bims-novged 2021-08-29 papers

on Non-viral vectors for gene delivery

Issue of 2021–08–29
fourteen papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

TNFα siRNA delivery by nanoparticles and photochemical internalization for psoriasis topical therapy.
Synergistic gene delivery by self-assembled nanocomplexes using fusion peptide and calcium phosphate.
Development and Characterization of High Efficacy Cell-Penetrating Peptide via Modulation of the Histidine and Arginine Ratio for Gene Therapy.
Self-assembly of an aptamer-decorated chimeric peptide nanocarrier for targeted cancer gene delivery.
Chimeric flaviviral RNA-siRNA molecules resist degradation by the exoribonuclease Xrn1 and trigger gene silencing in mammalian cells.
Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines.
Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery.
Controlled DNA Delivery Using Poly(lactide) Nanoparticles and Understanding the Binding Interactions.
Facile Construction of a Solely-DNA-Based System for Targeted Delivery of Nucleic Acids.
Dual DNA Transfection Using 1,6-Hexanedithiol-Conjugated Maleimide-Functionalized PU-PEI600 For Gene Correction in a Patient iPSC-Derived Fabry Cardiomyopathy Model.
Pathologically Responsive Mitochondrial Gene Therapy in an Allotopic Expression-Independent Manner Cures Leber's Hereditary Optic Neuropathy.
Synthesis and characterization of hybrid nanocarrier layered double hydroxide grafted by polyethylene glycol and gemcitabine.
Nanoliposomal Delivery of MicroRNA-203 Suppresses Migration of Triple-Negative Breast Cancer through Distinct Target Suppression.
Long Tracts of Guanines Drive Aggregation of RNA G-Quadruplexes in the Presence of Spermine.

J Control Release. 2021 Aug 23. pii: S0168-3659(21)00445-4. [Epub ahead of print]

TNFα siRNA delivery by nanoparticles and photochemical internalization for psoriasis topical therapy.

Isabella Suzuki, Margarete Moreno de Araujo, Vanderlei Salvador Bagnato, Maria Vitoria Lopes Badra Bentley.

  Psoriasis is a chronic inflammatory skin disease that presents increased expression of tumor necrosis factor α (TNFα), a proinflammatory cytokine. The discovery of RNA interference (RNAi), mediated by short interfering RNA (siRNA), made it possible for the expression of some genes to be eliminated. However, for its application, it is necessary to use carriers that can protect siRNA and release it in the target cells. Herein, we developed a delivery system for siRNA based on hybrid polymer-lipid nanoparticles (PLNs) and combined this system with photochemical internalization (PCI), photoactivating the photosensitizer TPPS2a, to optimize the endosomal escape of TNFα siRNA in the cytoplasm, aiming to use the system as a topical formulation to treat psoriasis. The PLNs composed of 2.0% of Compritol® 888 ATO (lipid), 1.5% of poloxamer 188 and 0.1% of the cationic polymer poly(allylamine hydrochloride) showed an average nanoparticle size of 142 nm, a zeta potential of +25 mV, and the ability to efficiently coencapsulate TPPS2a and complexed siRNA. In addition, these materials did not present cellular toxicity and showed high cellular uptake. In vitro delivery studies using porcine skin model revealed that the PLNs delivered siRNA and TPPS2a into the skin. The efficacy was verified using an in vivo psoriasis animal (hairless mouse) model induced by imiquimod (IMQ) cream. The results revealed that PLN-TPPS2a-TNFα siRNA combined with PCI resulted in a decrease in the levels of TNFα, showing the efficiency of the treatment to silence this cytokine in psoriatic lesions, which was accompanied by a reduction in the redness and scaling of the mouse skin. The results showed the potential of the developed PLNs in combined silencing gene therapy and PCI for topical treatment of psoriasis.

Keywords:  Antisense therapy; Hybrid nanoparticles; Photochemical internalization; Psoriasis; TNFα siRNA; Topical delivery

DOI:  https://doi.org/10.1016/j.jconrel.2021.08.039
J Control Release. 2021 Aug 20. pii: S0168-3659(21)00440-5. [Epub ahead of print]

Synergistic gene delivery by self-assembled nanocomplexes using fusion peptide and calcium phosphate.

