Correction: Chhalliyil et al. A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant. Foods 2020, 9, 1245.

The stated objective of the paper was to provide regulatory laboratories and industry laboratories a complete, legally robust method for the quantitative detection of SU canola [...].

A Real-Time Quantitative PCR Method Specific for Detection and Quantification of the First Commercialized Genome-Edited Plant.

Discussion regarding the regulatory status of genome-edited crops has focused on precision of editing and on doubts regarding the feasibility of analytical monitoring compliant with existing GMO regulations. Effective detection methods are important, both for regulatory enforcement and traceability in case of biosafety, environmental or socio-economic impacts. Here, we approach the analysis question for the first time in the laboratory and report the successful development of a quantitative PCR detection method for the first commercialized genome-edited crop, a canola with a single base pair edit conferring herbicide tolerance. The method is highly sensitive and specific (quantification limit, 0.05%), compatible with the standards of practice, equipment and expertise typical in GMO laboratories, and readily integrable into their analytical workflows, including use of the matrix approach. The method, validated by an independent laboratory, meets all legal requirements for GMO analytical methods in jurisdictions such as the EU, is consistent with ISO17025 accreditation standards and has been placed in the public domain. Having developed a qPCR method for the most challenging class of genome edits, single-nucleotide variants, this research suggests that qPCR-based method development may be applicable to virtually any genome-edited organism. This advance resolves doubts regarding the feasibility of extending the regulatory approach currently employed for recombinant DNA-based GMOs to genome-edited organisms.

Decreasing miRNA sequencing bias using a single adapter and circularization approach.

The ability to accurately quantify all the microRNAs (miRNAs) in a sample is important for understanding miRNA biology and for development of new biomarkers and therapeutic targets. We develop a new method for preparing miRNA sequencing libraries, RealSeq®-AC, that involves ligating the miRNAs with a single adapter and circularizing the ligation products. When compared to other methods, RealSeq®-AC provides greatly reduced miRNA sequencing bias and allows the identification of the largest variety of miRNAs in biological samples. This reduced bias also allows robust quantification of miRNAs present in samples across a wide range of RNA input levels.

Right- and left-loop short shRNAs have distinct and unusual mechanisms of gene silencing.

Small hairpin RNAs (shRNAs) having duplex lengths of 25-29 bp are normally processed by Dicer into short interfering RNAs (siRNAs) before incorporation into the RNA-induced silencing complex (RISC). However, shRNAs of ≤ 19 bp [short shRNAs (sshRNAs)] are too short for Dicer to excise their loops, raising questions about their mechanism of action. sshRNAs are designated as L-type or R-type according to whether the loop is positioned 3' or 5' to the guide sequence, respectively. Using nucleotide modifications that inhibit RNA cleavage, we show that R- but not L-sshRNAs require loop cleavage for optimum activity. Passenger-arm slicing was found to be important for optimal functioning of L-sshRNAs but much less important for R-sshRNAs that have a cleavable loop. R-sshRNAs could be immunoprecipitated by antibodies to Argonaute-1 (Ago1); complexes with Ago1 contained both intact and loop-cleaved sshRNAs. In contrast, L-sshRNAs were immunoprecipitated with either Ago1 or Ago2 and were predominantly sliced in the passenger arm of the hairpin. However, 'pre-sliced' L-sshRNAs were inactive. We conclude that active L-sshRNAs depend on slicing of the passenger arm to facilitate opening of the duplex, whereas R-sshRNAs primarily act via loop cleavage to generate a 5'-phosphate at the 5'-end of the guide strand.

Evidence for recycling of external guide sequences during cleavage of bipartite substrates in vitro by reconstituted archaeal RNase P.

