Kadri Toome

Detection of NASBA amplified bacterial tmRNA molecules on SLICSel designed microarray probes

BackgroundWe present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity.ResultsWe developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU.ConclusionsThe described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.

Fluorescent labeling of NASBA amplified tmRNA molecules for microarray applications

BackgroundHere we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions.ResultsTwo different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt.ConclusionWe have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.

Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

BackgroundThe hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities.ResultsWe demonstrate that adding specific DNA helper oligonucleotides (chaperones) to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule.ConclusionsThe sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.

Identification of a peptide recognizing cerebrovascular changes in mouse models of Alzheimer’s disease

Cerebrovascular changes occur in Alzheimer’s disease (AD). Using in vivo phage display, we searched for molecular markers of the neurovascular unit, including endothelial cells and astrocytes, in mouse models of AD. We identified a cyclic peptide, CDAGRKQKC (DAG), that accumulates in the hippocampus of hAPP-J20 mice at different ages. Intravenously injected DAG peptide homes to neurovascular unit endothelial cells and to reactive astrocytes in mouse models of AD. We identified connective tissue growth factor (CTGF), a matricellular protein that is highly expressed in the brain of individuals with AD and in mouse models, as the target of the DAG peptide. We also showed that exogenously delivered DAG homes to the brain in mouse models of glioblastoma, traumatic brain injury, and Parkinson’s disease. DAG may potentially be used as a tool to enhance delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation.Cerebrovascular changes and astrogliosis occur in Alzheimer’s disease (AD). Using an in vivo phage display technique, the authors identified a peptide that upon systematic administration, can home to brain endothelial cells and astrocytes in mouse models of AD at the early stages of the disease.

Publisher Correction: Identification of a peptide recognizing cerebrovascular changes in mouse models of Alzheimer’s disease

The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 1. The HTML has now been updated to include a corrected version of the Supplementary Information.

Abstract 1343: P32-targeting TT1 peptide delivers nanoparticles to intracranial glioblastomas

Targeted delivery of cancer therapeutics using affinity ligands can dramatically improve antitumor efficacy. Over the years a number of homing peptides that upon systemic injection accumulate in solid tumors have been identified by in vivo peptide phage display. In a quest to find homing peptides optimally suited for drug delivery to high-grade gliomas, our laboratories are using advanced mouse models of glioblastoma (GBM) to systematically audit known tumor-homing peptides and to perform new in vivo screens using peptide phage libraries. P32 is a mitochondrial chaperone that is aberrantly expressed on the cell surface in activated malignant and stromal cells in tumors. P32 is a receptor for widely used LypP-1 peptide and for recently identified TT1 peptide. Here we show that iron oxide nanoworms (IONW) functionalized with linear TT1 peptide (CKRGARST) strongly home to intracranial GBMs grafted in immune deficient mice. IONW are paramagnetic nanoparticles that are PEGylated to extend blood half-life, and have, because of their elongated shape, more effective targeting properties than spherical nanoparticles. Five hours after intravenous injection of IONW (7.5 mg/kg), macroscopic fluorescence imaging demonstrated robust homing of TT1-IONW in GBMs of murine origin (WT GBM and VEGF KO GBM from the G. Berger lab) and in a patient-derived glioma model (P13 model from the Bjerkvig lab). Confocal microscopy confirmed the presence of TT1 but not control IONW in gliomas, with TT1-IONW signal showing partial overlap with blood- and lymphatic vessel markers (CD31 and LYVE-1) in WT GBM and P13 gliomas, whereas lower level of colocalization with these markers was detected for mouse GBM not expressing VEGF. In addition, moderate colocalization between TT1-IONW and the macrophage marker CD11b was detected in the P13 tumors. Detailed phenotyping and functional characterization of TT1-positive macrophages is ongoing. Our data suggest that TT1 peptide has potential applications as a glioma-targeting vehicle. Currently, we are evaluating TT1-targeted IONWs as a contrast agent for glioma MRI and as carriers for cytotoxic compounds. Citation Format: Pille Saalik, Hedi Hunt, Allan Tobi, Anne-Mari Anton Willmore, Kadri Toome, Shweta Sharma, Ramana Kotamraju, Gabriele Bergers, Rolf Bjerkvig, Erkki Ruoslahti, Tambet Teesalu, Tambet Teesalu. P32-targeting TT1 peptide delivers nanoparticles to intracranial glioblastomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1343.

Abstract 705: In vivo audition of tumor homing peptides using high-throughput sequencing and q-PCR

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In vivo peptide phage display has been widely used to explore tumor vascular heterogeneity and to develop targeting ligands for the delivery of anticancer drugs and imaging agents. Numerous tumor homing peptides with various targeting specificities have been identified. To select a suitable targeting ligand for a particular tumor from an existing peptide panel, it is crucial to perform unbiased quantitative assessment of in vivo peptide homing. Here we describe an approach for parallel evaluation of in vivo tumor homing of peptide-displaying phage clones. Mice bearing orthotopic xenografts of gliomas, breast tumors, and prostate tumors were intravenously injected with equimolar mix of phage clones displaying 12 different tumor homing or control peptides. After 30 min circulation and removal of blood by perfusion, representation of each phage clone in tumors and control organs was determined by high throughput sequencing (HTS) and quantitative PCR (qPCR). The ratiometric analysis demonstrated a clear and reproducible homing signature for each tumor model. Calibrated copy number data obtained by qPCR correlated well with the HTS analysis. Selected phage clones were validated for tumor homing by conventional single-phage homing studies using immunofluorescence, phage titration, and qPCR. Our study shows that the ratiometric in vivo phage biodistribution analysis can be used to rapidly identify peptides that efficiently deliver payloads to particular tumor types. As the peptides are evaluated side-by-side in a same animal, inter-animal variability is eliminated and the number of animals required is dramatically decreased. The choice of analysis (HTS vs qPCR) depends on the complexity of phage pool to be audited. HTS enables the analysis of highly complex phage pools, but introduces a potential bias owing to multiple DNA amplification steps. On the other hand, qPCR allows precise quantification of the copy number of the target sequences, and is suitable for low complexity pools. Narrowing down a complex pool with HTS followed by precise evaluation with qPCR can be a potential combination of the two analytical methods. Citation Format: Kadri Toome, Tarmo Molder, Kuldar Koiv, Pille Saalik, Kazuki N. Sugahara, Erkki Ruoslahti, Tambet Teesalu. In vivo audition of tumor homing peptides using high-throughput sequencing and q-PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 705. doi:10.1158/1538-7445.AM2014-705