Sensitive detection of fat mass and obesity-associated protein based on terminal deoxynucleotidyl transferase-mediated signal amplification

Ma, Xuemei; Shi, Liyi; Xie, Haojie; Kuang, Deqi; Xu, Yetao; Ma, Jianhua; Yang, Jie; Sun, Lizhou

doi:10.1016/j.microc.2022.108131

Cited by 5 publications

(1 citation statement)

References 36 publications

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“…We envisioned these could aid in synthesis of aptamer libraries 5 . In addition, G/C-biased variants are desirable subjects for in vitro testing, since G-biased TdTs may improve the performance of biosensors that synthesize G-quadruplexes for signal generation 16,[46][47][48][49][50][51][52][53][54][55][56][57] or enable the use of these biosensors in crude biosamples (without needing to purify specimens to remove all 4 natural nucleotides). However, because G/C-biased variants were rare among the library members we profiled in our multiplex assay, we attempted to amplify G/C bias by creating a double mutant possessing mutations from two modestly G/C-biased variants isolated from libraries 2 and 3 (L398M from library 2, plus R454D and E457D from library 3).…”

Section: In Vitro Biases Of Tdt Mutants Match Those Determined In The...mentioning

confidence: 99%

A massively parallelin vivoassay of TdT mutants yields variants with altered nucleotide insertion biases

Carlson,

Loveless,

Milisavljevic

et al. 2024

Preprint

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Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase capable of template-independent extension of DNA with random nucleotides. TdT’sde novoDNA synthesis ability has found utility in DNA recording, DNA data storage, oligonucleotide synthesis, and nucleic acid labeling, but TdT’s intrinsic nucleotide biases limit its versatility in such applications. Here, we describe a multiplexed assay for profiling and engineering the bias and overall activity of TdT variants in high throughput. In our assay, a library of TdTs is encoded next to a CRISPR-Cas9 target site in HEK293T cells. Upon transfection of Cas9 and sgRNA, the target site is cut, allowing TdT to intercept the double strand break and add nucleotides. Each resulting insertion is sequenced alongside the identity of the TdT variant that generated it. Using this assay, 25,623 unique TdT variants, constructed by site-saturation mutagenesis at strategic positions, were profiled. This resulted in the isolation of several altered-bias TdTs that expanded the capabilities of our TdT-based DNA recording system, Cell History Recording by Ordered Insertion (CHYRON), by increasing the information density of recording through an unbiased TdT and achieving dual-channel recording of two distinct inducers (hypoxia and Wnt) through two differently biased TdTs. Select TdT variants were also testedin vitro, revealing concordance between each variant’sin vitrobias and thein vivobias determined from the multiplexed high throughput assay. Overall, our work, and the multiplex assay it features, should support the continued development of TdT-based DNA recorders,in vitroapplications of TdT, and further study of the biology of TdT.

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Section: In Vitro Biases Of Tdt Mutants Match Those Determined In The...mentioning

confidence: 99%

A massively parallelin vivoassay of TdT mutants yields variants with altered nucleotide insertion biases

Carlson,

Loveless,

Milisavljevic

et al. 2024

Preprint

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Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase

Gao

Song

Chen

et al. 2023

Analytica Chimica Acta

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Label-free and sensitive detection of N6-methyladenosine demethylase activity in crude cell extracts and clinical cancer tissues based on demethylation-triggered exponential signal amplification

Li,

Zhang,

Liu

et al. 2023

Analytica Chimica Acta

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Sensitive detection of fat mass and obesity-associated protein based on terminal deoxynucleotidyl transferase-mediated signal amplification

Cited by 5 publications

References 36 publications

A massively parallelin vivoassay of TdT mutants yields variants with altered nucleotide insertion biases

A massively parallelin vivoassay of TdT mutants yields variants with altered nucleotide insertion biases

Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase

Label-free and sensitive detection of N6-methyladenosine demethylase activity in crude cell extracts and clinical cancer tissues based on demethylation-triggered exponential signal amplification

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