Design rules of synthetic non-coding RNAs in bacteria.

One of the long-term goals of synthetic biology is to develop designable genetic parts with predictable behaviors that can be utilized to implement diverse cellular functions. The discovery of non-coding RNAs and their importance in cellular processing have rapidly attracted researchers' attention towards designing functional non-coding RNA molecules. These synthetic non-coding RNAs have simple design principles governed by Watson-Crick base pairing, but exhibit increasingly complex functions. Importantly, due to their specific and modular behaviors, synthetic non-coding RNAs have been widely adopted to modulate transcription and translation of target genes. In this review, we summarize various design rules and strategies employed to engineer synthetic non-coding RNAs. Specifically, we discuss how RNA molecules can be transformed into powerful regulators and utilized to control target gene expression. With the establishment of generalizable non-coding RNA design rules, the research community will shift its focus to RNA regulators from protein regulators.

Programmable control of bacterial gene expression with the combined CRISPR and antisense RNA system.

A central goal of synthetic biology is to implement diverse cellular functions by predictably controlling gene expression. Though research has focused more on protein regulators than RNA regulators, recent advances in our understanding of RNA folding and functions have motivated the use of RNA regulators. RNA regulators provide an advantage because they are easier to design and engineer than protein regulators, potentially have a lower burden on the cell and are highly orthogonal. Here, we combine the CRISPR system from Streptococcus pyogenes and synthetic antisense RNAs (asRNAs) in Escherichia coli strains to repress or derepress a target gene in a programmable manner. Specifically, we demonstrate for the first time that the gene target repressed by the CRISPR system can be derepressed by expressing an asRNA that sequesters a small guide RNA (sgRNA). Furthermore, we demonstrate that tunable levels of derepression can be achieved (up to 95%) by designing asRNAs that target different regions of a sgRNA and by altering the hybridization free energy of the sgRNA-asRNA complex. This new system, which we call the combined CRISPR and asRNA system, can be used to reversibly repress or derepress multiple target genes simultaneously, allowing for rational reprogramming of cellular functions.

Synthesis and antisense properties of 2'-O-(2S-methoxypropyl)-RNA-modified gapmer antisense oligonucleotides.

To ascertain whether increasing hydrophobicity can enhance the activity of second-generation antisense oligonucleotides (ASOs) in muscle, we investigated the antisense properties of 2'-O-(2S-methoxypropyl)-RNA (2S-MOP)-modified ASOs. Synthesis of the 2S-MOP 5-methyl uridine phosphoramidite was accomplished on a multi-gram scale by Lewis-acid-catalyzed ring opening of 5'-O-tert-butyldiphenylsilyl ether-protected 2,2'-anhydro-5-methyl uridine with 2S-methoxy-1-propanol. Synthesis of the 2S-MOP 5-methyl cytidine nucleoside from the corresponding 5-methyl uridine nucleoside was accomplished by formation and displacement of a 4-triazolide intermediate with aqueous ammonia. 2S-MOP-modified oligonucleotides were prepared on an automated DNA synthesizer and showed similar enhancements in duplex thermal stability as 2'-O-methoxyethyl RNA (MOE)-modified oligonucleotides. 2S-MOP-containing antisense oligonucleotides were evaluated in Balb-c mice and showed good activity for decreasing the expression levels of scavenger receptor B1 (Srb1) and phosphatase and tensin homologue (PTEN) mRNA in liver and muscle tissue.

Effective synthesis of 3'-deoxy-3'-azido nucleosides for antiviral and antisense ribonucleic guanidine (RNG) applications.

Two synthetic routes to 3'-deoxy-3'-azido nucleosides are described, one toward the synthesis of 3'-deoxy-3'-azidouridine and a second toward 3'-deoxy-3'-azidocytidine. The target compounds may serve as precursors to provide building blocks for use in automated synthesis of guanidine-linked RNA analogs (RNG) or oligonucleotide N3'→P5' phosphoramidates. Moreover, the synthetic approaches are adaptable to the general synthesis of 3'-substituted 3'-deoxynucleosides for development of new antiviral drugs.

[Mapping of the 3' end for artificial human microRNA].

The 3' end was exactly mapped for has-mir-30a-like artificial human microRNAs that specifically recognize the mRNA of the aml1/eto fusion oncogene. The results indicated that the intracellular microRNA pool was heterogeneous in linear size relative to the 3' end, which is necessary to consider in designing and using artificial microRNAs specific for mRNAs of other genes.

Rapid generation of microRNA sponges for microRNA inhibition.