RNA-mediated RNA cleavage events are being increasingly exploited to disrupt RNA function, an important objective in post-genomic biology. RNase P, a ribonucleoprotein enzyme that catalyzes the removal of 5'-leaders from precursor tRNAs, has previously been utilized for sequence-specific cleavage of cellular RNAs. In one of these strategies, borne out in bacterial and mammalian cell culture, an external guide sequence (EGS) RNA base-paired to a target RNA makes the latter a substrate for endogenous RNase P by rendering the bipartite target RNA-EGS complex a precursor tRNA structural mimic. In this study, we first obtained evidence that four different mesophilic and thermophilic archaeal RNase P holoenzymes, reconstituted in vitro using their respective constituent RNA and protein subunits, recognize and cleave such substrate-EGS complexes. We further demonstrate that these EGSs engage in multiple rounds of substrate recognition while assisting archaeal RNase P-mediated cleavage of a target RNA in vitro. Taken together, the EGS-based approach merits consideration as a gene knockdown tool in archaea.

miR-ID: a novel, circularization-based platform for detection of microRNAs.

MicroRNAs (miRNAs) are important regulators of gene expression and have great potential as biomarkers, prognostic indicators, and therapeutic targets. Determining the expression patterns of these molecules is essential for elucidating their biogenesis, regulation, relation to disease, and response to therapy. Although PCR-based assays are commonly used for expression profiling of miRNAs, the small size, sequence heterogeneity, and (in some cases) end modifications of miRNAs constrain the performance of existing PCR methods. Here we introduce miR-ID, a novel method that avoids these constraints while providing superior sensitivity and sequence specificity at a lower cost. It also has the unique ability to differentiate unmodified small RNAs from those carrying 2'-OMe groups at their 3'-ends while detecting both forms. miR-ID is comprised of the following steps: (1) circularization of the miRNA by a ligase; (2) reverse transcription of the circularized miRNA (RTC), producing tandem repeats of a DNA sequence complementary to the miRNA; and (3) qPCR amplification of segments of this multimeric cDNA using 5'-overlapping primers and a nonspecific dye such as SYBR Green. No chemically modified probes (e.g., TaqMan) or primers (e.g., LNA) are required. The circular RNA and multimeric cDNA templates provide unmatched flexibility in the positioning of primers, which may include straddling the boundaries between these repetitive miRNA sequences. miR-ID is based on new findings that are themselves of general interest, including reverse transcription of small RNA circles and the use of 5'-overlapping primers for detection of repetitive sequences by qPCR.

An RNA-aptamer-based assay for the detection and analysis of malachite green and leucomalachite green residues in fish tissue.

A robust screening assay employing solid phase extraction (SPE) followed by a novel aptamer-based procedure is presented for the rapid detection and semiquantitation of the triphenylmethane dye, Malachite Green (MG) and its primary metabolite Leucomalachite Green (LMG) in fish tissue. To the authors' knowledge, this is the first reported use of an RNA aptamer for the development of a diagnostic assay for the detection of chemical residues in food. The aptamer based screening assay is found to be highly specific for MG; but has negligible affinity for the LMG metabolite. However, because the LMG metabolite is lipophilic and known to be highly persistent in tissues, an oxidation step has been incorporated within the sample cleanup procedure to ensure that all LMG residues are converted to MG prior to measurement. This article provides evidence that an oligonucleotide aptamer can be used as an alternative recognition element to conventional antibodies with application to the detection of residues in food. Furthermore, this finding has the future potential to reduce the number of animals currently being used in the production of antibodies for immunodiagnostic kits.

Minimal-length short hairpin RNAs: the relationship of structure and RNAi activity.

