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PubMed2001

Effective inhibition of HIV-1 replication in cultured cells by external guide sequences and ribonuclease.

We examined the suppression effect of HIV-1 expression by cleavage of the HIV-1 RNA gene, using the catalytic RNA subunit RNase P and the 3'-half tRNA [External Guide Sequence (EGS)] in vivo. The vectors were designed to express an anti-HIV EGS, U5, which targets the 5' leader sequence. We constructed an EGS expression vector, that used the tRNA(met) or U6 promoter as an expression cassette for EGS. RNase P cleaves the targeted HIV-1 mRNA when they are in a complex with the EGS. To test the antiviral efficacy of these EGS vectors, we have cotransfected into COS cells with the HIV-1 proviral DNA (pNL4-3 Luc) and the plasmid expressing the EGS from the tRNA(met) or U6 promoter. HIV-1 expression was inhibited by the tRNA-EGS-1 and U6-EGS-1 from the tRNA(met) and U6 promoters, respectively. No difference in the inhibitory effects on HIV-1 expression between the tRNA(met) and U6 promoters could be detected.

Animals↗
PubMed2005

Short interfering RNAs as a tool for cancer gene therapy.

There are mainly two types of short RNAs that target complementary messengers in animals: small interfering RNAs and micro-RNAs. Both are produced by the cleavage of double-stranded RNA precursors by Dicer, a member of the Rnase III family of double-stranded specific endonucleases, and both guide the RNA-induced silencing complex to cleave specifically RNAs sharing sequence identity with them. In designing a particular RNA interference (RNAi), it is important to identify the sense/antisense combination that provides the most potent suppression of the target mRNA, and several rules have been established to give >90% gene expression inhibition. RNAi technology can be directed against cancer using a variety of strategies. These include the inhibition of overexpressed oncogenes, blocking cell division by interfering with cyclin E and related genes or promoting apoptosis by suppressing antiapoptotic genes. RNAi against multidrug resistance genes or chemoresistance targets may also provide useful cancer treatments. Studies investigating these approaches in preclinical models are also reviewed.

Apoptosis↗
PubMed2006

Determinants of specific RNA interference-mediated silencing of human beta-globin alleles differing by a single nucleotide polymorphism.

A single nucleotide polymorphism (SNP) in the sickle beta-globin gene (beta(S)) leads to sickle cell anemia. Sickling increases sharply with deoxy sickle Hb concentration and decreases with increasing fetal gamma-globin concentration. Measures that decrease sickle Hb concentration should have an antisickling effect. RNA interference (RNAi) uses small interfering (si)RNAs for sequence-specific gene silencing. A beta(S) siRNA with position 10 of the guide strand designed to align with the targeted beta(S) SNP specifically silences beta(S) gene expression without affecting the expression of the gamma-globin or normal beta-globin (beta(A)) genes. Silencing is increased by altering the 5' end of the siRNA antisense (guide) strand to enhance its binding to the RNA-induced silencing complex (RISC). Specific beta(S) silencing was demonstrated by using a luciferase reporter and full-length beta(S) cDNA transfected into HeLa cells and mouse erythroleukemia cells, where it was expressed in the context of the endogenous beta-globin gene promoter and the locus control region enhancers. When this strategy was used to target beta(E), silencing was not limited to the mutant gene but also targeted the normal beta(A) gene. siRNAs, mismatched with their target at position 10, guided mRNA cleavage in all cases except when two bulky purines were aligned. The specific silencing of the beta(S)-globin gene, as compared with beta(E), as well as studies of silencing SNP mutants in other diseases, indicates that siRNAs developed to target a disease-causing SNP will be specific if the mutant residue is a pyrimidine and the normal residue is a purine.

Base Sequence↗
PubMed2001

Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate.

Duplexes of 21-23 nucleotide (nt) RNAs are the sequence-specific mediators of RNA interference (RNAi) and post-transcriptional gene silencing (PTGS). Synthetic, short interfering RNAs (siRNAs) were examined in Drosophila melanogaster embryo lysate for their requirements regarding length, structure, chemical composition and sequence in order to mediate efficient RNAi. Duplexes of 21 nt siRNAs with 2 nt 3' overhangs were the most efficient triggers of sequence-specific mRNA degradation. Substitution of one or both siRNA strands by 2'-deoxy or 2'-O-methyl oligonucleotides abolished RNAi, although multiple 2'-deoxynucleotide substitutions at the 3' end of siRNAs were tolerated. The target recognition process is highly sequence specific, but not all positions of a siRNA contribute equally to target recognition; mismatches in the centre of the siRNA duplex prevent target RNA cleavage. The position of the cleavage site in the target RNA is defined by the 5' end of the guide siRNA rather than its 3' end. These results provide a rational basis for the design of siRNAs in future gene targeting experiments.

