Stability Of Hairpin Research Articles

Enhancement and inactivation effect of CRISPR/Cas12a via extending hairpin activators for detection of transcription factors.

An enhancement effect for the activation of CRISPR/Cas12a (CRISPR = clustered regularly interspaced short palindromic repeats; Cas = CRISPR-associated) was discovered. That was, a hairpin model with dangling 5' end complementary to crRNA (CRISPR RNA) greatly improved the activity of CRISPR/Cas12a after extention of two random sequences. But, the corresponding intact hairpin without PAM (protospacer adjacent motif) or suboptimal PAM sequences was completely inactive to CRISPR/Cas12a because of the superhigh stability of intact hairpin. According to the finding, a CRISPR/Cas12a-based strategy coupled with a signal reported system was designed for transcription factors detection. By using mono-labeled ssDNA (single-stranded DNA) as reporter and two newly synthesized N-C (nitrogen-doped carbon) nanosheets as scavenger to eliminate the fluorescent background, the strategy realized the detection of NF-ĸB p50 (p50 subunit of nuclear factor kappa-B) with a linear detection range of 0.8 - 2000.0pM and a LOD of 0.5pM. The discovery of "enhancement and inactivation effect" not only deepened insight into CRISPR/Cas12a but also broadened the practical application of CRISPR/Cas systems for the molecular detection and disease diagnostics.

Detection of Hofmeister effects at a single-molecule level

Metal cations are essential for the function of nucleic acids. Given their efficiency for DNA overwinding and the DNA hairpin folding/unfolding process coupled with DNA twist, we systematically investigated the effects of cation identities and increasing concentrations of NaCl, KCl, and LiCl from 50 mM to 300 mM on the folding/unfolding process by manipulating a DNA hairpin construct with magnetic tweezers. The critical forces and free energy of the unfolding transition of the DNA hairpin at a fixed concentration of KCl, NaCl, and LiCl followed the same ranking as , and are reversed with the unfolding rate ranking of {}\ ext{Na}^{+}>{}\ ext{Li}^{+}$ ?> . This highlights that the addition of Li+ in solution obviously enhances the stability of the DNA hairpin when compared to Na+, and K+, and their efficiency on the stability of the DNA hairpin was ranked as .

Genomic characterization and phylogenetic analysis of Aleutian mink disease virus identified in a sudden death mink case

Aleutian mink disease (AMD) is one of the most serious diseases in minks worldwide, it brings tremendous financial losses in mink farming. AMD virus (AMDV) has unusually high genetic diversity, its genomic structure remains unclear. In 2014, sudden death of breeding minks was occurred in northeast China. After clinical signs evaluation and virus isolation, AMDV was identified in all sudden death minks, we investigated the complete genomic sequence of AMDV-LM isolated from the sudden death case. The full-genome sequence of AMDV-LM was 7 nucleotides (nts) or 8 nts longer than isolates AMDV-BJ and AMDV-G. AMDV-LM contained two unique nucleotide changes in VP2 (G79T, T710C), which led to two amino acid changes G27W and L237S. For NS1, some unique point mutations, such as A374C, A428C, A463C, and T476A were found and resulted in four unique amino acid mutations at N24V, H125P, V143P, K155Q, and V159N, respectively. The predicted secondary structure of the 5′ terminal of AMDV-LM formed a large bubble formation near the 5′ end, which affected the stability of the U-shaped hairpin. Phylogenetic analysis demonstrated that AMDV-LM was closely related to Chinese isolates and confirmed that AMDV strains circulating in China had different origins of ancestors. This study was first to investigate the association of sudden death of adult breeding minks with AMDV infection. Our findings provide useful suggestions for evaluation of the pathogenic potential of AMDV, additional details on AMDV genome characterization were also presented. Future work should focus on the importance of AMDV-LM strain in mink infection.

Stabilization of DNA Duplexes and Hairpins by Charge-Transfer Interactions Using DAN:NDI Pairs.