Small hairpin RNAs (shRNAs) are widely used in RNAi studies and typically consist of a stem of 19-29 base pairs (bp), a loop of at least 4 nucleotides (nt), and a dinucleotide overhang at the 3' end. Compared with shRNAs with 21-29 bp stems, we have found that shRNAs with 19-bp or shorter stems (sshRNAs) possess some unique structure-activity features that depend on whether the antisense strand is positioned 5' or 3' to the loop (L- or R-type sshRNAs, respectively). L sshRNAs can have IC(50)s in the very low picomolar range, and sshRNAs with nominal loop sizes of 1 or 4 nt were at least as active as those with longer loops. L sshRNAs remained highly potent even when the 3' end of the antisense strand was directly linked with the 5' end of the sense strand. In this case, the sense strand can be shorter than the antisense strand, and the loop can be formed entirely by the 3' end of the antisense strand. Monomer sshRNAs are not processed by recombinant Dicers in vitro. Although they can form dimers that are sometimes Dicer substrates, their RNAi activity is not dependent on the formation of such structures. Our findings have implications for the mechanism of action of sshRNAs, and the ability to design highly potent shRNAs with minimal length is encouraging for the prospects of the therapeutic use of direct-delivered shRNAs.

Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.

We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.

shRNAs targeting hepatitis C: effects of sequence and structural features, and comparision with siRNA.

Hepatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) or short hairpin RNA (shRNA) can mediate degradation of a target RNA molecule in a sequence-specific manner. In this study, we screened in vitro-transcribed 25-bp shRNAs targeting the internal ribosome entry site (IRES) of HCV for the ability to inhibit IRES-driven gene expression in cultured cells. We identified a 44-nt region at the 3'-end of the IRES within which all shRNAs efficiently inhibited expression of an IRES-linked reporter gene. Subsequent scans within this region with 19-bp shRNAs identified even more potent molecules, providing effective inhibition at concentrations of 0.1 nM. Experiments varying features of the shRNA design showed that, for 25-bp shRNAs, neither the size of the loop (4-10 nt) nor the sequence or pairing status of the ends affects activity, whereas in the case of 19-bp shRNAs, larger loops and the presence of a 3'-UU overhang increase efficacy. A comparison of shRNAs and siRNAs targeting the same sequence revealed that shRNAs were of comparable or greater potency than the corresponding siRNAs. Anti-HCV activity was confirmed with HCV subgenomic replicons in a human hepatocyte line. The results indicate that shRNAs, which can be prepared by either transcription or chemical synthesis, may be effective agents for the control of HCV.

Ligation of the hairpin ribozyme in cis induced by freezing and dehydration.

Although reducing the temperature slows most chemical reactions, freezing can stimulate some reactions by mechanisms that are only partially understood. Here we show that freezing stimulates the self-ligation (circularization) of linear forms of the hairpin ribozyme (HPR) containing 2',3'-cyclic phosphate and 5'-OH termini. Divalent metal ions (M2+) are not required, but monovalent cations and anions at millimolar concentrations can have various effects on this reaction depending on the specific ion. Under optimal conditions, the observed rate of M2+-independent self-ligation reaches a peak (0.04 min(-1)) at -10 degrees C with a yield of -60% after 1 h. In contrast, no ligation occurs either at above 0 degrees C or in solutions that remain unfrozen when supercooled to subzero temperatures. Under freezing conditions, the cleavage-ligation equilibrium strongly favors ligation. Besides freezing, evaporation of the aqueous solvent as well as the presence of ethanol at levels of 40% or above can also induce M2+-independent HPR ligation at 25 degrees C. We argue that partial RNA dehydration, which is a common feature of freezing, evaporation, and the presence of ethanol, is a key factor supporting HPR ligation activity at both above- and below-freezing temperatures. In the context of the RNA world hypothesis, freezing-induced ligation is an attractive mechanism by which complex RNAs could have evolved under conditions in which RNA was relatively protected against degradation.

The RNA world on ice: a new scenario for the emergence of RNA information.

The RNA world hypothesis refers to a hypothetical era prior to coded peptide synthesis, where RNA was the major structural, genetic, and catalytic agent. Though it is a widely accepted scenario, a number of vexing difficulties remain. In this review we focus on a missing link of the RNA world hypothesis-primitive miniribozymes, in particular ligases, and discuss the role of these molecules in the evolution of RNA size and complexity. We argue that prebiotic conditions associated with freezing, rather than "warm and wet" conditions, could have been of key importance in the early RNA world.

