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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↗
PubMed2005

An algorithmic framework for genome-wide modeling and analysis of translation networks.

The sequencing of genomes of several organisms and advances in high throughput technologies for transcriptome and proteome analysis has allowed detailed mechanistic studies of transcription and translation using mathematical frameworks that allow integration of both sequence-specific and kinetic properties of these fundamental cellular processes. To understand how perturbations in mRNA levels affect the synthesis of individual proteins within a large protein synthesis network, we consider here a genome-scale codon-wide model of the translation machinery with explicit description of the processes of initiation, elongation, and termination. The mechanistic codon-wide description of the translation process and the large number of mRNAs competing for resources, such as ribosomes, requires the use of novel efficient algorithmic approaches. We have developed such an efficient algorithmic framework for genome-scale models of protein synthesis. The mathematical and computational framework was applied to the analysis of the sensitivity of a translation network to perturbation in the rate constants and in the mRNA levels in the system. Our studies suggest that the highest specific protein synthesis rate (protein synthesis rate per mRNA molecule) is achieved when translation is elongation-limited. We find that the mRNA species with the highest number of actively translating ribosomes exerts maximum control on the synthesis of every protein, and the response of protein synthesis rates to mRNA expression variation is a function of the strength of initiation of translation at different mRNA species. Such quantitative understanding of the sensitivity of protein synthesis to the variation of mRNA expression can provide insights into cellular robustness mechanisms and guide the design of protein production systems.

Algorithms↗
PubMed2026

Features affecting Cas9-induced editing efficiency and patterns in tomato: evidence from a large CRISPR dataset.

CRISPR/Cas9 is a cornerstone of plant genome editing, yet the determinants of editing efficiency for a given single-guide RNAs (sgRNAs) and DNA double-strand break (DSB) repair outcomes remain poorly understood, particularly in plants. Here, we generated a large experimental dataset comprising 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts, and quantified editing efficiency and repair footprints together with chromatin accessibility and transcriptional state in the same cellular context. Editing efficiency was consistently higher at targets in accessible chromatin and modestly higher in promoters and introns than in exons, whereas transcriptional activity had no detectable effect. Editing efficiencies were more similar among sgRNAs targeting the same gene than among different genes, revealing a local genomic influence on Cas9 activity. A distinct subset of sgRNAs achieved near-complete editing and produced characteristic repair footprints dominated by long deletions with extended microhomology tracts, indicative of microhomology-mediated end joining (MMEJ), resembling patterns associated with high-efficiency guides in human cells, and suggesting conserved sequence-driven repair biases across species. In contrast, widely used human-trained prediction models failed to accurately rank sgRNA performance in plants, highlighting the limits of cross-species predictability. Together, this dataset provides a resource for improving guide design and mechanistic understanding of plant DNA repair.

Solanum lycopersicum↗
PubMed2026

Suppression of HIV-1 replication in CEM-A cell cultures by trans-splicing group I introns targeting PAS/PBS sequences and conditionally expressing ΔN-Bax.

Anti-HIV group I introns containing antisense guide sequences directed against the HIV-1 primer activation signal and primer-binding site (PAS/PBS) were designed and evaluated. Because PAS/PBS sequences are present in the viral RNA species examined, these RNAs can serve as trans-splicing substrates. The introns were active against both artificial target RNAs and viral RNA generated during infection. Cleavage and degradation of targeted viral RNA may have contributed to suppression, whereas inclusion of a 3' exon encoding the proapoptotic protein ΔN-Bax was associated with increased programmed cell death and may have augmented suppression of viral replication. In cultured CEM-A cells, transgene expression of these introns markedly suppressed HIV-1 replication, with p24 levels falling below the assay detection limit in selected clones. RESULTS: RT-PCR and sequence analysis detected splice products containing the expected PAS/PBS junctions. In the dual-luciferase assay, intron expression reduced normalized Gaussia luciferase signal by approximately 70% relative to the negative control. Qualitative Annexin V imaging and caspase-3 assays were consistent with infection-dependent apoptosis after ΔN-Bax splice-product formation. Transient expression of each intron in HEK293T cells followed by infection with VSV-G-pseudotyped HIV-1NL4-3 at an MOI of 2 reduced p24 levels by approximately 50% at 4 days post-infection. Construct 128L produced the strongest RT-PCR band under the tested conditions and was selected for subsequent experiments. A canonical splice product and a low-abundance noncanonical splice product were detected; both involved the intended HIV-derived target RNA, although transcriptome-wide off-target splicing was not assessed. Heterogeneous transformed HEK293T populations showed an approximately 2-log10 reduction in p24. In selected clonal HEK293T and CEM-A lines, p24 was below the assay detection limit at the measured endpoints, including up to 90 days after infection in some CEM-A clones. CONCLUSIONS: PAS/PBS-targeting group I introns suppressed HIV-1-associated p24 production in the tested cell-culture models. Linking the introns to a ΔN-Bax 3' exon was associated with infection-dependent apoptosis and may further limit viral replication and spread. The use of highly conserved, functionally constrained target sequences may reduce the likelihood of escape, but viral evolution and transcriptome-wide off-target effects were not assessed. This conditional death-upon-infection strategy warrants further evaluation in primary-cell and in vivo models.

