Identification and verification of seed development related miRNAs in kernel almond by small RNA sequencing and qPCR.

Many studies have investigated the role of miRNAs on the yield of various plants, but so far, no report is available on the identification and role of miRNAs in fruit and seed development of almonds. In this study, preliminary analysis by high-throughput sequencing of short RNAs of kernels from the crosses between almond cultivars 'Sefid' × 'Mamaee' (with small and large kernels, respectively) and 'Sefid' × 'P. orientalis' (with small kernels) showed that the expressions of several miRNAs such as Pdu-miR395a-3p, Pdu-miR8123-5p, Pdu-miR482f, Pdu-miR6285, and Pdu-miR396a were significantly different. These miRNAs targeted genes encoding different proteins such as NYFB-3, SPX1, PGSIP3 (GUX2), GH3.9, and BEN1. The result of RT-qPCR revealed that the expression of these genes showed significant differences between the crosses and developmental stages of the seeds, suggesting that these genes might be involved in controlling kernel size because the presence of these miRNAs had a negative effect on their target genes. Pollen source can influence kernel size by affecting hormonal signaling and metabolic pathways through related miRNAs, a phenomenon known as xenia.

Identification of herbivore-elicited long non-coding RNAs in rice.

Long non-coding RNAs (lncRNAs) in plants are emerging as new players in biotic stress responses. Pathogen-associated lncRNAs have been broadly identified and functionally characterized in multiple species. However, herbivore-responsive lncRNAs in plants are poorly investigated. Our recent study revealed that lncRNAs also play roles in plant defense against herbivores in wild tobacco. Here, we identified armyworm (AW)-elicited lncRNAs in monocot rice by employing a similar approach. A total of 238 lncRNAs were found to be differentially expressed (DE) in AW-treated plants relative to control plants. The cis effect of these DE lncRNAs was predicted. Interestingly, one DE lncRNA was identified from the antisense transcripts of the jasmonate ZIM-domain gene JAZ10.

Single Cell Type Specific RNA Isolation and Gene Expression Analysis in Rice Using Laser Capture Microdissection (LCM)-Based Method.

The success of single cell type-specific gene expression or functional study largely depends on the efficient isolation of high-quality RNA from them. Laser capture microdissection (LCM) is an efficient technique that allows accessing and dissecting out a specific individual cell or cell type from a microscopic heterogeneous tissue in a minimally disruptive way. Here, we describe an efficient and inexpensive LCM-based method for the extraction of RNAs with high yield and integrity from laser-microdissected mesophyll and bundle sheath cells of rice leaf. The integrity of isolated RNA is assessed with bioanalyzer analysis, and the presence of mRNA of a specific gene is validated through RT-PCR. This RNA could further be used for uncovering single cell type-specific gene expression signature using next-generation transcriptome sequence or through regular RT-PCR.

Integrated transcriptome, small RNA and degradome sequencing approaches proffer insights into chlorogenic acid biosynthesis in leafy sweet potato.

Leafy sweet potato is rich in total phenolics (TP) which play key roles in health protection, the chlorogenic acid (CGA) constitutes the major components of phenolic compounds in leafy sweet potato. Unfortunately, the mechanism of CGA biosynthesis in leafy sweet potato is unclear. To dissect the mechanisms of CGA biosynthesis, we performed transcriptome, small RNA (sRNA) and degradome sequencing of one low-CGA content and one high-CGA content genotype at two stages. A total of 2,333 common differentially expressed genes (DEGs) were identified, and the enriched DEGs were related to photosynthesis, starch and sucrose metabolism and phenylpropanoid biosynthesis. The functional genes, such as CCR, CCoAOMT and HCT in the CGA biosynthetic pathway were down-regulated, indicating that the way to lignin was altered, and two possible CGA biosynthetic routes were hypothesized. A total of 38 DE miRNAs were identified, and 1,799 targets were predicated for 38 DE miRNAs by using in silico approaches. The target genes were enriched in lignin and phenylpropanoid catabolic processes. Transcription factors (TFs) such as apetala2/ethylene response factor (AP2/ERF) and Squamosa promoter binding protein-like (SPL) predicated in silico were validated by degradome sequencing. Association analysis of the DE miRNAs and transcriptome datasets identified that miR156 family negatively targeted AP2/ERF and SPL. Six mRNAs and six miRNAs were validated by qRT-PCR, and the results showed that the expression levels of the mRNAs and miRNAs were consistent with the sequencing data. This study established comprehensive functional genomic resources for the CGA biosynthesis, and provided insights into the molecular mechanisms involving in this process. The results also enabled the first perceptions of the regulatory roles of mRNAs and miRNAs, and offered candidate genes for leafy sweet potato improvements.