Animals↗
PubMed2004

General design and construction of RNase P ribozymes for gene-targeting applications.

RNase P ribozyme, such as M1 RNA, the catalytic RNA subunit of RNase P from Escherichia coli, cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural ptRNA substrate. When covalently linked with a guide sequence, the M1 ribozyme can function as a sequence-specific endonuclease, M1GS RNA, and cleave any target RNA sequences that basepair with the guide sequence. Using the mRNA coding for the major transcription regulatory protein ICP4 of herpes simplex virus 1 (HSV-1) as the model target, we describe in this chapter the general design and construction of M1GS ribozymes for gene-targeting applications. Specifically, methods are described in detail to determine ideal target regions of an mRNA for M1GS ribozymes and to construct highly active RNase P ribozymes that target these regions. Extensive protocols for in vitro synthesis of the ribozymes and for the cleavage assay of the ribozyme activity are also included. These methods are intended to provide general guidelines for the design and construction of M1GS ribozymes for gene-targeting applications.

Animals↗
PubMed2005

Highly active antiretroviral therapy (HAART) retreatment in patients on CD4-guided therapy achieved similar virologic suppression compared with patients on continuous HAART: the HIV Netherlands Australia Thailand Research Collaboration 001.4 study.

OBJECTIVE: To assess the safety of 2 intermittent treatment strategies compared with continuous therapy for patients with virologic suppression on highly active antiretroviral therapy (HAART) at baseline. DESIGN: Seventy-four nucleoside reverse transcriptase inhibitor (NRTI) and protease inhibitor (PI) pretreated patients with an HIV RNA level <50 copies at screening were randomized to continuous treatment, CD4-guided treatment, or week-on-week-off treatment with 2 NRTIs plus 1600 mg/100 mg of saquinavir/ritonavir once daily. At week 96 (end of the randomized phase of the study), all patients were given continuous HAART for 12 weeks to week 108. Primary outcomes were the proportion of patients with a CD4 count >350 cells/microL and HIV RNA level <400 copies/mL at week 108. METHODS: Patients were followed up every 12 weeks for CD4 count, HIV RNA level, and clinical and laboratory toxicities. In the CD4-guided arm, treatment was stopped and restarted using a CD4 count threshold (above or below 350 cells/microL or reduction of 30%). RESULTS: Seventy-four patients were enrolled with a median CD4 count of 644 cells/microL before the structured treatment interruption (STI). The week-on-week-off arm (n=26) was discontinued at week 72 because of high rates (46%) of HIV RNA rebound above 50 copies/mL. In the continuous arm, 25 (100%) of 25 patients and 24 (96%) of 25 patients had an HIV RNA level <400 copies/mL and <50 copies/mL, respectively, at week 108, and 96% had a CD4 count above 350 cells/microL, with a median CD4 count of 661 cells/microL. Patients in the CD4-guided arm had a significantly lower median CD4 count (489 cells/microL) than the patients in the continuous arm (P=0.03), but all had a CD4 count above 350 cells/microL and 1 had a new HIV-related illness. At week 108, 21 (91%) of 23 patients and 13 (57%) of 23 patients had an HIV RNA level <400 copies/mL and <50 copies/mL, respectively. Those who did not achieve an HIV RNA level <50 copies/mL had a higher HIV RNA load before retreatment, and 4 of 5 patients subsequently achieved viral suppression after an additional 12 weeks of HAART (week 120). Therefore, 17 (94%) of 18 evaluable CD4-guided arm patients achieved viral suppression after retreatment. Antiretroviral (ARV) side effects were similar in all arms. CD4-guided treatment had a 54% ARV cost savings. CONCLUSIONS: This pilot study suggests that CD4-guided HAART is a well-tolerated and cost-saving treatment strategy for patients with high pre-ARV and pre-STI CD4 counts. Week-on-week-off treatment had a high virologic failure rate and was discontinued. The HIV RNA suppression rate was similar in patients treated with continuous HAART and in those retreated with 12 to 24 weeks of HAART after CD4-guided therapy.

