Sequencing Mirror-Image DNA Chemically

Liu, Xianyu; Zhu, Ting

doi:10.1016/j.chembiol.2018.06.005

Cited by 9 publications

(5 citation statements)

References 24 publications

(37 reference statements)

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“…Another key technique required for realizing mirror-image selection is the sequencing of enriched l -DNA aptamers. One potential approach would be to amplify l -DNAs from single molecules (similar to cell-free cloning through limiting dilution 17 ) to isolate single l -DNA aptamers for downstream l -DNA sequencing 14 , 18 . However, the amplification efficiencies of the current mirror-image PCR systems based on mirror-image Dpo4 and Pfu DNA polymerase are not sufficient for amplifying single-molecule templates 14 – 16 .…”

Section: Resultsmentioning

confidence: 99%

Directed evolution and selection of biostable l-DNA aptamers with a mirror-image DNA polymerase

Chen

Zhu

2022

Nat Biotechnol

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Mirror-image aptamers made from chirally inverted nucleic acids are nuclease-resistant and exceptionally biostable, opening up opportunities for unique applications. However, the directed evolution and selection of mirror-image aptamers directly from large randomized l-DNA libraries has, to our knowledge, not been demonstrated previously. Here, we developed a ‘mirror-image selection’ scheme for the directed evolution and selection of biostable l-DNA aptamers with a mirror-image DNA polymerase. We performed iterative rounds of enrichment and mirror-image polymerase chain reaction (PCR) amplification of l-DNA sequences that bind native human thrombin, in conjunction with denaturing gradient gel electrophoresis (DGGE) to isolate individual aptamers and l-DNA sequencing-by-synthesis to determine their sequences. Based on the selected l-DNA aptamers, we designed biostable thrombin sensors and inhibitors, which remained functional in physiologically relevant nuclease-rich environments, even in the presence of human serum that rapidly degraded d-DNA aptamers. Mirror-image selection of biostable l-DNA aptamers directly from large randomized l-DNA libraries greatly expands the range of biomolecules that can be targeted, broadening their applications as biostable sensors, therapeutics and basic research tools.

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Section: Resultsmentioning

confidence: 99%

Directed evolution and selection of biostable l-DNA aptamers with a mirror-image DNA polymerase

Chen

Zhu

2022

Nat Biotechnol

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“…The emergence of rapid DNA sequencing techniques has significantly expedited advancements in biological and medical research. The traditional methods of DNA sequencing comprise: (1) the Chemical Method, also referred to as the Maxam–Gilbert method (Liu & Zhu, 2018 ); (2) the Chain Termination Method, also recognized as the Sanger dideoxy method (Kimoto et al., 2020 ); and (3) the next‐generation sequencing (NGS) technologies (Slatko et al., 2018 ). However, these detection techniques have several shortcomings and hazards, most notably their short reads, making it potentially difficult to detect a particular single nucleotide variation (SNV) at both the analytical and interpretation levels (Kumar et al., 2019 ).…”

Section: Discussionmentioning

confidence: 99%

A new genetic diagnosis strategy for paroxysmal kinesigenic dyskinesia: Targeted high‐throughput detection of PRRT2 gene c.649 locus

Wen,

Huang,

Huang

et al. 2024

Molec Gen & Gen Med

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BackgroundParoxysmal kinesigenic dyskinesia (PKD) is the most prevalent kind type of paroxysmal Dyskinesia, characterized by recurrent and transient episodes of involuntary movements. Most PKD cases were attributed to the proline‐rich transmembrane protein 2 (PRRT2) gene, in which the c.649 region is a hotspot for known mutations. Even though some patients with PKD have been genetically diagnosed using whole‐exome sequencing (WES) and Sanger sequencing, there are still cases of missed diagnoses due to the limitations of sequencing technology and analytic methods on throughput.MethodsPatients meeting the diagnosis criteria of PKD with negative results of PRRT2‐Sanger sequencing and WES were included in this study. Mutation screening and targeted high‐throughput sequencing were performed to analyze and verify the sequencing results of the potential mutations.ResultsSix patients with PKD with high mutation ratios of c.649dupC were screened using our targeted high‐throughput sequencing from 26 PKD patients with negative results of PRRT2‐Sanger sequencing and WES (frequency = 23.1%), which compensated for the comparatively shallow sequencing depth and statistical flaws in this region. Compared with the local normal population and other patients with PKD, the mutation ratios of c.649dupC of these six patients with PKD were much higher and also had truncated protein structures and differentially altered mRNA expression.ConclusionBased on the above studies, we emphasize the routine targeted high‐throughput sequencing of the c.649 site in the PRRT2 gene in so‐called genetic‐testing‐negative patients with PKD, and manually calculate the deletion and duplication mutations depth and ratios to lower the rate of clinical misdiagnosis.

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“…Blocking groups mainly based on nitrobenzyl 201 or substituted phenol groups 202 have been appended at virtually any position of the nucleosidic scaffold and the intended function of the photocaged DNA or RNA oligonucleotides can be restored by photoirradiation which removes the light sensitive groups. An alternative strategy involves a transient blockade of the function of RNA by the installation of an acetylation pattern on the 2′-OH groups of RNA.…”

Section: -191mentioning

confidence: 99%