In Situ Detection of Mature miRNAs in Plants Using LNA-Modified DNA Probes.

MicroRNAs (miRNAs) play important roles in development in plants, and some miRNAs show developmentally regulated organ- and tissue-specific expression patterns. Therefore, in situ detection of mature miRNAs is important for understanding the functions for both miRNAs and their targets. The construction of promoter-reporter fusions and examination of their in planta expression has been widely used and the results obtained thus far are rather informative; however, in some cases, the length of promoter that contains entire regulatory elements is difficult to determine. In addition, traditional in situ hybridization with the antisense RNA fragment as the probe usually fails to detect miRNAs, because the mature miRNAs are too short (~21-nucleotides) to exhibit stable hybridization signals. In recent years, the Locked nucleic acid (LNA) modified DNA probe has been successfully used in animals and plants to detect small RNAs. Here, we describe a modified protocol using LNA-modified DNA probes to detect mature miRNAs in plant tissues, including the design of LNA probes and detailed steps for the in situ hybridization experiment, using Arabidopsis miR165 as an example.

Guidelines for Setting Up a mRNA Sequencing Experiment and Best Practices for Bioinformatic Data Analysis.

RNA-sequencing, commonly referred to as RNA-seq, is the most recently developed method for the analysis of transcriptomes. It uses high-throughput next-generation sequencing technologies and has revolutionized our understanding of the complexity and dynamics of whole transcriptomes.In this chapter, we recall the key developments in transcriptome analysis and dissect the different steps of the general workflow that can be run by users to design and perform a mRNA-seq experiment as well as to process mRNA-seq data obtained by the Illumina technology. The chapter proposes guidelines for completing a mRNA-seq study properly and makes available recommendations for best practices based on recent literature and on the latest developments in technology and algorithms. We also remark the large number of choices available (especially for bioinformatic data analysis) in front of which the scientist may be in trouble.In the last part of the chapter we discuss the new frontiers of single-cell RNA-seq and isoform sequencing by long read technology.

CircPlant: An Integrated Tool for circRNA Detection and Functional Prediction in Plants.

The recent discovery of circular RNAs (circRNAs) and characterization of their functional roles have opened a new avenue for understanding the biology of genomes. circRNAs have been implicated to play important roles in a variety of biological processes, but their precise functions remain largely elusive. Currently, a few approaches are available for novel circRNA prediction, but almost all these methods are intended for animal genomes. Considering that the major differences between the organization of plant and mammal genomes cannot be neglected, a plant-specific method is needed to enhance the validity of plant circRNA identification. In this study, we present CircPlant, an integrated tool for the exploration of plant circRNAs, potentially acting as competing endogenous RNAs (ceRNAs), and their potential functions. With the incorporation of several unique plant-specific criteria, CircPlant can accurately detect plant circRNAs from high-throughput RNA-seq data. Based on comparison tests on simulated and real RNA-seq datasets from Arabidopsis thaliana and Oryza sativa, we show that CircPlant outperforms all evaluated competing tools in both accuracy and efficiency. CircPlant is freely available at http://bis.zju.edu.cn/circplant.

