Niels Tolstrup

NetOglyc: Prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility

The specificities of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases which link the carbohydrate GalNAc to the side-chain of certain serine and threonine residues in mucin type glycoproteins, are presently unknown. The specificity seems to be modulated by sequence context, secondary structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. Charged residues were disfavoured at position – 1 and +3. A jury of artificial neural networks was trained to recognize the sequence context and surface accessibility of 299 known and verified mucin type O-glycosylation sites extracted from O-GLYCBASE. The cross-validated NetOglyc network system correctly found 83% of the glycosylated and 90% of the non-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predictions of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based on the amino acid sequence. The server addresses are http://www.cbs.dtu.dk/services/NetOGlyc/ and netOglyc@cbs.dtu.dk.

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/.

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

Abstract 3987: Exosomal microRNA in cell-free urine samples as a source for liquid prostate cancer biopsy

MicroRNAs constitute a class of small cellular RNAs (typically 19-23 nt) that function as post-transcriptional regulators of gene expression. Current estimates indicate that more than one third of the human cellular transcriptome is regulated by this small class of RNA (∼2000 miRNA). MicroRNAs have been shown to be actively exported from tissues into the circulation through a variety of mechanisms including complexing with RNA binding proteins or HDL and through active exosome transport. Exosomes are nanovesicles secreted into the extracellular environment by a wide range of cell types under normal and pathological conditions. As the profile of exosomal microRNAs may be a fingerprint of the releasing cell type and because they are released in easily accessible body fluids such as blood and urine, their microRNA content holds potential as biomarkers for early detection of malignancy. Cell free urine samples are an obvious liquid biopsy source for microRNA markers in prostate cancer and could serve as diagnostic tools as well as treatment response markers. Yet microRNA levels in urine samples are extremely sparse and cannot be detected reliable by conventional sample preparation methods. It has been reported that tumour derived exosomes carrying genetic information specific for prostate cancer can be measured in urine samples following prostate massage. However, a methodology eliminating the need for prostate massage of every patient to be tested in a clinical setting is highly desirable. The purpose of this study was to combine a simple exosomal enrichment method with our highly sensitive LNA™-based qPCR platform for detection of microRNAs and apply this to a cohort consisting of more than 300 cell free urine samples from prostate cancer cases and controls. In conclusion, cell free urine samples holds potential as a liquid biopsy source for exosomal microRNA markers in prostate cancer, showing miRNA signatures of strong diagnostic potential. Analysis is pending and data will be presented. Citation Format: Thorarinn Blondal, Anni R. Thomsen, Jorg Krummheuer, Michael Borre, Jacob Fredsoe, Christa Haldrup, Ditte Andreasen, Maria W. Teilum, Niels Tolstrup, Karina D. Sorensen, Torben F. Orntoft, Peter Mouritzen. Exosomal microRNA in cell-free urine samples as a source for liquid prostate cancer biopsy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3987. doi:10.1158/1538-7445.AM2015-3987

Abstract PR14: Potent knock down of lncRNAs in vitro and in vivo with antisense LNA™ GapmeRs

Exiqon develops strategies for the optimal design of single stranded LNA™-enhanced antisense oligonucleotides (ASOs, also known as LNA™ GapmeRs) that catalyze RNaseH dependent degradation of target mRNAs and long non-coding RNAs (lncRNA). We have developed an empirically derived design algorithm to provide ASOs that achieve potent target knockdown with a high hit-rate. We describe the latest improvements to the design algorithm, including the incorporation of alignments to both spliced and unspliced transcriptomes in the Ensembl database, to provide maximal target specificity. Many lncRNAs are nuclear retained or have long residence time in the nucleus. This makes them hard to target by RNAi based methods. However since RNaseH is almost exclusively present in the nucleus nuclear RNAs are expected to be particularly sensitive LNA™ GapmeR targets. We have tested LNA™ GapmeRs to knockdown multiple different RNA targets in vitro – including mRNA and lncRNA targets residing in either cytoplasmic or nuclear compartments. Our results demonstrate that all of these targets were equally efficiently silenced irrespective of the type of RNA target and its subcellular localization. We also report highly efficient and long lasting knockdown of a nuclear retained lncRNA in a broad range of tissues in mice subjected to systemic adminstration of a LNA™ GapmeR. LNA GapmeRs are therefore excellent tools for lncRNA loss of function analysis in vitro and in vivo and provide an excellent platform for therapeutic targeting of lncRNA. Risk of hybridization based off-target activity with gapmers has traditionally been considered to be low. However in a recent publication1 it was shown that unless properly designed antisense off-target activity with gapmers can be quite significant. Surprisingly, particularly sensitive off-targets were frequently localized in introns of primary transcripts causing significant reduction of the amount of mature spliced mRNAs. Since RNaseH is confined to the nucleus we therefore speculated that the true target of gapmers might be unspliced primary transcripts in the nucleus rather than the spliced mature transcripts that are exported to the cytoplasm. We present data showing that intron targeting LNA GapmeRs often provide highly potent knockdown of the mature spliced RNA. These results have profound implications on therapeutic gapmer design. 1. Kamola PJ, Nucl. Acids Res. 2015, pii: gkv857 This abstract is also presented as Poster B45. Citation Format: Johnathan Lai, Asli Ozen, Peter Mouritzen, Niels Tolstrup, Niels Montano Frandsen. Potent knock down of lncRNAs in vitro and in vivo with antisense LNA™ GapmeRs. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR14.

