ABSTRACT
Nanobodies have emerged as promising tools in biomedicine due to their single-chain structure and inherent stability. They generally have convex paratopes, which potentially prefer different epitope sites in an antigen compared to traditional antibodies. In this study, a synthetic phage display nanobody library was constructed and used to identify nanobodies targeting a tumor-associated antigen, the human B7-H3 protein. Combining next-generation sequencing and single-clone validation, two nanobodies were identified to specifically bind B7-H3 with medium nanomolar affinities. Further characterization revealed that these two clones targeted a different epitope compared to known B7-H3-specific antibodies, which have been explored in clinical trials. Furthermore, one of the clones, dubbed as A6, exhibited potent antibody-dependent cell-mediated cytotoxicity (ADCC) against a colorectal cancer cell line with an EC50 of 0.67 nM, upon conversion to an Fc-enhanced IgG format. These findings underscore a cost-effective strategy that bypasses the lengthy immunization process, offering potential rapid access to nanobodies targeting unexplored antigenic sites.
ABSTRACT
Human DNA topoisomerase 1 (Top1) is a crucial enzyme responsible for alleviating torsional stress on DNA during transcription and replication, thereby maintaining genome stability. Previous researches had found that non-working Top1 interacted extensively with chromosomal DNA in human cells. However, the reason for its retention on chromosomal DNA remained unclear. In this study, we discovered a close association between Top1 and chromosomal DNA, specifically linked to the presence of G-quadruplex (G4) structures. G4 structures, formed during transcription, trap Top1 and hinder its ability to relax neighboring DNAs. Disruption of the Top1-G4 interaction using G4 ligand relieved the inhibitory effect of G4 on Top1 activity, resulting in a further reduction of R-loop levels in cells. Additionally, the activation of Top1 through the use of a G4 ligand enhanced the toxicity of Top1 inhibitors towards cancer cells. Our study uncovers a negative regulation mechanism of human Top1 and highlights a novel pathway for activating Top1.
Subject(s)
DNA Topoisomerases, Type I , G-Quadruplexes , Transcription, Genetic , Humans , DNA/chemistry , DNA Replication , DNA Topoisomerases, Type I/metabolism , Ligands , Topoisomerase I Inhibitors/pharmacologyABSTRACT
G-quadruplex is an essential element in gene transcription that serves as a promising drug target. Guanine-vacancy-bearing G-quadruplex (GVBQ) is a newly identified G-quadruplex that has distinct structural features from the canonical G-quadruplex. Potential GVBQ-forming motifs are widely distributed in gene promoter regions. However, whether GVBQ can form in genomic DNA and be an effective target for manipulating gene expression is unknown. Using photo-crosslinking, dimethyl sulfate footprinting, exonuclease digestion and in vitro transcription, we demonstrated the formation of a GVBQ in the G-rich nuclease hypersensitivity element within the human PDGFR-ß gene promoter region in both single-stranded and double-stranded DNA. The formation of GVBQ in dsDNA could be induced by negative supercoiling created by downstream transcription. We also found that the PDGFR-ß GVBQ was specifically recognized and stabilized by a new synthetic porphyrin guanine conjugate (mPG). Targeting the PDGFR-ß GVBQ in human cancer cells using the mPG could specifically alter PDGFR-ß gene expression. Our work illustrates that targeting GVBQ with mPG in human cells can regulate the expression level of a specific gene, thus indicating a novel strategy for drug development.
