C-rich Sequences Research Articles

Applying Machine Learning and SERS for Precise Typing of DNA Secondary Structures.

Surface-enhanced Raman spectroscopy (SERS) has been demonstrated as an effective method for elucidating secondary structural characteristics of DNA. However, the inherent complexity of the DNA conformation and the lack of SERS samples pose challenges for identifying numerous secondary structures. To address these issues, a synergistic method integrating machine learning with SERS was proposed so as to analyze the SERS spectra of 54 well-defined conformational oligonucleotides, namely, G-quadruplex (G4), i-motif (iM), double-strand (DS), and single-strand (SS) configurations. Principal component analysis (PCA) effectively segregated the oligonucleotides into three distinct conformational groups (G4s, iMs, and others). Furthermore, linear discriminant analysis (LDA), K-nearest neighbor (KNN), and support vector machine (SVM) approaches were utilized to improve the typing accuracy of 54 trained sequences. This enabled the correct classification of the structures of five untrained sequences, as well as the identification of the predominant conformations including G4, iM, and DS formed by two complementary G-rich and C-rich sequences in acidic and neutral pH conditions. The results of this study demonstrated the potential of the proposed methodology for rapid screening and prediction of secondary DNA conformations.

Transient interactions modulate the affinity of NF-κB transcription factors for DNA

The dimeric nuclear factor kappa B (NF-κB) transcription factors (TFs) regulate gene expression by binding to a variety of κB DNA elements with conserved G:C-rich flanking sequences enclosing a degenerate central region. Toward defining mechanistic principles of affinity regulated by degeneracy, we observed an unusual dependence of the affinity of RelA on the identity of the central base pair, which appears to be noncontacted in the complex crystal structures. The affinity of κB sites with A or T at the central position is ~10-fold higher than with G or C. The crystal structures of neither the complexes nor the free κB DNAs could explain the differences in affinity. Interestingly, differential dynamics of several residues were revealed in molecular dynamics simulation studies, where simulation replicates totaling 148 μs were performed on NF-κB:DNA complexes and free κB DNAs. Notably, Arg187 and Arg124 exhibited selectivity in transient interactions that orchestrated a complex interplay among several DNA-interacting residues in the central region. Binding and simulation studies with mutants supported these observations of transient interactions dictating specificity. In combination with published reports, this work provides insights into the nuanced mechanisms governing the discriminatory binding of NF-κB family TFs to κB DNA elements and sheds light on cancer pathogenesis of cRel, a close homolog of RelA.

Extrachromosomal telomere DNA derived from excessive strand displacements

Alternative lengthening of telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication, evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), including C-circles, are unique to ALT cells, their generation process remains undefined. Here, we introduce a method to detect single-stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single-stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear and circular C-rich ssDNAs are generated concurrently. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

Regulation of VEGF gene expression by bisacridine derivative through promoter i-motif for cancer treatment

BackgroundVascular endothelial growth factor (VEGF) is overexpressed in most malignant tumors, which has important impact on tumor angiogenesis and development. Its gene promoter i-motif structure formed by C-rich sequence can regulate gene expression, which is a promising new target for anti-tumor therapy. MethodsWe screened various compounds and studied their effects on VEGF through extensive experiments, including SPR, MST, TO displacement, FRET, CD, ESI-MS, NMR, MTT, clone formation, qPCR, Western blot, dual-luciferase reporter assay, immunofluorescence, cell scrape, apoptosis, transwell assay, and animal model. ResultsAfter extensive screening, bisacridine derivative B09 was found to have selective binding and stabilization to VEGF promoter i-motif, which could down-regulate VEGF gene expression. B09 showed potent inhibition on MCF-7 and HGC-27 cell proliferation and metastasis. B09 significantly inhibited tumor growth in xenograft mice model with HGC-27 cells, showing decreased VEGF expression analyzed through immunohistochemistry. ConclusionB09 could specifically regulate VEGF gene expression, possibly through interacting with promoter i-motif structure. As a lead compound, B09 could be further developed for innovative anti-cancer agent targeting VEGF.