Humans↗
PubMed2002

RNA cleavage by a mammalian tRNA 3'processing endoribonuclease (3'tRNase) reduces HIV-1 expression.

We examined the suppression of HIV-1 expression by cleavage of the HIV-1 RNA gene, using a mammalian tRNA 3'processing endoribonuclease (3'tRNase) as an External Guide Sequence oligozyme (EGS) in vivo. The enzyme can also recognize and cleave a target RNA that forms a pre-tRNA like complex with a substrate RNA. We designed an EGS to specifically recognize the packaging signal region (p.s.) and the upstream portion of the gag region (g.s.) of HIV-1 mRNA, and constructed EGS expression vectors that used the tRNA(met) promoter as an expression cassette for the EGS. Their cleavage activities were assessed in cell culture, using a transient assay system. The EGS efficiencies were determined by co-transfection of the EGS expression vectors and the HIV-1 gene plasmid vector (pNL-luc) into COS cells. The assay results showed a significant inhibition of the HIV-1 gag p24 antigen expression. In addition, we constructed MLV-based VSV-G pseudotyped retrovirus vectors that express the EGS. They were co-infected into MT-4 cells with the HIV-1 virus (NL4-3). These EGS expression retrovirus vectors exhibited a similar inhibitory effect.

Animals↗
PubMed2002

A randomized trial assessing the impact of phenotypic resistance testing on antiretroviral therapy.

OBJECTIVE: To compare the effect of treatment decisions guided by phenotypic resistance testing (PRT) or standard of care (SOC) on short-term virological response. DESIGN: A prospective, randomized, controlled clinical trial conducted in 25 university and private practice centers in the United States. PARTICIPANTS: A total of 272 subjects who failed to achieve or maintain virological suppression (HIV-1-RNA plasma level > 2000 copies/ml) with previous exposure to two or more nucleoside reverse transcriptase inhibitors and one protease inhibitor. INTERVENTIONS: Randomization was to antiretroviral therapy guided by PRT or SOC. MAIN OUTCOME MEASURES: The percentage of subjects with HIV-1-RNA plasma levels less than 400 copies/ml at week 16 (primary); change from baseline in HIV-1-RNA plasma levels and number of "active" (less than fourfold resistance) antiretroviral agents used (secondary). RESULTS: At week 16, using intent-to-treat (ITT) analysis, a greater proportion of subjects had HIV-1-RNA levels less than 400 copies/ml in the PRT than in the SOC arm (P = 0.036, ITT observed; P = 0.079, ITT missing equals failure). An ITT observed analysis showed that subjects in the PRT arm had a significantly greater median reduction in HIV-1-RNA levels from baseline than the SOC arm (P = 0.005 for 400 copies/ml; P = 0.049 for 50 copies/ml assay detection limit). Significantly more subjects in the PRT arm were treated with two or more "active" antiretroviral agents than in the SOC arm (P = 0.003). CONCLUSION: Antiretroviral treatment guided prospectively by PRT led to the increased use of "active" antiretroviral agents and was associated with a significantly better virological response.

Adult↗
PubMed2003

New approaches to transplant immunosuppression.