Electron-rich 1,5-dialkoxynaphthalene (DAN) and electron-deficient 1,8,4,5-naphthalenetetracarboxylic diimide (NDI) are known to interact through the formation of charge-transfer complexes. The introduction of DAN and NDI into various DNA duplexes and hairpins was investigated by ultraviolet (UV) melting curve analysis. The positioning of the DAN:NDI pair was found to strongly influence the stability of DNA duplex and hairpins. In particular, while the introduction of one DAN/NDI pair in front of each other in the center of a DNA duplex led to a decrease of the thermal stability (ΔTm - 6 °C), the addition of a second pair restored or even increased the stability. In contrast, the introduction of DAN:NDI pairs at the end of a duplex always induced a strong stabilization (ΔTm up to +20 °C). Finally, a DAN:NDI pair positioned in the loop of a hairpin induced a stronger stabilization than a T4 loop (ΔTm + 10 °C). Based on charge-transfer interactions, the strong stabilizations observed allow the preparation of highly stabilized DNA nanostructures opening the way to numerous applications in nanotechnology.

A common rule for the intermediate state caused by DNA mismatch in single-molecule experiments* *The National Natural Science Foundation of China under Grant Nos. 11864006, 11874309, 12164007, and 12204118, Guizhou Scientific and Technological Program (20185781, ZK(2021)004, ZK(2021)032 and 20185781-15), Guizhou Provincial Graduate Scientific Research Foundation (YJSKYJJ(2021)066). We are also thankful for the computing support of the State Key Laboratory of

Defective structures, such as DNA mismatches, occur in DNA with a high frequency in some biological processes. They are difficult to identify and have recently become the focus of single-molecule investigations. Three single-molecule experiments were successively conducted to detect the effects of DNA mismatch on the stability of DNA hairpins. However, there was no consensus regarding the results of the intermediate state caused by DNA mismatch. Based on the extended ox-DNA model, DNA mismatch was introduced to the stem of DNA hairpins with different stem lengths (12–20 bps) and 4T in hairpin loops. The intermediate state and its dependence on the position of the DNA mismatch in the stem from the hairpin loop were systematically studied. The results indicated that DNA mismatch definitely reduced the critical forces of DNA hairpins. At the same time, a common rule about the dependence of the intermediate state on the position of DNA mismatch was generalized in a phase diagram constructed in a phase space of a scaled position of DNA mismatch. Three segments on its diagonal line corresponded to the ranges of the scaled position of DNA mismatch [0, 0.55), [0.55, 0.85), and [0.85, 1], respectively. In the [0.55, 0.85) range, the extension probability distribution of DNA hairpins had unfolded, intermediate, and folded states. In contrast, in the other ranges [0, 0.55) and [0.85, 1], the extension probability distributions had unfolded and folded states. The scaled positions of DNA mismatch for the DNA hairpins used in the three single-molecule experiments (0.65, 0.4736, and 0.5) fell in the ranges [0.55, 0.85) and [0, 0.55). Obviously, the common rule generalized in the phase diagram not only clarifies the non-consensus between the three single-molecule experiments but also highlights the design of single-molecule experiments in the future.

Stability of DNA and RNA hairpins: a comparative study based on ox-DNA

Advances in single-molecule experiments on macromolecular crowding urgently need an efficient simulation method to resolve their discrepancies quantitatively. Ox-DNA model has been since reworked to treat the thermodynamics and mechanical properties of DNA/RNA hairpin at a stretching force. In hopping experiments, the critical forces of RNA hairpins at different temperatures are greater than those of DNA hairpins, in addition, the Gibbs free energy at a fixed temperature required to convert an RNA hairpin into a single-stranded molecule at zero force is obviously greater than that of DNA hairpin and gradually decreases by increasing the temperature. As far as force-ramping experiments are concerned, the first-rupture forces of RNA/DNA hairpins corresponding to the maximum probability density linearly pertain to the force-loading rate, with those of RNA hairpins being greater. The extended ox-DNA model could potentially identify the interaction between biologically inert polymer and RNA/DNA hairpins in crowded environments.

