A Redox-Reversible Switch of DNA Hydrogen Bonding and Structure.

Modulating molecular structure and function at the nanoscale drives innovation across wide-ranging technologies. Electrical control of the bonding of individual DNA base pairs endows DNA with precise nanoscale structural reconfigurability, benefiting efforts in DNA origami and actuation. Here, alloxazine DNA base surrogates were synthesized and incorporated into DNA duplexes to function as a redox-active switch of hydrogen bonding. Circular dichroism (CD) revealed that 24-mer DNA duplexes containing one or two alloxazines exhibited CD spectra and melting transitions similar to DNA with only canonical bases, indicating that the constructs adopt a B-form conformation. However, duplexes were not formed when four or more alloxazines were incorporated into a 24-mer strand. Thiolated duplexes incorporating alloxazines were self-assembled onto multiplexed gold electrodes and probed electrochemically. Square-wave voltammetry (SWV) revealed a substantial reduction peak centered at -0.272 V vs Ag/AgCl reference. Alternating between alloxazine oxidizing and reducing conditions modulated the SWV peak in a manner consistent with the formation and loss of hydrogen bonding, which disrupts the base pair stacking and redox efficiency of the DNA construct. These alternating signals support the assertion that alloxazine can function as a redox-active switch of hydrogen bonding, useful in controlling DNA and bioinspired assemblies.

Structure-based investigation of a DNA aptamer targeting PTK7 reveals an intricate 3D fold guiding functional optimization.

DNA aptamers have emerged as novel molecular tools in disease theranostics owing to their high binding affinity and specificity for protein targets, which rely on their ability to fold into distinctive three-dimensional (3D) structures. However, delicate atomic interactions that shape the 3D structures are often ignored when designing and modeling aptamers, leading to inefficient functional optimization. Challenges persist in determining high-resolution aptamer-protein complex structures. Moreover, the experimentally determined 3D structures of DNA molecules with exquisite functions remain scarce. These factors impede our comprehension and optimization of some important DNA aptamers. Here, we performed a streamlined solution NMR-based structural investigation on the 41-nt sgc8c, a prominent DNA aptamer used to target membrane protein tyrosine kinase 7, for cancer theranostics. We show that sgc8c prefolds into an intricate three-way junction (3WJ) structure stabilized by long-range tertiary interactions and extensive base-base stackings. Delineated by NMR chemical shift perturbations, site-directed mutagenesis, and 3D structural information, we identified essential nucleotides constituting the key functional elements of sgc8c that are centralized at the core of 3WJ. Leveraging the well-established structure-function relationship, we efficiently engineered two sgc8c variants by modifying the apical loop and introducing L-DNA base pairs to simultaneously enhance thermostability, biostability, and binding affinity for both protein and cell targets, a feat not previously attained despite extensive efforts. This work showcases a simplified NMR-based approach to comprehend and optimize sgc8c without acquiring the complex structure, and offers principles for the sophisticated structure-function organization of DNA molecules.

Effects of interrupting residues on DNA dumbbell structures formed by CCTG tetranucleotide repeats associated with myotonic dystrophy type 2.

Myotonic dystrophy type 2 (DM2) is a neurogenerative disease caused by caprylic/capric triglyceride (CCTG) tetranucleotide repeat expansions in intron 1 of the cellular nucleic acid-binding protein (CNBP) gene. Non-B DNA structures formed by CCTG repeats can promote genetic instability, whereas interrupting motifs of NCTG (N = A/T/G) within CCTG repeats help to maintain genomic stability. However, whether the interrupting motifs can affect DNA structures of CCTG repeats remains unclear. Here, we report that four CCTG repeats with an interrupting 3'-A/T/G residue formed dumbbell structures, whereas a non-interrupting 3'-C residue resulted in a multi-loop structure exhibiting conformational dynamics that may contribute to a higher tendency of escaping from DNA mismatch repair and causing repeat expansions. The results provide new structural insights into the genetic instability of CCTG repeats in DM2.

Monitoring of DNA structural changes after incorporation of the phenylpyrazole insecticide fipronil.