Subject(s)
G-Quadruplexes , Gene Expression Regulation , Promoter Regions, Genetic , Receptor, Platelet-Derived Growth Factor beta/genetics , Base Sequence , DNA/chemistry , DNA, Single-Stranded/chemistry , Humans , Porphyrins/chemistryABSTRACT
G-quadruplexes (G4s) formed by guanine-rich nucleic acids play a role in essential biological processes such as transcription and replication. Besides the >1.5 million putative G-4-forming sequences (PQSs), the human genome features >640 million single-nucleotide variations (SNVs), the most common type of genetic variation among people or populations. An SNV may alter a G4 structure when it falls within a PQS motif. To date, genome-wide PQS-SNV interactions and their impact have not been investigated. Herein, we present a study on the PQS-SNV interactions and the impact they can bring to G4 structures and, subsequently, gene expressions. Based on build 154 of the Single Nucleotide Polymorphism Database (dbSNP), we identified 5 million gains/losses or structural conversions of G4s that can be caused by the SNVs. Of these G4 variations (G4Vs), 3.4 million are within genes, resulting in an average load of >120 G4Vs per gene, preferentially enriched near the transcription start site. Moreover, >80% of the G4Vs overlap with transcription factor-binding sites and >14% with enhancers, giving an average load of 3 and 7.5 for the two regulatory elements, respectively. Our experiments show that such G4Vs can significantly influence the expression of their host genes. These results reveal genome-wide G4Vs and their impact on gene activity, emphasizing an understanding of genetic variation, from a structural perspective, of their physiological function and pathological implications. The G4Vs may also provide a unique category of drug targets for individualized therapeutics, health risk assessment, and drug development.
Subject(s)
DNA-Binding Proteins/ultrastructure , G-Quadruplexes , Genome, Human/genetics , Nucleic Acid Conformation , DNA-Binding Proteins/genetics , Gene Expression Regulation/genetics , Humans , Polymorphism, Single Nucleotide/genetics , Promoter Regions, Genetic/genetics , Protein Binding/genetics , Regulatory Sequences, Nucleic Acid/genetics , Transcription Initiation Site , Transcriptional Activation/geneticsABSTRACT
G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123 000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and â¼70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.
Subject(s)
G-Quadruplexes , Animals , Cell Line , DEAD-box RNA Helicases/genetics , DNA, Superhelical , DNA-Binding Proteins/metabolism , Genome , Humans , Mice , Recombinant Proteins/metabolism , Transcription, GeneticABSTRACT
Telomerase, a key enzyme involved in telomere homeostasis, is a major player involved in or required for sustained cell proliferation. It is expressed in â¼90% tumor but rarely in normal somatic cells. Therefore, telomerase serves as a diagnostic marker and therapeutic target of cancers. Although many methods are available for measuring telomerase activity, a convenient, fast, sensitive, and reliable method is still lacking for routine use in both clinics and research. Here, we present a single-enzyme sensitivity telomere repeat amplification protocol for quantifying telomerase activity. With multiple optimizations, the protocol pushes the ultimate detection limit down to a single telomerase complex, enabling measurement of telomerase activity of not only multiple cancerous/normal cell samples but also single cancer cells alone or even in the presence of 8000 normal cells. Implemented in a one-step mix-and-run format, the protocol offers a most sensitive, fast, accurate, and reproducible quantification of telomerase activity with linearity ranging from 20,000 HeLa cancer cells to a single telomerase complex. It requires minimal manual operation and experimental skill and is convenient for either low or high throughput of samples. We expect that the protocol should provide practical routine analyses of telomerase in both research and clinical applications. As an example, we demonstrate how telomerase activity evolves at the single-cell level and partitions in cell division in early mouse embryo development.
ABSTRACT
Stabilization of G-quadruplexes (G4s) formed in guanine-rich (G-rich) nucleic acids by small-molecule ligands has been extensively explored as a therapeutic approach for diseases such as cancer. Finding ligands with sufficient affinity and specificity toward G4s remains a challenge, and many ligands reported seemed to compromise between the two features. To cope with this challenge, we focused on targeting a particular type of G4s, i.e., the G-vacancy-bearing G-quadruplexes (GVBQs), by taking a structure complementation strategy to enhance both affinity and selectivity. In this approach, a G-quadruplex-binding peptide RHAU23 is guided toward a GVBQ by a guanine moiety covalently linked to the peptide. The filling-in of the vacancy in a GVBQ by the guanine ensures an exclusive recognition of GVBQ. Moreover, the synergy between the RHAU23 and the guanine dramatically improves both the affinity toward and stabilization of the GVBQ. Targeting a GVBQ in DNA by this bifunctional peptide strongly suppresses in vitro replication. This study demonstrates a novel and promising alternative targeting strategy to a distinctive panel of G4s that are as abundant as the canonical ones in the human genome.