Soon Gil Jeong, Yeong Chae Ryu, Byeong Hee Hwang.

  Gene therapy can be a promising therapeutic approach to cure the fundamental causes of incurable genetic diseases. Because virus carriers are costly and can cause inflammation and immunogenicity, efficient non-viral carriers need to be developed for broader gene therapy applications. Therefore, we designed novel synergistic nanocomplexes for efficient transfection incorporated by the fusion of nuclear localization signal and cell-penetrating peptides with calcium phosphate. Fusion peptides were able to package large plasmid DNAs into nanocomplexes spontaneously and efficiently. After optimization, S-R/CaP or S-S/CaP nanocomplexes significantly improved specific luciferase expression up to 2-fold compared to Lipofectamine® 2000. In addition, the large Cas9-encoding plasmids were transfected into HEK293T cells more efficiently than Lipofectamine® 2000. Furthermore, subcutaneously injected cells to mice maintained more stable protein expression until 10 days than Lipofectamine® 2000. Moreover, the biocompatibility was revealed by observing negligible cytotoxicity, histological difference, and inflammatory cytokine release. Consequently, the new chimeric strategy will be an efficient and safe gene carrier into cells and tissues to treat various genetic diseases through gene therapy.

Keywords:  Calcium phosphate; Gene delivery; Gene therapy; Nanocomplex; Peptide; Therapeutics

DOI:  https://doi.org/10.1016/j.jconrel.2021.08.034
Materials (Basel). 2021 Aug 19. pii: 4674. [Epub ahead of print]14(16):

Development and Characterization of High Efficacy Cell-Penetrating Peptide via Modulation of the Histidine and Arginine Ratio for Gene Therapy.

Yu Liu, Huan-Huan Wan, Duo-Mei Tian, Xiao-Jun Xu, Chang-Long Bi, Xiao-Yong Zhan, Bi-Hui Huang, Yun-Sheng Xu, Le-Ping Yan.

  Cell-penetrating peptides (CPPs), as non-viral gene delivery vectors, are considered with lower immunogenic response, and safer and higher gene capacity than viral systems. In our previous study, a CPP peptide called RALA (arginine rich) presented desirable transfection efficacy and owns a potential clinic use. It is believed that histidine could enhance the endosome escaping ability of CPPs, yet RALA peptide contains only one histidine in each chain. In order to develop novel superior CPPs, by using RALA as a model, we designed a series of peptides named HALA (increased histidine ratio). Both plasmid DNA (pDNA) and siRNA transfection results on three cell lines revealed that the transfection efficacy is better when histidine replacements were on the C-terminal instead of on the N-terminal, and two histidine replacements are superior to three. By investigating the mechanism of endocytosis of the pDNA nanocomplexes, we discovered that there were multiple pathways that led to the process and caveolae played the main role. During the screening, we discovered a novel peptide-HALA2 of high cellular transfection efficacy, which may act as an exciting gene delivery vector for gene therapy. Our findings also bring new insights on the development of novel robust CPPs.

Keywords:  RALA peptide; cell penetrating peptide; endosomal escaping; gene therapy; histidine

DOI:  https://doi.org/10.3390/ma14164674
Colloids Surf B Biointerfaces. 2021 Aug 16. pii: S0927-7765(21)00491-4. [Epub ahead of print]208 112047

Self-assembly of an aptamer-decorated chimeric peptide nanocarrier for targeted cancer gene delivery.

Sadegh Dehghani, Mona Alibolandi, Zeinab Amiri Tehranizadeh, Reza Kazemi Oskuee, Rahim Nosrati, Fatemeh Soltani, Mohammad Ramezani.

  In this study, we developed a peptide-based non-viral carrier decorated with aptamer to overcome the specific gene delivery barriers. The carrier (KLN/Apt) was designed to contain multiple functional segments, including 1) two tandem repeating units of low molecular weight protamine (LMWP) to condense DNA into stable nanosize particles and protect it from enzymatic digestion, 2) AS1411 aptamer as targeting moiety to target nucleolin and promote carrier internalization, 3) a synthetic pH-sensitive fusogenic peptide (KALA) for disrupting endosomal membranes and enhancing cytosol escape of the nanoparticles, and 4) a nuclear localization signal (NLS) for active cytoplasmic trafficking and nuclear delivery of DNA. The obtained results revealed the developed carrier capacity in terms of specific cell targeting, overcoming cellular gene delivery barriers, and mediating efﬁcient gene transfection. The KLN/pDNA/aptamer nanoparticles offer remarkable potential for the conceptual design and formation of promising multi-functionalized carriers towards the most demanding therapeutic applications.