Allosteric mechanism of transcription inhibition by NusG-dependent pausing of RNA polymerase.

NusG is a transcription elongation factor that stimulates transcription pausing in Gram+ bacteria including B. subtilis by sequence-specific interaction with a conserved pause-inducing -11TTNTTT-6 motif found in the non-template DNA (ntDNA) strand within the transcription bubble. To reveal the structural basis of NusG-dependent pausing, we determined a cryo-EM structure of a paused transcription complex (PTC)containing RNA polymerase (RNAP), NusG, and the TTNTTT motif in the ntDNA strand. The interaction of NusG with the ntDNA strand rearranges the transcription bubble by positioning three consecutive T residues in a cleft between NusG and the β-lobe domain of RNAP. We revealed that the RNAP swivel module rotation (swiveling), which widens (swiveled state) and narrows (non-swiveled state) a cleft between NusG and the β-lobe, is an intrinsic motion of RNAP and is directly linked to trigger loop (TL) folding, an essential conformational change of all cellular RNAPs for the RNA synthesis reaction. We also determined cryo-EM structures of RNAP escaping from the paused transcription state. These structures revealed the NusG-dependent pausing mechanism by which NusG-ntDNA interaction inhibits the transition from swiveled to non-swiveled states, thereby preventing TL folding and RNA synthesis allosterically. This motion is also reduced by the formation of an RNA hairpin within the RNA exit channel. Thus, the pause half-life can be modulated by the strength of the NusG-ntDNA interaction and/or the stability of the RNA hairpin. NusG residues that interact with the TTNTTT motif are widely conserved in bacteria, suggesting that NusG-dependent pausing is widespread.

On a barrier height problem for RNA branching

The branching of an RNA molecule is an important structural characteristic yet difficult to predict correctly, especially for longer sequences. Using plane trees as a combinatorial model for RNA folding, we consider the thermodynamic cost, known as the barrier height, of transitioning between branching configurations. Using branching skew as a coarse energy approximation, we characterize various types of paths in the discrete configuration landscape. In particular, we give sufficient conditions for a path to have both minimal length and minimal branching skew. The proofs offer some biological insights, notably the potential importance of both hairpin stability and domain architecture to higher resolution RNA barrier height analyses.

Evaluation of Thermal Stability of DNA Oligonucleotide Structures Embedded in Hydrogels

Understanding the self-assembly and hybridization properties of DNA oligonucleotides in confined spaces can help to improve their applications in biotechnology and nanotechnology. This study investigates the effects of spatial confinement in the pores of hydrogels on the thermal stability of DNA oligonucleotide structures. The preparation of oligonucleotides embedded in agarose gels was simple, whereas the preparation of oligonucleotides embedded in polyacrylamide gels was required to remove unpolymerized monomers. In the latter case, a method for rehydrating a washed dry gel with a buffer solution containing oligonucleotides was developed. Fluorescence measurements of oligonucleotides bearing fluorescent probes revealed no significant influence of the internal environment of the gel pores on the stability of DNA duplex, hairpin, and G-quadruplex structures. Moreover, the effects of poly(ethylene glycol) on the stability of DNA structures in the gels were similar to those in solutions. It is likely that the oligonucleotides are not strongly constrained in the gels and may be preferentially located in a water-rich environment in the gel matrix. The gel preparation was also applied to the assessment of the stability of DNA structures under the conditions of a reduced number of water molecules. The studies using hydrogels provide insights into the ability of self-assembly and hybridization of oligonucleotides in confined environments and under low-water-content conditions.

