RESUMO
The small molecule epiberberine (EPI) is a natural alkaloid with versatile bioactivities against several diseases including cancer and bacterial infection. EPI can induce the formation of a unique binding pocket at the 5' side of a human telomeric G-quadruplex (HTG) sequence with four telomeric repeats (Q4), resulting in a nanomolar binding affinity (KD approximately 26 nM) with significant fluorescence enhancement upon binding. It is important to understand (1) how EPI binding affects HTG structural stability and (2) how enhanced EPI binding may be achieved through the engineering of the DNA binding pocket. In this work, the EPI-binding-induced HTG structure stabilization effect was probed by a peptide nucleic acid (PNA) invasion assay in combination with a series of biophysical techniques. We show that the PNA invasion-based method may be useful for the characterization of compounds binding to DNA (and RNA) structures under physiological conditions without the need to vary the solution temperature or buffer components, which are typically needed for structural stability characterization. Importantly, the combination of theoretical modeling and experimental quantification allows us to successfully engineer Q4 derivative Q4-ds-A by a simple extension of a duplex structure to Q4 at the 5' end. Q4-ds-A is an excellent EPI binder with a KD of 8 nM, with the binding enhancement achieved through the preformation of a binding pocket and a reduced dissociation rate. The tight binding of Q4 and Q4-ds-A with EPI allows us to develop a novel magnetic bead-based affinity purification system to effectively extract EPI from Rhizoma coptidis (Huang Lian) extracts.
Assuntos
Berberina , Quadruplex G , Berberina/química , Berberina/análogos & derivados , Berberina/farmacologia , Humanos , DNA/química , Ácidos Nucleicos Peptídicos/químicaRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Marcação de Genes , Edição de RNA , Animais , Sequência de Bases , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Modelos Animais de Doenças , Éxons , Regulação Neoplásica da Expressão Gênica , Marcação de Genes/métodos , Terapia Genética/métodos , Humanos , Camundongos , Neoplasias/genética , Neoplasias/terapia , Oligonucleotídeos Antissenso/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Minor-groove base triples formed between stem 1 and loop 2 of the simian retrovirus type 1 (SRV-1) mRNA frameshifting pseudoknot are essential in stimulating -1 ribosomal frameshifting. How tertiary base triple formation affects the local stabilities of secondary structures (stem 1 and stem 2) and thus ribosomal frameshifting efficiency is not well understood. We made a short peptide nucleic acid (PNA) that is expected to invade stem 1 of the SRV-1 pseudoknot by PNA-RNA duplex formation to mimic the stem 1 unwinding process by a translating ribosome. In addition, we used a PNA for invading stem 2 in the SRV-1 pseudoknot. Our nondenaturing polyacrylamide gel electrophoresis data for the binding of PNA to the SRV-1 pseudoknot and mutants reveal that mutations in loop 2 disrupting base triple formation between loop 2 and stem 1 in the SRV-1 pseudoknot result in enhanced invasion by both PNAs. Our data suggest that tertiary stem 1-loop 2 base triple interactions in the SRV-1 pseudoknot can stabilize both of the secondary structural components, stem 1 and stem 2. Stem 2 stability is thus coupled to the structural stability of stem 1-loop 2 base triples, mediated through a long-range effect. The apparent dissociation constants of both PNAs are positively correlated with the pseudoknot mechanical stabilities and frameshifting efficiencies. The relatively simple PNA local invasion experiment may be used to characterize the energetic contribution of tertiary interactions and ligand binding in many other RNA and DNA structures.
Assuntos
Mudança da Fase de Leitura do Gene Ribossômico , Vírus dos Macacos de Mason-Pfizer/genética , RNA Viral/química , Eletroforese em Gel de Poliacrilamida Nativa , Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/genética , Ribossomos/genética , Ribossomos/virologiaRESUMO
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.