Polyvalent Aptamer-Functionalized NIR-II Quantum Dots for Targeted Theranostics in High PD-L1-Expressing Tumors.

Ag2S quantum dots (QDs) show superior optical properties in the NIR-II region and display significant clinical potential with favorable biocompatibility. However, inherent defects of low targeting and poor solubility necessitate practical modification methods to achieve the theranostics of Ag2S QDs. Herein, we used rolling circle amplification (RCA) techniques to obtain long single-stranded DNA containing the PD-L1 aptamer and C-rich DNA palindromic sequence. The C-rich DNA palindromic sequences can specifically chelate Ag2+ and thus serve as a template to result in biomimetic mineralization and formation of pApt-Ag2S QDs. These QDs enable specific targeting and illuminate hot tumors with high PD-L1 expression effectively, serving as excellent molecular targeted probes. In addition, due to the high NIR-II absorption of Ag2S QDs, pApt-Ag2S QDs exhibit remarkable photothermal properties. And besides, polyvalent PD-L1 aptamers can recognize PD-L1 protein and effectively block the inhibitory signal of PD-L1 on T cells, enabling efficient theranostics through the synergistic effect of photothermal therapy and immune checkpoint blocking therapy. Summary, we enhance the biological stability and antibleaching ability of Ag2S QDs using long single-stranded DNA as a template, thereby establishing a theranostic platform that specifically targets PD-L1 high-expressing inflamed tumors and demonstrates excellent performance both in vitro and in vivo.

Prediction of DNA i-motifs via machine learning.

i-Motifs (iMs), are secondary structures formed in cytosine-rich DNA sequences and are involved in multiple functions in the genome. Although putative iM forming sequences are widely distributed in the human genome, the folding status and strength of putative iMs vary dramatically. Much previous research on iM has focused on assessing the iM folding properties using biophysical experiments. However, there are no dedicated computational tools for predicting the folding status and strength of iM structures. Here, we introduce a machine learning pipeline, iM-Seeker, to predict both folding status and structural stability of DNA iMs. The programme iM-Seeker incorporates a Balanced Random Forest classifier trained on genome-wide iMab antibody-based CUT&Tag sequencing data to predict the folding status and an Extreme Gradient Boosting regressor to estimate the folding strength according to both literature biophysical data and our in-house biophysical experiments. iM-Seeker predicts DNA iM folding status with a classification accuracy of 81% and estimates the folding strength with coefficient of determination (R2) of 0.642 on the test set. Model interpretation confirms that the nucleotide composition of the C-rich sequence significantly affects iM stability, with a positive correlation with sequences containing cytosine and thymine and a negative correlation with guanine and adenine.

Preferential formation of Z-RNA over intercalated motifs in long noncoding RNA.

Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Noncanonical secondary structures that form in nucleic acids have known roles in regulating gene expression and include G-quadruplexes (G4s), intercalated motifs (iMs), and R-loops (RLs). In this paper, we used the computational tools G4-iM Grinder and QmRLFS-finder to predict the formation of each of these structures throughout the lncRNA transcriptome in comparison to protein-coding transcripts. The importance of the predicted structures in lncRNAs in biological contexts was assessed by combining our results with publicly available lncRNA tissue expression data followed by pathway analysis. The formation of predicted G4 (pG4) and iM (piM) structures in select lncRNA sequences was confirmed in vitro using biophysical experiments under near-physiological conditions. We find that the majority of the tested pG4s form highly stable G4 structures, and identify many previously unreported G4s in biologically important lncRNAs. In contrast, none of the piM sequences are able to form iM structures, consistent with the idea that RNA is unable to form stable iMs. Unexpectedly, these C-rich sequences instead form Z-RNA structures, which have not been previously observed in regions containing cytosine repeats and represent an interesting and underexplored target for protein-RNA interactions. Our results highlight the prevalence and potential structure-associated functions of noncanonical secondary structures in lncRNAs, and show G4 and Z-RNA structure formation in many lncRNA sequences for the first time, furthering the understanding of the structure-function relationship in lncRNAs.