A large HIV gp41 construct with trimer-of-hairpins structure exhibits V2E mutation-dominant attenuation of vesicle fusion and helicity very similar to V2E attenuation of HIV fusion and infection and supports: (1) hairpin stabilization of membrane apposition with larger distance for V2E; and (2) V2E dominance by an antiparallel β sheet with interleaved fusion peptide strands from two gp41 trimers

There is complete attenuation of fusion and infection mediated by HIV gp160 with gp41 subunit with V2E mutation, and also V2E dominance with WT/V2E mixtures. V2E is at the N-terminus of the ∼25-residue fusion peptide (Fp) which likely binds the target membrane. In this study, large V2E attenuation and dominance were observed for vesicle fusion induced by FP_HM, a large gp41 ectodomain construct with Fp followed by hyperthermostable hairpin with N- and C-helices, and membrane-proximal external region (Mper). FP_HM is a trimer-of-hairpins, the final gp41 structure during fusion. Vesicle fusion and helicity were measured for FP_HM using trimers with different fractions (f's) of WT and V2E proteins. Reductions in FP_HM fusion and helicity vs. fV2E were quantitatively-similar to those for gp160-mediated fusion and infection. Global fitting of all V2E data supports 6 WT gp41 (2 trimers) required for fusion. These data are understood by a model in which the ∼25 kcal/mol free energy for initial membrane apposition is compensated by the thermostable hairpin between the Fp in target membrane and Mper/transmembrane domain in virus membrane. The data support a structural model for V2E dominance with a membrane-bound Fp with antiparallel β sheet and interleaved strands from the two trimers. Relative to fV2E = 0, a longer Fp sheet is stabilized with small fV2E because of salt-bridge and/or hydrogen bonds between E2 on one strand and C-terminal Fp residues on adjacent strands, like R22. A longer Fp sheet results in shorter N- and C-helices, and larger separation during membrane apposition which hinders fusion.

A Meta-Analysis of gRNA Library Screens Enables an Improved Understanding of the Impact of gRNA Folding and Structural Stability on CRISPR-Cas9 Activity.

CRISPR systems are known to be inhibited by unwanted secondary structures that form within the guide RNA (gRNA). The minimum free energy of predicted secondary structures has been used in prediction algorithms. However, the types of structures as well as the degree to which a predicted structure can inhibit Cas9/gRNA activity is not well characterized. Here, we perform a meta-analysis of 39 published CRISPR-Cas9 data sets to understand better the role of secondary structures in inhibiting gRNA activity. We (1) identify two distinct inhibitory structures that can form, (2) measure the prevalence of these structures in existing gRNA library data sets, and (3) provide free energy cutoffs at which these structures become inhibitory. First, we show that hairpins that form within the targeting portion (spacer) of the gRNA, having a minimum free energy of <-5 kcal/mol, negatively impact gRNA activity. Second, we demonstrate that a longer hairpin can form between the spacer and the nexus portion of the gRNA scaffold. A duplex stability of this longer hairpin of <-15 kcal/mol negatively impacts gRNA activity. These cutoffs help to explain conflicting impacts of free energy values in different data sets, as well as provide a guideline for future gRNA designs.

RNA hairpin folding dynamics in a cell

RNA folding plays an essential role for temperature dependent functions in cells. For example, RNA thermometers regulate translation of heat shock and virulence genes by folding and unfolding. Here, we employed the fourU RNA thermometer motif of Salmonella, a hairpin—structured RNA to study in-cell folding stability of an RNA hairpin and its in vitro stability which is perturbed by crowding and sticking agents. To discover the solvation environment that accounts for cellular crowding and sticking we conduct a two-dimensional scan of mixtures of artificial crowding and sticking agents, PEG10K (polyethylene glycol, 0-300 mg/ml) and M-PER buffer (Mammalian Protein Extraction Reagent, 0-80%).

CCG•CGG interruptions in high‐penetrance SCA8 families increase RAN translation and protein toxicity

Spinocerebellar ataxia type 8 (SCA8), a dominantly inherited neurodegenerative disorder caused by a CTG•CAG expansion, is unusual because most individuals that carry the mutation do not develop ataxia. To understand the variable penetrance of SCA8, we studied the molecular differences between highly penetrant families and more common sporadic cases (82%) using a large cohort of SCA8 families (n = 77). We show that repeat expansion mutations from individuals with multiple affected family members have CCG•CGG interruptions at a higher frequency than sporadic SCA8 cases and that the number of CCG•CGG interruptions correlates with age at onset. At the molecular level, CCG•CGG interruptions increase RNA hairpin stability, and in cell culture experiments, increase p‐eIF2α and polyAla and polySer RAN protein levels. Additionally, CCG•CGG interruptions, which encode arginine interruptions in the polyGln frame, increase toxicity of the resulting proteins. In summary, SCA8 CCG•CGG interruptions increase polyAla and polySer RAN protein levels, polyGln protein toxicity, and disease penetrance and provide novel insight into the molecular differences between SCA8 families with high vs. low disease penetrance.

