ABSTRACT
In early mitosis, the duplicated chromosomes are held together by the ring-shaped cohesin complex1. Separation of chromosomes during anaphase is triggered by separase-a large cysteine endopeptidase that cleaves the cohesin subunit SCC1 (also known as RAD212-4). Separase is activated by degradation of its inhibitors, securin5 and cyclin B6, but the molecular mechanisms of separase regulation are not clear. Here we used cryogenic electron microscopy to determine the structures of human separase in complex with either securin or CDK1-cyclin B1-CKS1. In both complexes, separase is inhibited by pseudosubstrate motifs that block substrate binding at the catalytic site and at nearby docking sites. As in Caenorhabditis elegans7 and yeast8, human securin contains its own pseudosubstrate motifs. By contrast, CDK1-cyclin B1 inhibits separase by deploying pseudosubstrate motifs from intrinsically disordered loops in separase itself. One autoinhibitory loop is oriented by CDK1-cyclin B1 to block the catalytic sites of both separase and CDK19,10. Another autoinhibitory loop blocks substrate docking in a cleft adjacent to the separase catalytic site. A third separase loop contains a phosphoserine6 that promotes complex assembly by binding to a conserved phosphate-binding pocket in cyclin B1. Our study reveals the diverse array of mechanisms by which securin and CDK1-cyclin B1 bind and inhibit separase, providing the molecular basis for the robust control of chromosome segregation.
Subject(s)
CDC2 Protein Kinase/chemistry , CDC2 Protein Kinase/metabolism , Cyclin B1/chemistry , Cyclin B1/metabolism , Securin/chemistry , Securin/metabolism , Separase/chemistry , Separase/metabolism , Amino Acid Motifs , CDC2 Protein Kinase/antagonists & inhibitors , CDC2 Protein Kinase/ultrastructure , CDC2-CDC28 Kinases/chemistry , CDC2-CDC28 Kinases/metabolism , CDC2-CDC28 Kinases/ultrastructure , Cell Cycle Proteins/metabolism , Chromosome Segregation , Cryoelectron Microscopy , Cyclin B1/ultrastructure , DNA-Binding Proteins/metabolism , Humans , Models, Molecular , Phosphoserine/metabolism , Protein Binding , Protein Domains , Securin/ultrastructure , Separase/antagonists & inhibitors , Separase/ultrastructure , Substrate SpecificityABSTRACT
Stapled peptides with an enforced α-helical conformation have been shown to overcome major limitations in the development of short peptides targeting protein-protein interactions (PPIs). While the growing arsenal of methodologies to staple peptides facilitates their preparation, stapling methodologies are not broadly embraced in synthetic library screening. Herein, we report a strategy leveraged on hybridization of short PNA-peptide conjugates wherein nucleobase driven assembly facilitates ligation of peptide fragments and constrains the peptide's conformation into an α-helix. Using native chemical ligation, we show that a mixture of peptide fragments can be combinatorially ligated and used directly in affinity selection against a target of interest. This approach was exemplified with a focused library targeting the p-53/MDM2 interaction. One hundred peptides were obtained in a one-pot ligation reaction, selected by affinity against MDM2 immobilized on beads, and the best binders were identified by mass spectrometry.
Subject(s)
Peptide Nucleic Acids/metabolism , Peptides/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Humans , Nucleic Acid Hybridization , Peptide Library , Peptide Nucleic Acids/chemistry , Protein Binding/drug effects , Protein Conformation, alpha-Helical , Proto-Oncogene Proteins c-mdm2/chemistry , Tumor Suppressor Protein p53/chemistryABSTRACT
Life is orchestrated by biomolecules interacting in complex networks of biological circuitry with emerging function. Progress in different areas of chemistry has made the design of systems that can recapitulate elements of such circuitry possible. Herein we review prominent examples of networks, the methodologies available to translate an input into various outputs, and speculate on potential applications and directions for the field. The programmability of nucleic acid hybridization has inspired applications beyond its function in heredity. At the circuitry level, DNA provides a powerful platform to design dynamic systems that respond to nucleic acid input sequences with output sequences through diverse logic gates, enabling the design of ever more complex circuitry. In order to interface with more diverse biomolecular inputs and yield outputs other than oligonucleotide sequences, an array of nucleic acid conjugates have been reported that can engage proteins as their input and yield a turn-on of enzymatic activity, a bioactive small molecule, or morphological changes in nanoobjects. While the programmability of DNA makes it an obvious starting point to design circuits, other biosupramolecular interactions have also been demonstrated, and harnessing progress in protein design is bound to deliver further integration of macromolecules in artificial circuits.