Although considerable progress has been achieved using immunosuppressive drugs that inhibit lymphocyte activation and T-cell cytokine signal transduction pathways, the widespread tissue distribution of the molecular targets exploited to date, calcineurin, mammalian target of rapamycin, and inosine monophosphate dehydrogenase, engenders a constellation of collateral toxicities. One strategy to develop new immunosuppressants seeks to identify targets that are critical for and specific to the adaptive immune response. Three approaches have been used to guide this enterprise; molecular design based on steric resemblance of the antagonist to the natural ligand; construction of complementary DNA oligonucleotides that hybridize with the leader sequence of messenger RNA encoding the synthesis of the specific target, thereby preventing production of that protein; and functional comparisons based on similar inhibitory profiles of candidate compounds and a probe that blocks the target nonselectively. Use of these 3 technologies has led to identification of antagonists blocking selectins, intercellular adhesion molecule-1, or Janus kinase 3, respectively. These lead compounds have been tested for their effects on the alloimmune response and/or the ischemia-reperfusion injuries.

Humans↗
PubMedDate not supplied

Preoperative diagnosis of suspicious parathyroid adenomas by RT-PCR using mRNA extracted from leftover cells in a needle used for ultrasonically guided fine needle aspiration cytology.

OBJECTIVE: To determine the usefulness of reverse transcriptase-polymerase chain reaction (RT-PCR) detection of parathormone (PTH) gene mRNA in needle aspirates to confirm the parathyroid nature of suspicious cervical lesions in patients with hyperparathyroidism. STUDY DESIGN: Ultrasound-guided fine needle aspiration cytology (FNAC) was performed on 12 patients with suspected parathyroid adenomas. The aspirates were subjected to cytologic and chromogranin A examination. Messenger RNA was isolated from left-over cells within the needle, and RT-PCR was used to amplify mRNA encoding PTH. RESULTS: The 12 aspirates were positive by PTH RT-PCR, and molecular diagnosis was confirmed by histologic examination. The lower sensitivity of cytologic and immunocytochemical methods was demonstrated, respectively, by 67% and 50% of positive results. Sensitivity assays demonstrated that with our system, PTH RT-PCR products are obtained from as few as 10 pg of parathyroid mRNA. CONCLUSION: The present study showed that RT-PCR-based analysis of PTH gene transcripts in aspirates, obtained by US-guided FNAC of cervical lesions suspicious for parathyroid adenoma, is a feasible, sensitive and specific method for preoperative diagnosis of parathyroid adenomas.

Adenoma↗
PubMed2006

PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.

In the present study, we analyzed the kinetics of the PCR-suppression effect (PS-effect) and firstly demonstrate that lower annealing temperature enhances PS-effect. Furthermore, we controlled the average size of complex PCR products over a wide range, and simultaneously amplified targets from 0.25 to 10kb by regulating the degree of suppression in a single-primer PCR. In addition, we describe an improved template-switching full-length cDNA synthesis method that greatly reduces truncated cDNA. This study provides a general guide for the design of PS-effect related PCR and is useful for representative or long-transcript enriched cDNA library construction, especially when only a small amount of total RNA is available.

Base Sequence↗
PubMed1998

Inhibition of viral gene expression by human ribonuclease P.

External guide sequences (EGSs) are small RNA molecules which consist of a sequence complementary to a target mRNA and render the target RNA susceptible to degradation by ribonuclease P (RNase P). EGSs were designed to target the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1 for degradation. These EGSs were shown to be able to direct human RNase P to cleave the TK mRNA sequence efficiently in vitro. A reduction of about 80% in the expression level of both TK mRNA and protein was observed in human cells that steadily expressed an EGS, but not in cells that either did not express the EGS or produced a "disabled" EGS which carried a single nucleotide mutation that precluded RNase P recognition. Thus, EGSs may represent novel gene-targeting agents for inhibition of gene expression and antiviral activity.

Animals↗
PubMed2004

Rapid and sensitive p53 alteration analysis in biopsies from lung cancer patients using a functional assay and a universal oligonucleotide array: a prospective study.

