Journal: ACS Synthetic Biology

Article Title: Mammalian Genomic Manipulation with Orthogonal Bxb1 DNA Recombinase Sites for the Functional Characterization of Protein Variants

doi: 10.1021/acssynbio.3c00355

Figure Lengend Snippet: Excision of undesired bacterial sequence using flanking GA recombination sites. (A) Schematic of the recombination plasmid generated to test GA site-based excision of bacterial sequence. The top panel shows the construct before GA site recombination, while the bottom panels show the two products expected after recombination occurs. The brown arrow indicates the recombination site that was removed to make the control construct that is incapable of excision. (B) Schematic illustration of possible intermediate and final products, when the excision occurs either as a plasmid or as integrated genomic DNA. (C) Example flow cytometry plots showing the patterns of green and red fluorescence observed with the GA site flanked construct, or the control construct where paring of the plasmid cannot occur as one recombination site was deleted. (D) Example green/red ratiometric histograms for the flanked and control constructs, with the threshold value above which the highest 95% of control cell values measured shown as the dotted line. A constant value of 250 was added to all green MFI values to render all values positive. (E) Upon transfection of flanked construct, the fraction of red cells that were also green, indicative of failed excision. Red points are individual replicates, and the black bar is the geometric mean of four replicate experiments. (F) Fraction of Illumina sequencing reads corresponding to each amplicon product, with different primer sets, for cells transfected with the two constructs. The primer-binding sites and expected amplicons are shown in the schematic in panel A. The cells were selected with hygromycin prior to genomic DNA extraction and PCR. Abbreviations: PTEN, phosphatase and tensin homologue gene as transgenic cargo; mCherry, red fluorescent protein; IRES, internal ribosome entry site; Hpt, hygromycin phosphotransferase gene; UnaG, green fluorescent protein derived from eel; Pac, puromycin N -acetyltransferase; Bac ori, bacterial origin of replication; AmpR, ampicillin resistance gene; SV40 term, transcriptional terminator from simian virus 40; and TKterm, transcriptional terminator from herpes simplex virus thymidine kinase.

Article Snippet: The following plasmids were procured from Addgene and used as template DNA molecules to create the plasmid constructs used in this work: 1066 pBabe puroL PTEN was a gift from William Sellers (Addgene plasmid # 10785; http://n2t.net/addgene:10785 ; RRID:Addgene_10785); pCDNA3_UnaG-Flag-Sec61B was a gift from Hyun-Woo Rhee (Addgene plasmid # 83413; http://n2t.net/addgene:83413 ; RRID:Addgene_83413); pLEX-MCS-ASC-GFP was a gift from Christian Stehlik (Addgene plasmid # 73957; http://n2t.net/addgene:73957 ; RRID:Addgene_73957); pcDNA3.1(+)_SpyCatcher-6aa-sfCherry2(1–10) was a gift from Bo Huang (Addgene plasmid # 117484; http://n2t.net/addgene:117484 ; RRID:Addgene_117484); gRNA_Cloning Vector was a gift from George Church (Addgene plasmid # 41824; http://n2t.net/addgene:41824 ; RRID:Addgene_41824); pSFFV_sfCherry3C(1–10) was a gift from Bo Huang (Addgene plasmid # 117482; http://n2t.net/addgene:117482 ; RRID:Addgene_117482); pGP-CMV-jGCaMP7c variant 1513 was a gift from Douglas Kim & GENIE Project (Addgene plasmid # 105320; http://n2t.net/addgene:105320 ; RRID:Addgene_105320); and pLNCX2-STIM1 was a gift from Shengyu Yang (Addgene plasmid # 89817; http://n2t.net/addgene:89817 ; RRID:Addgene_89817).

