Isolation of Single‐Stranded DNA

Wakimoto, Yuji; Jiang, Jianming; Wakimoto, Hiroko

doi:10.1002/0471142727.mb0215s107

Cited by 8 publications

(9 citation statements)

References 19 publications

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“…Single-stranded Alu cDNA, Alu cDNA ISM− , 7SL cDNA, and 7SL cDNA ISM+ cDNA were isolated from biotinylated double-stranded PCR products synthesized from a linearized plasmid containing a consensus Alu Y or 7SL sequence using Dynabeads M-270 Streptavidin (Life Technologies) and then purified using QIAquick PCR purification kit (Qiagen catalog no. 28104) ( 46 ). Briefly, PCR products were biotinylated on one strand by synthesis with a biotinylated primer (forward 5′-biotin-GGGCCGGGCGCGGTG-3′ and reverse 5′-GTACCTTTAAAGAGACAGAGTCTCGC-3′ for Alu Y and forward 5′-biotin-CGTGCCTGTAGTCCCAGCTA-3′ and reverse 5′-AGACGGGGTCTCGCTATGTT-3′ for 7SL).…”

Section: Methodsmentioning

confidence: 99%

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity

et al. 2021

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Long interspersed nuclear element-1 (L1)-mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNAinduced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA-induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

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Section: Methodsmentioning

confidence: 99%

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity

et al. 2021

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“…For most experiments requiring long ssDNA (excluding GMP-compatible scale-up described separately below), a ssDNA isolation protocol adapted from Wakimoto et al using biotinylated primers and streptavidin-coated magnetic beads was used 34 . Amplicons were generated as described above using primers that include a 5' biotin modification (IDT) on either the forward or reverse PCR primer.…”

Section: Hdrt Template Preparationmentioning

confidence: 99%

Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing

Shy

Vykunta

et al. 2021

Preprint

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CRISPR-Cas9 offers unprecedented opportunities to modify genome sequences in primary human cells to study disease variants and reprogram cell functions for next-generation cellular therapies. CRISPR has several potential advantages over widely used retroviral vectors including: 1) site-specific transgene insertion via homology directed repair (HDR), and 2) reductions in the cost and complexity of genome modification. Despite rapid progress with ex vivo CRISPR genome engineering, many novel research and clinical applications would be enabled by methods to further improve knock-in efficiency and the absolute yield of live knock-in cells, especially with large HDR templates (HDRT). We recently reported that Cas9 target sequences (CTS) could be introduced into double-stranded DNA (dsDNA) HDRTs to improve knock-in, but yields and efficiencies were limited by toxicity at high HDRT concentrations. Here we developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA). We designed hybrid ssDNA HDRTs that incorporate CTS sites and were able to boost knock-in percentages by >5-fold and live cell yields by >7-fold relative to dsDNA HDRTs with CTS. Knock-in efficiency and yield with ssCTS HDRTs were increased further with small molecule inhibitor combinations to improve HDR. We demonstrate application of these methods across a variety of target loci, knock-in constructs, and primary human cell types to reach ultra-high HDR efficiencies (>80-90%) which we use for pathogenic gene variant modeling and universal gene replacement strategies for IL2RA and CTLA4 mutations associated with mendelian immune disorders. Finally, we develop a GMP-compatible method for fully non-viral CAR-T cell manufacturing, demonstrating knock-in efficiencies of 46-62% and generating yields of >1.5 x 10^9 CAR+ T cells, well above current doses for adoptive cellular therapies. Taken together, we present a comprehensive non-viral approach to model disease associated mutations and re-write targeted genome sequences to program immune cell therapies at a scale compatible with future clinical application.

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“…For instance, asymmetric PCR can generate ssDNA up to 15,000 nt in length 4 . Other approaches include PCR amplification using differentially modified primers: phosphorylated and unphosphorylated for lambda exonuclease digestion 5 , or biotinylated and non-biotinylated for streptavidin-bead separation 6 —resulting in isolation of long ssDNA strands. However, these techniques typically yield less than 1 microgram (μg) of ssDNA per 50-microliter (μL) reaction, making the production of milligram quantities costly and inefficient due to the extensive labor and high reagent consumption, underscoring the necessity for more scalable and economical ssDNA production methods.…”

Section: Introductionmentioning

confidence: 99%

Engineering an Escherichia coli strain for production of long single-stranded DNA

Shen,

Flood,

Zhang

et al. 2024

Preprint

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Long single-stranded DNA (ssDNA) is a versatile molecular reagent with applications including RNA-guided genome engineering and DNA nanotechnology, yet its production is typically resource-intensive. We introduce a novel method utilizing an engineered E. coli helper strain and phagemid system that simplifies long ssDNA generation to a straightforward transformation and purification procedure. Our method obviates the need for helper plasmids and their associated contamination by integrating M13mp18 genes directly into the E. coli chromosome. We achieved ssDNA lengths ranging from 504 to 20,724 nucleotides with titers up to 250 μg/L following alkaline-lysis purification. The efficacy of our system was confirmed through its application in primary T cell genome modifications and DNA origami folding. The reliability, scalability, and ease of our approach promises to unlock new experimental applications requiring large quantities of long ssDNA.

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Isolation of Single‐Stranded DNA

Cited by 8 publications

References 19 publications

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity

Hybrid ssDNA repair templates enable high yield genome engineering in primary cells for disease modeling and cell therapy manufacturing

Engineering an Escherichia coli strain for production of long single-stranded DNA

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