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.

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.

Aptamers are ligand-binding RNA or DNA molecules and have been widely examined as biosensors, diagnostic tools, and therapeutic agents. The application of aptamers as biosensors commonly requires an expression platform to produce a signal to report the aptamer-ligand binding event. Traditionally, aptamer selection and expression platform integration are two independent steps and the aptamer selection requires the immobilization of either the aptamer or the ligand. These drawbacks can be easily overcome through the selection of allosteric DNAzymes (aptazymes). Herein, we used the technique of Expression-SELEX developed in our laboratory to select for aptazymes that can be specifically activated by low concentrations of l-phenylalanine. We chose a previous DNA-cleaving DNAzyme known as II-R1 as the expression platform for its low cleavage rate and used stringent selection conditions to drive the selection of high-performance aptazyme candidates. Three aptazymes were chosen for detailed characterization and these DNAzymes were found to exhibit a dissociation constant for l-phenylalanine as low as 4.8 μM, a catalytic rate constant improvement as high as 20 000-fold in the presence of l-phenylalanine, and the ability to discriminate against closely related l-phenylalanine analogs including d-phenylalanine. This work has established the Expression-SELEX as an effective SELEX method to enrich high-quality ligand-responsive aptazymes.

Background Only 1.5% of the human genome encodes proteins, while most 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 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 provides evidence for the potential biological functions of some newly found ncRNA motifs. 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.