High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants

With the wide use of double-stranded RNA interference (RNAi) for the analysis of gene function in plants, a high-throughput system for making hairpin RNA (hpRNA) constructs is in great demand. Here, we describe a novel restriction-ligation approach that provides a simple but efficient construction of intron-containing hpRNA (ihpRNA) vectors. The system takes advantage of the type IIs restriction enzyme BsaI and our new plant RNAi vector pRNAi-GG based on the Golden Gate (GG) cloning. This method requires only a single PCR product of the gene of interest flanked with BsaI recognition sequence, which can then be cloned into pRNAi-GG at both sense and antisense orientations simultaneously to form ihpRNA construct. The process, completed in one tube with one restriction-ligation step, produced a recombinant ihpRNA with high efficiency and zero background. We demonstrate the utility of the ihpRNA constructs generated with pRNAi-GG vector for the effective silencing of various individual endogenous and exogenous marker genes as well as two genes simultaneously. This method provides a novel and high-throughput platform for large-scale analysis of plant functional genomics.     

Rapid one-step construction of hairpin RNA

Hairpin RNA (hpRNA) is commonly used for gene-function exploration and gene engineering. In this study, a novel method was developed to construct intron-containing hairpin RNA (ihpRNA) rapidly and efficiently based on Overlap Extension PCR (OE-PCR). This method, Mixed One-step OE-PCR (MOOE-PCR), can amplify two inverted repeats of DNA fragments and a spliceable intron in parallel, and then assemble them to generate ihpRNA constructs in the same tube without the purification of intermediate products. This method required a PCR process of 38-40 cycles and ordinary PCR reagents. A total of 10 ihpRNA constructs were amplified successfully using this method, with the stems ranging from 50 bp to 484 bp in length. Our results suggest that this novel method is a useful strategy for constructing ihpRNA.     

Optimization of a genome-wide disordered lentivector-based short hairpin RNA library

To obtain a whole genome library that suppresses the total diversity of human mRNAs, lentiviral vector constructs and a short hairpin RNA (shRNA) expression cassette were optimized. The optimization of the vector increased the virus titer in preparations by 15–20 times. A simple shRNA structure with a 21-bp stem proved to be the most effective. Lentivector-based shRNA expression constructs were obtained by using puro ^R, copGFP, or H-2K ^k as a selectable marker. The efficiency of the optimized library was demonstrated when screening for shRNAs reactivating the tumor suppressor p53 in HeLa cells. Cells carried a reporter construct ensuring p53-responsive synthesis of a fluorescent protein, which allowed selection of cells with reactivated p53 by flow cytometry.     

Construction of equalized short hairpin RNA library from human brain cDNA

Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.     

Construction of a long hairpin RNA expression library using Cre recombinase

A large number of genome-wide screens using RNA interference (RNAi) libraries have been utilized to determine the function of individual gene products involved in a variety of biological processes. In this study, we describe a new method to enzymatically generate a long hairpin RNA (lhRNA) expression library from a cDNA plasmid library using a nicking endonuclease, BcaBEST DNA polymerase, and Cre recombinase without excising the inserted DNA fragment from the plasmid vector. This method involves 5 steps: (1) conversion of an inserted DNA fragment in a plasmid into a direct repeat (DR); (2) purification of the plasmid containing the DR; (3) subcloning a lox71 cassette into the plasmid; (4) conversion of the DR in the plasmid into an inverted repeat (IR) using Cre recombinase; and (5) purification of the plasmid containing the IR. We also established an efficient method for inserting DNase I-digested DNA fragments into expression plasmids to enable construction of a cDNA plasmid library suitable as source materials to construct the lhRNA expression library. We confirmed that each of the lhRNA expression plasmids constructed using this method induced strong RNAi in a silkworm cell line, NIAS-Bm-oyanagi2.     

RNA interference-inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer-like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi-inducing hairpin RNAs (hpRNAs) into 22-, 24- and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plant's repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long-distance silencing signals and that hpRNA-induced silencing is suppressed by the expression of a virus-derived suppressor protein. These findings indicate that hpRNA-mediated RNAi in plants operates through the viral defence pathway.     

