Inhibiting Gene Expression with Locked Nucleic Acids (LNAs) That Target Chromosomal DNA†

ABSTRACT Mycoplasma gallisepticum, the cause of chronic respiratory infections in the avian host, possesses a family of M9/pMGA genes encoding an adhesin(s) associated with hemagglutination. Nucleotide sequences of M9/pMGA gene family members indicate extensive sequence similarity in the promoter regions of both the transcribed and silent genes. The mechanism that regulates M9/pMGA gene expression is unknown, but studies have revealed an apparent correlation between gene expression and the number of tandem GAA repeat motifs located upstream of the putative promoter. In this study, transposon Tn4001was used as a vector with the Escherichia coli lacZ gene as the reporter system to examine the role of the GAA repeats in M9/pMGA gene expression in M. gallisepticum. A 336-bp M9 gene fragment (containing the GAA repeat region, the promoter, and the translation start codon) was amplified by PCR, ligated with alacZ gene from E. coli, and inserted into the Tn4001-containing plasmid pISM2062. This construct was transformed into M. gallisepticum PG31. Transformants were filter cloned on agar supplemented with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) to monitor lacZ gene expression on the basis of blue/white color selection. Several cycles of filter cloning resulted in cell lineages in which lacZ gene expression alternated between the On and Off states in successive generations of progeny clones. The promoter regions of the M9-lacZ hybrid genes of individual progeny clones were amplified by PCR and sequenced. The only differences between the promoter regions of the blue and white colonies were in the number of GAA repeats. Clones that expressedlacZ had exactly 12 tandem copies of the GAA repeat. Clones that did not express lacZ invariably had either more than 12 (14 to 16) or fewer than 12 (5 to 11) GAA repeats. Southern analysis of M. gallisepticum chromosomal DNA confirmed that the phase-variable expression of the lacZ reporter gene was not caused by Tn4001 transposition. These data strongly indicate that changes in the length of the GAA repeat region are responsible for regulating M9/pMGA gene expression.

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Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.7b01198 ◽

2017 ◽

Vol 121 (12) ◽

pp. 2594-2602 ◽

Cited By ~ 24

Author(s):

Xiaoping Olson ◽

Shohei Kotani ◽

Bernard Yurke ◽

Elton Graugnard ◽

William L. Hughes

Keyword(s):

Nucleic Acids ◽

Strand Displacement ◽

Dna Strand Displacement ◽

Locked Nucleic Acids ◽

Dna Strand ◽

Kinetics Of

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OP0045 NLRP12 INVOLVES IN THE LUPUS WITH POSITIVE INTERFERON SIGNATURE

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.4215 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 23.2-24

Author(s):