Subject(s)
Guanine/chemistry , Peptides/chemistry , G-Quadruplexes , Humans , Ligands , Molecular StructureABSTRACT
A dual-functional peptide-PNA (peptide nucleic acid) conjugate consisting of a PNA G3-tract and an RHAU23 peptide is devised to target nucleic acids bearing three tandem guanine tracts (G-tracts). The PNA G3-tract joins the three G-tracts to form a stable bimolecular G-quadruplex (G4) and the resulting G4 is then bound by the RHAU23 moiety to form an extra stable G4-peptide complex. Owing to this synergistic dual structural enforcement, the conjugate accomplished extremely high selectivity and nM to sub-nM affinities towards its targets that are up to 1000 times greater than the small molecule G4 ligands. As a result, the conjugate impacts the tracking activity of motor proteins on DNA with superior selectivity and potency that are rarely seen in other G4-targeting approaches.
Subject(s)
DNA/chemistry , G-Quadruplexes , Peptide Nucleic Acids/chemistry , DEAD-box RNA Helicases/chemistry , DNA/metabolism , Electrophoretic Mobility Shift Assay , Guanine/chemistry , Humans , Nucleic Acid Conformation , Peptide Nucleic Acids/metabolism , Peptides/chemistry , Peptides/metabolism , Telomerase/geneticsABSTRACT
The particular enrichment of G-quadruplex-forming sequences near transcription start sites signifies the involvement of G-quadruplexes in the regulation of transcription. The characterization of G-quadruplex formation, which holds the key to understand the function it plays in physiological and pathological processes, is mostly performed under simplified in vitro experimental conditions. Formation of G-quadruplexes in cells, however, occurs in an environment far different from the ones in which the in vitro studies on G-quadruplexes are normally carried out. Therefore, the characteristics of G-quadruplex structures obtained under the in vitro conditions may not faithfully reveal how the G-quadruplexes would behave in a physiologically relevant situation. In this mini-review, we attempt to briefly summarize the differences in a few important characteristics, including kinetics, conformation, and stability of G-quadruplex formation observed under the two conditions to illustrate how the intracellular environment might affect the behavior of G-quadruplexes largely based on the previous work carried out in the authors' laboratory. We also propose that unstable G-quadruplex variants may be better drug target candidates to improve selectivity and potency.
Subject(s)
DNA/chemistry , G-Quadruplexes , Animals , Drug Discovery , G-Quadruplexes/drug effects , Humans , KineticsABSTRACT
In vitro transcription of double-stranded DNA (dsDNA) induces a formation of two types of G-quadruplexes, intramolecular DNA G-quadruplexes or DNA:RNA hybrid G-quadruplexes, in clusters of guanine tracts. The formation of G-quadruplexes can be characterized by native PEG polyacrylamide gel electrophoresis, DMS footprinting, and ligand-induced photocleavage footprinting. The type of G-quadruplexes can be distinguished by transcription with 7-deaza-GTP (dzGTP) or 4-thio-uridine-5'-triphosphate (4SU) to detect the participation of RNA transcript.
Subject(s)
DNA/chemistry , G-Quadruplexes , Animals , DNA Footprinting , Electrophoresis, Polyacrylamide Gel , HumansABSTRACT
DNA supercoiling is an important regulator of gene activity. The transmission of transcription-generated supercoiling wave along a DNA helix provides a way for a gene being transcribed to communicate with and regulate its neighboring genes. Currently, the dynamic behavior of supercoiling transmission remains unclear owing to the lack of a suitable tool for detecting the dynamics of supercoiling transmission. In this work, we established a torsion sensor that quantitatively monitors supercoiling transmission in real time in DNA. Using this sensor, we studied the transmission of transcriptionally generated negative supercoiling in linear and multi-way DNA duplexes. We found that transcription-generated dynamic supercoiling not only transmits along linear DNA duplex but also equally diverges at and proceeds through multi-way DNA junctions. We also show that such a process is regulated by DNA-protein interactions and non-canonical DNA structures in the path of supercoiling transmission. These results imply a transcription-coupled mechanism of dynamic supercoiling-mediated intra- and inter-chromosomal signal transduction pathway and their regulation in DNA.