The use of insecticides presents a risk to the environment because they can accumulate in the water, soil, air, and organisms, endangering human and animal health. It is therefore essential to investigate the effects of different groups of insecticides on individual biomacromolecules such as DNA. We studied fipronil, which belongs to the group of phenylpyrazole insecticides. The interaction of fipronil with calf thymus DNA was investigated using spectroscopic methods (absorption and fluorescence spectroscopy) complemented with infrared spectroscopy and viscosity measurement. Fluorescence emission spectroscopy showed the formation of a fipronil/DNA complex with a combined static and dynamic type of quenching. The binding constant was 4.15 × 103 L/mol. Viscosity changes were recorded to confirm/disconfirm the intercalation mode of interaction. A slight change in DNA viscosity in the presence of fipronil was observed. The phenylpyrazole insecticide does not cause significant conformational changes in DNA structure or increase of its chain length. We hypothesize that fipronil is incorporated into the minor groove of the DNA macromolecule via hydrogen interactions as indicated by FT-IR and CD measurements.

Amodiaquine Nonspecifically Binds Double Stranded and Three-Way Junction DNA Structures.

The 4-aminoquinoline class of compounds includes the important antimalarial compounds amodiaquine and chloroquine. Despite their medicinal importance, the mode of action of these compounds is poorly understood. In a previous study we observed these compounds, as well as quinine and mefloquine, tightly bind the DNA cocaine-binding aptamer. Here, we further explore the range of nucleic acid structures bound by these compounds. To gauge a wide range of binding affinities, we used isothermal titration calorimetry to explore high affinity binding (nM to tens of µM) and NMR spectroscopy to assay weak binding biding in the hundreds of micromolar range. We find that amodiaquine tightly binds all double stranded DNA structures explored. Mefloquine binds double stranded DNA duplex molecules tightly and weakly associates with a three-way junction DNA construct. Quinine and chloroquine only weakly bind duplex DNA but do not tightly bind any of the DNA constructs explored. A simulation of the free energy of binding of these ligands to the Dickerson-Drew dodecamer resulted in an excellent agreement between the simulated and experimental free energy. These results provide new insight into the DNA binding of clinically important antimalarial compounds and may play a role in future development of new antimalarials.

Self-Assembled DNA Composite-Engineered Mesenchymal Stem Cells for Improved Skin-Wound Repair.

The direct use of mesenchymal stem cells (MSCs) as therapeutics for skin injuries is a promising approach, yet it still faces several obstacles, including limited adhesion, retention, and engraftment of stem cells in the wound area, as well as impaired regenerative and healing functions. Here, DNA-based self-assembled composites are reported that can aid the adhesion of MSCs in skin wounds, enhance MSC viability, and accelerate wound closure and re-epithelialization. Rolling-circle amplification (RCA)-derived DNA flowers, equipped with multiple copies of cyclic Arg-Gly-Asp (cRGD) peptides and anti-von Willebrand factor (vWF) aptamers, act as robust scavengers of reactive oxygen species (ROS) and enable synergistic recognition and adhesion to stem cells and damaged vascular endothelial cells. These DNA structure-aided stem cells are retained at localized wound sites, maintain repair function, and promote angiogenesis and growth factor secretion. In both normal and diabetes-prone db/db mice models with excisional skin injuries, facile topical administration of DNA flower-MSCs elicits rapid blood vessel formation and enhances the sealing of the wound edges in a single dose. DNA composite-engineered stem cells warrant further exploration as a new strategy for the treatment of skin and tissue damage.

Single-molecule observation of G-quadruplex and R-loop formation induced by transcription.

Potential G-quadruplex forming sequences (PQS) are enriched in cancer-related genes and immunoglobulin class-switch recombination. They are prevalent in the 5'UTR of transcriptionally active genes, thereby contributing to the regulation of gene expression. We and others previously demonstrated that the PQS located in the non-template strand leads to an R-loop formation followed by a G-quadruplex (G4) formation during transcription. These structural changes increase mRNA production. Here, we present how single-molecule technique was used to observe cotranscriptional G4 and R-loop formation and to examine the impact on transcription, particularly for the initiation and elongation stages.

The transcription of the main gene associated with Treacher-Collins syndrome (TCOF1) is regulated by G-quadruplexes and cellular nucleic acid binding protein (CNBP).