Assuntos
Pareamento de Bases/genética , Halogênios/química , Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/química , RNA de Cadeia Dupla/síntese química , Uracila/análogos & derivados , Uracila/química , Bromouracila/química , Linhagem Celular Tumoral , Biologia Computacional/métodos , Simulação por Computador , Halogenação , Células HeLa , Humanos , Ligação de Hidrogênio , Sequências Repetidas Invertidas/genética , MicroRNAs/genética , Proteínas de Ligação a RNA/químicaRESUMO
Recognition of RNAs under physiological conditions is important for the development of chemical probes and therapeutic ligands. Nucleobase-modified dsRNA-binding PNAs (dbPNAs) are promising for the recognition of dsRNAs in a sequence and structure specific manner under near-physiological conditions. Guanidinium is often present in proteins and small molecules for the recognition of G bases in nucleic acids, in cell-penetrating carriers, and in bioactive drug molecules, which might be due to the fact that guanidinium is amphiphilic and has unique hydrogen bonding and stacking properties. We hypothesized that a simple guanidinium moiety can be directly incorporated into PNAs to facilitate enhanced molecular recognition of G-C pairs in dsRNAs and improved bioactivity. We grafted a guanidinium moiety directly into a PNA monomer (designated as R) using a two-carbon linker as guided by computational modeling studies. The synthetic scheme of the PNA R monomer is relatively simple compared to that of the previously reported L monomer. We incorporated the R residue into various dbPNAs for binding studies. dbPNAs incorporated with R residues are excellent in sequence specifically recognizing G-C pairs in dsRNAs over dsDNA and ssRNAs. We demonstrated that the R residue is compatible with unmodified T and C and previously developed modified L and Q residues in dbPNAs for targeting model dsRNAs, the influenza A viral panhandle duplex structure, and the HIV-1 frameshift site RNA hairpin. Furthermore, R residues enhance the cellular uptake of PNAs.
Assuntos
DNA/metabolismo , Guanidinas/química , Ácidos Nucleicos Peptídicos/metabolismo , RNA de Cadeia Dupla/metabolismo , Animais , Pareamento de Bases , Transporte Biológico , DNA/genética , HIV-1/química , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação de Ácido Nucleico , Orthomyxoviridae/química , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/genética , RNA de Cadeia Dupla/genética , RNA Viral/genética , RNA Viral/metabolismo , Spodoptera/químicaRESUMO
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 â¼2.3 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.
Assuntos
Sequências Repetidas Invertidas , MicroRNAs/genética , Ácidos Nucleicos Peptídicos/metabolismo , Ácido Úrico/análogos & derivados , Sequência de Bases , Ácidos Nucleicos Peptídicos/química , Ácido Úrico/metabolismoRESUMO
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.
Assuntos
Ácidos Nucleicos Peptídicos/síntese química , Ácidos Nucleicos Peptídicos/fisiologia , RNA/química , Pareamento de Bases/genética , Pareamento de Bases/fisiologia , Simulação por Computador , DNA/química , Modelos Biológicos , Conformação de Ácido Nucleico , Ácidos Nucleicos Peptídicos/química , RNA/metabolismo , RNA de Cadeia DuplaRESUMO
RNAs are emerging as important biomarkers and therapeutic targets. The strategy of directly targeting double-stranded RNA (dsRNA) by triplex-formation is relatively underexplored mainly due to the weak binding at physiological conditions for the traditional triplex-forming oligonucleotides (TFOs). Compared to DNA and RNA, peptide nucleic acids (PNAs) are chemically stable and have a neutral peptide-like backbone, and thus, they show significantly enhanced binding to natural nucleic acids. We have successfully developed nucleobase-modified dsRNA-binding PNAs (dbPNAs) to facilitate structure-specific and selective recognition of dsRNA over single-stranded RNA (ssRNA) and dsDNA regions at near-physiological conditions. The triplex formation strategy facilitates the targeting of not only the sequence but also the secondary structure of RNA. Here, we report the development of novel dbPNA-based fluorescent light-up probes through the incorporation of A-U pair-recognizing 5-benzothiophene uracil (btU). The incorporation of btU into dbPNAs does not affect the binding affinity toward dsRNAs significantly, in most cases, as evidenced by our nondenaturing gel shift assay data. The blue fluorescence emission intensity of btU-modified dbPNAs is sequence- and structure-specifically enhanced by dsRNAs, including the influenza viral RNA panhandle duplex and HIV-1-1 ribosomal frameshift-inducing RNA hairpin, but not ssRNAs or DNAs, at 200 mM NaCl, pH 7.5. Thus, dbPNAs incorporating btU-modified and other further modified fluorescent nucleobases will be useful biochemical tools for probing and detecting RNA structures, interactions, and functions.
Assuntos
Fluorescência , Ácidos Nucleicos Peptídicos/química , RNA/química , Uracila/química , Sequência de Bases , Sítios de Ligação , Estrutura Molecular , Conformação de Ácido Nucleico , Espectrometria de Fluorescência , Uracila/análogos & derivadosRESUMO
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.
Assuntos
Orthomyxoviridae/efeitos dos fármacos , Ácidos Nucleicos Peptídicos/química , Ácidos Nucleicos Peptídicos/farmacologia , RNA de Cadeia Dupla/metabolismo , RNA Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Dicroísmo Circular , Cães , Células Madin Darby de Rim Canino , Eletroforese em Gel de Poliacrilamida Nativa , Conformação de Ácido Nucleico , Orthomyxoviridae/genética , Orthomyxoviridae/fisiologia , RNA de Cadeia Dupla/químicaRESUMO
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.