The hairpin extension controls solvent access to the chromophore binding pocket in a bacterial phytochrome: a UV\u2013vis absorption spectroscopy study

Solvent access to the protein interior plays an important role in the function of many proteins. Phytochromes contain a specific structural feature, a hairpin extension that appears to relay structural information from the chromophore to the rest of the protein. The extension interacts with amino acids near the chromophore, and hence shields the chromophore from the surrounding solvent. We envision that the detachment of the extension from the protein surface allows solvent exchange reactions in the vicinity of the chromophore. This can facilitate for example, proton transfer processes between solvent and the protein interior. To test this hypothesis, the kinetics of the protonation state of the biliverdin chromophore from Deinococcus radiodurans bacteriophytchrome, and thus, the pH of the surrounding solution, is determined. The observed absorbance changes are related to the solvent access of the chromophore binding pocket, gated by the hairpin extension. We therefore propose a model with an “open” (solvent-exposed, deprotonation-active on a (sub)second time-scale) state and a “closed” (solvent-gated, deprotonation inactive) state, where the hairpin fluctuates slowly between these conformations thereby controlling the deprotonation process of the chromophore on a minute time scale. When the connection between the hairpin and the biliverdin surroundings is destabilized by a point mutation, the amplitude of the deprotonation phase increases considerably. In the absence of the extension, the chromophore deprotonates essentially without any “gating”. Hence, we introduce a straightforward method to study the stability and fluctuation of the phytochrome hairpin in its photostationary state. This approach can be extended to other chromophore-protein systems where absorption changes reflect dynamic processes of the protein.

Effect of loop sequence on unzipping of short DNA hairpins.

The dependence of stability on the sequence of a DNA hairpin has been investigated through atomistic simulations. For this, a sequence of 16 bases of a hairpin, which consists of a loop of four bases and a stem of six base pairs, has been considered. We have taken eight different sequences, where the first five base pairs were kept fixed in all sequences, whereas the loop sequence and the identity of the duplex base pair closing the loop have been varied. For these hairpin structures, force-induced melting (unzipping) studies were carried out to investigate the effect of the variables on the stability of hairpin. The temperature at which half of the base pairs are open is termed the melting temperature. We defined the unzipping force F_{h} (half of the base pairs are open) and showed that it may not provide the effect of closing the base pair or loop sequence on the stability of the DNA hairpin. In order to have a better understanding of the stability of a DNA hairpin, the closing base pair or hairpin loop must be open. This requires complete opening of the stem. We defined a force F_{c} at which all base pairs of the stem are open, and we showed that the F_{c} gives better understanding of DNA hairpin stability.

Quantify the combined effects of temperature and force on the stability of DNA hairpin

OxDNA, as a successful coarse-grain model, has been applied to reproduce the thermodynamic and mechanical properties of both single- and double-stranded DNA. In current simulation, oxDNA is extended to explore the combined effects of temperature and force on the stability of DNA hairpin and its free energy landscape. Simulations were carried out at different forces and temperatures, at each temperature, a 18-base-pair DNA hairpin dynamically transited between folded state and unfolded state, and the separation between two states is consistent with the full contour length of single-stranded DNA in the unfolded state. Two methods were used to identify the critical force of DNA hairpin at each temperature and the critical forces obtained from two methods were consistent with each other and gradually decreased with the increasing temperature from 300 K to 326 K. The critical force at 300 K is reasonably consistent with the single molecule result of DNA hairpin with the same stem length. The two-state free energy landscape can be elucidated from the probability distribution of DNA hairpin extension and its dependence on the force and temperature is totally different. The increasing temperature not only reduces the free energy barrier, but also alters the position of transition point along the extension coordinate, resulting in the reduction of folding distance and the extension of unfolding distance, but their sum is not obviously dependent on the temperature. Generally, an assumption that the location of transition state in two-state energy landscape is independent of the stretching force is used to analyze the data of the single molecule experiment, but current simulation results indicate that effects of stretching forces on the location of transition state in two-state energy landscape are dependent on temperature. At relatively high temperature, stretching force can also change the location of transition state in the free energy landscape.