Low RIN Value for RNA-Seq Library Construction from Long-Term Stored Seeds: A Case Study of Barley Seeds.

Seed aging is a complex biological process and its fundamentals and mechanisms have not yet been fully recognized. This is a key issue faced by research teams involved in the collection and storage of plant genetic resources in gene banks every day. Transcriptomic changes associated with seed aging in the dry state have barely been studied. The aim of the study was to develop an efficient protocol for construction of RNA-Seq libraries from long-term stored seeds with very low viability and low RNA integrity number (RIN). Here, barley seeds that have almost completely lost their viability as a result of long-term storage were used. As a control, fully viable seeds obtained in the course of field regeneration were used. The effectiveness of protocols dedicated to RNA samples with high and low RIN values was compared. The experiment concluded that library construction from low viable or long-term stored seeds with degraded RNA (RIN < 3) should be carried out with extraordinary attention due to the possibility of uneven degradation of different RNA fractions.

Identification and characterization of cadmium stress-related LncRNAs from Betula platyphylla.

Cadmium (Cd) is one of the most serious global environmental pollutants, which inhibits plant growth and interferes with their physiological processes. However, there have been few studies on the involvement of long noncoding RNAs (lncRNAs) in Cd tolerance. In the present study, we identified the lncRNAs from Betula platyphylla (birch) that respond to Cd stress. Thirty lncRNAs that were differentially expressed under Cd treatment were identified, including 16 upregulated and 14 downregulated lncRNAs. Nine differentially regulated lncRNAs were selected for further characterization. These lncRNAs were transiently overexpressed in birch plants to determine their roles in Cd tolerance. Among them, two lncRNAs conferred Cd tolerance and two induced sensitivity to Cd stress. We further determined the Cd tolerance of four target genes of the lncRNAs involved in Cd tolerance, including l-lactate dehydrogenase A (LDHA),heat shock protein (HSP18.1), yellow stripe-like protein (YSL9), and H/ACA ribonucleoprotein complex subunit 2-like protein (HRCS2L). Among them, HSP18.1 and LDHA showed improved tolerance to Cd stress, whereas the other two genes did not appear to be involved in Cd tolerance. These results suggested that lncRNAs can up- or downregulate their target genes to improve Cd tolerance. These results increase our understanding of lncRNA-mediated Cd tolerance.

Global identification and characterization of miRNA family members responsive to potassium deprivation in wheat (Triticum aestivum L.).

Potassium (K) is essential for plant growth and stress responses. MicroRNAs (miRNAs) are involved in adaptation to nutrient deprivation through modulating gene expression. Here, we identified the miRNAs responsive to K deficiency in Triticum aestivum based on high-throughput small RNA sequencing analyses. Eighty-nine miRNAs, including 68 previously reported ones and 21 novel ones, displayed differential expression under K deficiency. In Gene Ontology and Kyoto Encyclopedia and Genome analyses, the putative target genes of the differentially expressed miRNAs were categorized into functional groups associated with ADP-binding activity, secondary metabolic pathways, and biosynthesis and metabolism. Functional characterization of tae-miR408, an miRNA significantly down-regulated under K deficiency, revealed its important role in mediating low-K tolerance. Compared with wild type, transgenic tobacco lines overexpressing tae-miR408 showed significantly improved K uptake, biomass, photosynthesis, and reactive oxygen species scavenging under K deficiency. These results show that distinct miRNAs function in the plant response to K deficiency through regulating target genes involved in energy metabolism and various secondary metabolic pathways. Our findings shed light on the plant response to K deficiency mediated by miRNAs in T. aestivum. Distinct miRNAs, such as tae-miR408, are valuable targets for generating crop varieties with improved K-use efficiency.

Identification of candidate miRNAs related in storage root development of sweet potato by high throughput sequencing.