MicroRNA (miRNA) sponges are transcripts with repeated miRNA antisense sequences that can sequester miRNAs from endogenous targets. MiRNA sponges are valuable tools for miRNA loss-of-function studies both in vitro and in vivo. We developed a fast and flexible method to generate miRNA sponges and tested their efficiency in various assays. Using a single directional ligation reaction we generated sponges with 10 or more miRNA binding sites. Luciferase and AGO2-immuno precipitation (IP) assays confirmed effective binding of the miRNAs to the sponges. Using a GFP competition assay we showed that miR-19 sponges with central mismatches in the miRNA binding sites are efficient miRNA inhibitors while sponges with perfect antisense binding sites are not. Quantification of miRNA sponge levels suggests that this is at least in part due to degradation of the perfect antisense sponge transcripts. Finally, we provide evidence that combined inhibition of miRNAs of the miR-17∼92 cluster results in a more effective growth inhibition as compared to inhibition of individual miRNAs. In conclusion, we describe and validate a method to rapidly generate miRNA sponges for miRNA loss-of-function studies.

In situ hybridization of embryos with antisense RNA probes.

Detection of transcripts in situ is a rapid means by which gene expression can be characterized in many systems. In the nematode, Caenorhabditis elegans, the ease with which transgenics can be made and the general reliability of reporter fusion expression patterns, have made this technique comparatively less popular than in other systems. There are, however, still applications in which in situ hybridization is desired, such as for maternally expressed genes, or in related species without established transgene methods. The most frequently used method of in situ hybridization uses DNA probes and formaldehyde fixation. A newer approach that permits single-transcript detection has been reported and will not be described here (Raj and Tyagi, 2010). Rather, we describe an alternative protocol that uses RNA probes with a different fixative. This approach has been applied to C. elegans and related nematodes, providing reliable, sensitive detection of endogenous transcripts.

RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation.

Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA-RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners.

Using synthetic precursor and inhibitor miRNAs to understand miRNA function.

Although the majority of gene function studies center themselves around protein-encoding RNAs, the study of non-protein-encoding RNAs is becoming more widespread because of the discovery of hundreds of small RNA termed micro (mi) RNA that have regulator functions within cells. Currently, over 470 human miRNA genes are predicted to exist and are annotated within the "miRBase" public miRNA database ( http://microrna.sanger.ac.uk/ ). There is no denying that short interfering (si) and short hairpin (sh) RNAs have revolutionized how scientists approach understanding gene function; however, si and shRNAs are not effective for analyzing the function of miRNAs given that miRNAs are typically short (17-24 bases). In turn, new sets of agents that allow for the expression of miRNA above endogenous levels and inhibition of miRNAs have become a valuable technology for the study of these small regulatory RNAs. In this chapter, we provide step-by-step methods on how to utilize synthetic precursor and antisense inhibitor molecules for understanding miRNA function.

In situ hybridization technique for mRNA detection in whole mount Arabidopsis samples.

High throughput microarray transcription analyses provide us with the expression profiles for large amounts of plant genes. However, their tissue and cellular resolution is limited. Thus, for detailed functional analysis, it is still necessary to examine the expression pattern of selected candidate genes at a cellular level. Here, we present an in situ mRNA hybridization method that is routinely used for the analysis of plant gene expression patterns. The protocol is optimized for whole mount mRNA localizations in Arabidopsis seedling tissues including embryos, roots, hypocotyls and young primary leaves. It can also be used for comparable tissues in other species. Part of the protocol can also be automated and performed by a liquid handling robot. Here we present a detailed protocol, recommended controls and troubleshooting, along with examples of several applications. The total time to carry out the entire procedure is approximately 7 d, depending on the tissue used.

Multigene reverse transcription-PCR profiling of circulating tumor cells in hormone-refractory prostate cancer.