Keywords:  AS1411 aptamer; Gene delivery; Gene transfection; Low molecular weight protamine; Non-viral carrier

DOI:  https://doi.org/10.1016/j.colsurfb.2021.112047
Chembiochem. 2021 Aug 25.

Chimeric flaviviral RNA-siRNA molecules resist degradation by the exoribonuclease Xrn1 and trigger gene silencing in mammalian cells.

Cressida Harvey, Sven Klassa, Esteban Finol, Jonathan Hall, Alyssa Hill.

  RNA is an emerging platform for drug delivery, but the susceptibility of RNA to nuclease degradation remains a major barrier to its implementation in vivo. Here, we engineered flaviviral Xrn1-resistant RNA (xrRNA) motifs to host small interfering RNA (siRNA) duplexes. The xrRNA-siRNA molecules self-assemble in vitro, resist degradation by the conserved eukaryotic 5' to 3' exoribonuclease Xrn1, and trigger gene silencing in 293T cells. The resistance of the molecules to Xrn1 does not translate to stability in blood serum. Nevertheless, our results demonstrate that flavivirus-derived xrRNA motifs can confer Xrn1 resistance on a model therapeutic payload and set the stage for further investigations into using the motifs as building blocks in RNA nanotechnology.

Keywords:  RNA nanotechnology; exonuclease-resistant RNA; small interfering RNA

DOI:  https://doi.org/10.1002/cbic.202100434
J Control Release. 2021 Aug 18. pii: S0168-3659(21)00435-1. [Epub ahead of print]338 201-210

Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines.

Anna K Blakney, Paul F McKay, Kai Hu, Karnyart Samnuan, Nikita Jain, Andrew Brown, Anitha Thomas, Paul Rogers, Krunal Polra, Hadijatou Sallah, Jonathan Yeow, Yunqing Zhu, Molly M Stevens, Andrew Geall, Robin J Shattock.

  Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.

Keywords:  Immunogenicity; Lipid nanoparticle; Polyplex; Protein expression; Replicon; SARS-CoV-2; Self-amplifying RNA; Vaccine

DOI:  https://doi.org/10.1016/j.jconrel.2021.08.029
Pharmaceutics. 2021 Aug 19. pii: 1292. [Epub ahead of print]13(8):

Microfluidic Formulation of DNA-Loaded Multicomponent Lipid Nanoparticles for Gene Delivery.

Erica Quagliarini, Serena Renzi, Luca Digiacomo, Francesca Giulimondi, Barbara Sartori, Heinz Amenitsch, Valentina Tassinari, Laura Masuelli, Roberto Bei, Lishan Cui, Junbiao Wang, Augusto Amici, Cristina Marchini, Daniela Pozzi, Giulio Caracciolo.

  In recent years, lipid nanoparticles (LNPs) have gained considerable attention in numerous research fields ranging from gene therapy to cancer immunotherapy and DNA vaccination. While some RNA-encapsulating LNP formulations passed clinical trials, DNA-loaded LNPs have been only marginally explored so far. To fulfil this gap, herein we investigated the effect of several factors influencing the microfluidic formulation and transfection behavior of DNA-loaded LNPs such as PEGylation, total flow rate (TFR), concentration and particle density at the cell surface. We show that PEGylation and post-synthesis sample concentration facilitated formulation of homogeneous and small size LNPs with high transfection efficiency and minor, if any, cytotoxicity on human Embryonic Kidney293 (HEK-293), spontaneously immortalized human keratinocytes (HaCaT), immortalized keratinocytes (N/TERT) generated from the transduction of human primary keratinocytes, and epidermoid cervical cancer (CaSki) cell lines. On the other side, increasing TFR had a detrimental effect both on the physicochemical properties and transfection properties of LNPs. Lastly, the effect of particle concentration at the cell surface on the transfection efficiency (TE) and cell viability was largely dependent on the cell line, suggesting that its case-by-case optimization would be necessary. Overall, we demonstrate that fine tuning formulation and microfluidic parameters is a vital step for the generation of highly efficient DNA-loaded LNPs.

Keywords:  lipid nanoparticles; lipofectamine; microfluidics; transfection efficiency

DOI:  https://doi.org/10.3390/pharmaceutics13081292
J Phys Chem B. 2021 Aug 26.

Controlled DNA Delivery Using Poly(lactide) Nanoparticles and Understanding the Binding Interactions.

Sudipta Senapati, Anurag Upadhyaya, Somnath Dhruw, Debaprasad Giri, Pralay Maiti.

Cationic polymer-based gene delivery vectors suffer from several limitations such as low DNA-loading capacity, poor transfection, toxicity, environmental degradations, etc. Again, very limited works are available addressing the binding interactions in detail at the atomic level explaining the loading capacity, protection ability against harsh environments, and controlled release behavior of the DNA-encapsulated vehicles. Here, a poly(l-lactide) (PLA) nanoparticle-based controlled DNA release system is proposed. The developed vehicle possesses a high DNA-loading capacity and can release the loaded DNA in a controlled manner. Spectroscopic, physicochemical, and molecular simulation techniques (AM1 and atomistic molecular dynamics) have been employed to understand the binding interactions between PLA and DNA molecules enabling high DNA loading, protection against external harsh environments, and controlled DNA release behavior. Methyl thiazolyl tetrazolium (MTT) assay experiments confirm the biocompatible nature of the vehicle. Cellular uptake efficiency and endo-lysosomal escape capabilities have been investigated against HeLA cells. This study, therefore, demonstrates the development of a promising nonviral DNA delivery vector and includes a detailed investigation of the atomic-level interaction behavior between PLA and DNA molecules.

DOI: https://doi.org/10.1021/acs.jpcb.1c06520
Nanomaterials (Basel). 2021 Jul 30. pii: 1967. [Epub ahead of print]11(8):

Facile Construction of a Solely-DNA-Based System for Targeted Delivery of Nucleic Acids.

Ziwen Dai, Juan Li, Yongfang Lin, Zhigang Wang, Yang Huang.

  We designed a functional drug delivery system based solely on DNA. The whole system was built with only four DNA strands. Cyclization of DNA strands excluded the formation of byproducts. DNA aptamers were equipped to endow triangular DNA nanostructures with targeting ability. The homogeneity of materials enabled not only facile construction but also convenient loading of nucleic acid-based drugs with much ease.

Keywords:  DNA aptamer; DNA nanostructure; targeted drug delivery

DOI:  https://doi.org/10.3390/nano11081967
Front Cell Dev Biol. 2021 ;9 634190

Dual DNA Transfection Using 1,6-Hexanedithiol-Conjugated Maleimide-Functionalized PU-PEI600 For Gene Correction in a Patient iPSC-Derived Fabry Cardiomyopathy Model.

Chian-Shiu Chien, Yueh Chien, Yi-Ying Lin, Ping-Hsing Tsai, Shih-Jie Chou, Aliaksandr A Yarmishyn, Elham Rastegari, Ting-Xian Wang, Hsin-Bang Leu, Yi-Ping Yang, Mong-Lien Wang, Ying-Chun Jheng, Henkie Isahwan Ahmad Mulyadi Lai, Lo-Jei Ching, Teh-Ia Huo, Jong-Yuh Cherng, Chien-Ying Wang.

  Non-viral gene delivery holds promises for treating inherited diseases. However, the limited cloning capacity of plasmids may hinder the co-delivery of distinct genes to the transfected cells. Previously, the conjugation of maleimide-functionalized polyurethane grafted with small molecular weight polyethylenimine (PU-PEI600-Mal) using 1,6-hexanedithiol (HDT) could promote the co-delivery and extensive co-expression of two different plasmids in target cells. Herein, we designed HDT-conjugated PU-PEI600-Mal for the simultaneous delivery of CRISPR/Cas9 components to achieve efficient gene correction in the induced pluripotent stem cell (iPSC)-derived model of Fabry cardiomyopathy (FC) harboring GLA IVS4 + 919 G > A mutation. This FC in vitro model recapitulated several clinical FC features, including cardiomyocyte hypertrophy and lysosomal globotriaosylceramide (Gb3) deposition. As evidenced by the expression of two reporter genes, GFP and mCherry, the addition of HDT conjugated two distinct PU-PEI600-Mal/DNA complexes and promoted the co-delivery of sgRNA/Cas9 and homology-directed repair DNA template into target cells to achieve an effective gene correction of IVS4 + 919 G > A mutation. PU-PEI600-Mal/DNA with or without HDT-mediated conjugation consistently showed neither the cytotoxicity nor an adverse effect on cardiac induction of transfected FC-iPSCs. After the gene correction and cardiac induction, disease features, including cardiomyocyte hypertrophy, the mis-regulated gene expressions, and Gb3 deposition, were remarkably rescued in the FC-iPSC-differentiated cardiomyocytes. Collectively, HDT-conjugated PU-PEI600-Mal-mediated dual DNA transfection system can be an ideal approach to improve the concurrent transfection of non-viral-based gene editing system in inherited diseases with specific mutations.

Keywords:  CRISPR/Cas9; Fabry disease; GLA; cardiomyopathy; induced pluripotent stem cells; polyethyleneimine; polyurethane

DOI:  https://doi.org/10.3389/fcell.2021.634190
Adv Mater. 2021 Aug 25. e2103307

Pathologically Responsive Mitochondrial Gene Therapy in an Allotopic Expression-Independent Manner Cures Leber's Hereditary Optic Neuropathy.

Yi Wang, Li-Fan Hu, Peng-Fei Cui, Lian-Yu Qi, Lei Xing, Hu-Lin Jiang.

  Leber's hereditary optic neuropathy (LHON) is a rare inherited blindness caused by mutations in the mitochondrial DNA (mtDNA). The disorder is untreatable and tricky, as the existing chemotherapeutic agent Idebenone alleviates symptoms rather than overcoming the underlying cause. Although some studies have made progress on allotopic expression for LHON, in situ mitochondrial gene therapy remains challenging, which may simplify delivery procedures to be a promising therapeutic for LHON. LHON becomes more difficult to manage in the changed mitochondrial microenvironment, including increasing reactive oxygen species (ROS) and decreasing mitochondrial membrane potential (MMP). Herein, a pathologically responsive mitochondrial gene delivery vector named [triphenylphosphine-terminated poly(sulfur-containing thioketal undecafluorohexylamine histamine) and Ide-terminated poly(sulfur-containing thioketal undecafluorohexylamine histamine)] (TISUH) is reported to facilitate commendable in situ mitochondrial gene therapy for LHON. TISUH directly targets diseased mitochondria via triphenylphosphine and fluorination addressing the decreasing MMP. In addition, TISUH can be disassembled by high mitochondrial ROS levels to release functional genes for enhancing gene transfection efficiency and fundamentally correcting genetic abnormalities. In both traditional and gene-mutation-induced LHON mouse models, TISUH-mediated gene therapy shows satisfactory curative effect through the sustained therapeutic protein expression in vivo. This work proposes a novel pathologically responsive in situ mitochondrial delivery platform and provides a promising approach for refractory LHON as well as other mtDNA mutated diseases treatments.

Keywords:  Idebenone; Leber's hereditary optic neuropathy; fluorination; in situ mitochondrial gene therapy; pathologically responsive polymers

DOI:  https://doi.org/10.1002/adma.202103307
J Biomater Sci Polym Ed. 2021 Aug 25. 1-11

Synthesis and characterization of hybrid nanocarrier layered double hydroxide grafted by polyethylene glycol and gemcitabine.

Xue Li, Shuxin Xu, Haojiang Wang, Anjie Dong.

