Distinguishing G-Quadruplexes Stabilizer and Chaperone for c-MYC Promoter G-Quadruplexes through Single-Molecule Manipulation.

G-quadruplex (G4) selective stabilizing ligands can regulate c-MYC gene expression, but the kinetic basis remains unclear. Determining the effects of ligands on c-MYC promoter G4s' folding/unfolding kinetics is challenging due to the polymorphic nature of G4s and the high energy barrier to unfold c-MYC promoter G4s. Here, we used single-molecule magnetic tweezers to manipulate a duplex hairpin containing a c-MYC promoter sequence to mimic the transiently denatured duplex during transcription. We measured the effects of six commonly used G4s binding ligands on the competition between quadruplex and duplex structures, as well as the folding/unfolding kinetics of G4s. Our results revealed two distinct roles for G4s selective stabilization: CX-5461 is mainly acting as c-MYC G4s stabilizer, reducing the unfolding rate (ku) of c-MYC G4s, whereas PDS and 360A also act as G4s chaperone, accelerating the folding rates (kf) of c-MYC G4s. qRT-PCR results obtained from CA46 and Raji cell lines demonstrated that G4s stabilizing ligands can downregulate c-MYC expression, while G4s stabilizer CX-5461 exhibited the strongest c-MYC gene suppression. These results shed light on the potential of manipulating G4s' folding/unfolding kinetics by ligands for precise regulation of promoter G4-associated biological activities.

A TeZla micromixer for interrogating the early and broad folding landscape of G-quadruplex via multistage velocity descending.

Biomacromolecular folding kinetics involves fast folding events and broad timescales. Current techniques face limitations in either the required time resolution or the observation window. In this study, we developed the TeZla micromixer, integrating Tesla and Zigzag microstructures with a multistage velocity descending strategy. TeZla achieves a significant short mixing dead time (40 µs) and a wide time window covering four orders of magnitude (up to 300 ms). Using this unique micromixer, we explored the folding landscape of c-Myc G4 and its noncanonical-G4 derivatives with different loop lengths or G-vacancy sites. Our findings revealed that c-Myc can bypass folding intermediates and directly adopt a G4 structure in the cation-deficient buffer. Moreover, we found that the loop length and specific G-vacancy site could affect the folding pathway and significantly slow down the folding rates. These results were also cross-validated with real-time NMR and circular dichroism. In conclusion, TeZla represents a versatile tool for studying biomolecular folding kinetics, and our findings may ultimately contribute to the design of drugs targeting G4 structures.

Nonselective Intercalation of G-Quadruplex-Targeting Ligands into Double-Stranded DNA Quantified by Single-Molecule Stretching.

Most G-quadruplex (G4)-targeting ligands reported so far contain planar heteroaromatic groups and can intercalate into adjacent base pairs of double-stranded DNA (dsDNA). However, quantitative data on the binding number γ (ligands/bp) of G4 ligands that intercalate into long dsDNA remain lacking, which are essential for understanding the selectivity of G4 ligands. Here, using a single-molecule stretching assay based on the lengthening of dsDNA, we analyzed the dissociation constants and the binding number of eight most commonly used G4 ligands that intercalate into dsDNA. We showed that five ligands (CX-5461, BRACO-19, RHPS4, TrisQ, and Phen-DC3) intercalate into dsDNA avidly (Kd = 0.5-2.1 µM, saturated γ > 0.2 ligands/bp), which was similar to the typical dsDNA intercalator EB. Two bisquinolines, PDS and 360A, showed moderate intercalation ability (Kd = 22.5 and 48.7 µM) and γ < 0.01 ligands/bp in the presence of 1 µM ligands. Porphyrin NMM showed no intercalative binding even at 200 µM. Molecular docking and molecular dynamics simulations were carried out to further evaluate the intercalative binding of these G4 ligands with dsDNA by calculating the binding energies and π-π stacking probability.

Force-Dependent Intercalative Bulky DNA Adduct Formation Detected by Single-Molecule Stretching.

Quantitatively analyzing the binding topology and reactivity is essential for understanding the cytotoxic or tumorigenic activities of bulky DNA adducts formed by chemotherapeutic drugs or carcinogens. Biochemical methods require purification of DNA and discontinuous steps to digest or label the adducts and thus have difficulties in identifying the binding topology and are not suitable for detecting unstable adducts. Herein, we used a single-molecule stretching assay to characterize the number of intercalative adducts, the formation kinetics, and the mechanical properties of intercalative DNA adducts based on measuring adduct-induced DNA elongation. We analyzed various reactive conditions, including formaldehyde-mediated anthracycline-DNA adducts, UV light-catalyzed psoralen-DNA adducts, and liver S9 fraction-catalyzed aflatoxin B1-DNA adducts. We showed that adduct formation abilities are correlated with the noncovalent intercalation binding ability. External forces on double-stranded DNA increased the intercalation of ligands and can result in a 1.8- to 5.3-fold increase in DNA adduct formation.