Subject(s)
DNA, Superhelical/chemistry , DNA/chemistry , G-Quadruplexes , Transcription, Genetic , Base Sequence , Biosensing Techniques , DNA/genetics , DNA/metabolism , DNA, Superhelical/genetics , DNA, Superhelical/metabolism , Kinetics , Models, Genetic , Promoter Regions, Genetic/genetics , Protein Binding , Spectrometry, Fluorescence/methodsABSTRACT
Transcription induces formation of intramolecular G-quadruplex structures at the upstream region of a DNA duplex by an upward transmission of negative supercoiling through the DNA. Currently the regulation of such G-quadruplex formation remains unclear. Using plasmid as a model, we demonstrate that while it is the dynamic negative supercoiling generated by a moving RNA polymerase that triggers a formation of a G-quadruplex, the constitutional superhelicity determines the potential and range of the formation of a G-quadruplex by constraining the propagation of the negative supercoiling. G-quadruplex formation is maximal in negatively supercoiled and nearly abolished in relaxed plasmids while being moderate in nicked and linear ones. The formation of a G-quadruplex strongly correlates with the presence of an R-loop. Preventing R-loop formation virtually abolished G-quadruplex formation even in the negatively supercoiled plasmid. Enzymatic action and protein binding that manipulate supercoiling or its propagation all impact the formation of G-quadruplexes. Because chromosomes and plasmids in cells in their natural form are maintained in a supercoiled state, our findings reveal a physical basis that justifies the formation and regulation of G-quadruplexes in vivo. The structural features involved in G-quadruplex formation may all serve as potential targets in clinical and therapeutic applications.
Subject(s)
DNA/chemistry , G-Quadruplexes , Transcription, Genetic , DNA/genetics , DNA-Directed RNA Polymerases , Escherichia coli , Nucleic Acid Denaturation , Plasmids , Viral ProteinsABSTRACT
A guanine-vacancy-bearing G-quadruplex (GVBQ) interacts with guanine and derivatives by a structural complementation to form a more stable and intact G-quadruplex. Sensors using GVBQs are devised to detect guanine and other nucleobases, and their derivatives derived from structurally similar compounds. A strict requirement of Hoogsteen hydrogen bonds between the GVBQ and analyte in the structural complementation confers exceptional selectivity on the analyte. As such, subtle modifications on analytes affecting even a single hydrogen bond can preclude the recognition. In principle, the strategy may also be expanded to detect many planar cyclic compounds. Because nucleobases and derivatives/metabolites are involved in many physiological and pathological processes, this type of sensor may find applications in risk assessment of pathogenesis and therapeutics related to nucleic acid metabolism.
ABSTRACT
G-quadruplexes are implicated in many essential cellular processes and sequences with potential to form a G-quadruplex are widely present in DNA and RNA. However, it is difficult to know whether a sequence of interest naturally forms a G-quadruplex in living cells. Here we report the detection of a G-quadruplex in defined RNA sequences in living cells in a natural intracellular environment. A G-quadruplex forming sequence in a RNA transcript is tagged at proximity with an aptamer. The two structures are recognized respectively by two probe proteins each of which is fused with a split half of enhanced green fluorescent protein (eGFP). Simultaneous binding of the two proteins to RNA brings the two halves of eGFP into proximity, permitting them to reconstitute into a functional eGFP that emits fluorescence to signal the formation of a G-quadruplex in RNA. We show that a G-quadruplex can form in RNA and can be detected with sequence and structure specificity under both in vitro and in vivo conditions. The results, therefore, provide direct evidence for the formation of RNA G-quadruplexes in live cells and the method provides a useful tool to validate G-quadruplex formation in a specific sequence under a natural cellular condition.