BACKGROUND: Circulating tumor cells (CTCs) represent a surrogate source of tissue and conceptually represent a "real-time" biopsy. We previously reported that the number of CTCs mirrors disease progression in hormone-refractory prostate cancer (HRPC). To improve characterization of CTCs we further investigated whether in vitro transcription-based multigene reverse transcription-PCR expression profiles could be obtained from CTCs in HRPC. METHODS: We evaluated the expression of 37 genes with potential utility for epithelial cell characterization from antisense RNA libraries constructed from immunomagnetically enriched CTCs from 7.5-mL blood samples from healthy donors and patients with HRPC. RESULTS: In the control group 13 of 37 genes were not expressed. The most notable of the genes expressed in CTCs of 23 blood specimens drawn from 9 patients with metastatic prostate cancer were prostate-specific antigen (20 of 23; 87%), prostate-specific membrane antigen (17 of 23; 74%), androgen receptor (16 of 23; 70%), human glandular kallikrein 2 (7 of 23; 30%), epidermal growth factor receptor (4 of 23; 17%), and prostate-specific gene with homology to G protein receptor (2 of 23; 9%). The number of CTCs in these samples ranged from 4 to 283 in 7.5 mL of blood (mean, 87; median, 89). Expression of some of the genes was low in the control samples and higher in the patient samples. In all 23 samples, cytokeratin 19, epithelial cell adhesion molecule, or mucin 1 was expressed. Because of background expression in the controls, expression of 13 of the 37 genes, including HER-2, p53, and BCL-2, could not be measured in CTCs. CONCLUSION: Antisense RNA libraries can be constructed from CTCs and gene expression profiles of CTCs obtained from patients with HRPC. This could enhance the characterization of HRPC and facilitate the development of more effective therapies.

Diastereomeric process control in the synthesis of 2'-O-(2-methoxyethyl) oligoribonucleotide phosphorothioates as antisense drugs.

Coupling of 2'-O-methoxyethylsubstituted nucleoside phosphoramidites to 5'-hydroxyl group of a nucleoside or nucleotide on solid support is under stereochemical process control and is independent of scale, concentration, synthesizer, ratio of amidite diastereomers, solid support etc. However, activators and phosphate protecting groups do play a role in influencing the ratio of phosphorothioate diesters obtained by sulfurization of phosphite triesters.

The use of triisopropylsilyl-oxymethyl (TOM) in the synthesis of anti-telomerase 2-5A antisense compound RBI 011.

The 2-5A antisense compound RBI 011 targeting telomerase RNA was synthesized using the triisopropylsilyl-oxymethyl (TOM) group for the 3'-hydroxyl protection of 2',5'-linked RNA.

Applications of antisense and siRNAs during preclinical drug development.

A significantly greater number of candidate drug targets and compounds are now being generated during preclinical drug development. To date, however, such increases have not led to improvements in clinical success rates or reduced times to market. There is a need for better strategies to prioritize targets and drug candidates. Antisense and siRNA technologies offer exceptional speed and specificity to address this need. In particular, antisense and siRNAs are beginning to be used in combination with expression profiling to evaluate drug specificity and mechanism-of-action, aiding in the identification of better candidates earlier in the drug development process.

Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.

Short interfering RNAs (siRNAs) are double-stranded RNAs of approximately 21-25 nucleotides that have been shown to function as key intermediaries in triggering sequence-specific RNA degradation during posttranscriptional gene silencing in plants and RNA interference in invertebrates. siRNAs have a characteristic structure, with 5'-phosphate/3'-hydroxyl ends and a 2-base 3' overhang on each strand of the duplex. In this study, we present data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice. In each case, the interference by siRNAs was superior to the inhibition of gene expression mediated by single-stranded antisense oligonucleotides. The siRNAs seem to avoid the well documented nonspecific effects triggered by longer double-stranded RNAs in mammalian cells. These observations may open a path toward the use of siRNAs as a reverse genetic and therapeutic tool in mammalian cells.

Thermodynamics of RNA-RNA duplexes with 2- or 4-thiouridines: implications for antisense design and targeting a group I intron.

Antisense compounds are designed to optimize selective hybridization of an exogenous oligonucleotide to a cellular target. Typically, Watson-Crick base pairing between the antisense compound and target provides the key recognition element. Uridine (U), however, not only stably base pairs with adenosine (A) but also with guanosine (G), thus reducing specificity. Studies of duplex formation by oligonucleotides with either an internal or a terminal 2- or 4-thiouridine (s(2)U or s(4)U) show that s(2)U can increase the stability of base pairing with A more than with G, while s(4)U can increase the stability of base pairing with G more than with A. The latter may be useful when binding can be enhanced by tertiary interactions with a s(4)U-G pair. To test the effects of s(2)U and s(4)U substitutions on tertiary interactions, binding to a group I intron ribozyme from mouse-derived Pneumocystis carinii was measured for the hexamers, r(AUGACU), r(AUGACs(2)U), and r(AUGACs(4)U), which mimic the 3' end of the 5' exon. The results suggest that at least one of the carbonyl groups of the 3' terminal U of r(AUGACU) is involved in tertiary interactions with the catalytic core of the ribozyme and/or thio groups change the orientation of a terminal U-G base pair. Thus thio substitutions may affect tertiary interactions. Studies of trans-splicing of 5' exon mimics to a truncated rRNA precursor, however, indicate that thio substitutions have negligible effects on overall reactivity. Therefore, modified bases can enhance the specificity of base pairing while retaining other activities and, thus, increase the specificity of antisense compounds targeting cellular RNA.