Complete, gene-specific siRNA libraries: production and expression in mammalian cells.

Short interfering RNAs (siRNAs) are widely used to silence the expression of specific genes. Current practice for designing effective siRNAs is to use algorithms based on sequence-efficacy correlations; however, there are many highly effective sequences that these algorithms do not anticipate. To ensure that the best siRNAs are identified, all possible gene-specific siRNA sequences of appropriate lengths should be screened in cell culture. Synthesizing and testing all such sequences individually is costly. A potentially much easier alternative is to prepare a mixture of all these sequences (a gene-specific library), express them in cells, select cells having the desired phenotype, and identify the siRNA contained within the selected cells. Here we describe two new methods for preparing and expressing such libraries. The first uses cloned Dicer or RNase III to digest gene-specific RNA duplexes to siRNAs, which are then converted to the corresponding DNA sequences by attaching RNA primers and performing reverse transcription-PCR. The second method involves partial DNase I digestion of gene-specific DNA, purification of a 20-30-bp fraction, and amplification by attaching DNA adapters followed by PCR. DNA libraries specific for TNF-alpha, DsRed, and part of the hepatitis C virus genome, generated by methods, were inserted into siRNA expression vectors between convergent human U6 and H1 promoters. Randomly selected clones from each library together with vectors expressing the corresponding target genes were cotransfected into 293FT cells and assayed for target gene inhibition. About 10%-20% of siRNAs represented in these libraries show significant inhibition of their target genes. Most of these inhibitory sequences are not predicted by existing algorithms.

Hairpin ribozyme-catalyzed ligation in water-alcohol solutions.

The hairpin ribozyme (HPR) is a naturally existing RNA that catalyzes site-specific RNA cleavage and ligation. At 37 degrees C and in the presence of divalent metal ions (M(2+)), the HPR efficiently cleaves RNA substrates in trans. Here, we show that the HPR can catalyze efficient M(2+)-independent ligation in trans in aqueous solutions containing any of several alcohols, including methanol, ethanol, and isopropanol, and millimolar concentrations of monovalent cations. Ligation proceeds most efficiently in 60% isopropanol at 37 degrees C, whereas the reverse (cleavage) reaction is negligible under these conditions. We suggest that dehydration of the RNA is the key factor promoting HPR activity in water- alcohol solutions. Alcohol-induced ribozyme ligation may have practical applications.

Ligation activity of fragmented ribozymes in frozen solution: implications for the RNA world.

A vexing difficulty of the RNA world hypothesis is how RNA molecules of significant complexity could ever have evolved given their susceptibility to degradation. One way degradation might have been reduced is through low temperature. Here we report that truncated and fragmented derivatives of the hairpin ribozyme can catalyze ligation of a wide variety of RNA molecules to a given sequence in frozen solution despite having little or no activity under standard solution conditions. These results suggest that complex RNAs could have evolved in freezing environments on the early earth and perhaps elsewhere.

ROLL: a method of preparation of gene-specific oligonucleotide libraries.

The selection of nucleic acid sequences capable of specifically and efficiently hybridizing to target sequences is crucial to the success of many applications, including microarrays, PCR and other amplification procedures, antisense inhibition, ribozyme-mediated cleavage, and RNA interference (RNAi). Methods of selection using nucleotide sequence libraries have several advantages over rational approaches using defined sequences. However, the high complexity of completely random (degenerate) libraries and their high toxicity in cell-based assays make their use in many applications impractical. Gene-specific oligonucleotide libraries, which contain all possible sequences of a certain length occurring within a given gene, have much lower complexity and, thus, can significantly simplify and accelerate sequence screening. Here, we describe a new method for the preparation of gene-specific libraries using the ligation of randomized oligonucleotide probes hybridized adjacently on target polynucleotide templates followed by PCR amplification. We call this method random oligonucleotide ligated libraries (ROLL).