Adult↗
PubMed1993

Ligation-based DNA diagnostics.

A number of novel gene detection techniques all revolve around the ligation of synthetic nucleic acid probes. In such ligase-assisted gene detection reactions, specific DNA or RNA sequences are investigated by using them as guides for the covalent joining of pairs of probe molecules. The probes are designed to hybridize immediately next to each other on the target nucleic acid strand. Demonstration of ligated probes results in highly specific detection of and efficient distinction between similar sequence variants under standard reaction conditions. Accordingly, the principle has been applied in automated genetic screening procedures. Ligation reactions are also integral to a number of amplification procedures and they will be of value in an expanding range of genetic analyses.

DNA↗
PubMed2026

Rational and computation-assisted engineering of a compact and efficient CRISPR-Cas12f genome editor.

The CRISPR-Cas12f system is an ultracompact genome-editing platform, yet only a few orthologs exhibit robust activity in mammalian cells. Here, we systematically screened 23 Cas12f orthologs and identified two active nucleases, PspCas12f1 and TcCas12f1, capable of genome editing in human cells. Single guide RNA (sgRNA) scaffold optimization enhanced the basal activity of PspCas12f1. To further improve its performance, we combined structure-guided rational design with protein language model-assisted filtering. Candidate mutations predicted by SaProt were further screened based on structural proximity to the DNA-binding interface and electrostatic compatibility. This integrative strategy identified Q100R and E293R, whose combination yielded the optimized variant enPspCas12f1. enPspCas12f1 achieved genome-editing efficiencies comparable to SpCas9 across multiple endogenous loci while maintaining high specificity. Collectively, our results demonstrate that integrating protein language model-assisted filtering with structure-guided rational design provides an effective strategy for engineering PspCas12f1 and may facilitate the optimization of additional compact CRISPR nucleases.

CRISPR-Cas12f↗
PubMed2005

[Construction of a trans-splicing ribozyme for restoring EGFP truncation mutation].

Special designed group I intron ribozymes can specifically splice objective RNA, repair the mutant gene in RNA level. The specificity of ribozyme is determined by nucleotides specific internal guide sequence (IGS) introduced to the enzyme. In this study, fragment sequence containing Tetrahymena thermophilia intron I of 26S rRNA gene was cloned and cis-splicing activity of this ribozyme was confirmed by in vitro transcription. For evaluating the trans-splicing activity of this ribozyme, a truncated mutant Green Fluorescence Protein (GFP) vector, XYQ5/XYQ10- pEGFP-C2, was constructed. This vector deleted the 3' end 564bp fragment of EGFP coding sequence, led to the lost the activity of emitting green fluorescence. Trans-splicing ribozyme plasmids ptrans-rib-CMV2 for remedy of the truncated mutant EGFP was constructed by PCR and molecular cloning techniques. This vector utilizing cloned 26S rRNA intron 1 as core enzyme; selecting T-G site at 194bp of EGFP coding sequence as splicing receptor, designed an IGS which is inversely complement to the 188-193nt of EGFP mRNA; the 195-890bp fragment of EGFP coding sequence was ligated to the 3'-end of ribozyme core. The fragment containing these components was inserted to a eukayotic expression vector pRC-CMV2. Using linearized XYQ5/XYQ10- pEGFP-C2 and ptrans-rib-CMV2 as templates, truncated EGFP mRNA and the constructed ribozyme vector were transcribed and mixed to evaluate the trans-splicing activity. Analysis of in vitro transcription products mix by RT-PCR verified the existence of wild type EGFP mRNA molecule. Co-transfection of XYQ5/XYQ10- pEGFP-C2 with ptrans-rib-CMV2 to Hela cells proved this ribozyme restored green fluorescence within cell, but the efficiency was low.

Animals↗
PubMed2002

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

Base Sequence↗
PubMed2006

HIV-l and the microRNA-guided silencing pathway: an intricate and multifaceted encounter.

