fluorescently labeled rna substrates  (GenScript corporation)

Journal: mSystems

Article Title: Analysis of co-occurrence of type II toxin–antitoxin systems and antibiotic resistance determinants in Staphylococcus aureus

doi: 10.1128/msystems.00957-24

Figure Lengend Snippet: Assessment of RNase activity of MazF-Sa, PemK-Sa1, PemK-Sa6, and mutants against different substrates. ( A ) PemK-Sa6 and its E19R and F42T mutants do not exhibit detectable activity against phage MS2 RNA. Very low activity is observed for the double mutant. PemK-Sa1 displays a strong RNase activity. ( B ) RNase activity of PemK-Sa6 toxin and its mutants against fluorescently labeled oligo RNAs. PemK-Sa6 single mutants as well as PemK-Shae are enzymatically inactive. Double-mutant PemK-Sa6 (E19R + F42T) shows activity comparable to PemK-Sa1. ( C ) RNase activity of PemK-Sa1 and MazF-Sa against cat194 transcripts containing indicated numbers of target sequences (UAUU and UACAU, respectively). MazF-Sa displays residual unspecific activity visible as digestion of transcripts devoid of target UACAU sites.

Article Snippet: Short fluorescently labeled RNA substrates (GenScript) contained 6-carboxyfluorescein (6-FAM) at the 5′-end and 5-carboxytetramethylrhodamine (5-TAMRA) at the 3′-end.

Techniques: Activity Assay, Mutagenesis, Labeling

Journal: bioRxiv

Article Title: Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

doi: 10.1101/2023.05.10.540130

Figure Lengend Snippet: Trimeric SOSS1 complex suppresses the inhibitory effect of RPA in hSSB1- mediated binding of R-loops A) Graph representing quantification of EMSA experiments (n=3) conducted between hSSB1 and R-loop, RNA:DNA hybrid, 61-mer ssDNA, bubble DNA, 21-mer DNA, and 61-mer dsDNA respectively. B) Graph representing quantification of EMSA experiments (n=3) conducted between trimeric SOSS1 and R-loop, RNA:DNA hybrid, 61-mer ssDNA, bubble DNA, 21-mer DNA, and 61- mer dsDNA respectively. C) Scan of representative EMSA experiments (left) and bar chart graph (right) representing quantification of EMSA experiments (n=3) conducted between hSSB1 (at indicated concentrations) and ssDNA (61-mer) in the absence or presence of RPA. D) Scan of representative EMSA experiments (left) and bar chart graph (right) representing quantification of EMSA experiments (n=3) conducted between hSSB1 (at indicated concentrations) and R-loop in the absence or presence of RPA. E) Scan of representative EMSA experiments (left) and bar chart graph (right) representing quantification of EMSA experiments (n=3) conducted between the trimeric SOSS1 (at indicated concentrations) and ssDNA (61-mer) in the absence or presence of RPA. F) Scan of representative EMSA experiments (left) and bar chart graph (right) representing quantification of EMSA experiments (n=3) conducted between the trimeric SOSS1 (at indicated concentrations) and R-loop in the absence or presence of RPA.

Article Snippet: Oligonucleotides for preparing synthetic fluorescently-labelled (Cy3) RNA/DNA substrates were purchased from Sigma (HPLC purified) and their sequences are available in .

Techniques: Binding Assay

Journal: bioRxiv

Article Title: Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

doi: 10.1101/2023.05.10.540130

Figure Lengend Snippet: A) Scans of representative EMSA experiments of hSSB1 with 61-mer ssDNA. B) Scans of representative EMSA experiments of hSSB1 with 21-mer ssDNA. C) Scans of representative EMSA experiments of hSSB1 with 61-mer dsDNA. D) Scans of representative EMSA experiments of hSSB1 with RNA:DNA hybrids. E) Scans of representative EMSA experiments of hSSB1 with DNA bubble. F) Scans of representative EMSA experiments of hSSB1 with R-loops. G) Scans of representative EMSA experiments of hSSB1 with 61-mer ssDNA (black) and ssRNA (red). H) Graph representing quantification of EMSA experiments from G (n=3).

Article Snippet: Oligonucleotides for preparing synthetic fluorescently-labelled (Cy3) RNA/DNA substrates were purchased from Sigma (HPLC purified) and their sequences are available in .