Abstract C083: SMNDC1 alters the splicing of ERK to potentiate its activity in pancreatic cancer

Abstract While oncogenic KRAS is believed to drive pancreatic cancer by maintaining active ERK signaling, our studies suggest that ERK signaling can be also regulated in a KRAS-independent manner. We found that survival motor neuron domain containing 1 (SMNDC1), a poorly studied splicing factor is amplified or upregulated in 30% of pancreatic ductal adenocarcinomas (PDACs) and associated with lower survival rates in patients. To determine the role of SMNDC1 in PDACs, we conducted loss- and gain-of-function studies and found that this splicing factor drives tumor growth by promoting the retention of A/C-rich cassette exons, which otherwise are excluded or retained at a lower rate after RNA splicing in normal cells. Importantly, across tumors, SMNDC1 promoted the inclusion of the cassette exon 4 (E4) of MAPK3 (ERK1), which encodes the kinase-activating phosphorylation sites (Thr202/Tyr204). Forced exclusion of MAPK3-E4 using antisense oligonucleotide morpholino (ASO), inhibited tumor establishment, ERK1 phosphorylation, and expression of ERK-targets. We then tested if retention of E4 in MAPK3 by SMNDC1 is a mechanism to resist chemical inhibition of the KRAS-MEK-ERK pathway. We found that PDAC cells acutely treated with selective MEK inhibitors (MEKi), trametinib, or selumetinib increased SMNDC1 and MAPK3 E4 retention expression. In addition, PDAC cells deficient for SMNDC1 were 25-fold more sensitive to trametinib than isogenic cells expressing SMNDC1. To demonstrate that increased E4 retention drives MEKi resistance, we concomitantly treated SMNDC1 proficient PDAC cells with trametinib and E4 ASO, which led to an 8-fold increase in sensitivity compared to cells treated with trametinib and control ASO. Also, we found that resistance to standard-of-care chemotherapeutic agents to treat PDAC, Gemcitabine (GEM) and 5-fluorouracil (5-FU), was also associated with increased expression of SMNDC1 and retention of E4 in MAPK3 in multiple PDAC cells lines. To determine the biochemical mechanism that leads to E4 inclusion by SMNDC1, we performed high-throughput RNA Bind-n-Seq (RBNS) and e-CLIP of SMNDC1 in PDAC cells which revealed specific RNA-binding preference for C-rich polypyrimidine tract upstream of retained MAPK3-E4. To validate these findings, we constructed a dual-fluorescent reporter mini-gene of MAPK3-E4 splicing, with either wild-type sequences in E4 and polypyrimidine tract, or different point mutations at the predicted cis-elements. We found that retention of the E4 of MAPK3 is dependent on SMNDC1-binding to C-rich sequences at distant 3’ splice sites of PDAC cells. These data support that PDAC cells exploit a “splicing switch” to promote ERK kinase activity and resistance to therapies, independent of activating mutations in the EGFR/RAS/MAPK pathway and offer a druggable alternative to block oncogenic signaling and altered RNA splicing in PDAC. Citation Format: Md Afjalus Siraj, Yushan Zhang, Prabir Chakraborty, Robert Tseng, Li-Ting Ku, Grant Goda, Gilbert Giri, Deanne Yugawa, Mathew Wang, Shamik Das, Anindya Dey, Shailendra Dhar Dwivedi, Geeta Rao, Min Zhang, Da Yang, Md Nazir Hossen, Wei-Qun Ding, Kar-Ming Fung, Daniel Dominguez, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. SMNDC1 alters the splicing of ERK to potentiate its activity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C083.

The roles of DNA methylation on pH dependent i-motif(iM) formation in rice.

