Peter Stein Nielsen

OligoDesign: optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling

We report the development of new software, OligoDesign, which provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. LNAs constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Furthermore it includes routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. We have successfully used the OligoDesign tool to design a Caenorhabditis elegans LNA oligonucleotide microarray, which allows monitoring of the expression of a set of 120 potential marker genes for a variety of stress and toxicological processes and toxicologically relevant pathways. The OligoDesign program is freely accessible at http://lnatools.com/.

Transcriptional regulation of the Saccharomyces cerevisiae amino acid permease gene BAP2.

Abstract. Uptake of branched-chain amino acids by Saccharomyces cerevisiae from media containing a preferred nitrogen source is mediated by the permeases encoded by BAP2, BAP3, and VAP1/TAT1. The transcriptional activity of the BAP2 promoter is affected by a number of genes, including SSY1, which encodes an amino acid permease homologue that is necessary for transcription of BAP2. Other genes that control BAP2 encode known (Leu3p, Tup1p) and putative (Stp1p, Stp2p) transcription factors. We present evidence that the zinc-finger proteins Stp1p and Stp2p bind directly to the BAP2 promoter. Binding of Stp1p to the BAP2 promoter in vivo and in vitro indicates that the STP gene family indeed encodes transcription factors. The presence of a Leu3p binding site in the BAP2 promoter is required for full promoter activity on synthetic complete medium. The capacity of Leu3p to activate BAP2 transcription correlates with conditions that affect the level of α-isopropyl malate. The effect of a tup1 deletion on BAP2 transcription depends on SSY1. In an ssy1 strain, the phenotype of tup1 conforms to the well-established role of Tup1p as part of a repressor complex, but in the SSY1 strain deletion of TUP1 causes a decrease in transcription, indicating that Tup1p may also have an activating role at the BAP2 promoter. Our results thus suggest a complex interplay between several transcription factors in the expression of BAP2.

The ProbeLibrary™ - Expression profiling 99% of all human genes using only 90 dual-labeled real-time PCR Probes

While quantitative real-time RT-PCR is in principle a simple technique, the assay design remains fairly complex and designed assays often perform inadequately with respect to specificity, sensitivity, and reproducibility (1,2). The time spent on assay design, optimization, and validation often becomes a bottleneck in the implementation of new assays for large-scale expression profiling. Commercially available pre-validated real-time RT-PCR assays simplify the assay development process, but the time of delivery sometimes causes delays in experimental progress. Furthermore, prevalidated probe-based assays lack flexibility, due to the fact that these assays target a specific site in a given transcript. Consequently, quantification of another transcript or splice variant requires a different assay. Here we describe a novel, highly flexible concept for quantitative real-time RT-PCR, based on the development of a ProbeLibraryTM of 90 prevalidated real-time PCR detection probes, and a new web-based assay design software, enabling fast and easy design of optimal real-time PCR assays for gene expression analysis. By combining individual ProbeLibraryTM probes and target-specific PCR primers selected using the Primer3 software (3), the Assay Design Center software is able to design more than 644,000 different assays in the human transcriptome or target 98% of all human transcripts (Figure 1, Table 1). PRINCIPLES OF THE TECHNOLOGY

Efficient Poly(A) + RNA Selection Using LNA Oligo(T) Capture

This chapter describes a method for the isolation of intact polyadenylated mRNA using LNA oligo(T) capture. The method enables efficient isolation of poly(A)(+) RNA directly from guanidinium thiocyanate (GuSCN)-containing cell or tissue extract by combining the design of biotinylated LNA oligo(T) capture probes with subsequent immobilization of the captured poly(A)(+) RNA onto streptavidin-coated magnetic particles. In contrast to DNA oligo-dT and polyT PNA based mRNA isolation techniques, the LNA oligo(T) capture method allows poly(A) selection in the presence of 4 M GuSCN cell lysis buffer, which is needed for efficient inactivation of endogenous RNases. In addition, LNA oligo(T) facilitates highly efficient poly(A)(+) isolation at elevated temperatures compared to standard oligo(dT) technology. The successful use of the LNA oligo(T) capture method in recovery of mRNA from human cells and the subsequent use of the mRNA in northern blotting analysis, RT-PCR and qRT-PCR are demonstrated.