Techniques:

Journal: bioRxiv

Article Title: Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

doi: 10.1101/2023.05.10.540130

Figure Lengend Snippet: A) Scans of representative EMSA experiments of trimeric SOSS1 with 61-mer ssDNA. B) Scans of representative EMSA experiments of trimeric SOSS1 with RNA:DNA hybrids. C) Scans of representative EMSA experiments of trimeric SOSS1 with R-loops. D) Scans of representative EMSA experiments of trimeric SOSS1 with DNA bubble. E) Scans of representative EMSA experiments of trimeric SOSS1 with 21-mer ssDNA. F) Scans of representative EMSA experiments of trimeric SOSS1 with 61-mer dsDNA. G) Scans of representative EMSA experiments of trimeric SOSS1 with 61-mer ssDNA (black) and ssRNA (red). H) Graph representing quantification of EMSA experiments from G (n=3).

Article Snippet: Oligonucleotides for preparing synthetic fluorescently-labelled (Cy3) RNA/DNA substrates were purchased from Sigma (HPLC purified) and their sequences are available in .

Techniques:

Journal: bioRxiv

Article Title: Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

doi: 10.1101/2023.05.10.540130

Figure Lengend Snippet: A) Scans of representative EMSA experiments of RPA with 61-mer ssDNA. B) Scans of representative EMSA experiments of RPA with 21-mer ssDNA. C) Scans of representative EMSA experiments of RPA with 61-mer dsDNA. D) Scans of representative EMSA experiments of RPA with RNA:DNA hybrids. E) Scans of representative EMSA experiments of RPA with DNA bubble. F) Scans of representative EMSA experiments of RPA with R-loops. G) Scans of representative EMSA experiments of RPA with 61-mer ssDNA (black) and ssRNA (red). H) Graph representing quantification of EMSA experiments from A-G (n=3).

Article Snippet: Oligonucleotides for preparing synthetic fluorescently-labelled (Cy3) RNA/DNA substrates were purchased from Sigma (HPLC purified) and their sequences are available in .

Techniques:

Journal: bioRxiv

Article Title: Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

doi: 10.1101/2023.05.10.540130

Figure Lengend Snippet: A) Graph representing quantification of EMSA experiments of hSSB1 with 61-mer ssDNA in absence or presence of RPA at various concentrations (n=3). B) Graph representing quantification of EMSA experiments of hSSB1 with R-loop in absence or presence of RPA at various concentrations (n=3). C) Scans of representative EMSA experiments of hSSB1 with RNA:DNA hybrids in absence or presence of RPA at various concentrations (left) and graph representing quantification of EMSA experiments (n=3) (right). D) Scans of representative EMSA experiments of hSSB1 with 21-mer ssDNA in absence or presence of RPA at various concentrations (left) and graph representing quantification of EMSA experiments (n=3) (right).

Article Snippet: Oligonucleotides for preparing synthetic fluorescently-labelled (Cy3) RNA/DNA substrates were purchased from Sigma (HPLC purified) and their sequences are available in .

Techniques:

Journal: bioRxiv

Article Title: Phosphorylated trimeric SOSS1 complex and RNA polymerase II trigger liquid-liquid phase separation at double-strand breaks

doi: 10.1101/2023.05.10.540130

Figure Lengend Snippet: A) Graph representing quantification of EMSA experiments of trimeric SOSS1 with 61-mer ssDNA in absence or presence of RPA at various concentrations (n=3). B) Graph representing quantification of EMSA experiments of trimeric SOSS1 with R-loop in absence or presence of RPA at various concentrations (n=3). C) Scans of representative EMSA experiments of trimeric SOSS1 with RNA:DNA hybrids in absence or presence of RPA at various concentrations (left) and graph representing quantification of EMSA experiments (n=3) (right). D) Scans of representative EMSA experiments of trimeric SOSS1 with 21-mer ssDNA in absence or presence of RPA at various concentrations (left) and graph representing quantification of EMSA experiments (n=3) (right).

Article Snippet: Oligonucleotides for preparing synthetic fluorescently-labelled (Cy3) RNA/DNA substrates were purchased from Sigma (HPLC purified) and their sequences are available in .

Techniques: