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1. Cell Penetrating Peptides, Novel Vectors for Gene Therapy

   https://doi.org/10.3390/pharmaceutics12030225  

   Taylor, Rebecca E.; Zahid, Maliha (March 2020, Pharmaceutics)

   null (Ed.)

   Cell penetrating peptides (CPPs), also known as protein transduction domains (PTDs), first identified ~25 years ago, are small, 6–30 amino acid long, synthetic, or naturally occurring peptides, able to carry variety of cargoes across the cellular membranes in an intact, functional form. Since their initial description and characterization, the field of cell penetrating peptides as vectors has exploded. The cargoes they can deliver range from other small peptides, full-length proteins, nucleic acids including RNA and DNA, liposomes, nanoparticles, and viral particles as well as radioisotopes and other fluorescent probes for imaging purposes. In this review, we will focus briefly on their history, classification system, and mechanism of transduction followed by a summary of the existing literature on use of CPPs as gene delivery vectors either in the form of modified viruses, plasmid DNA, small interfering RNA, oligonucleotides, full-length genes, DNA origami or peptide nucleic acids. 

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2. Two distinct nucleic acid binding surfaces of Cdc5 regulate development

   https://doi.org/10.1042/BCJ20190502  

   Wang, Chao; Li, Mu; Li, Guorui; Liu, Xinsen; Zhao, Wensheng; Yu, Bin; Liu, Junfeng; Yang, Jun; Peng, You-Liang (November 2019, Biochemical Journal)

   Cell division cycle 5 (Cdc5) is a highly conserved nucleic acid binding protein among eukaryotes and plays critical roles in development. Cdc5 can simultaneously bind to DNA and RNA by its N-terminal DNA-binding domain (DBD), but molecular mechanisms describing its nucleic acid recognition and the regulation of development through its nucleic acid binding remain unclear. Herein, we present a crystal structure of the N-terminal DBD of MoCdc5 (MoCdc5-DBD) from the rice blast fungus Magnaporthe oryzae. Residue K100 of MoCdc5 is on the periphery of a positively charged groove that is formed by K42, K45, R47, and N92 and is evolutionally conserved. Mutation of K100 significantly reduces the affinity of MoCdc5-DBD to a Cdc5-binding element but not to a conventional myeloblastosis (Myb) domain-binding element, suggesting that K100 is a key residue of the high binding affinity to Cdc5-binding element. Another conserved residue (R31) is located close to the U6 RNA in the structure of the spliceosome, and its mutation dramatically reduces the binding capacity of MoCdc5-DBD for U6 RNA. Importantly, mutations in these key residues, including R31, K42, and K100 in AtCDC5, an Arabidopsis thaliana ortholog of MoCdc5, greatly impair the functions of AtCDC5, resulting in pleiotropic development defects and reduced levels of primary microRNA transcripts. Taken together, our findings suggest that Cdc5-DBD binds nucleic acids with two distinct binding surfaces, one for DNA and another for RNA, which together contribute to establishing the regulation mechanism of Cdc5 on development through nucleic acid binding. 

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3. High-throughput methods in aptamer discovery and analysis

   https://doi.org/10.1016/bs.mie.2019.02.009  

   Cole, Kyle; Luptak, Andrej (March 2019, Methods in enzymology)

   Aptamers are small, functional nucleic acids that bind a variety of targets, often with high specificity and affinity. Genomic aptamers constitute the ligand-binding domains of riboswitches, whereas synthetic aptamers find applications as diagnostic and therapeutic tools, and as ligand-binding domains of regulatory RNAs in synthetic biology. Discovery and characterization of aptamers has been limited by a lack of high-throughput approaches that uncover the target-binding domains and the biochemical properties of individual sequences. With the advent of high-throughput sequencing, large-scale analysis of in vitro selected populations of aptamers (and catalytic nucleic acids, such as ribozymes and DNAzmes) became possible. In recent years the development of new experimental approaches and software tools has led to significant streamlining of the selection-pool analysis. This article provides an overview of post-selection data analysis and describes high-throughput methods that facilitate rapid discovery and biochemical characterization of aptamers. 

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4. Zinc Alters the Supramolecular Organization of Nucleic Acid Complexes with Full-Length TIA1

   Yizhuo Yang, Keith J. (January 2023, bioRxiv)

   na (Ed.)