Treacle ribosome biogenesis factor 1 (TCOF1) is responsible for about 80% of mandibular dysostosis (MD) cases. We have formerly identified a correlation between TCOF1 and CNBP (CCHC-type zinc finger nucleic acid binding protein) expression in human mesenchymal cells. Given the established role of CNBP in gene regulation during rostral development, we explored the potential for CNBP to modulate TCOF1 transcription. Computational analysis for CNBP binding sites (CNBP-BSs) in the TCOF1 promoter revealed several putative binding sites, two of which (Hs791 and Hs2160) overlap with putative G-quadruplex (G4) sequences (PQSs). We validated the folding of these PQSs measuring circular dichroism and fluorescence of appropriate synthetic oligonucleotides. In vitro studies confirmed binding of purified CNBP to the target PQSs (both folded as G4 and unfolded) with Kd values in the nM range. ChIP assays conducted in HeLa cells chromatin detected the CNBP binding to TCOF1 promoter. Transient transfections of HEK293 cells revealed that Hs2160 cloned upstream SV40 promoter increased transcription of downstream firefly luciferase reporter gene. We also detected a CNBP-BS and PQS (Dr2393) in the zebrafish TCOF1 orthologue promoter (nolc1). Disrupting this G4 in zebrafish embryos by microinjecting DNA antisense oligonucleotides complementary to Dr2393 reduced the transcription of nolc1 and recapitulated the craniofacial anomalies characteristic of Treacher Collins Syndrome. Both cnbp overexpression and Morpholino-mediated knockdown in zebrafish induced nolc1 transcription. These results suggest that CNBP modulates the transcriptional expression of TCOF1 through a mechanism involving G-quadruplex folding/unfolding, and that this regulation is active in vertebrates as distantly related as bony fish and humans. These findings may have implications for understanding and treating MD.

High-Resolution NMR Structures of Intrastrand Hairpins Formed by CTG Trinucleotide Repeats.

The CAG and CTG trinucleotide repeat expansions cause more than 10 human neurodegenerative diseases. Intrastrand hairpins formed by trinucleotide repeats contribute to repeat expansions, establishing them as potential drug targets. High-resolution structural determination of CAG and CTG hairpins poses as a long-standing goal to aid drug development, yet it has not been realized due to the intrinsic conformational flexibility of repetitive sequences. We herein investigate the solution structures of CTG hairpins using nuclear magnetic resonance (NMR) spectroscopy and found that four CTG repeats with a clamping G-C base pair was able to form a stable hairpin structure. We determine the first solution NMR structure of dG(CTG)4C hairpin and decipher a type I folding geometry of the TGCT tetraloop, wherein the two thymine residues form a T·T loop-closing base pair and the first three loop residues continuously stack. We further reveal that the CTG hairpin can be bound and stabilized by a small-molecule ligand, and the binding interferes with replication of a DNA template containing CTG repeats. Our determined high-resolution structures lay an important foundation for studying molecular interactions between native CTG hairpins and ligands, and benefit drug development for trinucleotide repeat expansion diseases.

A trivalent aptasensor by using DNA tetrahedron as scaffold for label-free determination of antibiotics.

Owing to advantage in high sensitivity and fast response, aptamer based electrochemical biosensors have attracted much more attention. However, inappropriate interfacial engineering strategy leads to poor recognition performance, which ascribe to the following factors of immobilized oligonucleotide strand including steric hindrance, interchain entanglement, and unfavorable conformation. In this work, we proposed a DNA tetrahedron based diblock aptamer immobilized strategy for the construction of label-free electrochemical biosensor. The diblock aptamer sequence is composite of T-rich anchor domain and recognition domain, where T-rich domain enabling anchored on the edge of DNA tetrahedron via Hoogsteen hydrogen bond at neutral condition. The DNA tetrahedron scaffold offers an appropriate lateral space for target recognition of diblock aptamer. More importantly, this trivalent aptamer recognition interface can be regenerated by simply adjusting the pH environment to alkaline, resulting in the dissociation of diblock aptamer. Under the optimum condition, proposed electrochemical aptasensor manifested a satisfied sensitivity for aminoglycosides antibiotic, kanamycin with a limit of detection of 0.69 nM, which is 45-fold lower than traditional Au-S immobilization strategy. Moreover, the proposed aptasensor had also successfully been extended to ampicillin detection by changing the sequence of recognition domain in diblock aptamer. This work paves a new way for the rational design of aptamer-based electrochemical sensor.

