Journal: bioRxiv

Article Title: Phage-encoded CasPRs transcriptionally silence diverse CRISPR-Cas systems

doi: 10.64898/2026.02.23.707548

Figure Lengend Snippet: (A) ChlP-seq profiles of indicated his6-tagged CasPRs, plotted along the LS1 type l-B CRISPR-Cas locus. Plot represents the ratio of reads in CasPR ChIP samples to those in empty vector (EV) control lacking CasPRs. ChIP signal was normalized to reads from a total input DNA sample. (B) As in (A), but plotting the CasPRIIAI ChIP profile along the type ll-A CRISPR-Cas locus from L. seeligeri. (C). Weblogo assembled from alignment of 250 unique cas8b gene sequences, representing region bound by CasPRIBI. (D) EMSA with 50 nM of indicated 60 bp DNA substrate and 0, 250, 200, 150, 100, 75, 50 nM CasPRIBI, resolved on native acrylamide gels. (E) Sequence in the cas8b gene determined critical for CasPRIBI binding in EMSAs (See ). Binding measurements with 150 nM CasPRIBI and 50 nM indicated substrates containing 2 bp mutations with respect to the WT 30 bp CasPRIBI binding site. Percent binding relative to WT substrate shown for 4 biological replicates. Red nts exhibit diminished binding when mutated.

Article Snippet: Samples were resolved on 8% native acrylamide gels (29:1 acrylamide:bisacrylamide; Bio-Rad) containing 0.5 X TBE.

Techniques: CRISPR, Plasmid Preparation, Control, Sequencing, Binding Assay

Journal: bioRxiv

Article Title: Phage-encoded CasPRs transcriptionally silence diverse CRISPR-Cas systems

doi: 10.64898/2026.02.23.707548

Figure Lengend Snippet: (A) Coomassie gel indicating purified L. seeligeri CasPRIBI sample, with molecular weight 19.1 kDa. Molecular weight marker in kDa. (B) Size exclusion chromatogram of CasPRIBI along with size standards. (C) EMSA with 50 nM of indicated DNA substate containing nonspecific (ns) or CasPRIBI ChIP sequence and 250, 125, 62, 31, 16, 8, 4, 2, 1, or 0 nM CasPRIBI. (D) WT 120 bp cas8b sequence bound by CasPRIBI, with ChIP peak center underlined, and twelve 60-bp tiled substrates, each overlapping by 5 bp, that collectively cover the interval. Substrates in green were bound by CasPRIBI in EMSA, indicating a region critical for binding (between dashed lines). (E) EMSA with 50 nM of indicated 60 bp DNA substrate (from panel D) and 0, 250, 200, 150,100, 75, 50 nM CasPRIBI, resolved on native acrylamide gels. (F) Seven diverse natural variants of the CasPRIBI binding site that vary from the site in LS1 at sites in bold were tested for binding in vitro. Substrates in red exhbited severely reduced binding. Quantification of variant binding relative to LS1 is shown for four replicates. Binding sites in strains LS43 and LS51, whose in vivo CasPR sensitivity is measured in , are also shown.

Article Snippet: Samples were resolved on 8% native acrylamide gels (29:1 acrylamide:bisacrylamide; Bio-Rad) containing 0.5 X TBE.

Techniques: Purification, Molecular Weight, Marker, Sequencing, Binding Assay, In Vitro, Variant Assay, In Vivo

Journal: bioRxiv

Article Title: In Vivo Selection of anti-glioblastoma DNA aptamer-drug conjugates in an orthotopic patient-derived xenograft model

doi: 10.64898/2026.02.16.706148

Figure Lengend Snippet: A. Chemical structure of PEG4-Val-Cit-PAB-MMAE conjugated to the 5’ end of a DNA aptamer. B. A polyacrylamide denaturing gel post-stained with SYBR gold showing that MMAE-PAB-Val-Cit-PEG4 can be conjugated to DNA primers by trans-cyclooctene-tetrazine (TCO-Tz) click chemistry. C. Confirmation of MMAE structural stability by LC-MS following 20 rounds of thermal cycling conditions. ISTD= internal standard D. Confirmation that Taq polymerase could incorporate a 5’MMAE conjugated DNA primer. Two PCR reactions were run: one with an unmodified forward primer (lanes 2-6) and one with 5’MMAE conjugated forward primer (lanes 7-11). Samples were taken throughout the PCR reaction and run on a native polyacrylamide gel and post-stained with SYBR gold. p=primers, mp = 5’MMAE conjugated primer, and *=amplicon

Article Snippet: Solutions were subjected to electrophoresis through 8% native polyacrylamide gels (29:1 acrylamide:bisacrylamide Bio-Rad #1610146) and stained with SYBR gold (Invitrogen #S11494).

Techniques: Staining, Liquid Chromatography with Mass Spectroscopy, Amplification

Journal: bioRxiv

Article Title: In Vivo Selection of anti-glioblastoma DNA aptamer-drug conjugates in an orthotopic patient-derived xenograft model

doi: 10.64898/2026.02.16.706148

Figure Lengend Snippet: A. A schematic diagram of the in vivo SELEX with an ApDC library. B. Native polyacrylamide gel shift assay detecting the presence of MMAE with an anti-MMAE antibody in the naïve library used for Round 1 and the final library used for Round 10. -F = free library, -S = shifted library due to anti-MMAE antibody-ApDC complex C. Top 10 sequences by area under the curve at Round 10 from NGS. D. Biodistribution of the top 5 sequences identified at Round 10 in the Round 10 mouse.

Article Snippet: Solutions were subjected to electrophoresis through 8% native polyacrylamide gels (29:1 acrylamide:bisacrylamide Bio-Rad #1610146) and stained with SYBR gold (Invitrogen #S11494).

Techniques: In Vivo, Gel Shift

Journal: bioRxiv

Article Title: In Vivo Selection of anti-glioblastoma DNA aptamer-drug conjugates in an orthotopic patient-derived xenograft model

doi: 10.64898/2026.02.16.706148

Figure Lengend Snippet: Mice were injected I.P. with 350 µl of 1 µM ApDC/aptamer cocktail (50 pmol per sequence) or vehicle. The cocktail was composed of the 5 top ApDC candidates, a scrambled sequence of ApDC 1, and the negative control aptamer sequence used in our previous in vivo SLELEX. 4 h post-injection, the mice were euthanized and thoroughly perfused prior to organ harvest. Tumor was detected by GFP positive staining. Aptamers were isolated from organs and detected with sequence specific PCR primers. A. Denaturing polyacrylamide gel stained with SYBR gold confirming the size shift and purity of the ApDCs in the cocktail injected into the mice. B. The amount of ApDCs and negative control aptamer detected in the GFP positive brain tumor. C. Comparison of the levels of ApDCs detected in the brain tumor compared to other areas of the brain. D. Levels of aptamer detected in tissues sensitive to anti-tubulin agents like MMAE. E. Whole organ biodistribution of the candidates across 5 mice. *= comparison to ApDC 6. ‡ = comparison to Ap 7. *p<0.05; **p<0.01, ***p<0.001, and ****p<0.0001.

Article Snippet: Solutions were subjected to electrophoresis through 8% native polyacrylamide gels (29:1 acrylamide:bisacrylamide Bio-Rad #1610146) and stained with SYBR gold (Invitrogen #S11494).

Techniques: Injection, Sequencing, Negative Control, In Vivo, Staining, Isolation, Comparison