Sweet potato (Ipomoea batatas L.) is a food consumed worldwide, an industrial raw material and new energy crop. The storage root is the most economical part of the crop. However, the mechanism of storage root initiation and development is still unclear. In this study, conserved and novel miRNAs during storage root development were identified by high-throughput sequencing technology by constructing small RNA libraries from sweet potato fibrous roots (F) and storage roots at four different developmental stages (storage roots with different diameters: 1 cm, D1; 3 cm, D3; 5 cm, D5 and 10 cm, D10). A total of 61 known miRNAs and 471 novel miRNAs were identified. In addition, 145 differentially expressed miRNAs were identified in the F library compared with the four storage root libraries, with 30 known miRNAs and 115 novel miRNAs. Moreover, the targets of the differentially expressed miRNAs were predicted and their network was further investigated by GO analysis using our previous transcriptome data. The GO analysis revealed that antioxidant activity and binding process were the most enriched terms of the target genes. The secondary structure and expression of six candidate miRNAs including three conserved miRNAs and three novel miRNAs were investigated and their predicted targets were validated by qRT-PCR. The results showed that the expression levels of the miRNAs were all consistent with the sequencing data. Most of the miRNAs and their corresponding targets had obvious negative correlations. This study contributed to elucidating the potential miRNA mediated regulatory mechanism of storage root development in sweet potato. The specific differentially expressed miRNAs in sweet potato storage roots can be used to breed high-yield sweet potatoes and other tuberous root crops.

Direct SERDS sensing of molecular biomarkers in plants under field conditions.

Molecular biomarkers such as microRNAs (miRNAs) play important roles in regulating various developmental processes in plants. Understanding these pathways will help bioengineer designing organisms for efficient biomass accumulation. Current methods for RNA analysis require sample extraction and multi-step sample analysis, hindering work in field studies. Recent work in the incorporation of nanomaterials for plant bioengineering research is leading the way of an agri-tech revolution. As an example, surface-enhanced Raman scattering (SERS)-based sensors can be used to monitor RNA in vivo. However, the use of SERS in the field has been limited due to issues with observing Raman signal over complex background. To this end, shifted-excitation Raman difference spectroscopy (SERDS) offers an effective solution to extract the SERS signal from high background based on a physical approach. In this manuscript, we report the first application of SERDS on SERS sensors. We investigated this technique on SERS sensor developed for the detection of a microRNA biomarker, miR858. We tested the technique on in vitro samples and validated the technique by detecting the presence of exogenous miR858 in plants directly under ambient light in a growth chamber. The possibility of moving the detection of nucleic acid targets outside the constraints of laboratory setting enables numerous important bioengineering applications. Such applications can revolutionize biofuel production and agri-tech through the use of nanotechnology-based monitoring of plant growth, plant health, and exposure to pollution and pathogens.

Small RNA In Situ Hybridizations on Sections of Arabidopsis Embryos.

Small RNAs mediate posttranscriptional gene silencing in plants and animals. This often occurs in specific cell or tissue types and can be necessary for their differentiation. Determining small RNA (sRNA) localization patterns at cellular resolution can therefore provide information on the corresponding gene regulatory processes they are involved in. Recent improvements with in situ hybridization methods have allowed them to be applied to sRNAs. Here we describe an in situ hybridization protocol to detect sRNAs from sections of early staged Arabidopsis thaliana (Arabidopsis) embryos.

RNASeq analysis of giant cane reveals the leaf transcriptome dynamics under long-term salt stress.