  For the past few years, organic-inorganic hybrid nanocarriers have been widely explored for effective drug delivery and preferable disease treatments. In this article, hydrothermal method was utilized to prepare fine dispersed layered double hydroxide (Mg-Al LDH) suspension. Polyethylene glycol (PEG) was grafted on the surface of LDH lamella in order to improve the dispersibility of LDH. Besides, the anti-cancer drug gemcitabine was grafted on the surface of LDH lamellas through chemical grafting. Hence a novel new type of organic-inorganic hybrid drug delivery system LDH-mPEG-Gemcitabine was obtained. In addition, the siRNA was intercalated into the LDH interlamination by ion exchange method to realize drug and gene co-delivery. The loading capacity of LDH and LDH-mPEG-Gemcitabine was evaluated by agarose gel electrophoresis. The characterization by laser particle size analyzer, TEM, FT-IR, XRD, in vitro cell viability and in vitro drug release demonstrated that LDH-mPEG-Gemcitabine possessed fine dispersibility, uniform morphology and particle size, fine biocompatibility, ideal drug loading and releasing capacity and held great potential to be used as a desired co-delivery system for drug and gene.

Keywords:  Gemcitabine; Layered double hydroxide (LDH); drug and gene delivery; polyethylene glycol; siRNA

DOI:  https://doi.org/10.1080/09205063.2021.1967701
Noncoding RNA. 2021 Jul 27. pii: 45. [Epub ahead of print]7(3):

Nanoliposomal Delivery of MicroRNA-203 Suppresses Migration of Triple-Negative Breast Cancer through Distinct Target Suppression.

Shuxuan Song, Kelsey S Johnson, Henry Lujan, Sahar H Pradhan, Christie M Sayes, Joseph H Taube.

  Triple-negative breast cancers affect thousands of women in the United States and disproportionately drive mortality from breast cancer. MicroRNAs are small, non-coding RNAs that negatively regulate gene expression post-transcriptionally by inhibiting target mRNA translation or by promoting mRNA degradation. We have identified that miRNA-203, silenced by epithelial-mesenchymal transition (EMT), is a tumor suppressor and can promote differentiation of breast cancer stem cells. In this study, we tested the ability of liposomal delivery of miR-203 to reverse aspects of breast cancer pathogenesis using breast cancer and EMT cell lines. We show that translationally relevant methods for increasing miR-203 abundance within a target tissue affects cellular properties associated with cancer progression. While stable miR-203 expression suppresses LASP1 and survivin, nanoliposomal delivery suppresses BMI1, indicating that suppression of distinct mRNA target profiles can lead to loss of cancer cell migration.

Keywords:  EMT; breast cancer; microRNA; microRNA-203; nanoliposome; nanoparticle

DOI:  https://doi.org/10.3390/ncrna7030045
Biochemistry. 2021 Aug 27.

Long Tracts of Guanines Drive Aggregation of RNA G-Quadruplexes in the Presence of Spermine.

Allison M Williams, Raghav R Poudyal, Philip C Bevilacqua.

G-Quadruplexes (GQs) are compact, stable structures in DNA and RNA comprised of two or more tiers of quartets whose G-rich motif of tracts of two or more G's occurs commonly within genomes and transcriptomes. While thermodynamically stable in vitro, these structures remain difficult to study in vivo. One approach to understanding GQ in vivo behavior is to test whether conditions and molecules found in cells facilitate their folding. Polyamines are biogenic polycations that interact with RNA. Among common polyamines, spermine contains the highest charge and is found in eukaryotes, making it a good candidate for association with high-charge density nucleic acid structures like GQs. Using a variety of techniques, including ultraviolet-detected thermal denaturation, circular dichroism, size exclusion chromatography, and confocal microscopy, on an array of quadruplex sequence variants, we find that eukaryotic biological concentrations of spermine induce microaggregation of three-tiered G-rich sequences, but not of purely two-tiered structures, although higher spermine concentrations induce aggregation of even these. The formation of microaggregates can also be induced by addition of as little as a single G to a two-tiered structure; moreover, they form at biological temperatures, are sensitive to salt, and can form in the presence of at least some flanking sequence. Notably, GQ aggregation is not observed under prokaryotic-like conditions of no spermine and higher NaCl concentrations. The sequence, polyamine, and salt specificity of microaggregation reported herein have implications for the formation and stability of G-rich nucleic acid aggregates in vivo and for functional roles for understudied GQ sequences with only two quadruplex tiers.

DOI: https://doi.org/10.1021/acs.biochem.1c00467