcriptome to identify mRNAs containing triplet repeats and to classify them according to structural and functional criteria. Based on this knowledge, we discuss the putative wider role of triplet repeat RNA hairpins in human diseases. A hypothetical model is proposed in which long normal RNA hairpins formed by the repeats may also be involved in pathogenesis.

Sobczak, Krzysztof; de Mezer, Mateusz; Michlewski, Gracjan; Krol, Jacek; Krzyzosiak, Wlodzimierz J.

doi: 10.1093/nar/gkg766pmid: 14500809

The tandem repeats of trinucleotide sequences are present in many human genes and their expansion in specific genes causes a number of hereditary neurological disorders. The normal function of triplet repeats in transcripts is barely known and the role of expanded RNA repeats in the pathogenesis of Triplet Repeat Expansion Diseases needs to be more fully elucidated. Here we have described the structures formed by transcripts composed of AAG, CAG, CCG, CGG and CUG repeats, which were determined by chemical and enzymatic structure probing. With the exception of the repeated AAG motif, all studied repeats form hairpin structures and these hairpins show several alternative alignments. We have determined the molecular architectures of these co‐existing hairpin structures by using transcripts with GC‐clamps which imposed single alignments of hairpins. We have provided experimental evidence that CCUG repeats implicated in myotonic dystrophy type 2 also form hairpin structures with properties similar to that composed of the CUG repeats.

Maier, Holly; Ostraat, Rachel; Parenti, Sarah; Fitzsimmons, Daniel; Abraham, Lawrence J.; Garvie, Colin W.; Hagman, James

doi: 10.1093/nar/gkg785pmid: 14500810

Pax‐5, a member of the paired domain family of transcription factors, is a key regulator of B lymphocyte‐specific transcription and differentiation. A major target of Pax‐5‐mediated activation is the mb‐1 gene, which encodes the essential transmembrane signaling protein Ig‐α. Pax‐5 recruits three members of the Ets family of transcription factors: Ets‐1, Fli‐1 and GABPα (with GABPβ1), to assemble ternary complexes on the mb‐1 promoter in vitro. Using the Pax‐5:Ets‐1:DNA crystal structure as a guide, we defined amino acid requirements for transcriptional activation of endogenous mb‐1 genes using a novel cell‐based assay. Mutations in the β‐hairpin/β‐turn of the DNA‐binding domain of Pax‐5 demonstrated its importance for DNA sequence recognition and activation of mb‐1 transcription. Mutations of amino acids contacting Ets‐1 in the crystal structure reduced or blocked mb‐1 promoter activation. One of these mutations, Q22A, resulted in greatly reduced mb‐1 gene transcript levels, concurrent with the loss of its ability to recruit Fli‐1 to bind the promoter in vitro. In contrast, the mutation had no effect on recruitment of the related Ets protein GABPα (with GABPβ1). These data further define requirements for Pax‐5 function in vivo and reveal the complexity of interactions required for cooperative partnerships between transcription factors.

Blount, Kenneth F.; Tor, Yitzhak

doi: 10.1093/nar/gkg755pmid: 14500811

To quantitatively understand the binding affinity and target selectivity of small‐molecule RNA interactions, it is useful to have a rapid, highly reproducible binding assay that can be readily generalized to different RNA targets. To that end, an assay has been developed and validated for measuring the binding of low‐molecular weight ligands to RNA by monitoring the fluorescence of a covalently incorporated fluorophore. As a test system, the fluorescence of a pyrene‐derivatized HIV‐1 TAR (transactivating response element) RNA was measured upon titration with aminoglycoside antibiotics. The binding isotherms thus obtained fit well with a model for a 1:1 interaction and yield an accurate measure of the equilibrium dissociation constant. Among a series of natural aminoglycosides, the binding affinity correlates with the number of amines, supporting an electrostatic compensation model for binding. Furthermore, the ionic strength dependence confirms that much of the binding energy is electrostatic. Finally, by measuring the binding affinity in the presence of nucleic acid competitors, we confirm that although aminoglycosides show high RNA to DNA selectivity, their selectivity among different RNA targets is sub‐ optimal. We conclude that this newly developed assay can be generalized to measure the binding affinities and selectivities of a variety of small molecules to a specific RNA target.