Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA

Sato, Tokijiro; Sato, Yusuke; Nishizawa, Seiichi

doi:10.1021/jacs.6b05554

Cited by 78 publications

(87 citation statements)

References 30 publications

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“…We tested if the modified PNAs form pre-organized helix structure by CD spectroscopy as previously observed for a backbone-modified PNA (74). Our CD spectroscopic data suggest the formation of PNA·dsRNA triplexes (Supplementary Figure S29A), which is in agreement with previously reported results (23,28,38). However, a Br U-modified PNA alone does not show helicity, indicating that the PNAs are not preorganized as a helical structure for triplex formation.…”

Section: Substitution Of T With U or Halouracils In An Unmodified Pnasupporting

confidence: 88%

“…To recognize a Watson-Crick G-C base pair in an RNA duplex, one may design a major-groove C + ·G-C triple (Figure 1A). However, to form a Hoogsteen C + ·G pair often requires a relatively low pH (<6) for the protonation of the N3 atom of the C base (28,33,38). Modified PNA bases such as thio-pseudoisocytosine (L, Figure 1B) and 2-aminopyridine (M) have close-to-neutral pK a values for the corresponding nitrogen atom and thus allow enhanced recognition of a G-C pair in RNA duplexes through L·G-C and M·G-C base triple formation, respectively, at physiological pH (29,33,39).…”

Section: Introductionmentioning

confidence: 99%

“…An unmodified T base has been incorporated into PNAs to recognize a Watson-Crick A-U pair through major-groove T·A-U base triple formation ( Figure 1C) (13,20,28,38,39,42). Enhancing the recognition of Watson-Crick A-U base pairs is crucial due to the relatively high abundance of A-U pairs in dsRNAs.…”

Section: Introductionmentioning

confidence: 99%

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

Patil

Toh

Yuan

et al. 2018

Nucleic Acids Research

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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.

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Section: Substitution Of T With U or Halouracils In An Unmodified Pnasupporting

confidence: 88%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

Patil

Toh

Yuan

et al. 2018

Nucleic Acids Research

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show abstract

“…For example, it would be useful to integrate cyanine dyes with longer emission wavelengths than that of TO, such as TO3 derivatives, as the signaling unit in the probe design, which could reduce the background autofluorescence. As for the binding property, the use of triplex‐forming PNAs to target the RNA duplex near the overhanging structure would lead to improved binding affinity and selectivity of the probe to target siRNAs. We are now undertaking further studies in these directions.…”

Section: Resultsmentioning

confidence: 99%

Enhanced Binding Affinity of siRNA Overhang‐Binding Fluorescent Probes by Conjugation with Cationic Oligopeptides for Improved Analysis of the siRNA Delivery Process

et al. 2018

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Carrier‐mediated delivery of small interfering RNAs (siRNAs) into the living cells is important for the realization of siRNA therapeutics that can silence target genes through RNA interference. We recently proposed a new strategy for analyzing the siRNA delivery process based on affinity labeling with a peptide nucleic acid (PNA)‐based fluorescent probe (PyAATO; Py: pyrene, A: adenine; TO: thiazole orange) capable of selectively binding to the overhanging structures of siRNAs. We have prepared new probes with improved binding affinity by conjugation with a cationic oligopeptide. The probe, carrying six lysine residues (PyAATO‐Lys6 (Lys6)), displayed a 39‐fold increase in affinity, compared with that of the parent probe containing no oligopeptides. Thermodynamic characterization revealed that enhanced affinity resulted from the favorable polyelectrolyte effect, due to the electrostatic interaction between lysine residues and phosphate anions of the RNA duplexes near the overhanging structure. Lys6 showed the improved imaging ability of the carrier‐mediated siRNA delivery process in living cells, in which 20 nm siRNA could be analyzed and was considered to show the minimal off‐target effects.

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“…Recently,t he group of Roznersa nd our group reportedf luorescent probesb ased on triplex-forming peptide nucleic acid (TFP) for sensing native double-stranded structures of RNA. [10,11] Homopyrimidine peptiden ucleic acid (PNA) was reported to strongly bind to the complementary homopurine tracts within dsRNA, and the binding affinity for dsRNA is higher than that for dsDNA. [12] This unique binding preference can be used ford evelopment of dsRNA-selective fluorescent probes.…”

Section: Introductionmentioning

confidence: 99%

Optimization of the Alkyl Linker of TO Base Surrogate in Triplex‐Forming PNA for Enhanced Binding to Double‐Stranded RNA

Sato

Nishizawa

2017

Chemistry A European J

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A series of triplex-forming peptide nucleic acid (TFP) probes carrying a thiazole orange (TO) base surrogate through an alkyl linker was synthesized, and the interactions between these so-called tFIT probes and purine-rich sequences within double-stranded RNA (dsRNA) were examined. We found that the TO base surrogate linker significantly affected both the binding affinity and the fluorescence response upon triplex formation with the target dsRNA. Among the probes examined, the TO base surrogate connected through the propyl linker in the tFIT probes increased the binding affinity by a factor of ten while maintaining its function as the fluorescent universal base. Isothermal titration calorimetry experiments revealed that the increased binding affinity resulted from the gain in the binding enthalpy, which could be explained by the enhanced π-stacking interaction between the TO base surrogate and the dsRNA part of the triplex. We expect that these results will provide a molecular basis for designing strong binding tFIT probes for fluorescence sensing of various kinds of purine-rich dsRNAs sequences including those carrying a pyrimidine-purine inversion. The obtained data also offers a new insight into further development of the universal bases incorporated in TFP.

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Triplex-Forming Peptide Nucleic Acid Probe Having Thiazole Orange as a Base Surrogate for Fluorescence Sensing of Double-stranded RNA

Cited by 78 publications

References 30 publications

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

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

Enhanced Binding Affinity of siRNA Overhang‐Binding Fluorescent Probes by Conjugation with Cationic Oligopeptides for Improved Analysis of the siRNA Delivery Process

Optimization of the Alkyl Linker of TO Base Surrogate in Triplex‐Forming PNA for Enhanced Binding to Double‐Stranded RNA

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