ABSTRACT
G-quadruplex structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and nanodevices. G-quadruplexes are normally composed of four Gn (n ≥ 3) tracts assembled into a core of multiple stacked G-quartet layers. By dimethyl sulfate footprinting, circular dichroism spectroscopy, thermal melting, and photo-cross-linking, here we describe a unique type of intramolecular G-quadruplex that forms with one G2 and three G3 tracts and bears a guanine vacancy (G-vacancy) in one of the G-quartet layers. The G-vacancy can be filled up by a guanine base from GTP or GMP to complete an intact G-quartet by Hoogsteen hydrogen bonding, resulting in significant G-quadruplex stabilization that can effectively alter DNA replication in vitro at physiological concentration of GTP and Mg(2+). A bioinformatic survey shows motifs of such G-quadruplexes are evolutionally selected in genes with unique distribution pattern in both eukaryotic and prokaryotic organisms, implying such G-vacancy-bearing G-quadruplexes are present and play a role in gene regulation. Because guanine derivatives are natural metabolites in cells, the formation of such G-quadruplexes and guanine fill-in (G-fill-in) may grant an environment-responsive regulation in cellular processes. Our findings thus not only expand the sequence definition of G-quadruplex formation, but more importantly, reveal a structural and functional property not seen in the standard canonical G-quadruplexes.
Subject(s)
G-Quadruplexes , Guanine/analogs & derivatives , Guanine/chemistry , Circular Dichroism , DNA/chemistry , DNA ReplicationABSTRACT
DNA with four guanine tracts can fold into G-quadruplexes that are targets of transcription regulation. We recently found that hybrid DNA:RNA G-quadruplexes (HQs) can form during inâ vitro transcription. However, it is unclear whether they can form in cells. Evidence is presented supporting their formation in plasmids in bacterial cells. The formation of the HQs is indicated by a unique pattern of prematurely terminated transcripts under two conditions where the RNA transcripts do or do not participate in G-quadruplex assembly and further supported by a number of chemical and biochemical analysis. HQs dominate over the intramolecular DNA G-quadruplexes (DQ) in mediating the transcription termination when both structures are able to form. These findings provide the first evidence of HQ formation in cells and suggest that the competition/conversion between HQ and DQ may regulate transcription and serve as drug target in pharmaceutical applications.
Subject(s)
G-Quadruplexes , Transcription Termination, Genetic/physiology , DNA/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Plasmids/chemistry , Plasmids/metabolism , RNA/chemistryABSTRACT
G-quadruplexes are implicated in important cellular processes. Previous studies mostly focused on intramolecular G-quadruplexes of three or more G-quartets. Those composed of two G-quartets were only shown to form in single-stranded oligonucleotides. On the basis of electrophoresis, DMS footprinting, fluorescence labeling, and photo-cross-linking, we detected the formation of DNA:RNA hybrid G-quadruplexes (HQs) of two G-quartets during the transcription of DNA duplexes. These HQs have a lifetime on the minute scale and are stabilized by a stabilizing ligand. They are far shorter-lived than the HQs of three G-quartets, which last for hours. The occurrence of putative formation motifs of such HQs shows a transcription-dependent strand-biased selection, thus supporting their formation and function in genomes. They are present in almost all human genes in large amounts. We speculate that the two-G-quartet HQs may be a distinct type of G-quadruplexes that may play a role in timely responsive processes and for purposes of fine-tuning.