PURPOSE: Molecular profiling of alterations associated with lung cancer holds the promise to define clinical parameters such as response to treatment or survival. Because <5% of small cell lung cancers and <30% of non-small cell lung cancers are surgically resectable, molecular analysis will perforce rely on routinely available clinical samples such as biopsies. Identifying tumor mutations in such samples will require a sensitive and robust technology to overcome signal from excess amounts of normal DNA. EXPERIMENTAL DESIGN: p53 mutation status was assessed from the DNA and RNA of biopsies collected prospectively from 83 patients with lung cancer. Biopsies were obtained either by conventional bronchoscopy or computed tomography-guided percutaneous biopsy. Matched surgical specimens were available for 22 patients. Three assays were used: direct sequencing; a functional assay in yeast; and a newly developed PCR/ligase detection reaction/Universal DNA array assay. RESULTS: Using the functional assay, p53 mutation was found in 62% of biopsies and 64% of surgical specimens with a concordance of 80%. The sensitivity of the functional assay was determined to be 5%. Direct sequencing confirmed mutations in 92% of surgical specimens but in only 78% of biopsies. The DNA array confirmed 100% of mutations in both biopsies and surgical specimens. Using this newly developed DNA array, we demonstrate the feasibility of directly identifying p53 mutations in clinical samples containing <5% of tumor cells. CONCLUSIONS: The versatility and sensitivity of this new array assay should allow additional development of mutation profiling arrays that could be applied to biological samples with a low tumor cell content such as bronchial aspirates, bronchoalveolar lavage fluid, or serum.

Aged↗
PubMed2026

CRISPR/Cas- and Argonaute-Based In Vivo Nucleic-Acid Imaging Technologies: Strategies, Challenges, and Perspectives.

Live-cell monitoring of sequence-specific nucleic acids is essential to understanding genome organization, RNA regulation, and disease progression. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) systems provide programmable, guide-directed recognition of DNA or RNA and are increasingly used as platforms for in vivo bioimaging. This review summarizes the structural and mechanistic features of representative CRISPR and Ago effectors and discusses design strategies for sensitive, specific, and multiplexed imaging of genomic loci, extrachromosomal DNA, and endogenous RNA in living cells. We compare the analytical performance and limitations of CRISPR- and Ago-based imaging, with particular emphasis on the major technical and biological challenges affecting their accuracy, applicability, and reliability. Finally, this review offers insights into developing high-resolution and user-friendly bioimaging platforms for fundamental biology and future translational applications.

CRISPR↗
PubMed2026

CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae.

Constructing target-gene mutants with a common genetic background is crucial for elucidating gene function in antimicrobial resistance (AMR) research. Taking advantage of the single-guide RNA (sgRNA) and protospacer adjacent motif (PAM) sequence (3'-NGG) specificity of the Cas9 protein in the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system and codon degeneracy, the authors design a repair template that incorporates the desired point mutation while excluding the PAM sequence disrupted by a synonymous substitution, thereby preventing re-cleavage by CRISPR/Cas9. This method employs two available plasmids, pCasKP-apr and pSGKP-spe, offering straightforward operation and high screening specificity. As a result, the approach enables efficient generation of genetically defined mutant strains of Klebsiella pneumoniae (K. pneumoniae) and is readily adaptable to routine laboratory settings. Furthermore, the protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.

Klebsiella pneumoniae↗
PubMed1998

A novel snoRNA (U73) is encoded within the introns of the human and mouse ribosomal protein S3a genes.

The mouse ribosomal protein S3a-encoding gene (mRPS3a) was cloned and sequenced in this study. mRPS3a shares identical exon/intron structure with its human counterpart. Both genes are split to six exons and exhibit remarkable conservation of the promoter region (68.8% identity in the 250 bp upstream of cap site) and coding region (the proteins differ in two amino acids). mRPS3a displays many features common to other r-protein genes, including the CpG-island at 5'-end of the gene, cap site within an oligopyrimidine tract and no consensus TATA or CAAT boxes. However, mRPS3a represents a rare subclass of r-protein genes that possess a long coding sequence in the first exon. Comparison of human and mouse S3a genes revealed sequence fragments with striking similarity within introns 3 and 4. Here we demonstrate that these sequences encode for a novel small nucleolar RNA (snoRNA) designated U73. U73 contains C, D and D' boxes and a 12-nucleotide antisense complementarity to the 28S ribosomal RNA. These features place U73 into the family of intron-encoded antisense snoRNAs that guide site-specific 2'-O-ribose methylation of pre-rRNA. We propose that U73 is involved in methylation of the G1739 residue of the human 28S rRNA. In addition, we present the mapping of human ribosomal protein S3a gene (hRPS3a) and internally nested U73 gene to the human chromosome 4q31.2-3.

Amino Acid Sequence↗
PubMed2026

Creating bottom-up RNA transfer vehicles from synthetic protein assemblies.