Regulation of VP30-Dependent Transcription by RNA Sequence and Structure in the Genomic Ebola Virus Promoter.

Viral transcription and replication of Ebola virus (EBOV) is balanced by transcription factor VP30, an RNA binding protein. An RNA hairpin at the transcription start site (TSS) of the first gene (NP hairpin) in the 3'-leader promoter is thought to mediate the VP30 dependency of transcription. Here, we investigated the constraints of VP30 dependency using a series of monocistronic minigenomes with sequence, structure and length deviations from the native NP hairpin. Hairpin stabilizations decreased while destabilizations increased transcription in the absence of VP30, but in all cases, transcription activity was higher in the presence versus absence of VP30. This also pertains to a mutant that is unable to form any RNA secondary structure at the TSS, demonstrating that the activity of VP30 is not simply determined by the capacity to form a hairpin structure at the TSS. Introduction of continuous 3'-UN5 hexamer phasing between promoter elements PE1 and PE2 by a single point mutation in the NP hairpin boosted VP30-independent transcription. Moreover, this point mutation, but also hairpin stabilizations, impaired the relative increase of replication in the absence of VP30. Our results suggest that the native NP hairpin is optimized for tight regulation by VP30 while avoiding an extent of hairpin stability that impairs viral transcription, as well as for enabling the switch from transcription to replication when VP30 is not part of the polymerase complex.IMPORTANCE A detailed understanding is lacking how the Ebola virus (EBOV) protein VP30 regulates activity of the viral polymerase complex. Here, we studied how RNA sequence, length and structure at the transcription start site (TSS) in the 3'-leader promoter influence the impact of VP30 on viral polymerase activity. We found that hairpin stabilizations tighten the VP30 dependency of transcription but reduce transcription efficiency and attenuate the switch to replication in the absence of VP30. Upon hairpin destabilization, VP30-independent transcription - already weakly detectable at the native promoter - increases, but never reaches the same extent as in the presence of VP30. We conclude that the native hairpin structure involving the TSS (i) establishes an optimal balance between efficient transcription and tight regulation by VP30, (ii) is linked to hexamer phasing in the promoter, and (iii) favors the switch to replication when VP30 is absent.

Exploring the Thermodynamics of 7-Amino Actinomycin D-Induced Single-Stranded DNA Hairpin by Spectroscopic Techniques and Computational Simulations.

NMR studies have indicated that the anti-tumor therapeutic agent actinomycin D (ACTD) can induce seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT3GTGG-3' to form a hairpin structure with tandem GT mismatches at the stem region next to a loop of three stacked thymine bases. In an effort to uncover the preference of binding sequence and to elucidate the thermodynamics properties of the binding, a combination of spectroscopic techniques and computational simulation studies was performed with d(CCGTTnGTGG) and d(CCGAAnGAGG) (denoted as GTTn and GAAn, respectively; n = 3, 5, and 7) sequences. In the presence of 7-amino actinomycin D (7AACTD), all the six oligomers formed stable hairpin structures. The GTT5-7AACTD/GAA5-7AACTD hairpin structure was more stable than the corresponding GTTn-7AACTD and GAAn-7AACTD (n = 3, 7). No significant ΔG difference was observed between GTTn-7AACTD and GAAn-7AACTD complexes with the same loop length. In agreement with the 7AACTD-induced hairpin stability results, the binding affinity of GTTn and GAAn with 7AACTD increased from n = 3 to n = 5 and then decreased when n is 7. Moreover, GTTn and GAAn with the same loop length showed comparable binding affinities to 7AACTD. Furthermore, molecular dynamics simulations found that van der Waals interactions between GTTn/GAAn and 7AACTD were the primary attractive forces for 7AACTD binding, and the electrostatic interactions between the carbonyl groups of 7AACTD and bases in the hairpin were the major unfavorable forces. These findings furthered our understanding that 7AACTD is sensitive to the loop size and sequence as well as tandem GT/GA mismatches of their deoxyribonucleic acid (DNA) targets. A deep understanding of the thermodynamics and the molecular recognition mechanism of 7AACTD with ssDNAs would further the development of ACTD-like antitumor agents.