Poly(A)-associated RNA in plants

The RNA associated with poly(A) sequences from Euglena gracilis and Vicia faba has been isolated by binding to millipore filters and characterized by sedimentation velocity centrifugation and electrophoretic mobility. Poly(A)-associated RNA as isolated in solution was highly aggregated. When denatured, it sedimented as a broad peak with a mean value of 16-18 S. This RNA was shown to be covalently linked to poly(A) sequences which are 150-250 nucleotides long. Our size estimates for plant poly(A) and poly(A)-associated RNA are similar to those obtained for animal cells.     

High-resolution insertion-site analysis by linear amplification-mediated PCR (LAM-PCR)

Integrating vector systems used in clinical gene therapy have proven their therapeutic potential in the long-term correction of immunodeficiencies. The integration loci of such vectors in the cellular genome represent a molecular marker unique for each transduced cell and its clonal progeny. To gain insight into the physiology of gene-modified hematopoietic repopulation and vector-related influences on clonal contributions, we have previously introduced a technology--linear amplification-mediated (LAM) PCR--for detecting and sequencing unknown DNA flanking sequences down to the single cell level (Supplementary Note online). LAM-PCR analyses have enabled qualitative and quantitative measurements of the clonal kinetics of hematopoietic regeneration in gene transfer studies, and uncovered the clonal derivation of non-leukemogenic and leukemogenic insertional side effects in preclinical and clinical gene therapy studies. The reliability and robustness of this method results from the initial preamplification of the vector-genome junctions preceding nontarget DNA removal via magnetic selection. Subsequent steps are carried out on a semisolid streptavidin phase, including synthesis of double complementary strands, restriction digest, ligation of a linker cassette onto the genomic end of the fragment and exponential PCR(s) with vector- and linker cassette-specific primers. LAM-PCR can be adjusted to all unknown DNA sequences adjacent to a known DNA sequence. Here we describe the use of LAM-PCR analyses to identify 5' long terminal repeat (LTR) retroviral vector adjacent genomic sequences.     

Alternative methods for the efficient construction of short hairpin RNA expression vectors

Short hairpin RNA (shRNA)-mediated RNA interference has become a basic technique in modern molecular biology and biochemistry for studying gene function and biological pathways. Here, we report two alternative and efficient methods to construct shRNA expression vectors based respectively on multiple-step sequential PCR and primer extension–homologous recombination (PE-HR). Neither method requires synthesizing long oligonucleotides containing hairpin sequences as used in traditional approaches. The hairpin sequences may produce mutations during oligo synthesis, pose problems in annealing, and lead to inefficient cloning. The PE-HR method further provides rapid and economical construction of shRNA expression vectors without needing the ligation procedure.     

NMR (13)C-relaxation study of base and sugar dynamics in GCAA RNA hairpin tetraloop

Intramolecular dynamics of a 14-mer RNA hairpin including GCAA tetraloop was investigated by (13)C NMR relaxation. R(1) and R(1rho) relaxation rates were measured for all protonated base carbons as well as for C1' carbons of ribose sugars at several magnetic field strengths. The data has been interpreted in the framework of modelfree analysis [G. Lipari and A. Szabo. J Am Chem Soc 104, 4546-4559 (1982); G. Lipari and A. Szabo. J Am Chem Soc 104, 4559-4570 (1982)] characterizing the internal dynamics of the molecule by order parameters and correlation times for fast motions on picosecond to nanosecond time scale and by contributions of the chemical exchange. The fast dynamics reveals a rather rigid stem and a significantly more flexible loop. The cytosine and the last adenine bases in the loop as well as all the loop sugars exhibit a significant contribution of conformational equilibrium on microsecond to millisecond time scale. The high R(1rho) values detected on both base and sugar moieties of the loop indicate coordinated motions in this region. A semiquantitative analysis of the conformational equilibrium suggests the exchange rates on the order of 10(4) s(-1). The results are in general agreement with dynamics studies of GAAA loops by NMR relaxation and fluorescent spectroscopy and support the data on the GCAA loop dynamics obtained by MD simulations.     

Selection of potent non-toxic inhibitory sequences from a randomized HIV-1 specific lentiviral short hairpin RNA library

RNA interference (RNAi) has been considered as an efficient therapeutic approach against the human immunodeficiency virus type 1 (HIV-1). However, to establish a durable inhibition of HIV-1, multiple effective short hairpin RNAs (shRNAs) need to be stably expressed to prevent the emergence of viral escape variants. In this study, we engineered a randomized lentiviral H1-promoter driven shRNA-library against the viral genome. Potent HIV-1 specific shRNAs were selected by ganciclovir treatment of cell lines stably expressing the cDNA of Herpes Simplex Virus thymidine kinase (HSV-TK) fused to HIV-1 nucleotide sequences. More than 50% of 200 selected shRNAs inhibited an HIV-1 based luciferase reporter assay by more than 70%. Stable expression of some of those shRNAs in an HIV-1 permissive HeLa cell line inhibited infection of wild-type HIV-1 by more than 90%. The combination of a randomized shRNA-library directed against HIV-1 with a live cell selection procedure yielded non-toxic and highly efficient HIV-1 specific inhibitory sequences that could serve as valuable candidates for gene therapy studies.     

