G4-PROTAC: targeted degradation of a G-quadruplex binding protein.

G-quadruplex (G4) binding proteins regulate important biological processes, but their interaction networks are poorly understood. We report the first use of G4 as a warhead of a proteolysis-targeting chimera (G4-PROTAC) for targeted degradation of a G4-binding protein (RHAU/DHX36). G4-PROTAC provides a new way to explore G4-protein networks and to develop potential therapeutics.

Targeting RNA editing of antizyme inhibitor 1: A potential oligonucleotide-based antisense therapy for cancer.

Dysregulated adenosine-to-inosine (A-to-I) RNA editing is implicated in various cancers. However, no available RNA editing inhibitors have so far been developed to inhibit cancer-associated RNA editing events. Here, we decipher the RNA secondary structure of antizyme inhibitor 1 (AZIN1), one of the best-studied A-to-I editing targets in cancer, by locating its editing site complementary sequence (ECS) at the 3' end of exon 12. Chemically modified antisense oligonucleotides (ASOs) that target the editing region of AZIN1 caused a substantial exon 11 skipping, whereas ECS-targeting ASOs effectively abolished AZIN1 editing without affecting splicing and translation. We demonstrate that complete 2'-O-methyl (2'-O-Me) sugar ring modification in combination with partial phosphorothioate (PS) backbone modification may be an optimal chemistry for editing inhibition. ASO3.2, which targets the ECS, specifically inhibits cancer cell viability in vitro and tumor incidence and growth in xenograft models. Our results demonstrate that this AZIN1-targeting, ASO-based therapeutics may be applicable to a wide range of tumor types.

RNA Secondary Structure-Based Design of Antisense Peptide Nucleic Acids for Modulating Disease-Associated Aberrant Tau Pre-mRNA Alternative Splicing.

Alternative splicing of tau pre-mRNA is regulated by a 5' splice site (5'ss) hairpin present at the exon 10-intron 10 junction. Single mutations within the hairpin sequence alter hairpin structural stability and/or the binding of splicing factors, resulting in disease-causing aberrant splicing of exon 10. The hairpin structure contains about seven stably formed base pairs and thus may be suitable for targeting through antisense strands. Here, we used antisense peptide nucleic acids (asPNAs) to probe and target the tau pre-mRNA exon 10 5'ss hairpin structure through strand invasion. We characterized by electrophoretic mobility shift assay the binding of the designed asPNAs to model tau splice site hairpins. The relatively short (10-15 mer) asPNAs showed nanomolar binding to wild-type hairpins as well as a disease-causing mutant hairpin C+19G, albeit with reduced binding strength. Thus, the structural stabilizing effect of C+19G mutation could be revealed by asPNA binding. In addition, our cell culture minigene splicing assay data revealed that application of an asPNA targeting the 3' arm of the hairpin resulted in an increased exon 10 inclusion level for the disease-associated mutant C+19G, probably by exposing the 5'ss as well as inhibiting the binding of protein factors to the intronic spicing silencer. On the contrary, the application of asPNAs targeting the 5' arm of the hairpin caused an increased exon 10 exclusion for a disease-associated mutant C+14U, mainly by blocking the 5'ss. PNAs could enter cells through conjugation with amino sugar neamine or by cotransfection with minigene plasmids using a commercially available transfection reagent.

Incorporating 2-Thiouracil into Short Double-Stranded RNA-Binding Peptide Nucleic Acids for Enhanced Recognition of A-U Pairs and for Targeting a MicroRNA Hairpin Precursor.

Chemically modified short peptide nucleic acids (PNAs) recognize RNA duplexes under near physiological conditions by major-groove PNA·RNA-RNA triplex formation and show great promise for the development of RNA-targeting probes and therapeutics. Thymine (T) and uracil (U) are often incorporated into PNAs to recognize A-U pairs through major-groove T·A-U and U·A-U base triple formation. Incorporation of a modified nucleobase, 2-thiouracil (s2U), into triplex-forming oligonucleotides stabilizes both DNA and RNA triplexes. Thiolation of uracil causes a decrease in the dehydration energy penalty for triplex formation as well as a decrease in the pKa of the N3 atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions, similar to the previously reported thiolation effect of pseudoisocytosine (J to L substitution). Here, we incorporated s2U into short PNAs, followed by binding studies of a series of s2U-modified PNAs. We demonstrated by nondenaturing polyacrylamide gel electrophoresis and thermal melting experiments that s2U and L incorporated into dsRNA-binding PNAs (dbPNAs) enhance the recognition of A-U and G-C pairs, respectively, in RNA duplexes in a position-independent manner, with no appreciable binding to the DNA duplex. Combining s2U and L modifications in dbPNAs facilitates enhanced recognition of dsRNAs and maintains selective binding to dsRNAs over ssRNAs. We further demonstrated through a cell-free assay the application of the s2U- and L-modified dbPNAs (8-mer, with a molecular mass of ∼2.3 kDa) in the inhibition of the pre-microRNA-198 maturation in a substrate-specific manner. Thus, s2U-modified dbPNAs may be generally useful for the enhanced and selective recognition of RNA duplexes and for the regulation of RNA functions.