Characterization of G-Quadruplexes Folding/Unfolding Dynamics and Interactions with Proteins from Single-Molecule Force Spectroscopy.

G-quadruplexes (G4s) are stable secondary nucleic acid structures that play crucial roles in many fundamental biological processes. The folding/unfolding dynamics of G4 structures are associated with the replication and transcription regulation functions of G4s. However, many DNA G4 sequences can adopt a variety of topologies and have complex folding/unfolding dynamics. Determining the dynamics of G4s and their regulation by proteins remains challenging due to the coexistence of multiple structures in a heterogeneous sample. Here, in this mini-review, we introduce the application of single-molecule force-spectroscopy methods, such as magnetic tweezers, optical tweezers, and atomic force microscopy, to characterize the polymorphism and folding/unfolding dynamics of G4s. We also briefly introduce recent studies using single-molecule force spectroscopy to study the molecular mechanisms of G4-interacting proteins.

Mechanical diversity and folding intermediates of parallel-stranded G-quadruplexes with a bulge.

A significant number of sequences in the human genome form noncanonical G-quadruplexes (G4s) with bulges or a guanine vacancy. Here, we systematically characterized the mechanical stability of parallel-stranded G4s with a one to seven nucleotides bulge at various positions. Our results show that G4-forming sequences with a bulge form multiple conformations, including fully-folded G4 with high mechanical stability (unfolding forces > 40 pN), partially-folded intermediates (unfolding forces < 40 pN). The folding probability and folded populations strongly depend on the positions and lengths of the bulge. By combining a single-molecule unfolding assay, dimethyl sulfate (DMS) footprinting, and a guanine-peptide conjugate that selectively stabilizes guanine-vacancy-bearing G-quadruplexes (GVBQs), we identified that GVBQs are the major intermediates of G4s with a bulge near the 5' or 3' ends. The existence of multiple structures may induce different regulatory functions in many biological processes. This study also demonstrates a new strategy for selectively stabilizing the intermediates of bulged G4s to modulate their functions.

Noninvasive Differentiation of Obstructive Azoospermia and Nonobstructive Azoospermia Using Multimodel Diffusion Weighted Imaging.

RATIONALE AND OBJECTIVES: To evaluate the diagnostic performance of parameters derived from multimodel diffusion weighted imaging (monoexponential, stretched-exponential diffusion weighted imaging and diffusion kurtosis imaging [DKI]) from noninvasive magnetic resonance imaging in distinguishing obstructive azoospermia (OA) from nonobstructive azoospermia (NOA). MATERIALS AND METHODS: Forty-six patients with azoospermia were prospectively enrolled and classified into two groups (21 OA patients and 25 NOA patients). The multimodel parameters of diffusion-weighted imaging (DWI; apparent diffusion coefficient [ADC], distributed diffusion coefficient [DDC], diffusion heterogeneity [α], diffusion kurtosis diffusivity [Dapp], and diffusion kurtosis coefficient [Kapp]) were derived. The diagnostic performance of these parameters for the differentiation of OA and NOA patients were evaluated using receiver operating characteristic analysis. The area under the curve (AUC) was calculated to evaluate the diagnostic accuracy of each parameter. RESULTS: All the parameters (ADC, α, DDC, Dapp, and Kapp) values were significantly different between OA and NOA (P < 0.001 for all). For the differentiation of OA from NOA, Kapp showed the highest AUC value (0.965), followed by DDC (0.946), Dapp (0.933), ADC (0.922), and α (0.887). Kapp had a significantly higher AUC than the conventional ADC (P < 0.05). CONCLUSION: Parameters derived from multimodels of DWI have the potential for the noninvasive differentiation of OA and NOA. The Kapp value derived from the DKI model might serve as a useful imaging marker for the differentiation of azoospermia.

Oxygen-Challenge Blood Oxygen Level-Dependent Magnetic Resonance Imaging for Evaluation of Early Change of Hepatocellular Carcinoma to Chemoembolization: A Feasibility Study.