I-motifs (iMs) are four-stranded non-B DNA structures containing C-rich DNA sequences. The formation of iMs is sensitive to pH conditions and DNA methylation, although the extent of which is still unknown in both humans and plants. To investigate this, we here conducted iMab antibody-based immunoprecipitation and sequencing (iM-IP-seq) along with bisulfite sequencing using CK (original genomic DNA without methylation-relatedtreatments) and hypermethylated or demethylated DNA at both pH 5.5 and 7.0 in rice, establishing a link between pH, DNA methylation and iM formation on a genome-wide scale. We found that iMs folded at pH 7.0 displayed higher methylation levels than those formed at pH 5.5. DNA demethylation and hypermethylation differently influenced iM formation at pH 7.0 and 5.5. Importantly, CG hypo-DMRs (differentially methylated regions) and CHH (H=A, C and T) hyper-DMRs alone or coordinated with CG/CHG hyper-DMRs may play determinant roles in the regulation of pH dependent iM formation. Thus, our study shows that the nature of DNA sequences alone or combined with their methylation status plays critical roles in determining pH-dependent formation of iMs. It therefore deepens the understanding of the pH and methylation dependent modulation of iM formation, which has important biological implications and practical applications.

A beginner's handbook to identify and characterize i-motif DNA.

Genomic DNA exhibits an innate ability to manifest diverse sequence-dependent secondary structures, serving crucial functions in gene regulation and cellular equilibrium. While extensive research has confirmed the formation of G-quadruplex structures by guanine-rich sequences in vitro and in cells, recent investigations have turned the quadruplex community's attention to the cytosine (C)-rich complementary strands that can adopt unique tetra-stranded conformation, termed as intercalated motif or i-motif. I-motifs are stabilized by hemi-protonated C:CH+ base pairs under acidic conditions. Initially, the in vivo occurrence of i-motifs was underestimated because their formation is favored at non-physiological pH. However, groundbreaking research utilizing the structure-specific iMab antibody and high-throughput sequencing have recently detected their conserved dispersion throughout the genome, challenging previous assumptions. Given the evolving nature of this research field, it becomes imperative to conduct independent in vitro experiments aimed at identifying potential i-motif formation in C-rich sequences and consolidating the findings to address the properties of i-motifs. This chapter serves as an introductory guide for the swift identification of novel i-motifs, where we present an experimental framework for investigating and characterizing i-motif sequences in vitro. In this chapter, we selected a synthetic oligonucleotide (C7T3) sequence and outlined appropriate methodologies for annealing the i-motif structure into suitable buffers. Then, we validated its formation by CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance) spectroscopy. Finally, we provided a thorough account of the step-by-step procedures to investigate the effect of i-motif formation on the stalling or retardation of DNA replication using high resolution primer extension assays.

Simplified rolling circle amplification driven in situ generation of electrochemiluminescent silver nanoclusters for APE1 activity monitoring

Apurinic/apyrimidinic endonuclease 1 (APE1) plays an essential part in the base excision repair whose abnormal expression is closely correlated to the occurrence of tumors. Therefore, a sensitive assay of APE1 is crucial for the early diagnosis of cancer. Herein, we developed a simplified rolling circle amplification (RCA) strategy to drive in situ production of electrochemiluminescent (ECL) silver nanoclusters (Ag NCs) for APE1 activity monitoring. Unlike conventional RCA, which required locker DNA and padlock to form the circular template and hybridize with primer to initiate amplification, we proposed a simplified RCA that employed hairpin (HP) and padlock to directly form the circular template to initiate the amplification. Through reasonable design of the amplification templates, the RCA products were cytosine-rich (C-rich) sequences for silver ions adsorption, which were electrochemically reduced to Ag NCs. Combined with Cu-based metal-organic frameworks (Cu-MOFs) as co-reaction accelerator to facilitate the reduction of S2O82-, the ternary ECL system (Ag NCs/S2O82-/Cu-MOFs) demonstrated strong ECL intensity. The simplified RCA strategy combined with the ternary ECL sensing platform achieved the sensitive analysis of APE1 with a linear range of 1 × 10−8 to 1 × 10−2 U/μL and a detection limit of 2.29 × 10−9 U/μL, which presented a novel approach for sensitive monitoring of APE1 activity.

Comparative analysis of the myoglobin gene in whales and humans reveals evolutionary changes in regulatory elements and expression levels.