BACKGROUND: To compensate for the lack of information about the molecular mechanism involved in Arundo donax L. response to salt stress, we de novo sequenced, assembled and analyzed the A. donax leaf transcriptome subjected to two levels of long-term salt stress (namely, S3 severe and S4 extreme). RESULTS: The picture that emerges from the identification of differentially expressed genes is consistent with a salt dose-dependent response. Hence, a deeper re-programming of the gene expression occurs in those plants grew at extreme salt level than in those subjected to severe salt stress, probably representing for them an "emergency" state. In particular, we analyzed clusters related to salt sensory and signaling, transcription factors, hormone regulation, Reactive Oxygen Species (ROS) scavenging, osmolyte biosynthesis and biomass production, all of them showing different regulation either versus untreated plants or between the two treatments. Importantly, the photosynthesis is strongly impaired in samples treated with both levels of salinity stress. However, in extreme salt conditions, a dramatic switch from C3 Calvin cycle to C4 photosynthesis is likely to occur, this probably being the more impressive finding of our work. CONCLUSIONS: Considered the distinct response to salt doses, genes either involved in severe or in extreme salt response could constitute useful markers of the physiological status of A. donax to deepen our understanding of its biology and productivity in salinized soil. Finally, many of the unigenes identified in the present study have the potential to be used for the development of A. donax varieties with improved productivity and stress tolerance, in particular the knock out of the GTL1 gene acting as negative regulator of water use efficiency has been proposed as good target for genome editing.

Reconstruction of the full-length transcriptome atlas using PacBio Iso-Seq provides insight into the alternative splicing in Gossypium australe.

BACKGROUND: Gossypium australe F. Mueller (2n = 2x = 26, G2 genome) possesses valuable characteristics. For example, the delayed gland morphogenesis trait causes cottonseed protein and oil to be edible while retaining resistance to biotic stress. However, the lack of gene sequences and their alternative splicing (AS) in G. australe remain unclear, hindering to explore species-specific biological morphogenesis. RESULTS: Here, we report the first sequencing of the full-length transcriptome of the Australian wild cotton species, G. australe, using Pacific Biosciences single-molecule long-read isoform sequencing (Iso-Seq) from the pooled cDNA of ten tissues to identify transcript loci and splice isoforms. We reconstructed the G. australe full-length transcriptome and identified 25,246 genes, 86 pre-miRNAs and 1468 lncRNAs. Most genes (12,832, 50.83%) exhibited two or more isoforms, suggesting a high degree of transcriptome complexity in G. australe. A total of 31,448 AS events in five major types were found among the 9944 gene loci. Among these five major types, intron retention was the most frequent, accounting for 68.85% of AS events. 29,718 polyadenylation sites were detected from 14,536 genes, 7900 of which have alternative polyadenylation sites (APA). In addition, based on our AS events annotations, RNA-Seq short reads from germinating seeds showed that differential expression of these events occurred during seed germination. Ten AS events that were randomly selected were further confirmed by RT-PCR amplification in leaf and germinating seeds. CONCLUSIONS: The reconstructed gene sequences and their AS in G. australe would provide information for exploring beneficial characteristics in G. australe.

The R2R3 MYB transcription factor MdMYB30 modulates plant resistance against pathogens by regulating cuticular wax biosynthesis.

BACKGROUND: The MYB transcription factor family is one of the largest transcriptional factor families in plants and plays a multifaceted role in plant growth and development. However, MYB transcription factors involved in pathogen resistance in apple remain poorly understood. RESULTS: We identified a new MYB family member from apple, and named it MdMYB30. MdMYB30 was localized to the nucleus, and was highly expressed in young apple leaves. Transcription of MdMYB30 was induced by abiotic stressors, such as polyethylene glycol and abscisic acid. Scanning electron microscopy and gas chromatograph-mass spectrometry analyses demonstrated that ectopically expressing MdMYB30 in Arabidopsis changed the wax content, the number of wax crystals, and the transcription of wax-related genes. MdMYB30 bound to the MdKCS1 promoter to activate its expression and regulate wax biosynthesis. MdMYB30 also contributed to plant surface properties and increased resistance to the bacterial strain Pst DC3000. Furthermore, a virus-based transformation in apple fruits and transgenic apple calli demonstrated that MdMYB30 increased resistance to Botryosphaeria dothidea. Our findings suggest that MdMYB30 plays a vital role in the accumulation of cuticular wax and enhances disease resistance in apple. CONCLUSIONS: MdMYB30 bound to the MdKCS1 gene promoter to activate its transcription and regulate cuticular wax content and composition, which influenced the surface properties and expression of pathogenesis-related genes to resistance against pathogens. MdMYB30 appears to be a crucial element in the formation of the plant cuticle and confers apple with a tolerance to pathogens.