RATIONALE AND OBJECTIVES: To investigate the feasibility of oxygen-challenge blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) at 3T for evaluating the early change of blood oxygenation before and after transcatheter arterial embolization (TACE) in patients with hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Thirty HCC patients with cirrhosis (HCC group, n = 30) and 30 healthy volunteers (control group, n = 30) were included in this study. Patients in the HCC group underwent BOLD before and 1 month after TACE. Oxygen was administered via a mask. Differences between pre- and post-O2 T2* values were evaluated using a pairwise t-test. Analysis of variance was performed to assess the statistical differences in the T2* values measured in HCC group pre-TACE and post-TACE and in healthy volunteers. RESULTS: In the HCC group, the pre- and post-O2 T2* values of the cancerous area before TACE were 26.03 ± 3.30 and 26.84 ± 3.42 msec, respectively, and both decreased significantly to 8.67 ± 1.76 and 8.82 ± 1.80 msec, respectively, at 1 month after TACE (p < 0.001). The respective pre- and post-O2 T2* values of the noncancerous area increased significantly from 14.96 ± 2.32 and 15.33 ± 2.28 msec at baseline to 16.38 ± 2.22 and 16.89 ± 2.24 msec at 1 month after TACE (p < 0.001). No significant response to BOLD was observed in the control group (p = 0.059). CONCLUSION: Oxygen-challenge BOLD MRI is feasible to assess post-TACE changes in HCC patients.

Using Magnetic Tweezers to Unravel the Mechanism of the G-quadruplex Binding and Unwinding Activities of DHX36 Helicase.

Single-molecule manipulation methods are useful techniques to probe the interactions of proteins and nucleic acid structures. Here, we describe the magnetic tweezers-based single-molecule investigation of the binding of helicases to G-quadruplex structures and their ATP-dependent unwinding activity, using DHX36 (also known as RHAU and G4R1) helicase and a DNA G-quadruplex structure for an example. We specifically emphasize on the principle and method to probe the interactions between DHX36 and the DNA G-quadruplex in different intermediate states during an ATPase cycle of DHX36, based on detecting the DHX36-induced changes in the lifetime of the DNA G-quadruplex under tension. The principle of the measurement can be broadly extended to the studies of other DNA or RNA G-quadruplex helicases.

High Mechanical Stability and Slow Unfolding Rates Are Prevalent in Parallel-Stranded DNA G-Quadruplexes.

Guanine-rich repeat sequences are known to adopt diverse G-quadruplex (G4) topologies. Determining the unfolding rates of individual G4 species is challenging due to the coexistence of multiple G4 conformations in a solution. Here, using single-molecule magnetic tweezers, we systematically measured the unfolding force distributions of 4 oncogene promoter G4s, 12 model sequences with two 1-nucleotide (nt) thymine loops that predominantly adopt parallel-stranded G4 structures, and 6 sequences forming multiple G4 structures. All parallel-stranded G4s reveal an unfolding force peak at 40-60 pN, which is associated with extremely slow unfolding rates on the order of 10-5-10-7 s-1. In contrast, nonparallel G4s and partially folded intermediate states reveal an unfolding force peak <40 pN. These results suggest a strong correlation between the parallel-stranded G4s folding topology and the slow unfolding rates and provide important insights into the mechanism that govern the stability and the transition kinetics of G4s.

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.

Discrimination between benign and malignant testicular lesions using volumetric apparent diffusion coefficient histogram analysis.

PURPOSE: This study aimed to evaluate the role of volumetric apparent diffusion coefficient (ADC) histogram analysis in discriminating between benign and malignant testicular masses. METHODS: In this retrospective study, fifty-nine patients with 61 pathologically confirmed testicular masses were consecutively enrolled, including 18 benign lesions and 43 malignant lesions. All patients conducted preoperative magnetic resonance imaging (MRI) with diffusion-weighted imaging. Eighteen volumetric histogram parameters were extracted from the ADC map of each lesion. Comparisons were conducted by an independent t-test or Mann-Whitney U test, where appropriate. The classification performance of the parameters that showed significant differences between benign and malignant testicular disease were evaluated via receiver operating characteristic (ROC) curve analysis. RESULTS: Among the 18 histogram parameters we extracted, the energy, total energy, and range of ADC of testicular malignancies were all significantly increased compared with those of benignities. The minimum ADC and 10th percentile ADC of testicular malignancies were both significantly reduced compared with those of benignities. The minimum ADC value achieved the highest diagnostic performance in distinguishing between testicular benignities and malignancies, with an area under the ROC curve (AUC) of 0.822, sensitivity of 81.40 %, and specificity of 77.78 %. CONCLUSIONS: Volumetric ADC histogram analysis might be a useful tool to preoperatively discriminate between benign and malignant testicular masses.

Sight and switch off: Nerve density visualization for interventions targeting nerves in prostate cancer.