Cetacea and other diving mammals have undergone numerous adaptations to their aquatic environment, among them high levels of the oxygen-carrying intracellular hemoprotein myoglobin in skeletal muscles. Hypotheses regarding the mechanisms leading to these high myoglobin levels often invoke the induction of gene expression by exercise, hypoxia, and other physiological gene regulatory pathways. Here we explore an alternative hypothesis: that cetacean myoglobin genes have evolved high levels of transcription driven by the intrinsic developmental mechanisms that drive muscle cell differentiation. We have used luciferase assays in differentiated C2C12 cells to test this hypothesis. Contrary to our hypothesis, we find that the myoglobin gene from the minke whale, Balaenoptera acutorostrata, shows a low level of expression, only about 8% that of humans. This low expression level is broadly shared among cetaceans and artiodactylans. Previous work on regulation of the human gene has identified a core muscle-specific enhancer comprised of two regions, the "AT element" and a C-rich sequence 5' of the AT element termed the "CCAC-box". Analysis of the minke whale gene supports the importance of the AT element, but the minke whale CCAC-box ortholog has little effect. Instead, critical positive input has been identified in a G-rich region 3' of the AT element. Also, a conserved E-box in exon 1 positively affects expression, despite having been assigned a repressive role in the human gene. Last, a novel region 5' of the core enhancer has been identified, which we hypothesize may function as a boundary element. These results illustrate regulatory flexibility during evolution. We discuss the possibility that low transcription levels are actually beneficial, and that evolution of the myoglobin protein toward enhanced stability is a critical factor in the accumulation of high myoglobin levels in adult cetacean muscle tissue.

Keratin 19 binds and regulates cytoplasmic HNRNPK mRNA targets in triple-negative breast cancer.

Heterogeneous nuclear ribonucleoprotein K (HNRNPK) regulates pre-mRNA processing and long non-coding RNA localization in the nucleus. It was previously shown that shuttling of HNRNPK to the cytoplasm promotes cell proliferation and cancer metastasis. However, the mechanism of HNRNPK cytoplasmic localization, its cytoplasmic RNA ligands, and impact on post-transcriptional gene regulation remain uncharacterized. Here we show that the intermediate filament protein Keratin 19 (K19) directly interacts with HNRNPK and sequesters it in the cytoplasm. Correspondingly, in K19 knockout breast cancer cells, HNRNPK does not localize in the cytoplasm, resulting in reduced cell proliferation. We comprehensively mapped HNRNPK binding sites on mRNAs and showed that, in the cytoplasm, K19-mediated HNRNPK-retention increases the abundance of target mRNAs bound to the 3' untranslated region (3'UTR) at the expected cytidine-rich (C-rich) sequence elements. Furthermore, these mRNAs protected by HNRNPK in the cytoplasm are typically involved in cancer progression and include the p53 signaling pathway that is dysregulated upon HNRNPK knockdown (HNRNPK KD) or K19 knockout (KRT19 KO). This study identifies how a cytoskeletal protein can directly regulate gene expression by controlling the subcellular localization of RNA-binding proteins to support pathways involved in cancer progression.

Modulation of the tetrameric I-motif folding of C-rich Tetrahymena telomeric sequences by hexitol nucleic acid (HNA) modifications

I-motifs are non-canonical DNA structures consisting of two parallel strands held together by hemiprotonated cytosine-cytosine+ base pairs, which intercalate to form a ordered column of stacked base pairs. This unique structure covers potential relevance in various fields, including gene regulation and biotechnological applications. A unique structural feature of I-motifs (iM), is the presence of sugar-sugar interactions through their extremely narrow minor grooves. Consistently, oligonucleotides containing pentose derivatives such as ribose, 2′-deoxyribose, arabinose, and 2′-deoxy-2′-fluoroarabinose highlighted a very different attitude to fold into iM. On the other hand, there is significant attention focused on exploring sugar-modifications that can increase nucleic acids resistance to nuclease degradation, a crucial requirement for therapeutic applications.An interesting example, not addressed in the iM field yet, is represented by hexitol nucleic acid (HNA), a metabolically stable six-membered ring analogue compatible with A-like double helix formation. Herein, we selected two DNA C-rich Tetrahymena telomeric sequences whose tetrameric iMs were already resolved by NMR and we investigated the iM folding of related HNA and RNA oligonucleotides by circular dichroism, differential scanning calorimetry and NMR. The comparison of their behaviours vs the DNA counterparts provided interesting insights into the influence of the sugar on iM folding. In particular, ribose and hexitol prevented iM formation. However, by clustering the hexitol-containing residues at the 3′-end, it was possible to modulate the distribution of the different topological species described for the DNA iMs. These data open new avenues for the exploitation of sugar modifications for I-motif characterization and applications.