Nanopore Sequencing Accurately Identifies the Mutagenic DNA Lesion O6 -Carboxymethyl Guanine and Reveals Its Behavior in Replication.

O6 -carboxymethylguanine (O6 -CMG) is a highly mutagenic alkylation product of DNA, triggering transition mutations relevant to gastrointestinal cancer. However, precise localization of a single O6 -CMG with conventional sequencing platforms is challenging. Here nanopore sequencing (NPS), which directly senses single DNA bases according to their physiochemical properties, was employed to detect O6 -CMG. A unique O6 -CMG signal was observed during NPS and a single-event call accuracy of >95 % was achieved. Moreover, O6 -CMG was found to be a replication obstacle for Phi29 DNA polymerase (Phi29 DNAP), suggesting this lesion could cause DNA sequencing biases in next generation sequencing (NGS) approaches.

A Short Chemically Modified dsRNA-Binding PNA (dbPNA) Inhibits Influenza Viral Replication by Targeting Viral RNA Panhandle Structure.

RNAs play critical roles in diverse catalytic and regulatory biological processes and are emerging as important disease biomarkers and therapeutic targets. Thus, developing chemical compounds for targeting any desired RNA structures has great potential in biomedical applications. The viral and cellular RNA sequence and structure databases lay the groundwork for developing RNA-binding chemical ligands through the recognition of both RNA sequence and RNA structure. Influenza A virion consists of eight segments of negative-strand viral RNA (vRNA), all of which contain a highly conserved panhandle duplex structure formed between the first 13 nucleotides at the 5' end and the last 12 nucleotides at the 3' end. Here, we report our binding and cell culture anti-influenza assays of a short 10-mer chemically modified double-stranded RNA (dsRNA)-binding peptide nucleic acid (PNA) designed to bind to the panhandle duplex structure through novel major-groove PNA·RNA2 triplex formation. We demonstrated that incorporation of chemically modified PNA residues thio-pseudoisocytosine (L) and guanidine-modified 5-methyl cytosine (Q) previously developed by us facilitates the sequence-specific recognition of Watson-Crick G-C and C-G pairs, respectively, at physiologically relevant conditions. Significantly, the chemically modified dsRNA-binding PNA (dbPNA) shows selective binding to the dsRNA region in panhandle structure over a single-stranded RNA (ssRNA) and a dsDNA containing the same sequence. The panhandle structure is not accessible to traditional antisense DNA or RNA with a similar length. Conjugation of the dbPNA with an aminosugar neamine enhances the cellular uptake. We observed that 2-5 µM dbPNA-neamine conjugate results in a significant reduction of viral replication. In addition, the 10-mer dbPNA inhibits innate immune receptor RIG-I binding to panhandle structure and thus RIG-I ATPase activity. These findings would provide the foundation for developing novel dbPNAs for the detection of influenza viral RNAs and therapeutics with optimal antiviral and immunomodulatory activities.

General Recognition of U-G, U-A, and C-G Pairs by Double-Stranded RNA-Binding PNAs Incorporated with an Artificial Nucleobase.

Chemically modified peptide nucleic acids (PNAs) show great promise in the recognition of RNA duplexes by major-groove PNA·RNA-RNA triplex formation. Triplex formation is favored for RNA duplexes with a purine tract within one of the RNA duplex strands, and is severely destabilized if the purine tract is interrupted by pyrimidine residues. Here, we report the synthesis of a PNA monomer incorporated with an artificial nucleobase S, followed by the binding studies of a series of S-modified PNAs. Our data suggest that an S residue incorporated into short 8-mer dsRNA-binding PNAs (dbPNAs) can recognize internal Watson-Crick C-G and U-A, and wobble U-G base pairs (but not G-C, A-U, and G-U pairs) in RNA duplexes. The short S-modified PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. Interestingly, replacement of the C residue in an S·C-G triple with a 5-methyl C results in the disruption of the triplex, probably due to a steric clash between S and 5-methyl C. Previously reported PNA E base shows recognition of U-A and A-U pairs, but not a U-G pair. Thus, S-modified dbPNAs may be uniquely useful for the general recognition of RNA U-G, U-A, and C-G pairs. Shortening the succinyl linker of our PNA S monomer by one carbon atom to have a malonyl linker causes a severe destabilization of triplex formation. Our experimental and modeling data indicate that part of the succinyl moiety in a PNA S monomer may serve to expand the S base forming stacking interactions with adjacent PNA bases.