EST-SSR marker development based on RNA-sequencing of E. sibiricus and its application for phylogenetic relationships analysis of seventeen Elymus species.

BACKGROUND: Elymus L. is the largest genus in the tribe Triticeae Dumort., encompassing approximately 150 polyploid perennial species widely distributed in the temperate regions of the world. It is considered to be an important gene pool for improving cereal crops. However, a shortage of molecular marker limits the efficiency and accuracy of genetic breeding for Elymus species. High-throughput transcriptome sequencing data is essential for gene discovery and molecular marker development. RESULTS: We obtained the transcriptome dataset of E. sibiricus, the type species of the genus Elymus, and identified a total of 8871 putative EST-SSRs from 6685 unigenes. Trinucleotides were the dominant repeat motif (4760, 53.66%), followed by dinucleotides (1993, 22.47%) and mononucleotides (1876, 21.15%). The most dominant trinucleotide repeat motif was CCG/CGG (1119, 23.5%). Sequencing of PCR products showed that the sequenced alleles from different Elymus species were homologous to the original SSR locus from which the primer was designed. Different types of tri-repeats as abundant SSR motifs were observed in repeat regions. Two hundred EST-SSR primer pairs were designed and selected to amplify ten DNA samples of Elymus species. Eighty-seven pairs of primer (43.5%) generated clear and reproducible bands with expected size, and showed good transferability across different Elymus species. Finally, thirty primer pairs successfully amplified ninety-five accessions of seventeen Elymus species, and detected significant amounts of polymorphism. In general, hexaploid Elymus species with genomes StStHHYY had a relatively higher level of genetic diversity (H = 0.219, I = 0.330, %P = 63.7), while tetraploid Elymus species with genomes StStYY had low level of genetic diversity (H = 0.182, I = 0.272, %P = 50.4) in the study. The cluster analysis showed that all ninety-five accessions were clustered into three major clusters. The accessions were grouped mainly according to their genomic components and origins. CONCLUSIONS: This study demonstrated that transcriptome sequencing is a fast and cost-effective approach to molecular marker development. These EST-SSR markers developed in this study are valuable tools for genetic diversity, evolutionary, and molecular breeding in E. sibiricus, and other Elymus species.

Clubroot resistance gene Rcr6 in Brassica nigra resides in a genomic region homologous to chromosome A08 in B. rapa.

BACKGROUND: Clubroot, caused by Plasmodiophora brassicae Woronin, is a very important disease of Brassica species. Management of clubroot relies heavily on genetic resistance. In a cross of Brassica nigra lines PI 219576 (highly resistant, R) × CR2748 (highly susceptible, S) to clubroot, all F1 plants were resistant to clubroot. There was a 1:1 ratio of R:S in the BC1 and 3R:1S in the F2, which indicated that a single dominant gene controlled clubroot resistance in PI 219576. This gene was designated Rcr6. Mapping of Rcr6 was performed using genome sequencing information from A-genome of B. rapa and B-genome of B. nigra though bulked segregant RNA sequencing (BSR-Seq) and further mapping with Kompetitive Allele Specific PCR (KASP) analysis. RESULTS: Reads of R and S bulks from BSR-Seq were initially aligned onto B. rapa (A-genome; B. nigra has the B-genome) where Rcr6 was associated with chromosome A08. KASP analysis showed that Rcr6 was flanked by SNP markers homologous to the region of 14.8-15.4 Mb of chromosome A08. There were 190 genes annotated in this region, with five genes (Bra010552, Bra010588, Bra010589, Bra010590 and Bra010663) identified as encoding the toll-interleukin-1 receptor / nucleotide-binding site / leucine-rich-repeat (TIR-NBS-LRR; TNL) class of proteins. The reads from BSR-Seq were then aligned into a draft B-genome of B. nigra, where Rcr6 was mapped on chromosome B3. KASP analysis indicated that Rcr6 was located on chromosome B3 in a 0.5 Mb region from 6.1-6.6 Mb. Only one TNL gene homologous to the B. rapa gene Bra010663 was identified in the target region. This gene is a likely candidate for Rcr6. Subsequent analysis of the Rcr6 equivalent region based on a published B. nigra genome was performed. This gene is located into chromosome B7 of the published B-genome, homologous to BniB015819. CONCLUSION: Rcr6 was the first gene identified and mapped in the B-genome of Brassica species. It resides in a genomic region homologous to chromosome A08 of A-genome. Based on this finding, it could possibly integrate into A08 of B. napus using marker assisted selection with SNP markers tightly linked to Rcr6 developed in this study.