Probing of metal-binding domains of RNA hairpin loops by laser-induced lanthanide(III) luminescence

Direct laser excitation of aqueous Eu(III) bound to specific RNA fragments was used to probe the metal-binding sites of the anticodon loop of tRNA(Phe) from E. coli and of a tetraloop containing a GNRA consensus sequence. Binding of Mg(II) or Eu(III) to either RNA fragment resulted in a higher melting transition, but no global change in structure was observed. Aqueous Eu(III) exhibits a single weak excitation peak at 17273 cm(-1), the intensity of which increased upon addition of the tRNA loop fragment. Analysis of incremental increases in the luminescence intensity upon complexation with the tRNA loop indicated a stoichiometry of one high-affinity Eu(III)-binding site per loop fragment, with a Kd of 1.3 +/- 0.2 microM. Competition experiments between Eu(III) and Mg(II) were consistent with the two metal ions binding to a common site and with an approximately 30-fold lesser affinity of the tRNA loop for Mg(II) than for Eu(III). The rate of luminescence decay following excitation of Eu(III) bound to the tRNA loop corresponded to displacement of up to 4-5 (of a possible 9) waters of hydration on binding to the tRNA loop. By comparison, Eu(III) binds to the DNA analogue of the tRNA loop with an 8-fold lesser affinity and one fewer direct coordination site than to the RNA sequence, suggesting that a 2'OH of RNA is one of the direct ligands. In contrast with the absence of a shift in the excitation peak of aqueous Eu(III) upon formation of the tRNA loop complex, direct excitation of Eu(III) bound to a GNRA tetraloop fragment resulted in a substantially blue-shifted excitation peak (17290 cm(-1)). The tetraloop fragment also has a single Eu(III)-binding site, with a Kd of 12 +/- 3 microM. The bound Eu(III) was competed by Mg(II), although the relative affinity for Mg(II) was approximately 150-450-fold less than that for Eu(III). The Eu(III)-binding site of the tetraloop site is highly dehydrated, with approximately 7 water molecules displaced upon binding by RNA ligands, suggesting that the blue-shift of the excitation peak is the result of Eu(III) specifically bound in a nonpolar site within the GNRA loop structure.     

Structural characterization of a six-nucleotide RNA hairpin loop found in Escherichia coli, r(UUAAGU)

The binding region of the Escherichia coli S2 ribosomal protein contains a conserved UUAAGU hairpin loop. The structure of the hairpin formed by the oligomer r(GCGU4U5A6A7G8U9CGCA), which has an r(UUAAGU) hairpin loop, was determined by NMR and molecular modeling techniques as part of a study aimed at characterizing the structure and thermodynamics of RNA hairpin loops. Thermodynamic data obtained from melting curves for this RNA oligomer show that it forms a hairpin in solution with the following parameters: DeltaH degrees = -42.8 +/- 2.2 kcal/mol, DeltaS degrees = -127.6 +/- 6.5 eu, and DeltaG degrees (37) = -3.3 +/- 0.2 kcal/mol. Two-dimensional NOESY WATERGATE spectra show an NOE between U imino protons, which suggests that U4 and U9 form a hydrogen bonded U.U pair. The U5(H2') proton shows NOEs to both the A6(H8) proton and the A7(H8) proton, which is consistent with formation of a "U" turn between nucleotides U5 and A6. An NOE between the A7(H2) proton and the U9(H4') proton shows the proximity of the A7 base to the U9 sugar, which is consistent with the structure determined for the six-nucleotide loop. In addition to having a hydrogen-bonded U.U pair as the first mismatch and a U turn, the r(UUAAGU) loop has the G8 base protruding into the solvent. The solution structure of the r(UUAAGU) loop is essentially identical to the structure of an identical loop found in the crystal structure of the 30S ribosomal subunit where the guanine in the loop is involved in tertiary interactions with RNA bases from adjacent regions [Wimberly, B. T., Brodersen, D. E., Clemons, W. M., Morgan-Warren, R. J., Carter, A. P., Vonrhein, C., Hartsch, T., and Ramakrishnan, V. (2000) Nature 407, 327-339]. The similarity of the solution and solid-state structures of this hairpin loop suggests that formation of this hairpin may facilitate folding of 16S RNA.     

