Organism-Specific rRNA Capture System for Application in Next-Generation Sequencing

<div><p>RNA-sequencing is a powerful tool in studying RNomics. However, the highly abundance of ribosomal RNAs (rRNA) and transfer RNA (tRNA) have predominated in the sequencing reads, thereby hindering the study of lowly expressed genes. Therefore, rRNA depletion prior to sequencing is often performed in order to preserve the subtle alteration in gene expression especially those at relatively low expression levels. One of the commercially available methods is to use DNA or RNA probes to hybridize to the target RNAs. However, there is always a concern with the non-specific binding and unintended removal of messenger RNA (mRNA) when the same set of probes is applied to different organisms. The degree of such unintended mRNA removal varies among organisms due to organism-specific genomic variation. We developed a computer-based method to design probes to deplete rRNA in an organism-specific manner. Based on the computation results, biotinylated-RNA-probes were produced by <i>in vitro</i> transcription and were used to perform rRNA depletion with subtractive hybridization. We demonstrated that the designed probes of 16S rRNAs and 23S rRNAs can efficiently remove rRNAs from <i>Mycobacterium smegmatis</i>. In comparison with a commercial subtractive hybridization-based rRNA removal kit, using organism-specific probes is better in preserving the RNA integrity and abundance. We believe the computer-based design approach can be used as a generic method in preparing RNA of any organisms for next-generation sequencing, particularly for the transcriptome analysis of microbes.</p> </div

Workflow illustrating the procedures for Organism-Specific Probe Selection and rRNA depletion.

<p><b>A</b>. Workflow of Organism-Specific Probe Selection (OSPS) program. OSPS was used to screen for unique sequences of 16S rRNAs and 23S rRNAs that have no significant similarity to other transcripts in the same organism. <b>B</b>. Overall procedures of rRNA depletion. Sequences for probes were amplified and cloned into an in-house pT1 system, and the RNA probes were which by <i>in </i><i>vitro</i> transcribed with biotinylated UTP and tested for rRNA depletion efficiency. The best probes were selected and combined for further rRNA depletion.</p

Integrity of mRNA after rRNA depletion was determined by real-time PCR.

<p>The expression level of rRNA and 14 genes of different abundance were measured before and after rRNA depletion using MSMEG_5072 gene as a housekeeping control. Abundance of selected genes and the gene ID were shown. <b>A</b>. Relative fold change of selected mRNA and rRNA after depletion using MICROB<i>Express</i>™ Bacterial mRNA Enrichment. <b>B</b>. Fold change of selected mRNA and rRNA after depletion using probes designed by OSPS.</p

Comparative Studies of the Cellular Uptake, Subcellular Localization, and Cytotoxic and Phototoxic Antitumor Properties of Ruthenium(II)–Porphyrin Conjugates with Different Linkers

Six water-soluble free-base porphyrin-Ru­(II) conjugates, <b>1</b>–<b>3</b>, and Zn­(II) porphyrin-Ru­(II) conjugates, <b>4</b>–<b>6</b>, with different linkers between the hydrophobic porphyrin moiety and the hydrophilic Ru­(II)-polypyridyl complex, have been synthesized. The linear and two-photon-induced photophysical properties of these conjugates were measured and evaluated for their potential application as dual in vitro imaging and photodynamic therapeutic (PDT) agents. Conjugates <b>1</b>–<b>3</b>, with their high luminescence and singlet oxygen quantum yields, were selected for further study of their cellular uptake, subcellular localization, and cytotoxic and photocytotoxic (under linear and two-photon excitation) properties using HeLa cells. Conjugate <b>2</b>, with its hydrophobic phenylethynyl linker, was shown to be highly promising for further development as a bifunctional probe for two-photon (NIR) induced PDT and in vitro imaging. Cellular uptake and subcellular localization properties were shown to be crucial to its PDT efficacy