Techniques: Sequencing, Plasmid Preparation, Generated, Construct, Control, Flow Cytometry, Fluorescence, Transfection, Illumina Sequencing, Amplification, Binding Assay, DNA Extraction, Transgenic Assay, Derivative Assay, Virus

Journal: Scientific Reports

Article Title: Engineering Structurally Interacting RNA (sxRNA)

doi: 10.1038/srep45393

Figure Lengend Snippet: Bait-reporter DNA templates for T7-transcription were created by subcloning designed sxRNA bait and bait-reporter sequences into a pUC57 based plasmid using demarcated restriction sites (GenScript). The region for the insertion corresponds to the 3′UTR of the transcribed, artificial mRNA. Figure 4 depicts vector bait reporter vector 2 (BRV-2), used for all bait reporters other than BR-1a and BR-1b, that both use bait reporter vector 1. BRV-1 has 64 bases 3′ of the luciferase stop codon and 5′ of the switch insert, whereas BRV-2 has only 12 bases in this position and their composition differs from BRV-1 (see ). Restriction site denoted with asterisk is used to linearize plasmid to allow for polymerase “runoff” based termination of transcription. The prototypic architecture of a final, capped, T7-transcribed sxRNA (mRNA) is represented at the bottom.

Article Snippet: DNA template molecules (see for sequence details) were designed and produced commercially (GenScript, USA). sxRNA vector constructs were linearized using BtgI (New England Biolabs Inc.) following the standard protocol.

Techniques: Subcloning, Plasmid Preparation, Luciferase

Journal: GeoHealth

Article Title: Artificial Space Weathering to Mimic Solar Wind Enhances the Toxicity of Lunar Dust Simulants in Human Lung Cells

doi: 10.1029/2023GH000840

Figure Lengend Snippet: Simulant‐induced mitochondrial DNA damage. (a) Freshly‐ground LMS‐1 or LHS‐1, with or without prior reducing treatment, were added at 1.5 mg/cm 2 to cells in 6‐well plates, and after 1 hr, total DNA was immediately extracted for the Polymerase Chain Reaction (PCR) assay. (b) Protection by antioxidant supplementation. Reduced and freshly‐ground reduced LMS‐1 or LHS‐1 were added after N‐acetylcysteine (NAC) pretreatment where indicated, and after 1 hr, total DNA was immediately extracted for the PCR assay. * denotes p ‐values <0.01 compared to controls or comparisons in specified groups. The error bars are standard deviations ( n = 3).

Article Snippet: For “long” mitochondrial PCR (amplifying about half of the mitochondrial DNA molecule, 15 ng of DNA template was mixed with LongAmp buffer and 100 U/mL of LongAmp Taq polymerase [New England Biolabs #M0323S], 300 μM each of the four deoxynucleoside triphosphates, and 400 nM each of the forward and reverse primers [see Table ], in a total volume of 50 μL).

Techniques: Polymerase Chain Reaction

Journal: eLife

Article Title: Single-molecule tracking of the transcription cycle by sub-second RNA detection

doi: 10.7554/eLife.01775

Figure Lengend Snippet: ( A ) Probability distribution of Mfold-calculated free energies of self-folding of randomly selected, single-stranded 19-mer RNA (left) and DNA (right) oligonucleotides, composed of three or four bases. Results of analysis of three independent sets are shown as ‘+’, ‘x’, and ‘○’. About 100,000 three-letter sequences, and about 300,000 four-letter sequences were analyzed in each set. ( B ) Lempel–Ziv complexity analysis of three-letter 19-mer oligonucleotides (one set of ∼100,000 AUC sequences), four-letter 19-mer oligonucleotides (one set of ∼300,000 AUGC sequences), and all tiling 19-mers from the exome of the human chromosome 22. ( C ) Single-molecule measurements of hybridization rates of fastFISH probe-target pairs, and the effect of G-residues in the targets on the rates. Left: schematic of experiment. Cy3-labeled 90-base RNA oligonucleotides containing a single target sequence were immobilized on a glass surface through a biotin moiety at the 3′ end. Atto633-labeled DNA probes were injected into the imaging flow cell, and their hybridization was detected using TIRF/CoSMoS to obtain the probe arrival time T wait . Right: table of RNA target sequences, Mfold-calculated free energies of self-folding of RNA targets ( ΔG target ), DNA probes ( ΔG probe ), combined energies of targets and probes, and on-rates calculated based on probe T wait and concentrations. ( D ) Self-quenching approach to reduce fluorescence background from unbound DNA probes in TIRF imaging of probe-target hybridization. Left: schematic of experiment. A quencher (e.g., Iowa Black FQ) is placed on one end of a DNA probe, and a fluorophore (e.g., Cy3) is placed on the opposite end of the DNA probe. The short persistence length of single-stranded DNA ( l o ∼0.8 nm, ; ) enables quenching of Cy3. Upon hybridization to the target, the distance between Cy3 and Iowa Black FQ increases due to the larger l o ∼50 nm of the DNA-RNA duplex, leading to an increase of Cy3 fluorescence. Middle: representative TIRF image and a corresponding three-dimensional plot of target-hybridized F1 probes acquired in the presence of unbound, self-quenched F1 probe at 100 nM. Right: same set of molecules imaged in the presence of unbound, unquenched F1 probe at 100 nM. DOI: http://dx.doi.org/10.7554/eLife.01775.003