High Sensitivity of Human Translesion DNA Synthesis Polymerase κ to Variation in O6-Carboxymethylguanine Structures.

Carboxymethylation of DNA, including the formation of the DNA adduct O6-carboxymethylguanine ( O6-CMG), is associated with lifestyle factors, such as diet. It can impede replicative polymerases (Pols) and lead to replication fork stalling, or an alternative means for replication to proceed by translesion DNA synthesis (TLS). TLS requires specialized DNA Pols characterized by open and preformed active sites capable of preferential bypass of alkylated DNA adducts but that have high error rates, leading to mutations. Human TLS Pols can bypass O6-CMG with varying degrees of accuracy, but it is not known how the chemical structure of the O6-CMG adduct influences polymerase proficiency or fidelity. To better understand how adduct structure determines dNTP selection at lesion sites, we prepared DNA templates with a series of O6-CMG structural analogs and compared the primer extension patterns of Y- and X-family Pols in response to these modifications. The results indicate that the structure of the DNA adduct had a striking effect on dNTP selection by Pol κ and that an increased steric size influences the fidelity of Pol η, whereas Pol ι and ß function were only marginally affected. To test the hypothesis that specific hydrogen bonding interactions between the templating base and the incoming dNTP are a basis of this selection, we modeled the structural analogs with incoming dNTP in the Pol κ active site. These data indicate that the base pairing geometry and stabilization by a dense hydrogen bonding network are important molecular features for dNTP incorporation, providing a basis for understanding error-free bypass of O6-CMG by Pol κ.

Incorporating uracil and 5-halouracils into short peptide nucleic acids for enhanced recognition of A-U pairs in dsRNAs.

Double-stranded RNA (dsRNA) structures form triplexes and RNA-protein complexes through binding to single-stranded RNA (ssRNA) regions and proteins, respectively, for diverse biological functions. Hence, targeting dsRNAs through major-groove triplex formation is a promising strategy for the development of chemical probes and potential therapeutics. Short (e.g., 6-10 mer) chemically-modified Peptide Nucleic Acids (PNAs) have been developed that bind to dsRNAs sequence specifically at physiological conditions. For example, a PNA incorporating a modified base thio-pseudoisocytosine (L) has an enhanced recognition of a G-C pair in an RNA duplex through major-groove L·G-C base triple formation at physiological pH, with reduced pH dependence as observed for C+·G-C base triple formation. Currently, an unmodified T base is often incorporated into PNAs to recognize a Watson-Crick A-U pair through major-groove T·A-U base triple formation. A substitution of the 5-methyl group in T by hydrogen and halogen atoms (F, Cl, Br, and I) causes a decrease of the pKa of N3 nitrogen atom, which may result in improved hydrogen bonding in addition to enhanced base stacking interactions. Here, we synthesized a series of PNAs incorporating uracil and halouracils, followed by binding studies by non-denaturing polyacrylamide gel electrophoresis, circular dichroism, and thermal melting. Our results suggest that replacing T with uracil and halouracils may enhance the recognition of an A-U pair by PNA·RNA2 triplex formation in a sequence-dependent manner, underscoring the importance of local stacking interactions. Incorporating bromouracils and chlorouracils into a PNA results in a significantly reduced pH dependence of triplex formation even for PNAs containing C bases, likely due to an upshift of the apparent pKa of N3 atoms of C bases. Thus, halogenation and other chemical modifications may be utilized to enhance hydrogen bonding of the adjacent base triples and thus triplex formation. Furthermore, our experimental and computational modelling data suggest that PNA·RNA2 triplexes may be stabilized by incorporating a BrUL step but not an LBrU step, in dsRNA-binding PNAs.

Selective Binding to mRNA Duplex Regions by Chemically Modified Peptide Nucleic Acids Stimulates Ribosomal Frameshifting.

Minus-one programmed ribosomal frameshifting (-1 PRF) allows the precise maintenance of the ratio between viral proteins and is involved in the regulation of the half-lives of cellular mRNAs. Minus-one ribosomal frameshifting is activated by several stimulatory elements such as a heptameric slippery sequence (X XXY YYZ) and an mRNA secondary structure (hairpin or pseudoknot) that is positioned 2-8 nucleotides downstream from the slippery site. Upon -1 RF, the ribosomal reading frame is shifted from the normal zero frame to the -1 frame with the heptameric slippery sequence decoded as XXX YYY Z instead of X XXY YYZ. Our research group has developed chemically modified peptide nucleic acid (PNA) L and Q monomers to recognize G-C and C-G Watson-Crick base pairs, respectively, through major-groove parallel PNA·RNA-RNA triplex formation. L- and Q-incorporated PNAs show selective binding to double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). The sequence specificity and structural selectivity of L- and Q-modified PNAs may allow the precise targeting of desired viral and cellular RNA structures, and thus may serve as valuable biological tools for mechanistic studies and potential therapeutics for fighting diseases. Here, for the first time, we demonstrate by cell-free in vitro translation assays using rabbit reticulocyte lysate that the dsRNA-specific chemically modified PNAs targeting model mRNA hairpins stimulate -1 RF (from 2% to 32%). An unmodified control PNA, however, shows nonspecific inhibition of translation. Our results suggest that the modified dsRNA-binding PNAs may be advantageous for targeting structured RNAs.