MicroRNAs (miRNAs) are approximately 21-24 nucleotide RNAs that mediate repression of messenger RNA (mRNA) translation through recognition of specific miRNA binding sites usually located in the 3' non-translated region. Designed to simulate miRNAs, small interfering RNAs represent a powerful genetic approach to potently inhibit gene expression by mediating cleavage of the intended mRNA target. This strategy has been applied successfully to suppress replication of several viruses, including human immunodeficiency virus type 1 (HIV-1). However, recent evidences indicate that viral RNAs may themselves be processed, to some extent, by the endogenous miRNA biosynthetic machinery in mammalian cells, extending previous observations in plants. The resulting viral miRNAs may exert regulatory effects towards host and/or viral genes that may influence viral replication and modulate the course of infection. Viral miRNA generation and/or action may be limited by counteraction through inhibitory viral RNAs and/or proteins. This review article will focus on the relationship between HIV-1 and miRNA-guided RNA silencing, and discuss the different aspects of their interaction. As we learn more about the mechanism and importance of small RNA-based antiviral systems, a more intricate picture of the interaction between HIV-1 and a proven antiviral defense mechanism in lower eukaryotes is emerging.

Animals↗
PubMed1992

The gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis contains a group I intron.

The nucleotide sequence of the gene coding for small ribosomal subunit RNA in the basidiomycete Ustilago maydis was determined. It revealed the presence of a group I intron with a length of 411 nucleotides. This is the third occurrence of such an intron discovered in a small subunit rRNA gene encoded by a eukaryotic nuclear genome. The other two occurrences are in Pneumocystis carinii, a fungus of uncertain taxonomic status, and Ankistrodesmus stipitatus, a green alga. The nucleotides of the conserved core structure of 101 group I intron sequences present in different genes and genome types were aligned and their evolutionary relatedness was examined. This revealed a cluster including all group I introns hitherto found in eukaryotic nuclear genes coding for small and large subunit rRNAs. A secondary structure model was designed for the area of the Ustilago maydis small ribosomal subunit RNA precursor where the intron is situated. It shows that the internal guide sequence pairing with the intron boundaries fits between two helices of the small subunit rRNA, and that minimal rearrangement of base pairs suffices to achieve the definitive secondary structure of the 18S rRNA upon splicing.

Base Sequence↗
PubMed2006

Encoding folding paths of RNA switches.

RNA co-transcriptional folding has long been suspected to play an active role in helping proper native folding of ribozymes and structured regulatory motifs in mRNA untranslated regions (UTRs). Yet, the underlying mechanisms and coding requirements for efficient co-transcriptional folding remain unclear. Traditional approaches have intrinsic limitations to dissect RNA folding paths, as they rely on sequence mutations or circular permutations that typically perturb both RNA folding paths and equilibrium structures. Here, we show that exploiting sequence symmetries instead of mutations can circumvent this problem by essentially decoupling folding paths from equilibrium structures of designed RNA sequences. Using bistable RNA switches with symmetrical helices conserved under sequence reversal, we demonstrate experimentally that native and transiently formed helices can guide efficient co-transcriptional folding into either long-lived structure of these RNA switches. Their folding path is controlled by the order of helix nucleations and subsequent exchanges during transcription, and may also be redirected by transient antisense interactions. Hence, transient intra- and inter-molecular base pair interactions can effectively regulate the folding of nascent RNA molecules into different native structures, provided limited coding requirements, as discussed from an information theory perspective. This constitutive coupling between RNA synthesis and RNA folding regulation may have enabled the early emergence of autonomous RNA-based regulation networks.

Base Sequence↗
PubMed2004

Design of a highly reactive HDV ribozyme sequence uncovers facilitation of RNA folding by alternative pairings and physiological ionic strength.