Probing the Competition between Duplex, G-Quadruplex and i-Motif Structures of the Oncogenic c-Myc DNA Promoter Region.

Development of probe systems that provide unique spectral signatures for duplex, G-quadruplex (GQ) and i-motif (iM) structures is very important to understand the relative propensity of a G-rich-C-rich promoter region to form these structures. Here, we devise a platform using a combination of two environment-sensitive nucleoside analogs namely, 5-fluorobenzofuran-modified 2'-deoxyuridine (FBF-dU) and 5-fluoro-2'-deoxyuridine (F-dU) to study the structures adopted by a promoter region of the c-Myc oncogene. FBF-dU serves as a dual-purpose probe containing a fluorescent and 19 F NMR label. When incorporated into the C-rich sequence, it reports the formation of different iMs via changes in its fluorescence properties and 19 F signal. F-dU incorporated into the G-rich ON reports the formation of a GQ structure whose 19 F signal is clearly different from the signals obtained for iMs. Rewardingly, the labeled ONs when mixed with respective complementary strands allows us to determine the relative population of different structures formed by the c-Myc promoter by the virtue of the probe's ability to produce distinct and resolved 19 F signatures for different structures. Our results indicate that at physiological pH and temperature the c-Myc promoter forms duplex, random coil and GQ structures, and does not form an iM. Whereas at acidic pH, the mixture largely forms iM and GQ structures. Taken together, our system will complement existing tools and provide unprecedented insights on the population equilibrium and dynamics of nucleic acid structures under different conditions.

Genome-wide mapping of i-motifs reveals their association with transcription regulation in live human cells.

i-Motifs (iMs) are four-stranded DNA structures that form at cytosine (C)-rich sequences in acidic conditions in vitro. Their formation in cells is still under debate. We performed CUT&Tag sequencing using the anti-iM antibody iMab and showed that iMs form within the human genome in live cells. We mapped iMs in two human cell lines and recovered C-rich sequences that were confirmed to fold into iMs in vitro. We found that iMs in cells are mainly present at actively transcribing gene promoters, in open chromatin regions, they overlap with R-loops, and their abundance and distribution are specific to each cell type. iMs with both long and short C-tracts were recovered, further extending the relevance of iMs. By simultaneously mapping G-quadruplexes (G4s), which form at guanine-rich regions, and comparing the results with iMs, we proved that the two structures can form in independent regions; however, when both iMs and G4s are present in the same genomic tract, their formation is enhanced. iMs and G4s were mainly found at genes with low and high transcription rates, respectively. Our findings support the in vivo formation of iM structures and provide new insights into their interplay with G4s as new regulatory elements in the human genome.

Structural characterization of a cytosine-rich potential quadruplex forming sequence in the EGFR promoter

I-motifs are tetra-helixes that may form in cytosine-rich strands. They are based on cytosine–cytosine+ base pairs that require the N3 hemi-protonation of the nucleobases, and therefore, the stability of these non-canonical DNA arrangements depends on pH. These structures are promising targets for the development of new cancer therapies since they are enriched in the promoters of oncogenes where they can play a role in the regulation of transcription. The proximal promoter of the EGFR oncogene has multiple regions with a significant potential to form such a tetra-helix arrangement. Here, we present the thermodynamic characterization of a C-rich sequence located 37 nucleotides upstream of the transcription starting site of EGFR. We confirmed the ability of this sequence to fold into an I-motif. By applying a global analysis of calorimetric and spectroscopic data, we derived the dependency of the apparent standard Gibbs free energy change associated with the I-motif folding upon temperature and pH. The results showed that, in contrast to in silico prediction, only 4 CC+ base pairs formed while additional GC and TT base pairings were detected in the I-motif. Noteworthy, a single residue mutation at G14 largely shifts the equilibrium toward the formation of multimeric species.