Microfluidic bead-based biosensor: Ultrasensitive ctDNA detection based on duplex-functional split-DNAzyme and dendritic enzyme-free signal amplification.

Circulating tumor DNA (ctDNA) is a crucial cancer biomarker for early or noninvasive monitoring, which is essential for developing ultrasensitive and selective assays in cancer diagnosis and treatment. Herein, a cascade signal amplification of duplex-functional split-DNAzyme and dendritic probes was proposed for ultrasensitive and specific detection of nasopharyngeal carcinoma-associated Epstein-Barr virus (EBV) DNA on microfluidic microbead array chips. With the assistance of Pb2+, the duplex-functional split-DNAzyme recognizes EBV DNA and then rapidly cleaves the substrate strand. Subsequently, the released target could be recycled, and its exposed capture probe, triggered the dendritic enzyme-free signal amplification. As the enhanced mass transfer capability, target recycling, and dendritic DNA structure signal amplification inherent to microfluidic bead arrays were integrated, it achieved an excellent detection limit of 0.36 fM and a wide linear range of 1 fM∼103 fM. Further, it was applied to content detect simulated samples of EBV DNA, recovery ranged from 97.2 % to 108.1 %, and relative standard deviation (RSD) from 3.3 % to 5.9 %, exhibiting satisfactory recovery results. The developed microfluidic biosensor was a high-sensitivity and anti-interference system for ctDNA analysis, with minimal reagent volumes (microlitres) required. Thus, it is a promising platform for ctDNA at the lowest level at their earliest incidence.

Probing a Major DNA Weakness: Resolving the Groove and Sequence Selectivity of the Diimine Complex Λ-[Ru(phen)2 phi]2.

The grooves of DNA provide recognition sites for many nucleic acid binding proteins and anticancer drugs such as the covalently binding cisplatin. Here we report a crystal structure showing, for the first time, groove selectivity by an intercalating ruthenium complex. The complex Λ-[Ru(phen)2 phi]2+ , where phi=9,10-phenanthrenediimine, is bound to the DNA decamer duplex d(CCGGTACCGG)2 . The structure shows that the metal complex is symmetrically bound in the major groove at the central TA/TA step, and asymmetrically bound in the minor groove at the adjacent GG/CC steps. A third type of binding links the strands, in which each terminal cytosine base stacks with one phen ligand. The overall binding stoichiometry is four Ru complexes per duplex. Complementary biophysical measurements confirm the binding preference for the Λ-enantiomer and show a high affinity for TA/TA steps and, more generally, TA-rich sequences. A striking enantiospecific elevation of melting temperatures is found for oligonucleotides which include the TATA box sequence.

Simultaneous and sensitive detection of SARS-CoV-2 proteins spike and nucleocapsid based on long-range SERS biosensor.

BACKGROUND: Early diagnosis of SARS-CoV-2 infection is still critical to control COVID-19 outbreak. Traditional polymerase chain reaction, enzyme-linked immunosorbent assay or lateral flow immunoassay performed poorly on detection times, sample preparation process and accuracy. Surface-enhanced Raman scattering (SERS)-based detection has emerged as a powerful analytical technique, which overcomes the above limitations. However, due to the near-field effect of traditional substrate, it is difficult to monitor the binding event of aptamers with proteins. It is obvious that a novel SERS substrate thatsupportedextended and stronger electromagnetic fields was required to hold long-range effects and allow for binding event testing. RESULTS: Driven by this challenge, we reported a long-range SERS-active substrate, which was built by inserting bowtie nanoaperture arrays in a refractive-index-symmetric environment and Au mirror surfaces, for SARS-CoV-2 protein binding event detection. Then, a double-π structure aptasensor was simply designed through the hybridization of spike (S) and nucleocapsid (N) proteins aptamers, and a corresponding complementary strand. This kind of double-π structure would dissociate when targets proteins S and N existed and led to the SERS responses decreased, which established the detection basis of our system. What's more, due to two Raman labels were involved, both proteins S and N can be sensed simultaneously. Our proposed method showed improved sensitivity with a low limit of detection for multiplex detection (1.6 × 10-16 g/mL for protein S and 1.0 × 10-16 g/mL for protein N) over a wide concentration range. SIGNIFICANCE: This represents the first long-range SERS apatasensor platform for detection of S and N proteins simultaneously. Our method showed high sensitivity, selectivity, reproducibility, stability and remarkable recoveries in human in saliva and serum samples, which is particularly important for the early diagnostics of COVID as well as for future unknown coronavirus.

Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter.

Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reactivation or viral latency.

Effect of Gold Nanoparticle Radiosensitization on DNA Damage Using a Quartz Tuning Fork Sensor.

The development of sensor technology enables the creation of DNA-based biosensors for biomedical applications. Herein, a quartz tuning fork (QTF) sensing system was employed as a transducer for biomedical applications to address indirect DNA damage associated with gold nanoparticles (GNPs) and enhance the effectiveness of low-dose gamma radiation in radiation therapy. The experiment included two stages, namely during and after irradiation exposure; shift frequencies (Δf) were measured for 20 min in each stage. During the irradiation stage, the QTF response to DNA damage was investigated in a deionized aqueous solution with and without 100 nm GNPs at different concentrations (5, 10, 15, and 20 µg/mL). Upon exposure to gamma radiation for 20 min at a dose rate of 2.4 µGy/min, the ratio of Δf/ΔT indicates increased fork displacement frequencies with or without GNPs. Additionally, DNA damage associated with high and low GNP concentrations was evaluated using the change in the resonance frequency of the QTF. The results indicate that GNPs at 15 and 10 µg/mL were associated with high damage-enhancement ratios, while saturation occurred at 20 µg/mL. At 15 µg/mL, significant radiotherapy enhancement occurred compared to that at 10 µg/mL at 10 min after exposure. In the post-irradiation stage, the frequency considerably differed between 15 and 10 µg/mL. Finally, these results significantly depart from the experimental predictions in the post-radiation stage. They exhibited no appreciable direct effect on DNA repair owing to the absence of an environment that promotes DNA repair following irradiation. However, these findings demonstrate the potential of enhancing damage by combining GNP-mediated radiation sensitization and biosensor technology. Thus, QTF is recommended as a reliable measure of DNA damage to investigate the dose enhancement effect at various GNP concentrations.

Temperature-induced DNA density transition in phage λ capsid revealed with contrast-matching SANS.

Structural details of a genome packaged in a viral capsid are essential for understanding how the structural arrangement of a viral genome in a capsid controls its release dynamics during infection, which critically affects viral replication. We previously found a temperature-induced, solid-like to fluid-like mechanical transition of packaged λ-genome that leads to rapid DNA ejection. However, an understanding of the structural origin of this transition was lacking. Here, we use small-angle neutron scattering (SANS) to reveal the scattering form factor of dsDNA packaged in phage λ capsid by contrast matching the scattering signal from the viral capsid with deuterated buffer. We used small-angle X-ray scattering and cryoelectron microscopy reconstructions to determine the initial structural input parameters for intracapsid DNA, which allows accurate modeling of our SANS data. As result, we show a temperature-dependent density transition of intracapsid DNA occurring between two coexisting phases-a hexagonally ordered high-density DNA phase in the capsid periphery and a low-density, less-ordered DNA phase in the core. As the temperature is increased from 20 °C to 40 °C, we found that the core-DNA phase undergoes a density and volume transition close to the physiological temperature of infection (~37 °C). The transition yields a lower energy state of DNA in the capsid core due to lower density and reduced packing defects. This increases DNA mobility, which is required to initiate rapid genome ejection from the virus capsid into a host cell, causing infection. These data reconcile our earlier findings of mechanical DNA transition in phage.

Genomic Instability of G-Quadruplex Sequences in Escherichia coli: Roles of DinG, RecG, and RecQ Helicases.

