Lingyan Huang

Noninvasive imaging of Staphylococcus aureus infections with a nuclease-activated probe

Technologies that enable the rapid detection and localization of bacterial infections in living animals could address an unmet need for infectious disease diagnostics. We describe a molecular imaging approach for the specific, noninvasive detection of S. aureus based on the activity of the S. aureus secreted nuclease, micrococcal nuclease (MN). Several short synthetic oligonucleotides, rendered resistant to mammalian serum nucleases by various chemical modifications and flanked with a fluorophore and quencher, were activated upon degradation by purified MN and in S. aureus culture supernatants. A probe consisting of a pair of deoxythymidines flanked by several 2′-O-methyl–modified nucleotides was activated in culture supernatants of S. aureus but not in culture supernatants of several other pathogenic bacteria. Systemic administration of this probe to mice bearing S. aureus muscle infections resulted in probe activation at the infection sites in an MN-dependent manner. This new bacterial imaging approach has potential clinical applicability for infections with S. aureus and several other medically important pathogens.

Small RNA regulation of ovule development in the cotton plant, G. hirsutum L

BackgroundThe involvement of small RNAs in cotton fiber development is under explored. The objective of this work was to directly clone, annotate, and analyze small RNAs of developing ovules to reveal the candidate small interfering RNA/microRNAs involved in cotton ovule and fiber development.ResultsWe cloned small RNA sequences from 0–10 days post anthesis (DPA) developing cotton ovules. A total of 6691 individual colonies were sequenced from 11 ovule small RNA libraries that yielded 2482 candidate small RNAs with a total of 583 unique sequence signatures. The majority (362, 62.1%) of these 583 sequences were 24 nt long with an additional 145 sequences (24.9%) in the 21 nt to 23 nt size range. Among all small RNA sequence signatures only three mirBase-confirmed plant microRNAs (miR172, miR390 and ath-miR853-like) were identified and only two miRNA-containing clones were recovered beyond 4 DPA. Further, among all of the small RNA sequences obtained from the small RNA pools in developing ovules, only 15 groups of sequences were observed in more than one DPA period. Of these, only five were present in more than two DPA periods. Two of these were miR-172 and miR-390 and a third was identified as 5.8S rRNA sequence. Thus, the vast majority of sequence signatures were expressed in only one DPA period and this included nearly all of the 24 nt sequences. Finally, we observed a distinct DPA-specific expression pattern among our clones based upon sequence abundance. Sequences occurring only once were far more likely to be seen in the 0 to 2 DPA periods while those occurring five or more times were the majority in later periods.ConclusionThis initial survey of small RNA sequences present in developing ovules in cotton indicates that fiber development is under complex small RNA regulation. Taken together, the results of this initial small RNA screen of developing cotton ovules is most consistent with a model, proposed by Baulcombe, that there are networks of small RNAs that are induced in a cascade fashion by the action of miRNAs and that the nature of these cascades can change from tissue to tissue and developmental stage to developmental stage.

Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)

The “RNA revolution” that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding of RNA-dependent gene regulation. New generation high-throughput sequencing technologies further accelerated the studies of this “tiny world” and provided their global characterization and validation in many biological systems with sequenced genomes. Nevertheless, for the many “yet-unsequenced” plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. In this paper, we present a description of existing small RNA cloning methods as well as next-generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small RNA survey in the “still unsequenced” allotetraploid cotton genome.

piRNA-like RNAs in the marsupial Monodelphis domestica identify transcription clusters and likely marsupial transposon targets

PIWI-interacting RNAs (piRNAs) are a recently discovered class of small noncoding RNAs that have been detected in human, mouse, rat, zebrafish, and Drosophila genomes. We have utilized a size-directed small-RNA cloning procedure to clone and map more than 300 candidate piRNA-like small RNAs in the genome of the marsupial species Monodelphis domestica. Our results are consistent with those from other species in that the piRNA-like candidate sequences range in size from 28 to 31 nucleotides, show a pronounced preference for uridine at the 5′ end, are transcribed from a few large clusters, appear to target transposons, and display virtually no sequence conservation.

SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2′OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.

Imaging the Directed Transport of Single Engineered RNA Transcripts in Real-Time Using Ratiometric Bimolecular Beacons

The relationship between RNA expression and cell function can often be difficult to decipher due to the presence of both temporal and sub-cellular processing of RNA. These intricacies of RNA regulation can often be overlooked when only acquiring global measurements of RNA expression. This has led to development of several tools that allow for the real-time imaging of individual engineered RNA transcripts in living cells. Here, we describe a new technique that utilizes an oligonucleotide-based probe, ratiometric bimolecular beacon (RBMB), to image RNA transcripts that were engineered to contain 96-tandem repeats of the RBMB target sequence in the 3′-untranslated region. Binding of RBMBs to the target RNA resulted in discrete bright fluorescent spots, representing individual transcripts, that could be imaged in real-time. Since RBMBs are a synthetic probe, the use of photostable, bright, and red-shifted fluorophores led to a high signal-to-background. RNA motion was readily characterized by both mean squared displacement and moment scaling spectrum analyses. These analyses revealed clear examples of directed, Brownian, and subdiffusive movements.

An X chromosome MicroRNA Cluster in the Marsupial Species Monodelphis domestica

MicroRNAs (miRNAs) are an important class of posttranscriptional gene expression regulators. In the course of mapping novel marsupial-specific miRNAs in the genome of the gray short-tailed opossum, Monodelphis domestica, we encountered a cluster of 39 actual and potential miRNAs spanning 102 kb of the X chromosome. Analysis of the cluster revealed that 37 of the 39 miRNAs are predicted to form thermodynamically stable hairpins, and at least 3 members have been directly cloned from M. domestica tissues. The sequence characteristics of these miRNAs suggest that they all descended from a single common ancestor. Further, 2 distinct families appear to have diversified from the ancestral sequence through different duplication mechanisms: one through a series of simple tandem duplications and the other through a recurrent transposon-mediated duplication process.

Correction: SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

Figures 4 and 5 were incorrectly switched. The image currently appearing as Figure 4 belongs with the title and legend of Figure 5, and the image currently appearing as Figure 5 belongs with the title and legend of Figure 4. The titles and legends themselves are in correct order. In addition, there was an error in Figure 3. Please see the correct Figure 3 here: