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Purpose Formalin-fixed, paraffin-embedded (FFPE) tumor tissue, while standard for biological preservation, can significantly impact nucleic-acid quality via degradation and cross-linking. The low quality of FFPE-treated samples, coupled with the presence of native small molecules inhibitors in various tissue types, yields significant challenges for PCR-based technologies. Alternatively, NanoString RNA assays combine straightforward workflows with multiplexed detection of up to 800 targets via hybridization-based chemistry that is not reliant on enzymatic processes, and thus may offer advantages in a melanin-rich environment. For utilization in a melanoma disease setting, we evaluated the impact of different melanin-levels in FFPE samples on the performance of NanoString mRNA and miRNA assays Methods One FFPE cell line block was sectioned and spiked with zero or a range of biologically-relevant levels of melanin, both before and after total nucleic acid isolation with the Qiagen AllPrep FFPE procedure. Total RNA content was measured by NanoDrop and fluorescent-based methods. Abundance of individual RNA molecules was assessed by the NanoString IO360 gene expression assay and the NanoString Human v3 miRNA panel. Results Melanin concentration was observed to directly impact quantification results, with higher concentrations correlating to elevated estimates of total RNA yield by NanoDrop, but reduced RNA by fluorescent-based measurement. Different melanin concentrations did not impact RNA expression measurements in the enzyme-free IO360 gene expression assay. For the v3 miRNA assay, however, increased melanin concentration resulted in significant reduction in miRNA signal, but as observed with the IO360 assay, did not affect endogenous mRNA transcript detection. The reduction in miRNA levels was significantly greater for the samples spiked with melanin post-extraction compared with those aliquots spiked prior to isolation. Tumor inflammation signature (TIS) scoring was not impacted by elevated melanin levels. Conclusions NanoString hybridization reactions exhibited minimal sensitivity to melanin content. Conversely, NanoString assays requiring enzymatic processing yielded substantially reduced counts at elevated melanin concentrations. Isolation procedures were effective in removing lower levels of melanin, but could not fully mitigate melanin impact at all tested concentrations. Significantly, classification of tumor immune-environment by NanoString was not affected by melanin, potentially making TIS scoring an attractive tool for biomarker analysis of checkpoint inhibitor therapies in melanoma. Citation Format: Alexis Kurmis, Eileen Kelly, Nancy Valencia, Justin Wahl, Christian Laing, Nathan Riccitelli, Reinhold Pollner. Effects of melanin on NanoString gene and miRNA expression assays for utilization in biomarker analysis for melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1568.

Cluster BE1 Streptomyces bacteriophages belong to the Siphoviridae , with genome sizes over 130 kbp, and they contain direct terminal repeats of approximately 11 kbp. Eight newly isolated closely related cluster BE1 phages contain 43 to 48 tRNAs, one transfer-messenger RNA (tmRNA), and 216 to 236 predicted open reading frames (ORFs), but few of their genes are shared with other phages, including those infecting Streptomyces species.

Introduction The presence of MRD in patients with AML who are in morphologic remission has been shown to be a powerful predictor of eventual relapse. FMS-like tyrosine kinase 3 (FLT3) internal tandem duplications (ITD) confer a negative prognostic impact by increasing risk of relapse. The ability to detect FLT3-ITD mutations in remission bone marrow specimens is hampered by the limited sensitivity at 1% of PCR-based assays. To address such limitations, we developed a novel NGS-based MRD assay for the detection of FLT3-ITD mutations. Method Genomic DNA was isolated from bone marrow (BM) aspirates or peripheral blood (PB) samples. PCR was performed to amplify exons 13 to 15 of the FLT3 gene. Highly diverse NGS libraries were then generated and sequenced using Illumina’s sequencer. Using a custom bioinformatics approach, unique FLT3-ITD mutations of varying lengths were identified and mutant allelic frequency calculated. The assay was validated using clinical samples and contrived samples. For accuracy assessment, 30 samples (including PB or BM remnant patient DNA and cell line DNA, above DNA diluted in normal DNA) were included. Data were compared with a Fragment Size Analysis by Capillary Electrophoresis assay. To assess precision, the assay was validated at multiple levels evaluating intra-assay, inter-assay, inter-operator, inter-instrument and inter-reagent lot precision. DNA samples from selected mutant cell lines representing different FLT3-ITD lengths were spiked into normal DNA to evaluate assay sensitivity and linearity. Summary The ideal DNA input range was established as 300 ng to 500 ng. In all FLT3-ITD-positive cell line samples covering diverse FLT3-ITD lengths (6 bp to 156 bp), the FLT3-ITD MRD NGS assay showed 100% concordance with the reference assay. The assay is also capable of detecting ITDs as expected after the samples were diluted in normal DNA to mimic samples from patients in remission. All acceptance criteria for the different precision parameters were met. The lower limit of detection of 0.013% regardless of ITD length was established but the assay was capable of detecting FLT3-ITD mutations at a level as low as 0.003% without false-positive results. The assay was linear (R2 = 0.958) down to FLT3-ITD allele frequency levels of 0.035% or the lower limit of quantitation. Conclusion Analytical validation results established the role of this NGS-based MRD assay for the clinical management of FLT3-ITD AML. The FLT3-ITD MRD NGS assay demonstrated high sensitivity and high specificity by detecting the unique length of each patient’s mutation with no false-positive results in expected negative samples. This assay might be helpful in defining the depth of remission, identifying persistent disease, and helping to guide decision making in the use of FLT3 inhibitors as continuation therapy. Citation Format: Wenge Shi, Christian Laing, Wei Ding, Marc Mycoco, Jelveh Lameh, Reinhold Pollner. Development of a novel next-generation sequencing (NGS)-based assay for measurable residual disease (MRD) in FLT3-ITD acute myeloid leukemia (AML) and its potential clinical application in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2299.