Nerve density is associated with prostate cancer (PCa) aggressiveness and prognosis. Thus far, no visualization methods have been developed to assess nerve density of PCa in vivo. We compounded propranolol-conjugated superparamagnetic iron oxide nerve peptide nanoparticles (PSN NPs), which achieved the nerve density visualization of PCa with high sensitivity and high specificity, and facilitated assessment of nerve density and aggressiveness of PCa using magnetic resonance imaging and magnetic particle imaging. Moreover, PSN NPs facilitated targeted therapy for PCa. PSN NPs increased the survival rate of mice with orthotopic PCa to 83.3% and decreased nerve densities and proliferation indexes by more than twofold compared with the control groups. The present study, thus, developed a technology to visualize the nerve density of PCa and facilitate targeted neural drug delivery to tumors to efficiently inhibit PCa progression. Our study provides a potential basis for clinical imaging and therapeutic interventions targeting nerves in PCa.

Detecting the Formation Kinetics of Doxorubicin-DNA Interstrand Cross-link at the Single-Molecule Level and Clinically Relevant Concentrations of Doxorubicin.

Doxorubicin (DOX) ranks among the most effective anticancer agents. Increasing the formation of covalent DOX-DNA interstrand cross-links can improve the anticancer activity of DOX. However, due to the low stability of the DOX-DNA cross-links to heat and alkali, DOX can be extensively lost during isolation procedures of biochemical methods, thus reducing the apparent clinical relevance of this mechanism. Here, we developed a drug label-free, single-molecule magnetic tweezers assay that can detect a single DOX-DNA cross-link on the basis of the significant increase of the unzipping forces of DNA hairpins upon drug binding. Using this assay, we measured the DOX concentration-dependent cross-linking rates at clinically relevant concentrations of DOX. We report an ∼26-fold higher formaldehyde concentration dependence of cross-linking rates than previously reported and 0.9 ± 0.8 cross-links/103 bp at the clinically relevant concentrations of 70 nM DOX and 50 µM formaldehyde. Our results suggest a much higher cross-link formation ability than previous bulk measurements have reported and suggest that the cross-linking mechanism has promising therapeutic potential. This general method can be used to detect the formation kinetics of other DNA lesions or DNA adducts that affect DNA duplex stability.

T2-Weighted Image-Based Radiomics Signature for Discriminating Between Seminomas and Nonseminoma.

Objective: To evaluate the performance of a T2-weighted image (T2WI)-based radiomics signature for differentiating between seminomas and nonseminomas. Materials and Methods: In this retrospective study, 39 patients with testicular germ-cell tumors (TGCTs) confirmed by radical orchiectomy were enrolled, including 19 cases of seminomas and 20 cases of nonseminomas. All patients underwent 3T magnetic resonance imaging (MRI) before radical orchiectomy. Eight hundred fifty-one radiomics features were extracted from the T2WI of each patient. Intra- and interclass correlation coefficients were used to select the features with excellent stability and repeatability. Then, we used the minimum-redundancy maximum-relevance (mRMR) and the least absolute shrinkage and selection operator (LASSO) algorithms for feature selection and radiomics signature development. Receiver operating characteristic curve analysis was used to evaluate the diagnostic performance of the radiomics signature. Results: Five features were selected to build the radiomics signature. The radiomics signature was significantly different between the seminomas and nonseminomas (p < 0.01). The area under the curve (AUC), sensitivity, and specificity of the radiomics signature for discriminating between seminomas and nonseminomas were 0.979 (95% CI: 0.873-1.000), 90.00 (95% CI: 68.3-98.8), and 100.00 (95% CI: 82.4-100.0), respectively. Conclusion: The T2WI-based radiomics signature has the potential to non-invasively discriminate between seminomas and nonseminomas.

A Universal Assay for Making DNA, RNA, and RNA-DNA Hybrid Configurations for Single-Molecule Manipulation in Two or Three Steps without Ligation.

Despite having a great variety of topologies, most DNA, RNA, and RNA-DNA hybrid (RDH) configurations for single-molecule manipulation are composed of several single-stranded (ss) DNA and ssRNA strands, with functional labels at the two ends for surface tethering. On this basis, we developed a simple, robust, and universal amplification-annealing (AA) assay for making all these configurations in two or three steps without inefficient digestion and ligation reactions. As examples, we made ssDNA, short ssDNA with double-stranded (ds) DNA handles, dsDNA with ssDNA handles, replication-fork shaped DNA/RDH/RNA, DNA holiday junction, three-site multiple-labeled and nicked DNA, torsion-constrained RNA/RDH, and short ssRNA with RDH handles. In addition to single-molecule manipulation techniques including optical tweezers, magnetic tweezers, and atomic force microscopy, these configurations can be applied in other surface-tethering techniques as well.

