High-throughput sequencing SELEX for the determination of DNA-binding protein specificities in vitro

Pantier, Raphaël; Chhatbar, Kashyap; Alston, Grace; Lee, Heng Yang; Bird, Adrian

doi:10.1016/j.xpro.2022.101490

Cited by 4 publications

(3 citation statements)

References 15 publications

(28 reference statements)

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“…SELEX coupled with high-throughput sequencing (HT-SELEX) was performed as previously described (Pantier et al, 2022), in triplicate experiments. Oligonucleotides were ordered from Integrated DNA Technologies.…”

Section: Ht-selexmentioning

confidence: 99%

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Structure of SALL4 zinc finger domain reveals link between AT-rich DNA binding and Okihiro syndrome

et al. 2023

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Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome, and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.

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Section: Ht-selexmentioning

confidence: 99%

“…All possible canonical k-mer sequences (k = 6) were searched individually in SELEX libraries at different cycles using eme_selex (Pantier et al, 2022). A canonical sequence of a k-mer pair is the lexicographically smaller of the two reverse complementary sequences.…”

Section: Ht-selex Analysismentioning

confidence: 99%

Structure of SALL4 zinc finger domain reveals link between AT-rich DNA binding and Okihiro syndrome

et al. 2023

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“…Protein-bound DNAs are separated from unbound DNAs through affinity purification and the resulting DNAs are amplified by PCR. This process is repeated until most DNAs remaining after PCR associate with the tested transcription factor; the resulting DNAs are then analyzed through high-throughput sequencing technologies ( 7 , 8 ). Separation of protein-bound DNAs in SELEX-seq has also been achieved through electromobility shift assays (EMSAs) ( 9 ) and filter-binding assays ( 10 ).…”

Section: Introductionmentioning

confidence: 99%