Hormone and RNA-seq analyses reveal the mechanisms underlying differences in seed vigour at different maize ear positions.

KEY MESSAGE: ABA/GA4 ratio, stress resistance, carbon and nitrogen metabolism, and chromatin structure play important roles in vigour differences of seeds located at different maize ear positions. Seed vigour, which ensures rapid and uniform field emergence across diverse environments, differs at different maize ear positions. However, little is known regarding the associated mechanisms. In this study, we determined that seed vigour, stress resistance, and carbon and nitrogen metabolism were higher in seeds from middle and bottom section of the ear, while the ABA/GA4 ratio in the embryos was significantly lower. Compared with the seeds subjected to repeated pollination during silking, less variation in seed vigour and the ABA/GA4 ratio in the embryos was observed in seeds at different ear positions subjected to single pollination after complete silking. This indicated that single pollination can reduce, but not eliminate, the differences in seed vigour at different ear positions. RNA-seq analysis indicated that the seed vigour differences at the different locations of the maize ears of the single pollinated treatment were related to carbon and nitrogen metabolism. In contrast, the differences in seed vigour under repeated pollination were related to chromatin structure. The present study contributes to our understanding of the mechanisms underlying differences in seed vigour at different positions on the maize ear.

Characterization of the Photophysical Behavior of DFHBI Derivatives: Fluorogenic Molecules that Illuminate the Spinach RNA Aptamer.

( Z)-5-(3,5-Difluoro-4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one (DFHBI) and its analogues are fluorogenic molecules that bind the Spinach aptamer (a small RNA molecule), which was selected for imaging RNA. They are extremely weakly fluorescent in liquid solvents. It had been hypothesized that photoisomerization is a very efficient nonradiative process of deactivation. We show, consistent with the results of other studies, that if the isomerization is impeded, the fluorescence signal is enhanced significantly. In addition, we provide a thorough characterization of the photophysical behavior of DFHBI and its derivatives, notably that of ( Z)-5-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-3-((perfluorophenyl)methyl)-3,5-dihydro-4 H-imidazol-4-one (PFP-DFHBI) in various solvent environments. Solvent-dependent studies were performed with various mixtures of solvents. The results suggest that hydrogen bonding or strong interactions of the solvents with the phenolic-OH group change the absorption band near 420-460 nm and the nature of emission near 430 and 500 nm through various degrees of stabilization and the transformation between the neutral and the anionic species at both ground and excited states. These observations are confirmed by using a methoxy-substituted molecule (( Z)-5-(4-methoxybenzylidene)-2,3-dimethyl-3,5-dihydro-4 H-imidazol-4-one), where the 420-460 nm band is absent in the presence of methanol and the spectra are similar to those of PFP-DFHBI in noninteracting solvents, such as acetonitrile and dichloromethane. Thus, in addition to the major role of photoisomerization as a nonradiative process of deactivation of the excited state, the fluorescence of DFHBI-type molecules is very sensitively dependent upon the pH of the medium as well as upon solvent-specific interactions, such as hydrogen-bonding ability and polarity.