•SICLOPPS allows the rapid generation of cyclic peptide libraries in cells.•A variety of cyclic peptide ring sizes may be generated with SICLOPPS.•SICLOPPS libraries contain up to ∼109 unique ...members.•SICLOPPS can be combined with any cell-based assay for inhibitor identification.•SICLOPPS has been employed against a multitude of ‘difficult’ targets in various organisms.
Cyclic peptide libraries have demonstrated significant potential when employed against challenging targets such as protein–protein interactions. While a variety of methods for library generation exist, genetically encoded libraries hold several advantages over their chemically synthesized counterparts; they are more readily accessible and allow straightforward hit deconvolution. One method for the intracellular generation of such libraries is split-intein circular ligation of peptides and proteins (SICLOPPS). Here we detail and discuss the deployment of SICLOPPS libraries for the identification of cyclic peptide inhibitors of a variety of targets.
The chemical synthesis of oligonucleotides and their enzyme-mediated assembly into genes and genomes has significantly advanced multiple scientific disciplines. However, these approaches are not ...without their shortcomings; enzymatic amplification and ligation of oligonucleotides into genes and genomes makes automation challenging, and site-specific incorporation of epigenetic information and/or modified bases into large constructs is not feasible. Here we present a fully chemical one-pot method for the assembly of oligonucleotides into a gene by click-DNA ligation. We synthesize the 335 base-pair gene that encodes the green fluorescent protein iLOV from ten functionalized oligonucleotides that contain 5'-azide and 3'-alkyne units. The resulting click-linked iLOV gene contains eight triazoles at the sites of chemical ligation, and yet is fully biocompatible; it is replicated by DNA polymerases in vitro and encodes a functional iLOV protein in Escherichia coli. We demonstrate the power and potential of our one-pot gene-assembly method by preparing an epigenetically modified variant of the iLOV gene.
Cyclic peptides are an emerging class of molecular therapeutics that are increasingly viewed as ideal backbones for modulation of protein–protein interactions. A split‐intein based method, termed ...SICLOPPS, enables the rapid generation of genetically encoded cyclic peptide libraries of around a hundred million members. Here we review recent approaches using SICLOPPS for the discovery of bioactive compounds.
A real eye‐opener, SICLOPPS is a plasmid‐based method for the production of genetically encoded cyclic peptide libraries of over a hundred million members, which has been adopted and adapted by multiple groups. Here an overview of the various instances of SICLOPPS being used for the identification of cyclic peptide inhibitors of a given protein target is provided.
In this article we describe the production and screening of a genetically encoded library of 10
lanthipeptides in Escherichia coli using the substrate-tolerant lanthipeptide synthetase ProcM. This ...plasmid-encoded library was combined with a bacterial reverse two-hybrid system for the interaction of the HIV p6 protein with the UEV domain of the human TSG101 protein, which is a critical protein-protein interaction for HIV budding from infected cells. Using this approach, we identified an inhibitor of this interaction from the lanthipeptide library, whose activity was verified in vitro and in cell-based virus-like particle-budding assays. Given the variety of lanthipeptide backbone scaffolds that may be produced with ProcM, this method may be used for the generation of genetically encoded libraries of natural product-like lanthipeptides containing substantial structural diversity. Such libraries may be combined with any cell-based assay to identify lanthipeptides with new biological activities.
We present a simple strategy for the synthesis of main chain oligonucleotide rotaxanes with precise control over the position of the macrocycle. The novel DNA-based rotaxanes were analyzed to assess ...the effect of the mechanical bond on their properties.
Split intein circular ligation of peptides and proteins (SICLOPPS) is a genetically encoded method for the intracellular production of cyclic peptide libraries of around 100 million (108) members ...that utilizes the Synechocystis sp PCC6803 (Ssp) DnaE split inteins. However, Ssp inteins are relatively slow splicing and intolerant of amino acid variation around the splice junction, potentially limiting the utility and composition of SICLOPPS libraries. In contrast, Nostoc punctiforme (Npu) DnaE split inteins not only splice significantly faster, they are also much more tolerant of amino acid variation around their splice junctions. Here, we report the use of engineered Npu inteins in SICLOPPS for the generation of cyclic peptide libraries and cyclic proteins. Despite their superior splicing characteristics, however, we observed a high level of toxicity from the Npu SICLOPPS constructs in E. coli. The observed toxicity was overcome though incorporation of an SsrA tag to target the spliced Npu inteins to the ClpXP complex for degradation. The resulting traceless Npu SICLOPPS inteins showed no toxicity to E. coli, demonstrating their potential for the production of cyclic peptide libraries for use in a variety of high-throughput screens.