Y. P. Tsao ◽

F. Y. Tseng ◽

C. W. Chao ◽

M. H. Chen ◽

S. T. Chen

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Animal Models ◽

Nucleic Acids ◽

Disease Activity ◽

Interferon Signature ◽

Healthy Donors ◽

Healthy Control ◽

Lupus Disease Activity

Background:Systemic lupus erythematous (SLE) is a systemic autoimmune disease with diverse etiological factors. It was recognized that interferon (IFN) signature involved in the progress of SLE. NLRP12 (NOD-like receptor family (NLR) pyrin domain containing 12) is a pyrin containing NLR protein that we had linked its new biological function to the cross-regulation of Toll like receptor (TLRs) and Rig-I like receptor (RIG-I) pathways. NLPR12 acts as an innate immune check-point in regulating type I IFNs expression during TLRs and RIG-I activation. The importance of NLRP12 in lupus disease activity remained to be elucidated.Objectives:To clarify the role of NLRP12 in regulating the interferon signature.Methods:Peripheral blood mononuclear cells (PBMCs) were collected from SLE patients and healthy donors for analysis of NLRP12 and IFN-α gene expression by RT-QPCR. PBMCs were applied for Chromatin immuneprecipitation (ChIP) assay and electrical mobility shift assay (EMSA) to determine the putative transcription factor that regulates NLRP12 expression. An involvement of epigenetic regulation of NLRP12 expression in SLE patients was also analyzed. Bone marrow derived dendritic cells (BMDCs) were collected from wild type mouse and Nlrp12 knocked-out mice. Another CD14+ monocytes were isolated from 10 cases of lupus patients and 8 cases of healthy control, following by stimulating different type of nucleic acids, and IFN-α and IL-6 were measured with ELISA assay. CD14+ monocytes in lupus patients were also pre-treated with IFNAR2 antibody for further nucleic acid stimulation. Two mice models were applied for evaluation the role of Nlrp12: intraperitoneal injection of TMPD (2,6,10,14-tetramethylpentadecane, or pristane) in C57BL/6 mice and Faslpr mice. Both models were conducted with and without Nlrp12 knockout.Results:NLRP12 expression was significantly lower in PBMC isolated from SLE patients compared to healthy donors. The inverse correlation was observed in NLRP12 and IFNA gene expression as well as NLRP12 expression and amount of double-stranded DNA autoantibody in SLE patients. NLRP12 expression showed negative correlations with IFN-α treatment, as well as herpes simplex virus-1 (HSV-1) infection. Results from ChIP and EMSA analysis indicated a potential transcription factor 1 (TF-1) regulating NLRP12 promoter activity. TF-1 lead to transcriptional suppression of NLRP12 in SLE PBMC, and it was gradually induced after IFN treatment. Recruitment of TF-1 to NLRP12 promoter in SLE PBMC compared to the healthy PBMC was detected, and increased when treating with IFN. Human CD14+ monocytes collected from lupus and healthy control stimulating with different type of nucleic acids revealing significant increasing level of IFN-α and IL-6 in lupus patients. Among animal models, both pristine induced mice and Faslpr mice revealed increasing autoantibodies production and severity of glomerulonephritis in Nlrp12-/- group in comparison with Nlrp12+/+ ones, indicating the role of NLRP12 in maintaining positive interferon signature as well as disease activity.Conclusion:Expression level of NLRP1.2 has been demonstrated to be a biomarker of disease activity in SLE patients. The NLRP12 was involved in the interferon signature, which was also negatively regulated by TF-1. Both clinical samples and animal models revealed NLRP12 in maintaining the positive interferon signature, indicating the possible role of exacerbating factor for lupus disease activity.Disclosure of Interests:None declared

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Silencing Antibiotic Resistance with Antisense Oligonucleotides

Biomedicines ◽

10.3390/biomedicines9040416 ◽

2021 ◽

Vol 9 (4) ◽

pp. 416

Author(s):

Saumya Jani ◽

Maria Soledad Ramirez ◽

Marcelo E. Tolmasky

Keyword(s):

Antibiotic Resistance ◽

Nucleic Acids ◽

Antisense Oligonucleotides ◽

Bacterial Infections ◽

Genetic Disorders ◽

Antibiotic Resistance Genes ◽

Short Review ◽

Rnase H ◽

Biological Processes ◽

Locked Nucleic Acids

Antisense technologies consist of the utilization of oligonucleotides or oligonucleotide analogs to interfere with undesirable biological processes, commonly through inhibition of expression of selected genes. This field holds a lot of promise for the treatment of a very diverse group of diseases including viral and bacterial infections, genetic disorders, and cancer. To date, drugs approved for utilization in clinics or in clinical trials target diseases other than bacterial infections. Although several groups and companies are working on different strategies, the application of antisense technologies to prokaryotes still lags with respect to those that target other human diseases. In those cases where the focus is on bacterial pathogens, a subset of the research is dedicated to produce antisense compounds that silence or reduce expression of antibiotic resistance genes. Therefore, these compounds will be adjuvants administered with the antibiotic to which they reduce resistance levels. A varied group of oligonucleotide analogs like phosphorothioate or phosphorodiamidate morpholino residues, as well as peptide nucleic acids, locked nucleic acids and bridge nucleic acids, the latter two in gapmer configuration, have been utilized to reduce resistance levels. The major mechanisms of inhibition include eliciting cleavage of the target mRNA by the host’s RNase H or RNase P, and steric hindrance. The different approaches targeting resistance to β-lactams include carbapenems, aminoglycosides, chloramphenicol, macrolides, and fluoroquinolones. The purpose of this short review is to summarize the attempts to develop antisense compounds that inhibit expression of resistance to antibiotics.

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