Article Snippet: In ensemble measurements, 10 nM DNA template (same PCR fragment as used in single-molecule assays), 0.17 μCi/μl α- 32 P-ATP (Perkin Elmer, Waltham, MA) and different concentrations of RNAP were also included in the reaction.

Techniques: Hybridization, Labeling, Sequencing, Injection, Imaging, Fluorescence

Journal: eLife

Article Title: Single-molecule tracking of the transcription cycle by sub-second RNA detection

doi: 10.7554/eLife.01775

Figure Lengend Snippet: ( A ) Schematic of experiment. ( B ) Merged video montages (5 × 5 pixel region of interest) centered at a single, previously photobleached DNA molecule. Interactions of RNAP (false colored in red) and probe (false colored in green) with the DNA locus were imaged simultaneously at 2.5 Hz. Four representative transcriptional events are shown. ( C ) Fluorescence time traces corresponding to the montage shown in Panel B (RNAP and probe are shown in red and green, respectively). The first transcriptional event is zoomed in and shown on top. ( D ) Calculation of the time delay of probe binding with respect to RNAP binding ( ΔT on ) from heat maps of all RNAP-DNA (top) and all probe-DNA binding events (middle) post-synchronized by RNAP binding ( T on , N DNA = 112, N events = 469). Baseline of zero intensity indicates no RNAP/probe binding. Time point t = 0 corresponds to the frame immediately before RNAP binding (red dashed line). On the bottom are the weight-averaged, normalized signal intensities of RNAP and probe binding calculated based on the heat-maps of all RNAP-DNA (red) and probe-DNA (green) binding events. ( E ) Calculation of the time delay of probe dissociation with respect to RNAP run-off ( ΔT off ) from heat maps of all RNAP-DNA and probe-DNA dissociation events post-synchronized by RNAP dissociation ( T off ). Time point t = 0 corresponds to the frame immediately before RNAP dissociation (red dashed line). ( F ) Calculation of the efficiency of real-time RNA detection (left), and of the fraction of events in which RNA was released from DNA upon RNAP run-off (right). Productive RNAP-DNA interactions were post-synchronized by RNAP dissociation as shown in Panel E, and probability distribution of probe signal intensity was plotted for the time point immediately before RNAP run-off (left, t = 0, for the efficiency of RNA detection) and the time point immediately after RNAP run-off (right, t = 0.4 s, for the fraction of RNA released upon run-off). Fits of distributions to sums of two Gaussian functions are shown in blue ( R 2 = 0.92). The higher mean-value Gaussian (peak 1) corresponds to the events with the probe signal present and the lower mean value (peak 2) corresponds to the events without the probe signal present. DOI: http://dx.doi.org/10.7554/eLife.01775.011

Article Snippet: In ensemble measurements, 10 nM DNA template (same PCR fragment as used in single-molecule assays), 0.17 μCi/μl α- 32 P-ATP (Perkin Elmer, Waltham, MA) and different concentrations of RNAP were also included in the reaction.

Techniques: Fluorescence, Binding Assay, RNA Detection

Journal: eLife

Article Title: Single-molecule tracking of the transcription cycle by sub-second RNA detection

doi: 10.7554/eLife.01775

Figure Lengend Snippet: ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 P-ATP, resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009

Article Snippet: In ensemble measurements, 10 nM DNA template (same PCR fragment as used in single-molecule assays), 0.17 μCi/μl α- 32 P-ATP (Perkin Elmer, Waltham, MA) and different concentrations of RNAP were also included in the reaction.