Identification of new driver and passenger mutations within APOBEC-induced hotspot mutations in bladder cancer

BackgroundAPOBEC-driven mutagenesis and functional positive selection of mutated genes may synergistically drive the higher frequency of some hotspot driver mutations compared to other mutations within the same gene, as we reported for FGFR3 S249C. Only a few APOBEC-associated driver hotspot mutations have been identified in bladder cancer (BCa). Here, we systematically looked for and characterised APOBEC-associated hotspots in BCa.MethodsWe analysed 602 published exome-sequenced BCas, for part of which gene expression data were also available. APOBEC-associated hotspots were identified by motif-mapping, mutation signature fitting and APOBEC-mediated mutagenesis comparison. Joint analysis of DNA hairpin stability and gene expression was performed to predict driver or passenger hotspots. Aryl hydrocarbon receptor (AhR) activity was calculated based on its target genes expression. Effects of AhR knockout/inhibition on BCa cell viability were analysed.ResultsWe established a panel of 44 APOBEC-associated hotspot mutations in BCa, which accounted for about half of the hotspot mutations. Fourteen of them overlapped with the hotspots found in other cancer types with high APOBEC activity. They mostly occurred in the DNA lagging-strand templates and the loop of DNA hairpins. APOBEC-associated hotspots presented systematically a higher prevalence than the other mutations within each APOBEC-target gene, independently of their functional impact. A combined analysis of DNA loop stability and gene expression allowed to distinguish known passenger from known driver hotspot mutations in BCa, including loss-of-function mutations affecting tumour suppressor genes, and to predict new candidate drivers, such as AHR Q383H. We further characterised AHR Q383H as an activating driver mutation associated with high AhR activity in luminal tumours. High AhR activity was also found in tumours presenting amplifications of AHR and its co-receptor ARNT. We finally showed that BCa cells presenting those different genetic alterations were sensitive to AhR inhibition.ConclusionsOur study identified novel potential drivers within APOBEC-associated hotspot mutations in BCa reinforcing the importance of APOBEC mutagenesis in BCa. It could allow a better understanding of BCa biology and aetiology and have clinical implications such as AhR as a potential therapeutic target. Our results also challenge the dogma that all hotspot mutations are drivers and mostly gain-of-function mutations affecting oncogenes.

Single-Molecule Mechanical Unfolding of AT-Rich Chromosomal Fragile Site DNA Hairpins: Resolving the Thermodynamic and Kinetic Effects of a Single G-T Mismatch.

Chromosomal fragile sites (CFSs) contain AT-rich sequences that tend to form hairpins on lagging strands in DNA replication, making them hotspots for chromosomal rearrangements in cancers. Here, we investigate the structural stability of the AT-rich CFS DNA hairpins with a single non-AT base pair using magnetic tweezers. Strikingly, a single G-T mismatched base pair in the short CFS DNA hairpin gives a 38.7% reduction of the unfolding Gibbs free energy and a 100-fold increase of the transition kinetics compared to a single G-C matched base pair, which are deviated from the theoretical simulations. Our study reveals the unique features of CFSs to provide profound insights into chromosomal instability and structure-specific genome targeting therapeutics for genetic disorder-related diseases.