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA·RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

Incorporating a guanidine-modified cytosine base into triplex-forming PNAs for the recognition of a C-G pyrimidine-purine inversion site of an RNA duplex.

RNA duplex regions are often involved in tertiary interactions and protein binding and thus there is great potential in developing ligands that sequence-specifically bind to RNA duplexes. We have developed a convenient synthesis method for a modified peptide nucleic acid (PNA) monomer with a guanidine-modified 5-methyl cytosine base. We demonstrated by gel electrophoresis, fluorescence and thermal melting experiments that short PNAs incorporating the modified residue show high binding affinity and sequence specificity in the recognition of an RNA duplex containing an internal inverted Watson-Crick C-G base pair. Remarkably, the relatively short PNAs show no appreciable binding to DNA duplexes or single-stranded RNAs. The attached guanidine group stabilizes the base triple through hydrogen bonding with the G base in a C-G pair. Selective binding towards an RNA duplex over a single-stranded RNA can be rationalized by the fact that alkylation of the amine of a 5-methyl C base blocks the Watson-Crick edge. PNAs incorporating multiple guanidine-modified cytosine residues are able to enter HeLa cells without any transfection agent.

Efficient Cellular Entry of (r-x-r)-Type Carbamate-Plasmid DNA Complexes and Its Implication for Noninvasive Topical DNA Delivery to Skin.

Arginine-rich cell penetrating peptides are powerful tools for in vitro as well as in vivo delivery of a wide plethora of biomolecules. However, presence of consecutive arginine residues leads to enhanced amenability for proteolytic degradation as well as steric hindrances for membrane interactions which compromise its bioavailability. In order to overcome these limitations we previously reported a safe and stable octaarginine based oligomer, i.e., (r-x-r)4-carbamate, where the backbone amide linkages were replaced by carbamate linkages and 6-aminohexanoic acid based spacer moieties were incorporated for better flexibility, hydrophobicity, optimal spacing of guanidinium groups, and protection against proteolytic cleavage; resulting in improved transfection efficiency over its amide counterpart. In the present work we have investigated the mechanism behind this enhanced transfection efficiency and, based on our observations, demonstrate how the synergistic effect of rationalized oligomer designing, complex characteristics, and cell type contributes to overall effective intracellular delivery. Our results indicate that the (r-x-r)4-carbamate-plasmid DNA complexes primarily utilize lipid raft dependent pathway of cellular entry more than other pathways, and this possibly facilitates their increased entry in the lipid raft rich milieu of skin cells. We also emphasize the utility of oligomer (r-x-r)4-carbamate as an efficient carrier for topical delivery of nucleic acids in skin tissue. This carrier can be utilized for safe, efficient, and noninvasive delivery of therapeutically relevant macromolecular hydrophilic cargo like nucleic acids to skin.

Second generation, arginine-rich (R-X'-R)(4)-type cell-penetrating α-ω-α-peptides with constrained, chiral ω-amino acids (X') for enhanced cargo delivery into cells.

The syntheses of novel N-aminoalkyl proline-derived spacers (X') in polycationic (R-X'-R)-motif cell-penetrating α-ω-α-peptides are described as improved molecular transporters and their structural features studied by CD. FACS analysis shows enhanced cellular uptake and confocal microscopy indicates predominantly cytoplasmic localization. The oligomers are efficient at transporting pDNA into cells. The chirality together with the hydrophobicity and flexibility derived from the spacer chain are found to have marked influence on the cell-penetrating and cargo delivery properties of the cell-penetrating peptides (CPPs). The peptides containing N-(3-aminopropyl)-D-proline spacers are found to be the best at cell penetration and cargo delivery in the present study.

Highly efficient (R-X-R)-type carbamates as molecular transporters for cellular delivery.

The (R-X-R) motif-containing arginine-rich peptides are among the most effective cell-penetrating peptides. The replacement of amide linkages in the (R-X-R) motif by carbamate linkages as in (r-ahx-r)(4) or (r-ahx-r-r-apr-r)(2) increases the efficacy of such oligomers several-fold. Internalization of these oligomers in mammalian cell lines occurs by an energy-independent process. These oligomers show efficient delivery of biologically active plasmid DNA into CHO-K1 cells.