Subject(s)
DNA/chemistry , Small Molecule Libraries/chemistry , Macromolecular Substances/chemistryABSTRACT
Fluorescence-based oligonucleotide (ON) hybridization probes greatly aid the detection and profiling of RNA sequences in cells. However, certain limitations such as target accessibility and hybridization efficiency in cellular environments hamper their broad application because RNAs can form complex and stable structures. In this context, we have developed a robust hybridization probe suitable for imaging RNA in cells by combining the properties of 1)â a new microenvironment-sensitive fluorescent nucleobase analogue, obtained by attaching the Lucifer chromophore (1,8-naphthalimide) at the 5-position of uracil, and 2)â a peptide nucleic acid (PNA) capable of forming stable hybrids with RNA. The fluorescence of the PNA base analogue labeled with the Lucifer chromophore, when incorporated into PNA oligomers and hybridized to complementary and mismatched ONs, is highly responsive to its neighboring base environment. Notably, the PNA base reports the presence of an adenine repeat in an RNA ON with reasonable enhancement in fluorescence. This feature of the emissive analogue enabled the construction of a poly(T) PNA probe for the efficient visualization of polyadenylated [poly(A)] RNAs in cells-poly(A) being an important motif that plays vital roles in the lifecycle of many types of RNA. Our results demonstrate that such responsive fluorescent nucleobase analogues, when judiciously placed in PNA oligomers, could generate useful hybridization probes to detect nucleic acid sequences in cells and also to image them.
Subject(s)
Fluorescent Dyes/chemistry , Peptide Nucleic Acids/chemistry , Poly A/chemistry , RNA/chemistryABSTRACT
Double-stranded segments of a genome that can potentially form G-quadruplex (GQ) and/or i-motif (iM) structures are considered to be important regulatory elements. Hence, the development of a common probe that can detect GQ and iM structures and also distinguish them from a duplex structure will be highly useful in understanding the propensity of such segments to adopt duplex or non-canonical four-stranded structures. Here, we describe the utility of a conformation-sensitive fluorescent nucleoside analog, which was originally developed as a GQ sensor, in detecting the iM structures of C-rich DNA oligonucleotides (ONs). The analog is based on a 5-(benzofuran-2-yl)uracil scaffold, which when incorporated into C-rich ONs (e.g., telomeric repeats) fluorescently distinguishes an iM from random coil and duplex structures. Steady-state and time-resolved fluorescence techniques enabled the determination of transition pH for the transformation of a random coil to an iM structure. Furthermore, a qualitative understanding on the relative population of duplex and GQ/iM forms under physiological conditions could be gained by correlating the fluorescence, CD and thermal melting data. Taken together, this sensor could provide a general platform to profile double-stranded promoter regions in terms of their ability to adopt four-stranded structures, and also could support approaches to discover functional GQ and iM binders.