A triazole mimic of a DNA phosphodiester linkage has been produced by templated chemical ligation of oligonucleotides functionalized with 5'-azide and 3'-alkyne. The individual azide and alkyne ...oligonucleotides were synthesized by standard phosphoramidite methods and assembled using a straightforward ligation procedure. This highly efficient chemical equivalent of enzymatic DNA ligation has been used to assemble a 300-mer from three 100-mer oligonucleotides, demonstrating the total chemical synthesis of very long oligonucleotides. The base sequences of the DNA strands containing this artificial linkage were copied during PCR with high fidelity and a gene containing the triazole linker was functional in Escherichia coli.
Recent advances in chemical biology and the advantages presented by in vivo screening have highlighted the need for a robust and flexible biologically synthesized small-molecule library. Herein we ...describe a method for the biosynthesis of cyclic peptide libraries of up to 10(8) members in Escherichia coli using split-intein circular ligation of peptides and proteins (SICLOPPS). The method utilizes split-intein chemistry to cyclize randomized peptide sequences. The cyclic peptide library can potentially be of any size and the peptide itself may contain unlimited random residues. However, the library size is limited by the transformation efficiency of E. coli and random residues are generally limited to five, but additional amino acids can be used in the cyclic peptide backbone, varying the structure and ring size of the cyclic peptide. SICLOPPS libraries have been combined with a bacterial reverse two-hybrid system in our labs and used in the identification of inhibitors of several protein-protein interactions. This protocol is expected to take around 3-4 weeks to implement.
The budding of HIV from infected cells is driven by the protein–protein interaction between the p6 domain of the HIV Gag protein and the UEV domain of the human TSG101 protein. We report the ...development of a cyclic peptide inhibitor of the p6/UEV interaction, from a non cell-permeable parent that was identified in a SICLOPPS screen. Amino acids critical for the activity of the parent cyclic peptide were uncovered using alanine-scanning, and a series of non-natural analogues synthesized and assessed. The most potent molecule disrupts the p6/UEV interaction with an IC50 of 6.17 ± 0.24 μM by binding to UEV with a K d of 11.9 ± 2.8 μM. This compound is cell permeable and active in a cellular virus-like particle budding assay with an IC50 of ∼2 μM. This work further demonstrates the relative simplicity with which the potency and activity of cyclic peptides identified from SICLOPPS libraries can be optimized.
Click DNA ligation promises an alternative to the current enzymatic approaches for DNA assembly, with the ultimate goal of using efficient chemical reactions for the total chemical synthesis and ...assembly of genes and genomes. Such an approach would enable the incorporation of various chemically modified bases throughout long stretches of DNA, a feat not possible with current polymerase‐based methods. An unequivocal requirement for this approach is the biocompatibility of the resulting triazole‐linked DNA. The correct function of this unnatural DNA linker in human cells is demonstrated here by using a click‐linked gene encoding the fluorescent protein mCherry. Reverse transcription of mRNA isolated from these cells and subsequent sequencing of the mCherry cDNA shows error‐free transcription. Nucleotide excision repair (NER) is shown to not play a role in the observed biocompatibility by using a NER‐deficient human cell line. This is the first example of a non‐natural DNA linker being functional in a eukaryotic cell.
Just a click away: Human cells are shown to correctly transcribe through a non‐natural DNA‐backbone linker. Triazole‐linked DNA encoding the red fluorescent protein mCherry is shown to be functional in human cells without the need for nucleotide excision repair. These results challenge the idea that a phosphodiester backbone is essential for the biological function of DNA and open up the possibility of total chemical synthesis of genes by click DNA ligation.
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