Computational design of sequence-specific DNA-binding proteins
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PROVIDER: PRJNA993300 | ENA |
REPOSITORIES: ENA
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Project description:Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove.
2024-07-05 | GSE237017 | GEO
Project description:There are numerous binders of the pro-survival BCL2 family proteins such as BCL2, MCL1, and BCL-XL, but development of potent and selective binders of their pro-apoptotic counterparts BAK and BAX has remained a major unsolved challenge. We use computational protein design to generate 13 kDa binders of BAK and BAX with 400 pM and 3 nM affinity, orders of magnitude higher than any existing native or designed binder, and with greater than 100-fold specificity against pro-survival BCL2 family members. The crystal structure of the BAKᐧɑBAK2 complex is very close to the computational design model, with the binder making specific interactions extending out from the canonical BH3-binding groove. Liposome- and cell-based analyses reveal that ɑBAK2 inhibits membrane permeabilization when in excess of BAK, but activates BAK when BAK is in excess. Structural analyses indicate that binding of ɑBAK2 results in partial unfolding and exposure of BAK’s BH3 domain. Similar to ɑBAK2, ɑBAX2 activates BAX at low concentrations and does not activate BAX at high concentrations. This work provides valuable insight into design of small molecule or protein inhibitors of BAK and BAX; inhibition requires high affinity binding as well as a saturating concentration of binder at the site of action. Our designs are the first binders with the high specificity required for efficient modulation of apoptosis via direct interaction with BAK and BAX and they provide highly selective molecular probes for addressing outstanding cell biological questions about cell death.
2025-05-01 | GSE266473 | GEO
Project description:Computational design of potent and selective BAK and BAX binders
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Project description:RNA-binding proteins that lack canonical RNA-binding domains are rarely sequence-specific
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Project description:CLIP1 and DMD are two novel RNA-binding proteins through computational prediction and experimental validation
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Project description:Thousands of RNA-binding proteins (RBPs) crosslink to cellular mRNA. Among these are numerous unconventional RBPs (ucRBPs)—proteins that associate with RNA but lack known RNA-binding domains (RBDs). The vast majority of ucRBPs have uncharacterized RNA-binding specificities. We analyzed 492 human ucRBPs for intrinsic RNA-binding in vitro and identified 23 that bind specific RNA sequences. Most (17/23), including 8 ribosomal proteins, were previously associated with RNA-related function. We identified the RBDs responsible for sequence-specific RNA-binding for several of these 23 ucRBPs and surveyed whether corresponding domains from homologous proteins also display RNA sequence specificity. CCHC-zf domains from seven human proteins recognized specific RNA motifs, indicating that this is a major class of RBD. For Nudix, HABP4, TPR, RanBP2-zf, and L7Ae domains, however, only isolated members or closely related homologs yielded motifs, consistent with RNA-binding as a derived function. The lack of sequence specificity for most ucRBPs is striking, and we suggest that many may function analogously to chromatin factors, which often crosslink efficiently to cellular DNA, presumably via indirect recruitment. Finally, we show that ucRBPs tend to be highly abundant proteins and suggest their identification in RNA interactome capture studies could also result from weak nonspecific interactions with RNA.
2023-03-31 | GSE215198 | GEO
Project description:Integrin α5β1 is crucial for cell attachment and migration in development and tissue regeneration, and α5β1 binding proteins could have considerable utility in regenerative medicine and next-generation therapeutics. We use computational protein design to create de novo α5β1-specific modulating miniprotein binders, called NeoNectins, that bind to and stabilize the open state of α5β1. When immobilized onto titanium surfaces and throughout 3D hydrogels, the NeoNectins outperform native fibronectin and RGD peptide in enhancing cell attachment and spreading, and NeoNectin-grafted titanium implants outperformed fibronectin and RGD-grafted implants in animal models in promoting tissue integration and bone growth. NeoNectins should be broadly applicable for tissue engineering and biomedicine.
2025-08-01 | GSE272058 | GEO
Project description:Since RBPs play important roles in the cell, it’s particularly important to find new RBPs. We performed iRIP-seq and CLIP-seq to verify two proteins, CLIP1 and DMD, predicted by RBPPred whether are RBPs or not. The experimental results confirm that these two proteins have RNA-binding activity. We identified significantly enriched binding motifs UGGGGAGG, CUUCCG and CCCGU for CLIP1 (iRIP-seq), DMD (iRIP-seq) and DMD (CLIP-seq), respectively. The computational KEGG and GO analysis show that the CLIP1 and DMD share some biological processes and functions. Besides, we found that the SNPs between DMD and its RNA partners may associate with Becker muscular dystrophy, Duchenne muscular dystrophy, Dilated cardiomyopathy 3B and Cardiovascular phenotype. Among the thirteen cancers data, CLIP1 and another 300 genes always co-occur, and 123 of these 300 genes interact with CLIP1. These cancers may be associated with the mutations in both CLIP1 and the genes it interacts with.
2021-12-15 | GSE128318 | GEO