The hepatitis delta virus (HDV) ribozyme is a self-cleaving RNA that resides in the HDV genome and regulates its replication. The native fold of the ribozyme is complex, having two pseudoknots. Earlier work implicated four non-native pairings in slowing pseudoknot formation: Alt 1, Alt 2, Alt 3, and Alt P1. The goal of the present work was design of a kinetically simplified and maximally reactive construct for in vitro mechanistic and structural studies. The initial approach chosen was site-directed mutagenesis in which known alternative pairings were destabilized while leaving the catalytic core intact. Based on prior studies, the G11C/U27Delta double mutant was prepared. However, biphasic kinetics and antisense oligonucleotide response trends opposite those of the well-studied G11C mutant were observed suggesting that new alternative pairings with multiple registers, termed Alt X and Alt Y, had been created. Enzymatic structure mapping of oligonucleotide models supported this notion. This led to a model wherein Alt 2 and the phylogenetically conserved Alt 3 act as "folding guides", facilitating folding of the major population of the RNA molecules by hindering formation of the Alt X and Alt Y registers. Attempts to eliminate the strongest of the Alt X pairings by rational design of a quadruple mutant only resulted in more complex kinetic behavior. In an effort to simultaneously destabilize multiple alternative pairings, studies were carried out on G11C/U27Delta in the presence of urea or increased monovalent ion concentration. Inclusion of physiological ionic strength allowed the goal of monophasic, fast-folding (kobs approximately 60 min(-1)) kinetics to be realized. To account for this, a model is developed wherein Na+, which destabilizes secondary and tertiary structures in the presence of Mg2+, facilitates native folding by destabilizing the multiple alternative secondary structures with a higher-order dependence.

Catalytic Domain↗
PubMed2003

Efficient and specific repair of sickle beta-globin RNA by trans-splicing ribozymes.

Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle beta-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle beta-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5' end of the ribozyme and the beginning of the 3' exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%-50% of the sickle beta-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle beta-globin RNAs over wild-type beta-globin RNAs, although the wild-type beta-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle beta-globin RNAs in mammalian cells with greatly improved specificity.

Anemia, Sickle Cell↗
PubMed2005

Mammalian RNAi: a practical guide.

Silencing of gene expression by RNA interference (RNAi) has become a powerful tool for the functional annotation of the Caenorhabditis elegans and Drosophila melanogaster genomes. Recent advances in the design and delivery of targeting molecules now permit efficient and highly specific gene silencing in mammalian systems as well. RNAi offers a simple, fast, and cost-effective alternative to existing gene targeting technologies both in cell-based and in vivo settings. Synthetic small interfering RNA (siRNA) and retroviral short hairpin RNA (shRNA) libraries targeting thousands of human and mouse genes are publicly available for high-throughput genetic screens, and knockdown animals can be rapidly generated by lentivirus-mediated transgenesis. RNAi also holds great promise as a novel therapeutic approach. This review provides insight into the current gene silencing techniques in mammalian systems.

Animals↗
PubMed2005

A guide to issues in microarray analysis: application to endometrial biology.

Within the last decade, the development of DNA microarray technology has enabled the simultaneous measurement of thousands of gene transcripts in a biological sample. Conducting a microarray study is a multi-step process; starting with a well-defined biological question, moving through experimental design, target RNA preparation, microarray hybridisation, image acquisition and data analysis--finishing with a biological interpretation requiring further study. Advances continue to be made in microarray quality and methods of statistical analysis, improving the reliability and therefore appeal of microarray analysis for a wide range of biological questions. The purpose of this review is to provide both an introduction to microarray methodology, as well as a practical guide to the use of microarrays for gene expression analysis, using endometrial biology as an example of the applications of this technology. While recommendations are based on previous experience in our laboratory, this review also summarises the methods currently considered to be best practice in the field.

Animals↗
PubMed2005

In vitro construction of effective M1GS ribozymes targeting HCMV UL54 RNA segments.

Seven sequence-specific ribozymes (M1GS RNAs) derived in vitro from the catalytic RNA subunit of Escherichia coli RNase P and targeting the mRNAs transcribed by the UL54 gene encoding the DNA polymerase of human cytomegalovirus were screened from 11 ribozymes that were designed based on four rules: (1) the NCCA-3' terminal must be unpaired with the substrate; (2) the guide sequence (GS) must be at least 12 nt in length; (3) the eighth nucleotide must be U, counting from the site -1; and (4) around the cleavage site, the sites -1/+1/+2 must be U/G/C or C/G/C. Further investigation of the factors affecting the cleavage effect and the optimal ratio for M1GS/substrate was carried out. It was determined that the optimal ratio for M1GS/substrate was 2:1 and too much M1GS led to substrate degrading. As indicated above, several M1GS that cleaved HCMV UL54 RNA segments in vitro were successfully designed and constructed. Our studies support the use of ribozyme M1GS as antisense molecules to silence HCMV mRNA in vitro, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.

Cloning, Molecular↗