Subject(s)
DNA/genetics , G-Quadruplexes/drug effects , Genome/genetics , Oligonucleotides/genetics , RNA/genetics , Humans , PrevalenceABSTRACT
Human mitochondrial DNA contains a distinctive guanine-rich motif denoted conserved sequence block II (CSB II) that stops RNA transcription, producing prematurely terminated transcripts to prime mitochondrial DNA replication. Recently, we reported a general phenomenon that DNA:RNA hybrid G-quadruplexes (HQs) readily form during transcription when the non-template DNA strand is guanine-rich and such HQs in turn regulate transcription. In this work, we show that transcription of mitochondrial DNA leads to the formation of a stable HQ or alternatively an unstable intramolecular DNA G-quadruplex (DQ) at the CSB II. The HQ is the dominant species and contributes to the majority of the premature transcription termination. Manipulating the stability of the DQ has little effect on the termination even in the absence of HQ; however, abolishing the formation of HQs by preventing the participation of either DNA or RNA abolishes the vast majority of the termination. These results demonstrate that the type of G-quadruplexes (HQ or DQ) is a crucial determinant in directing the transcription termination at the CSB II and suggest a potential functionality of the co-transcriptionally formed HQ in DNA replication initiation. They also suggest that the competition/conversion between an HQ and a DQ may regulate the function of a G-quadruplex-forming sequence.
Subject(s)
DNA, Mitochondrial/chemistry , G-Quadruplexes , Terminator Regions, Genetic , Transcription Termination, Genetic , Base Sequence , Conserved Sequence , DNA Replication , DNA, Mitochondrial/biosynthesis , Humans , Mutation , Oligonucleotides/chemistry , Plasmids/genetics , RNA/chemistry , RNA, MitochondrialABSTRACT
We recently reported that a DNA:RNA hybrid G-quadruplex (HQ) forms during transcription of DNA that bears two or more tandem guanine tracts (G-tract) on the nontemplate strand. Putative HQ-forming sequences are enriched in the nearby 1000 nt region right downstream of transcription start sites in the nontemplate strand of warm-blooded animals, and HQ regulates transcription under both in vitro and in vivo conditions. Therefore, knowledge of the mechanism of HQ formation is important for understanding the biological function of HQ as well as for manipulating gene expression by targeting HQ. In this work, we studied the mechanism of HQ formation using an in vitro T7 transcription model. We show that RNA synthesis initially produces an R-loop, a DNA:RNA heteroduplex formed by a nascent RNA transcript and the template DNA strand. In the following round of transcription, the RNA in the R-loop is displaced, releasing the RNA in single-stranded form (ssRNA). Then the G-tracts in the RNA can jointly form HQ with those in the nontemplate DNA strand. We demonstrate that the structural cascade R-loop â ssRNA â HQ offers opportunities to intercept HQ formation, which may provide a potential method to manipulate gene expression.
Subject(s)
DNA/genetics , G-Quadruplexes , Guanine/chemistry , RNA/genetics , Transcription, Genetic/genetics , DNA/chemistry , DNA/metabolism , Guanine/metabolism , RNA/chemistry , RNA/metabolism , Ribonucleases/chemistry , Ribonucleases/metabolismABSTRACT
Recently, we reported the co-transcriptional formation of DNA:RNA hybrid G-quadruplex (HQ) structure by the non-template DNA strand and nascent RNA transcript, which in turn modulates transcription under both in vitro and in vivo conditions. Here we present bioinformatic analysis on putative HQ-forming sequences (PHQS) in the genomes of eukaryotic organisms. Starting from amphibian, PHQS motifs are concentrated in the immediate 1000-nt region downstream of transcription start sites, implying their potential role in transcription regulation. Moreover, their occurrence shows a strong bias toward the non-template versus the template strand. PHQS has become constitutional in genes in warm-blooded animals, and the magnitude of the strand bias correlates with the ability of PHQS to form HQ, suggesting a selection based on HQ formation. This strand bias is reversed in lower species, implying that the selection of PHQS/HQ depended on the living temperature of the organisms. In comparison with the putative intramolecular G-quadruplex-forming sequences (PQS), PHQS motifs are far more prevalent and abundant in the transcribed regions, making them the dominant candidates in the formation of G-quadruplexes in transcription. Collectively, these results suggest that the HQ structures are evolutionally selected to function in transcription and other transcription-mediated processes that involve guanine-rich non-template strand.