Abstract B40: A two-microRNA signature in urinary exosomes for diagnosis of prostate cancer

Antibody based detection of circulating prostate specific antigen (PSA) remains an important test in the diagnosis of prostate cancer. Unfortunately the test identifies a high number of false positives which require further testing to confirm the presence of cancer including several biopsies taken from the prostate itself. Whereas the performance of biopsies on individuals is associated with discomfort and risk of serious side effects, the invasive test also represents an expense to health care systems. Given the high number of false positives, the PSA test is therefore the direct cause of unnecessary suffering of healthy individuals and costs to health systems. To investigate the possibilities for developing a non-invasive and more specific test, we have developed different technologies enabling the detection of prostate cancer microRNA biomarkers in exosomes derived from urine from non-prostate massaged men. The first of these technologies is a highly sensitive LNA™-based qPCR platform for microRNA detection, which enables profiling in biofluids where microRNA levels are extremely low. At this point we have already applied this platform on thousands of biofluid samples including serum/plasma and urine to establish normal reference ranges for circulating microRNAs as well as to identify biomarkers of disease. The second technology is a simple exosome precipitation system which only requires low speed centrifugation to harvest urine exosomes. We have combined our developed technologies and applied this on cell-free urine from two different cohorts of approximately 220 patients each, to identify a number of differentially regulated microRNAs in urine from prostate cancer bearing individuals. Prostate cancer specific signatures have been obtained by different combinations of these differentially regulated microRNAs. The smallest signature is composed of two microRNAs which allows constructing receiver operating characteristic curves with areas under the curve well above 0.8. Citation Format: Peter Mouritzen, Jacob Christian Fredsoe, Thorarinn Blondal, Anne Karin Rasmussen, Michael Borre, Christa Haldrup, Ditte Andreasen, Niels Tolstrup, Torben Falck Orntoft, Karina Dalsgaard Sorensen. A two-microRNA signature in urinary exosomes for diagnosis of prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B40.

MicroRNA in biofluids—Robust biomarkers for disease, toxicology, or injury studies: The case of minimally invasive colorectal cancer detection.

20 Background: MicroRNAs function as post-transcriptional regulators of gene expression. Their high relative stability in common clinical source materials (FFPE blocks, plasma, serum, urine, saliva, etc.) and the ability of microRNA expression profiles to accurately classify discrete tissue types and specific disease states have positioned microRNAs as promising new biomarkers for diagnostic application. Furthermore microRNAs have been shown to be rapidly released from tissues into the circulation with the development of pathology. METHODS Thousands of biofluid samples were profiled including blood derived plasma/serum and urine using a genome-wide LNA-based microRNA qPCR platform, which has unparalleled sensitivity and robustness even in biofluids with extremely low microRNA levels. Only a single RT reaction is required to conduct full miRNome profiling thereby facilitating high-throughput profiling without the need for pre-amplification. RESULTS Normal reference ranges for circulating microRNAs were determined in several biofluids, allowing development of qPCR arrays containing only relevant microRNA subsets present in various biofluids together with tissue specific microRNA markers. Procedures were developed to control pre-analytical variables, for quality checking and qualifying biofluid samples in particular serum and plasma but also urine and other biofluids. An extensive QC system was implemented in order to secure technical excellence and reveal any unwanted bias in the dataset. We currently screen and validate microRNAs biomarkers for cancer with the aim of developing minimal invasive tests to be applied in early detection population screens. CONCLUSIONS The qPCR panels support development of robust biomarkers in disease, toxicology, and injury studies. We will demonstrate how panels may be quickly and robustly applied in biomarker discovery/validation projects using the specific case early detection of colorectal cancer in blood. Close attention is required on pre-analytical parameters. Hemolysis and cellular contamination affect miRNA profiles in biofluids and control is required.