Simultaneous quantitation of substance P-encoding preprotachykinin alternatively spliced mRNAs and substance P receptor NK-1 mRNA by an RNase protection assay.

Tachykinins form a family of peptides with neurotransmitter/neuromodulator function. Four tachykinins, substance P, neurokinin A, neuropeptide gamma and neuropeptide K, are encoded by the same PreProTachykinin (PPT) gene. Alternatively spliced mRNAs encode different combinations of these peptides (Brown, E.R., Harlan, R.E., Krause, J.E., Gonadal steroid regulation of substance P (SP) and SP-encoding mRNA in the rat anterior pituitary and hypothalamus, Endocrinology, 126 (1990) 330-340; Krause, J.E., Chirgwin, J.M., Carter, M.S., Xu, Z.S., Hershey, A.D., Three rat preprotachykinin mRNAs encode the neuropeptides substance P and neurokinin A, Proc. Natl. Acad. Sci. USA, 84 (1987) 881-885). The proportion of PPT mRNAs varies from tissue to tissue (Carter, M.S., Krause, J.E., Structure, expression, and some regulatory mechanisms of the rat preprotachykinin gene encoding substance P, neurokinin A, neuropeptide K, and neuropeptide gamma, J. Neurosci., 10 (1990) 2203-2214), and within the rat hypothalamus according to the estrous cycle-related hormonal status (Gautreau, A., Duval, P., Kerdelhué, B., Variations in substance P-encoding preprotachykinin and substance P receptor NK-1 mRNA transcripts in the rat hypothalamus throughout the estrous cycle: a correlation between amounts of beta-preprotachykinin and NK-1 mRNA, Mol. Brain Res., (1997) in press). Tachykinin receptors as well as tachykinins are regulated at the mRNA level. A fully quantitative method is needed to deal with the complex physiological regulation of the tachykinin system. Here, we describe an RNase protection assay that allows the simultaneous quantitation of alternatively spliced PPT mRNAs, Substance P receptor NK-1 mRNA, and glyceraldehyde-3-phosphodehydrogenase (GAPDH) mRNA as an internal control, in the rat hypothalamus. The advantages of this method are its high sensitivity (0.1 pg) and a wide range of linearity (more than 3 orders of magnitude). Moreover, this protocol provides guidelines to set up a quantitative multiprobe RNase protection assay for other genes.

The design of antisense RNA.

Antisense nucleic acids comprise short-chain synthetic oligonucleotides, often oligodeoxyribonucleotides (ODN) of less than approximately 30 nucleotides and substantially longer sequences formed by ribonucleic acids (RNA). Both groups differ with respect to several properties, including their generation, the mode of delivery, and their structure-function relationship. Long-chain antisense RNA transcribed in vitro or endogenously from recombinant genes fold into three-dimensional structures. The pairing reaction with their complementary target strand occurs via largely unknown annealing mechanisms and, depending on the phylogenetic cellular background, in different cellular compartments. The annealing pathway is influenced by a variety of biologic and biochemical parameters that are as yet poorly understood. However, the basal biochemical mechanisms underlying the relationship between RNA structure and efficient annealing could allow one to derive more general rules for the design of in vivo effective antisense RNA in a way that is not dependent on specific cell types. Here, some of the criteria are discussed that are currently thought to have major impact on the design of long-chain antisense RNA.

A reliable external control for ribonuclease protection assays.

A method is described for generating an external spiked human RNA control to enhance the reliability of assessment of gene expression in tumour extracts. Spiking with an external standard RNA controls for all subsequent steps of analysis on a lane by lane basis and allows for uniform comparison of the gene of interest as a fraction of total RNA, particularly when multiple samples are not available. The antisense probe that is being used to detect endogenous gene expression is also used as an external control. A sense riboprobe is made from the same vector. Because of the flanking RNA polymerase sites incorporated in both probes, hybridization with the sense riboprobe at a much lower concentration than the antisense probe generates a larger product that can be readily separated from the endogenous protected fragment. This method is generally applicable to any riboprobe that has a T3 and T7 RNA polymerase site and allows any externally added riboprobe use for assessing endogenous gene expression to be used as the external spike control.