Guanine-rich DNA can fold into highly stable four-stranded DNA structures called G-quadruplexes (G4). Originally identified in sequences from telomeres and oncogene promoters, they can alter DNA metabolism. Indeed, G4-forming sequences represent obstacles for the DNA polymerase, with important consequences for cell life as they may lead to genomic instability. To understand their role in bacterial genomic instability, different G-quadruplex-forming repeats were cloned into an Escherichia coli genetic system that reports frameshifts and complete or partial deletions of the repeat when the G-tract comprises either the leading or lagging template strand during replication. These repeats formed stable G-quadruplexes in single-stranded DNA but not naturally supercoiled double-stranded DNA. Nevertheless, transcription promoted G-quadruplex formation in the resulting R-loop for (G3T)4 and (G3T)8 repeats. Depending on genetic background and sequence propensity for structure formation, mutation rates varied by five orders of magnitude. Furthermore, while in vitro approaches have shown that bacterial helicases can resolve G4, it is still unclear whether G4 unwinding is important in vivo. Here, we show that a mutation in recG decreased mutation rates, while deficiencies in the structure-specific helicases DinG and RecQ increased mutation rates. These results suggest that G-quadruplex formation promotes genetic instability in bacteria and that helicases play an important role in controlling this process in vivo.

Shedding Light on the Photophysics and Photochemistry of I-Motifs Using Quantum Mechanical Calculations.

I-motifs are non-canonical DNA structures formed by intercalated hemiprotonated (CH·C)+ pairs, i.e., formed by a cytosine (C) and a protonated cytosine (CH+), which are currently drawing great attention due to their biological relevance and promising nanotechnological properties. It is important to characterize the processes occurring in I-motifs following irradiation by UV light because they can lead to harmful consequences for genetic code and because optical spectroscopies are the most-used tools to characterize I-motifs. By using time-dependent DFT calculations, we here provide the first comprehensive picture of the photoactivated behavior of the (CH·C)+ core of I-motifs, from absorption to emission, while also considering the possible photochemical reactions. We reproduce and assign their spectral signatures, i.e., infrared, absorption, fluorescence and circular dichroism spectra, disentangling the underlying chemical-physical effects. We show that the main photophysical paths involve C and CH+ bases on adjacent steps and, using this basis, interpret the available time-resolved spectra. We propose that a photodimerization reaction can occur on an excited state with strong C→CH+ charge transfer character and examine some of the possible photoproducts. Based on the results reported, some future perspectives for the study of I-motifs are discussed.

Profound Non-Randomness in Dinucleotide Arrangements within Ultra-Conserved Non-Coding Elements and the Human Genome.

Long human ultra-conserved non-coding elements (UCNEs) do not have any sequence similarity to each other or other characteristics that make them unalterable during vertebrate evolution. We hypothesized that UCNEs have unique dinucleotide (DN) composition and arrangements compared to the rest of the genome. A total of 4272 human UCNE sequences were analyzed computationally and compared with the whole genomes of human, chicken, zebrafish, and fly. Statistical analysis was performed to assess the non-randomness in DN spacing arrangements within the entire human genome and within UCNEs. Significant non-randomness in DN spacing arrangements was observed in the entire human genome. Additionally, UCNEs exhibited distinct patterns in DN arrangements compared to the rest of the genome. Approximately 83% of all DN pairs within UCNEs showed significant (>10%) non-random genomic arrangements at short distances (2-6 nucleotides) relative to each other. At the extremes, non-randomness in DN spacing distances deviated up to 40% from expected values and were frequently associated with GpC, CpG, ApT, and GpG/CpC dinucleotides. The described peculiarities in DN arrangements have persisted for hundreds of millions of years in vertebrates. These distinctive patterns may suggest that UCNEs have specific DNA conformations.

Why are G-quadruplexes good at preventing protein aggregation?

Maintaining a healthy protein folding environment is essential for cellular function. Recently, we found that nucleic acids, G-quadruplexes in particular, are potent chaperones for preventing protein aggregation. With the aid of structure-function and NMR analyses of two G-quadruplex forming sequences, PARP-I and LTR-III, we uncovered several contributing factors that affect G-quadruplexes in preventing protein aggregation. Notably, three factors emerged as vital in determining holdase activity of G-quadruplexes: their structural topology, G-quadruplex accessibility and dynamics, and oligomerization state. These factors together appear to largely dictate whether a G-quadruplex is able to prevent partially misfolded proteins from aggregating. Understanding the physical traits that govern the ability of G-quadruplexes to modulate protein aggregation will help elucidate their possible roles in neurodegenerative disease.