Optical, structural, and biological properties of silver nanoclusters formed within the loop of a C-12 hairpin sequence.

Silver nanoclusters (AgNCs) are the next-generation nanomaterials representing supra-atomic structures where silver atoms are organized in a particular geometry. DNA can effectively template and stabilize these novel fluorescent AgNCs. Only a few atoms in size - the properties of nanoclusters can be tuned using only single nucleobase replacement of C-rich templating DNA sequences. A high degree of control over the structure of AgNC could greatly contribute to the ability to fine-tune the properties of silver nanoclusters. In this study, we explore the properties of AgNCs formed on a short DNA sequence with a C12 hairpin loop structure (AgNC@hpC12). We identify three types of cytosines based on their involvement in the stabilization of AgNCs. Computational and experimental results suggest an elongated cluster shape with 10 silver atoms. We found that the properties of the AgNCs depend on the overall structure and relative position of the silver atoms. The emission pattern of the AgNCs depends strongly on the charge distribution, while all silver atoms and some DNA bases are involved in optical transitions based on molecular orbital (MO) visualization. We also characterize the antibacterial properties of silver nanoclusters and propose a possible mechanism of action based on the interactions of AgNCs with molecular oxygen.

DNA-RNA hybrid G-quadruplex tends to form near the 3′ end of telomere overhang

Telomeric repeat-containing RNA (TERRA) has been suggested to participate in telomere maintenance. TERRA consisting of UUAGGG repeats is capable of forming an intermolecular G-quadruplex (GQ) with single-stranded TTAGGG-repeat DNA in the telomere 3′ overhang. To explore the structural features and potential functions of this DNA-RNA hybrid GQ (HGQ), we used single-molecule FRET to study the folding patterns of DNA with four to seven telomeric tandem repeats annealed with a short RNA consisting of two or five telomeric repeats. Our data highlight that RNA prefers to form DNA-RNA HGQ near the 3′ end of telomeric DNA. Furthermore, the unfolding of secondary structures by a complementary C-rich sequence was observed for DNA GQ but not for DNA-RNA HGQ, which demonstrated the enhanced stability of the telomere 3′ end via hybridization with RNA. These conformational and physical properties of telomeric DNA-RNA HGQ suggest that TERRA might limit access to the 3′ end of the telomeric DNA overhang, which is known to be critical for the interaction with telomerase and other telomere-associated proteins.

Antibody-powered lighting-up fluorescence immunosensor based on hemin/G-quadruplex-quenched DNA-hosted dual silver nanoclusters as emitters

Immunosensors with many merits such as simplicity, high sensitivity and selectivity are intriguing for the detection of antibodies, especially bivalent antibody (e.g. anti-digoxin antibody, DigA). In this work, we report a sensitive immunosensor for DigA based on the switchable fluorescence of DNA-templated silver nanoclusters (AgNCs) modulated by hemin/G-quadruplex (hGq). To explore the fluorescing performance of one hGq-quenching dual-AgNCs (d-AgNCs), here we design a functional hosting DNA strand (HGH) encoding with one centered G-rich segment for forming hGq complex in the presence of hemin and K+, two terminated C-rich template sequences for the identical clustering of d-AgNCs, and two flanked symmetric domains for specific recognizing and linking. Because of the photoexcited electron transfer process to the stacked hGq, the originally presynthesized d-AgNCs colocalized in two ends are non-emissive, which are retainable after complementarily hybridizing with two recognizable single strands that are both tagged with a digoxigenin hapten, a Fab fragment. Upon introducing DigA, the steric strain associated with the affinity binding of two haptens results in the opening and stretch of hGq in a coil spacer, thereby lighting-up the d-AgNCs fluorescence. Based on one-step signal switch stimulated by targeting antibody, this label-free immunosensor strategy is of high specificity and sensitivity with low picomolar detection limit, as well as simplification, rapidness and cost-effectiveness.