The application of riboswitch sequencing for human gut bacterial classification and identification.

Bacteria, especially gut bacteria play important roles in human health and diseases. The classification of many bacterial genera by the 16S ribosomal RNA (rRNA) has failed due to its low inter-species resolution. Given the wide distribution of riboswitches in bacteria, they may help 16S rRNA differentiate closely related species. We found that among 28 groups of species that could not be distinguished by 16S rRNA, eight of them could be separated by the TPP riboswitch and other riboswitches. Moreover, the species in the 16S rRNA database and these riboswitch databases overlap, therefore, using riboswitch databases can help 16S rRNA better identify species. In addition, we used Klenow DNA polymerase and a pair of short primers to facilitate the library construction of TPP riboswitches for sequencing. The sequencing results showed that the TPP riboswitch could detect the major phyla similar to those detected by 16S rRNA. Therefore, the TPP riboswitch and other riboswitch classes could potentially be applied to gut bacteria classification.

Identification of 11 candidate structured noncoding RNA motifs in humans by comparative genomics.

BACKGROUND: Only 1.5% of the human genome encodes proteins, while large part of the remaining encodes noncoding RNAs (ncRNA). Many ncRNAs form structures and perform many important functions. Accurately identifying structured ncRNAs in the human genome and discovering their biological functions remain a major challenge. RESULTS: Here, we have established a pipeline (CM-line) with the following features for analyzing the large genomes of humans and other animals. First, we selected species with larger genetic distances to facilitate the discovery of covariations and compatible mutations. Second, we used CMfinder, which can generate useful alignments even with low sequence conservation. Third, we removed repetitive sequences and known structured ncRNAs to reduce the workload of CMfinder. Fourth, we used Infernal to find more representatives and refine the structure. We reported 11 classes of structured ncRNA candidates with significant covariations in humans. Functional analysis showed that these ncRNAs may have variable functions. Some may regulate circadian clock genes through poly (A) signals (PAS); some may regulate the elongation factor (EEF1A) and the T-cell receptor signaling pathway by cooperating with RNA binding proteins. CONCLUSIONS: By searching for important features of RNA structure from large genomes, the CM-line has revealed the existence of a variety of novel structured ncRNAs. Functional analysis suggests that some newly discovered ncRNA motifs may have biological functions. The pipeline we have established for the discovery of structured ncRNAs and the identification of their functions can also be applied to analyze other large genomes.

Integration of an Expression Platform in the SELEX Cycle to Select DNA Aptamer Binding to a Disease Biomarker.

Aptamers can be developed for biosensors, diagnostic tools, and therapeutic reagents. These applications usually require a fusion of aptamers and expression platforms. However, the fusion process is usually time-consuming and laborious. In this study, we integrated the deoxyribozyme (I-R3) as an expression platform in the SELEX cycle (called Expression-SELEX) to select aptazymes that can sense diverse molecules. We used the Maple syrup urine disease (MSUD) biomarker L-allo-isoleucine to test the selection model. After five rounds of screening, the cleavage products were sufficiently enriched to be visualized on polyacrylamide gel electrophoresis (PAGE) gel. Through high-throughput sequencing analysis, several candidates were identified. One such candidate, IR3-I-DNA, binds L-allo-isoleucine with a dissociation constant (K D) of 0.57 mM. When the ligand was present, the cleavage fraction of IR3-I-DNA increased from 0.3 to 0.5, and its K obs value improved from 1.38 min-1 to 1.97 min-1. Our selection approach can also be applied to produce aptazymes that can bind to variable ligands and be used more directly as biosensors.