Techniques: SDS Page, Labeling, Purification, Staining, Fluorescence, In Vitro, Activity Assay, Autoradiography

Journal: eLife

Article Title: Single-molecule tracking of the transcription cycle by sub-second RNA detection

doi: 10.7554/eLife.01775

Figure Lengend Snippet: ( A ) Schematic of experiment. ( B ) Co-localization analysis of RNAP-DNA interactions. Left: null promoter DNA template in the presence of NTPs. Center: wild type promoter DNA template in the presence of NTPs. Right: wild type promoter DNA template in the absence of NTPs. ( C ) Representative data. Top: video montages of RNAP interactions with template containing consensus promoter for a 5 × 5 pixel region of interest centered at a single, photobleached DNA molecule (1 pixel = 200 nm, imaged at 12.5 Hz). Bottom: fluorescence time traces corresponding to the montages shown on top. Baseline of zero intensity indicates no binding. Left: experiment carried out in the presence of NTPs. Right: experiment carried out in the absence of NTPs. Yellow arrows indicate the first frames of RNAP binding events. ( D ) Dwell time probability distributions of RNAP-DNA binding events. Left: experiment carried out in the presence of NTP. Fitting to a sum of single exponential and Gaussian functions is shown in blue. Right: experiment carried out in the absence of NTPs. Fitting to a single exponential function is shown in blue. ( E ) Dependence of the peak dwell time of RNAP-DNA interactions on the length of the transcribed DNA segment: schematic of experiment. DNA templates containing transcribed segments spanning from +1 to +295 (red), +633 (blue), or +910 (black) were separately immobilized, and interactions of labeled RNAP were recorded at 2.5 Hz. ( F ) Dwell time probability distributions of RNAP-DNA interactions for the three DNA templates shown in ( E ). N = 749 for the +1…+295 template (red), N = 716 for the +1 … +633 template (blue), and N = 213 for the +1 … 910 DNA template (black). The peak dwell times were calculated by fitting the distributions to a sum of single exponential and Gaussian functions. ( G ) Plot of the peak dwell time of RNAP-DNA interactions vs the length of the transcribed DNA segment. ( H ) Decay of intensity of RNAP fluorescence signal during productive RNAP-DNA interactions as an indicator of elongation by RNAP. All RNAP-DNA interactions having dwell times longer than 0.8 s (experiments in F ) were post-synchronized by RNAP binding (t = 0, circles) and by RNAP run-off (squares), and weight-averaged plots of RNAP binding and run-off were plotted for DNA templates having transcribed segments of different lengths (red − 295 bp; blue − 633 bp; black − 910 bp). Time offsets between RNAP binding and run-off were set at peak dwell lifetimes, T 1 , for the respective DNA templates measured in ( F ). DOI: http://dx.doi.org/10.7554/eLife.01775.008

Article Snippet: In ensemble measurements, 10 nM DNA template (same PCR fragment as used in single-molecule assays), 0.17 μCi/μl α- 32 P-ATP (Perkin Elmer, Waltham, MA) and different concentrations of RNAP were also included in the reaction.

Techniques: Fluorescence, Binding Assay, Labeling

Journal: eLife

Article Title: Single-molecule tracking of the transcription cycle by sub-second RNA detection

doi: 10.7554/eLife.01775

Figure Lengend Snippet: Double-stranded, biotinylated, labeled DNA molecules were immobilized on a passivated glass surface, imaged with TIRF microscope in the presence of oxygen scavengers, and photobleaching time-traces of fluorophores were recorded at 2.5 Hz. The same laser intensities were used as in single-molecule transcription experiments (532 nm at 300 W cm −2 to excite Cy3, and 640 nm at 100 W cm −2 to excite Cy5 and Atto633). The times of photobleaching of individual molecules were binned, plotted as probability histograms (red crosses at bin centers), and the histograms were fit to single exponential function (black circles at bin centers) to estimate mean photobleaching lifetimes ( T ). DOI: http://dx.doi.org/10.7554/eLife.01775.010

Article Snippet: In ensemble measurements, 10 nM DNA template (same PCR fragment as used in single-molecule assays), 0.17 μCi/μl α- 32 P-ATP (Perkin Elmer, Waltham, MA) and different concentrations of RNAP were also included in the reaction.

Techniques: Labeling, Microscopy