   T-Cell Intracellular Antigen-1 (TIA1) is a 43 kDa multi-domain RNA-binding protein involved in stress granule formation during eukaryotic stress response, and has been implicated in neurodegenerative diseases including Welander distal myopathy and amyotrophic lateral sclerosis. TIA1 contains three RNA recognition motifs (RRMs), which are capable of binding nucleic acids and a C-terminal Q/N-rich prion-related domain (PRD) which has been variously described as intrinsically disordered or prion inducing and is believed to play a role in promoting liquid-liquid phase separation connected with the assembly of stress granule formation. Motivated by the fact that our prior work shows RRMs 2 and 3 are well-ordered in an oligomeric full-length form, while RRM1 and the PRD appear to phase separate, the present work addresses whether the oligomeric form is functional and competent for binding, and probes the consequences of nucleic acid binding for oligomerization and protein conformation change. New SSNMR data show that ssDNA binds to full-length oligomeric TIA1 primarily at the RRM2 domain, but also weakly at the RRM3 domain, and Zn2+ binds primarily to RRM3. Binding of Zn2+ and DNA was reversible for the full-length wild type oligomeric form, and did not lead to formation of amyloid fibrils, despite the presence of the C-terminal prion-related domain. While TIA1:DNA complexes appear as long “daisy chained” structures, the addition of Zn2+ caused the structures to collapse. We surmise that this points to a regulatory role for Zn2+. By occupying various “half” binding sites on RRM3 Zn2+ may shift the nucleic acid binding off RRM3 and onto RRM2. More importantly, the use of different half sites on different monomers may introduce a mesh of crosslinks in the supramolecular complex rendering it compact and markedly reducing the access to the nucleic acids (including transcripts) from solution. 

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5. Competition between Nucleic Acids and Intrinsically Disordered Regions within Proteins

   https://doi.org/10.1021/acs.accounts.5c00261  

   Wang, Xi; Levy, Yaakov; Iwahara, Junji (July 2025, Accounts of Chemical Research)

   Intrinsically disordered regions (IDRs) are important components of protein functionality, with their charge distribution serving as a key factor in determining their roles. Notably, many proteins possess IDRs that are highly negatively charged, characterized by sequences rich in aspartate (D) or glutamate (E) residues. Bioinformatic analyses indicate that negatively charged low-complexity IDRs are significantly more common than their positively charged counterparts rich in arginine (R) or lysine (K). For instance, sequences of 10 or more consecutive negatively charged residues (D or E) are present in 268 human proteins. In contrast, corresponding sequences of 10 or more consecutive positively charged residues (K or R) are present in only 12 human proteins. Interestingly, about 50% of proteins containing D/E tracts function as DNA-binding or RNA-binding proteins. Negatively charged IDRs can electrostatically mimic nucleic acids and dynamically compete with them for the DNA-binding domains (DBDs) or RNA-binding domains (RBDs) that are positively charged. This leads to a phenomenon known as autoinhibition, in which the negatively charged IDRs inhibit binding to nucleic acids by occupying the binding interfaces within the proteins through intramolecular interactions. Rather than merely reducing binding activity, negatively charged IDRs offer significant advantages for the functions of DNA/RNA-binding proteins. The dynamic competition between negatively charged IDRs and nucleic acids can accelerate the target search processes for these proteins. When a protein encounters DNA or RNA, the electrostatic repulsion force between the nucleic acids and the negatively charged IDRs can trigger conformational changes that allow the nucleic acids to access DBDs or RBDs. Additionally, when proteins are trapped at high-affinity non-target sites on DNA or RNA ("decoys"), the electrostatic repulsion from the negatively charged IDRs can rescue the proteins from these traps. Negatively charged IDRs act as gatekeepers, rejecting nonspecific ligands while allowing the target to access the molecular interfaces of DBDs or RBDs, which increases binding specificity. These IDRs can also promote proper protein folding, facilitate chromatin remodeling by displacing other proteins bound to DNA, and influence phase separation, affecting local pH. The functions of negatively charged IDRs can be regulated through protein-protein interactions, post-translational modifications, and proteolytic processing. These characteristics can be harnessed as tools for protein engineering. Some frame-shift mutations that convert negatively charged IDRs into positively charged ones are linked to human diseases. Therefore, it is crucial to understand the physicochemical properties and functional roles of negatively charged IDRs that compete with nucleic acids. 

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