Validation of SE-EPI-based T2 mapping for characterization of prostate cancer: a new method compared with the traditional CPMG method.

PURPOSE: We aim to compare the results of spin echo-echo planar imaging (SE-EPI)-based T2 mapping with those of the conventional Carr-Purcell-Meiboom-Gill (CPMG) method and to investigate the potential validity of SE-EPI-T2 mapping for the characterization of prostate cancer (PCa). METHODS: Our retrospective study included 42 PCa patients and 42 noncancer patients who underwent 3.0T MRI with b values ranging from 0 to 2000 s/mm2 and echo times (TEs) ranging from 32 to 100 ms before biopsies. Bland-Altman analysis was used to compare the agreement between the two methods. The correlations between CPMG-T2 values and SE-EPI-T2 values at different b values were determined by Spearman's rho analysis or Pearson analysis. The Mann-Whitney U test and two-sample t tests were used to analyze the differences between the cancerous and noncancerous groups. RESULTS: Substantial agreement regarding the measurements was observed between the two methods. The average correlation between the CPMG-T2 values and SE-EPI-T2 values was moderate and positive, and the best correlations were found at b = 200 s/mm2 in the noncancer group (r = 0.557, P = 0.000) and at b = 100 s/mm2 in the cancer group (r = 0.537, P = 0.000). In addition, statistically significant differences were found between the noncancer and cancer groups in T2 values and ADC values (diff TEs) (P = 0.000). CONCLUSIONS: Substantial agreement in the measurements was found between the SE-EPI method and CPMG method. SE-EPI-based T2 mapping has potential clinical value for the prostate and can be considered an alternative to the traditional CPMG-T2 mapping method.

Multi-parametric MRI-based radiomics signature for discriminating between clinically significant and insignificant prostate cancer: Cross-validation of a machine learning method.

PURPOSE: To evaluate the performance of a multi-parametric MRI (mp-MRI)-based radiomics signature for discriminating between clinically significant prostate cancer (csPCa) and insignificant PCa (ciPCa). MATERIALS AND METHODS: Two hundred and eighty patients with pathology-proven PCa were enrolled and were randomly divided into training and test cohorts. Eight hundred and nineteen radiomics features were extracted from mp-MRI for each patient. The minority group in the training cohort was balanced via the synthetic minority over-sampling technique (SMOTE) method. We used minimum-redundancy maximum-relevance (mRMR) selection and the LASSO algorithm for feature selection and radiomics signature building. The classification performance of the radiomics signature for csPCa and ciPCa was evaluated by receiver operating characteristic curve analysis in the training and test cohorts. RESULTS: Nine features were selected for the radiomics signature building. Significant differences in the radiomics signature existed between the csPCa and ciPCa groups in both the training and test cohorts (p < 0.01 for both). The AUC, sensitivity and specificity of the radiomics signature were 0.872 (95% CI: 0.823-0.921), 0.883, and 0.753, respectively, in the training cohort, and 0.823 (95% CI: 0.669-0.976), 0.841, and 0.727, respectively, in the test cohort. CONCLUSION: Mp-MRI-based radiomics signature have the potential to noninvasively discriminate between csPCa and ciPCa.

Folding/unfolding kinetics of G-quadruplexes upstream of the P1 promoter of the human BCL-2 oncogene.

The G-rich Pu39 region of the P1 promoter of the oncogene BCL-2, an apoptosis regulator, can fold into multiple G-quadruplex (G4) structures. Bcl2-2345 and Bcl2-1245 are two major G4 species forming with high thermal stability and distinct topologies in the Pu39 region, but their folding/unfolding kinetics have not yet been investigated. Here, we used magnetic tweezers to measure the mechanical stability and the folding/unfolding kinetics of the Bcl2-2345 and Bcl2-1245 G4 structures. We report that the hybrid-stranded Bcl2-2345 G4 had a lower mechanical stability than the parallel-stranded Bcl2-1245 G4. We observed that the Bcl2-2345 G4 is a kinetically favored structure, whereas the Bcl2-1245 G4, with a slow unfolding rate, may function as a kinetic barrier for transcription. We also determined that in addition to the Bcl2-2345 and Bcl2-1245 G4s, other stable DNA secondary structures, such as a hybrid-stranded Bcl2-1234 G4, can also form in the Pu39 sequence. The characterization of the folding/unfolding kinetics of specific G4s reported here sheds light on the participation of G4s during gene transcription and provides information for designing G4-targeting small molecules that could modulate BCL-2 gene expression.