Motif finding in DNA, RNA and proteins plays an important role in life science research. Recent patents concerning motif finding in biomolecular data are recorded in the DNA Patent Database which serves as a resource for policy makers and members of the general public interested in fields like genomics, genetics and biotechnology. In this paper, we present a computational approach to mining for RNA tertiary motifs in genomic sequences. Specifically, we describe a method, named CSminer, and show, as a case study, the application of CSminer to genome-wide search for coaxial helical stackings in RNA 3-way junctions. A coaxial helical stacking occurs in an RNA 3-way junction where two separate helical elements form a pseudocontiguous helix and provide thermodynamic stability to the RNA molecule as a whole. Experimental results demonstrate the effectiveness of our approach.

RNA junctions are secondary structure elements formed when three or more helices come together. They are present in diverse RNA molecules with various fundamental functions in the cell. To better understand the intricate architecture of three-dimensional RNAs, we analyze currently solved 3D RNA junctions in terms of basepair interactions and three-dimensional configurations. First, we study basepair interaction diagrams for solved RNA junctions with five to ten helices and discuss common features. Second, we compare these higher-order junctions to those containing three or four helices and identify global motif patterns such as coaxial-stacking and parallel and perpendicular helical configurations. These analyses show that higher order junctions organize their helical components in parallel and helical configurations similar to lower order junctions. Their sub-junctions also resemble local helical configurations found in three and four-way junctions, and are stabilized by similar long-range interaction preferences such as A-minor interactions. Furthermore, loop regions within junctions are high in adenine but low in cytosine. And, in agreement with previous studies, we suggest that coaxial stacking between helices likely forms when the common single stranded loop is small in size; however, other factors such as stacking interactions involving non-canonical basepairs and proteins can greatly determine or disrupt coaxial stacking. Finally, we introduce the ribo-base interactions: when combined with the along-groove packing motif, these ribo-base interactions form novel motifs involved in perpendicular helix-helix interactions. Overall, these analyses suggest recurrent tertiary motifs that stabilize junction architecture, pack helices, and help form helical configurations that occur as sub-elements of larger junction networks. The frequent occurrence of similar helical motifs suggest Nature’s finite and perhaps limited repertoire of RNA helical conformation preferences. More generally, studies of RNA junctions and tertiary building blocks can ultimately help in the difficult task of RNA 3D structure prediction.

RNA junctions are important structural elements of RNA molecules. They are formed when three or more helices come together in three-dimensional space. Recent studies have focused on the annotation and prediction of coaxial helical stacking (CHS) motifs within junctions. Here we exploit such predictions to develop an efficient alignment tool to handle RNA secondary structures with CHS motifs. Specifically, we build upon our Junction-Explorer software for predicting coaxial stacking and RNAJAG for modelling junction topologies as tree graphs to incorporate constrained tree matching and dynamic programming algorithms into a new method, called CHSalign, for aligning the secondary structures of RNA molecules containing CHS motifs. Thus, CHSalign is intended to be an efficient alignment tool for RNAs containing similar junctions. Experimental results based on thousands of alignments demonstrate that CHSalign can align two RNA secondary structures containing CHS motifs more accurately than other RNA secondary structure alignment tools. CHSalign yields a high score when aligning two RNA secondary structures with similar CHS motifs or helical arrangement patterns, and a low score otherwise. This new method has been implemented in a web server, and the program is also made freely available, at http://bioinformatics.njit.edu/CHSalign/., Comment: 45 pages, 5 figures

RNA molecules are important cellular components involved in many fundamental biological processes. Understanding the mechanisms behind their functions requires RNA tertiary structure knowledge. Although modeling approaches for the study of RNA structures and dynamics lag behind efforts in protein folding, much progress has been achieved in the past two years. Here, we review recent advances in RNA folding algorithms, RNA tertiary motif discovery, applications of graph theory approaches to RNA structure and function, and in silico generation of RNA sequence pools for aptamer design. Advances within each area can be combined to impact many problems in RNA structure and function.