Selection of targets and the most efficient hairpin ribozymes for inactivation of mRNAs using a self-cleaving RNA library

The identification of proficient target sites within long RNA molecules, as well as the most efficient ribozymes for each, is a major concern for the use of ribozymes as gene suppressers. In vitro selection methods using combinatorial libraries are powerful tools for the rapid elucidation of interactions between macromolecules, and have been successfully used for different types of ribozyme study. This paper describes a new method for selecting effective target sites within long RNAs using a combinatorial library of self-cleaving hairpin ribozymes that includes all possible specificities. The method also allows the identification of the most appropriate ribozyme for each identified site. Searching for targets within the lacZ gene with this strategy yielded a clearly accessible site. Sequence analysis of ribozymes identified two variants as the most appropriate for this site. Both selected ribozymes showed significant inhibitory activity in the cell milieu.     

Phenotypic inheritance induced by hairpin RNA in Drosophila

Phenotypic inheritance induced by RNA has been documented in mouse and Caenorhabditis elegans. Here we report a similar inheritance in Drosophila. Mutant phenotypes of eye defects and antenna duplication generated from the crossing of one RNA interference （RNAi） transgenic line harboring one hairpin RNA transgene with a GAL4 driver line were inherited independently of the GAL4 driver. Hairpin RNA injection experiments demonstrated that the hairpin RNA could induce heritable mutant-like phenotypes on the eye and antenna. The penetrance of mutant phenotypes was reduced when the mutants were crossed to agol and piwi mutants. Our data suggest that hairpin RNA can induce phenotypic inheritance in Drosophila.     

Rolling circle replication of DNA in yeast mitochondria.

The conformation of mitochondrial DNA (mtDNA) from yeasts has been examined by pulsed field gel electrophoresis and electron microscopy. The majority of mtDNA from Candida (Torulopsis) glabrata (mtDNA unit size, 19 kb) exists as linear molecules ranging in size from 50 to 150 kb or 2-7 genome units. A small proportion of mtDNA is present as supercoiled or relaxed circular molecules. Additional components, detected by electron microscopy, are circular molecules with either single- or double-stranded tails (lariats). The presence of lariats, together with the observation that the majority of mtDNA is linear and 2-7 genome units in length, suggests that replication occurs by a rolling circle mechanism. Replication of mtDNA in other yeasts is thought to occur by the same mechanism. For Saccharomyces cerevisiae, the majority of mtDNA is linear and of heterogeneous length. Furthermore, linear DNA is the chief component of a plasmid, pMK2, when it is located in the mitochondrion of baker's yeast, although only circular DNA is detected when this plasmid occurs in the nucleus. The implications of long linear mtDNA for hypotheses concerning the ploidy paradox and the mechanism of the petite mutation are discussed.     

shRNAPred (version 1.0): An open source and standalone software for short hairpin RNA (shRNA) prediction

The small hairpin RNAs (shRNA) are useful in many ways like identification of trait specific molecular markers, gene silencing and characterization of a species. In public domain, hardly there exists any standalone software for shRNA prediction. Hence, a software shRNAPred (1.0) is proposed here to offer a user-friendly Command-line User Interface (CUI) to predict 'shRNA-like' regions from a large set of nucleotide sequences. The software is developed using PERL Version 5.12.5 taking into account the parameters such as stem and loop length combinations, specific loop sequence, GC content, melting temperature, position specific nucleotides, low complexity filter, etc. Each of the parameters is assigned with a specific score and based on which the software ranks the predicted shRNAs. The high scored shRNAs obtained from the software are depicted as potential shRNAs and provided to the user in the form of a text file. The proposed software also allows the user to customize certain parameters while predicting specific shRNAs of his interest. The shRNAPred (1.0) is open access software available for academic users. It can be downloaded freely along with user manual, example dataset and output for easy understanding and implementation. AVAILABILITY: The database is available for free at http://bioinformatics.iasri.res.in/EDA/downloads/shRNAPred_v1.0.exe.