Evolution guides biological systems to populate ecological niches, with viruses among the most successful examples of this principle. Viruses evolved over billions of years to efficiently transfer genetic information. Although viruses are highly diverse, most have converged towards remarkable similarity in the size and shape of their capsids1,2. By contrast, generative models for protein design enable the creation of protein architectures that are absent from nature3-5. Here we investigate whether protein assemblies designed by artificial intelligence can be functionalized to construct nucleic acid transport vehicles that are independent of evolutionary trajectories. By combining natural protein domains with synthetic protein assemblies, we create more than 100 bottom-up RNA transfer vehicles with unique sizes and shapes. These vehicles surpass the RNA transfer efficiency of widely used delivery vehicles by several orders of magnitude. In addition, we demonstrate that their tropism can be programmed by incorporation of computationally designed peptide binders and use them to deliver therapeutically relevant cargo RNAs into a wide range of cellular models. We show the in vivo biodistribution of one of these vehicles in a mouse at near-single-cell resolution, confirm its safety, and use it to perform a gene-editing treatment strategy for Duchenne muscular dystrophy in patient-derived cells and a pig. Our work demonstrates how proteins created by generative artificial intelligence can be harnessed for the rational engineering of RNA transport systems with the desired properties by overcoming the limitations of natural protein diversity.

Journal Article↗
PubMed2006

Lower genitourinary tract sources of seminal HIV.

OBJECTIVE: To investigate genital tract sources of HIV-1, we conducted extensive genitourinary sampling of 23 seropositive men without urethritis who shed HIV in their seminal plasma. DESIGN: Semen was collected, then samples were obtained for HIV RNA in blood plasma, urethral fluid, pre-prostate massage fluid/urine (PMF/U) and post-PMF/U, and expressed prostatic secretions. Systematic transrectal ultrasound-guided prostate biopsies obtained from multiple prostate areas were evaluated for HIV RNA and DNA. RESULTS: Seminal HIV RNA levels correlated with HIV RNA levels in urethral fluid and post-PMF/U and with prostate biopsies HIV DNA, but not with expressed prostatic secretions HIV RNA. However, only the HIV RNA level in post-PMF/U independently predicted that in semen (2.77-fold change in semen for each 10-fold change in post-PMF/U; 95% confidence interval, 1.0-7.7) accounting for one third of the seminal HIV RNA level variation, irrespective of adjustment for antiretroviral therapy. CONCLUSIONS: These data indicate that distal genitourinary sources other than the prostate appear to be the major source of seminal HIV in men without clinical urethritis or prostatitis. Because the HIV RNA level in blood plasma is not reliable as an independent clinical predictor of virus levels in seminal plasma, these findings also extend the concept that the male genital tract is a distinct virological compartment from blood.

Adult↗
PubMed2006

Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins.

Uridine (U)-insertion/deletion RNA editing in trypanosome mitochondria involves an initial cleavage of the preedited mRNA at specific sites determined by the annealing of partially complementary guide RNAs. An involvement of two RNase III-containing core editing complex (L-complex) proteins, MP90 (KREPB1) and MP61 (KREPB3) in, respectively, U-deletion and U-insertion editing, has been suggested, but these putative enzymes have not been characterized or expressed in active form. Recombinant MP90 proteins from Trypanosoma brucei and Leishmania major were expressed in insect cells and cytosol of Leishmania tarentolae, respectively. These proteins were active in specifically cleaving a model U-deletion site and not a U-insertion site. Deletion or mutation of the RNase III motif abolished this activity. Full-round guide RNA (gRNA)-mediated in vitro U-deletion editing was reconstituted by a mixture of recombinant MP90 and recombinant RNA editing exonuclease I from L. major, and recombinant RNA editing RNA ligase 1 from L. tarentolae. MP90 is designated REN1, for RNA-editing nuclease 1.

Amino Acid Sequence↗
PubMed2004

RNAi expression mediated inhibition of HCV replication.

The RNA interference (RNAi) mechanism is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into a cell causes the specific degradation of an mRNA containing the same sequence. The 21-23 nt guide RNAs, generated by RNase III cleavage from longer dsRNAs, are associated with sequence-specific mRNA degradation. Here, we show that vector derived dsRNA specifically inhibit the replication of HCV RNA in HCV replicon cells. We designed a long dsRNA targeted to the full length HCV IRES region (1-377 nt). Real Time RT-PCR was performed with a TaqMan RT-PCR, to solely amplify and enable quantification of HCV RNA. Our results indicated HCV replication reduction to near background levels in a sequence-specific manner by the long-dsRNAs in the HCV replicon cells. Our results support the potential of using siRNA gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.

Cell Line, Tumor↗