ABSTRACT
Fluorescent oligomers that are resistant to enzymatic degradation and report their binding to target oligonucleotides (ONs) by changes in fluorescence properties are highly useful in developing nucleic-acid-based diagnostic tools and therapeutic strategies. Here, we describe the synthesis and photophysical characterization of fluorescent peptide nucleic acid (PNA) building blocks made of microenvironment-sensitive 5-(benzofuran-2-yl)- and 5-(benzothiophen-2-yl)-uracil cores. The emissive monomers, when incorporated into PNA oligomers and hybridized to complementary ONs, are minimally perturbing and are highly sensitive to their neighboring base environment. In particular, benzothiophene-modified PNA reports the hybridization process with significant enhancement in fluorescence intensity, even when placed in the vicinity of guanine residues, which often quench fluorescence. This feature was used in the turn-on detection of G-quadruplex-forming promoter DNA sequences of human proto-oncogenes (c-myc and c-kit). Furthermore, the ability of benzothiophene-modified PNA oligomer to report the presence of an abasic site in RNA enabled us to develop a simple fluorescence hybridization assay to detect and estimate the depurination activity of ribosome-inactivating protein toxins. Our results demonstrate that this approach with responsive PNA probes will provide new opportunities to develop robust tools to study nucleic acids.
Subject(s)
Fluorescence , G-Quadruplexes , Oncogenes/genetics , Peptide Nucleic Acids/analysis , Peptide Nucleic Acids/chemistry , Ribosome Inactivating Proteins/metabolism , Humans , Molecular Structure , Peptide Nucleic Acids/chemical synthesisABSTRACT
Peptide nucleic acid (PNA) stands as one of the most successful artificial oligonucleotide mimetics. Salient features include the stability of hybridization complexes (either as duplexes or triplexes), metabolic stability, and ease of chemical modifications. These features have enabled important applications such as antisense agents, gene editing, nucleic acid sensing and as a platform to program the assembly of PNA-tagged molecules. Here, we review recent advances in these areas.
Subject(s)
Diagnosis , Peptide Nucleic Acids/chemistry , Therapeutics , Animals , Antisense Elements (Genetics) , Biosensing Techniques , Gene Editing , Humans , Molecular Probes , Nucleic Acids/analysis , Nucleic Acids/chemistryABSTRACT
The ability to bind strongly to complementary nucleic acid sequences, invade complex nucleic acid structures, and resist degradation by cellular enzymes has made peptide nucleic acid (PNA) oligomers as very useful hybridization probes in molecular diagnosis. For such applications, the PNA oligomers have to be labeled with appropriate reporters as they lack intrinsic labels that can be used in biophysical assays. Although solid-phase synthesis is commonly used to attach reporters onto PNA, development of milder and modular labeling methods will provide access to PNA oligomers labeled with a wider range of biophysical tags. Here, we describe the establishment of a postsynthetic modification strategy based on bioorthogonal chemical reactions in functionalizing PNA oligomers in solution with a variety of tags. A toolbox composed of alkyne- and azide-modified monomers were site-specifically incorporated into PNA oligomers and postsynthetically click-functionalized with various tags, ranging from sugar, amino acid, biotin, to fluorophores, by using copper(I)-catalyzed azide-alkyne cycloaddition, strain-promoted azide-alkyne cycloaddition, and Staudinger ligation reactions. As a proof of utility of this method, fluorescent PNA hybridization probes were developed and used in imaging human telomeres in chromosomes and poly(A) RNAs in cells. Taken together, this simple approach of generating a wide range of functional PNA oligomers will expand the use of PNA in molecular diagnosis.
ABSTRACT
Given the biological and therapeutic significance of telomeres and other G-quadruplex forming sequences in human genome, it is highly desirable to develop simple methods to study these structures, which can also be implemented in screening formats for the discovery of G-quadruplex binders. The majority of telomere detection methods developed so far are laborious and use elaborate assay and instrumental setups, and hence, are not amenable to discovery platforms. Here, we describe the development of a simple homogeneous fluorescence turn-on method, which uses a unique combination of an environment-sensitive fluorescent nucleobase analogue, the superior base pairing property of PNA, and DNA-binding and fluorescence quenching properties of graphene oxide, to detect human telomeric DNA repeats of varying lengths. Our results demonstrate that this method, which does not involve a rigorous assay setup, would provide new opportunities to study G-quadruplex structures.