Synthesis of Pyrrolidinyl PNA and Its Site-Specific Labeling at Internal Positions by Click Chemistry

Ditmangklo, Boonsong; Muangkaew, Penthip; Supabowornsathit, Kotchakorn; Vilaivan, Tirayut

doi:10.1007/978-1-0716-0243-0_3

Cited by 7 publications

(14 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Synthetic DNA/RNA targets used in this study were purchased from Pacific Science (Thailand). The Fmoc-protected acpcPNA monomers were synthesized following a reported protocol. , The details for the preparation of the FAM-labeled PNA probes are provided in the Supporting Information (Table S1). The sequences of the PNA probes and the DNA/RNA targets used in this study are shown in Table .…”

Section: Methodsmentioning

confidence: 99%

Enhancing Peptide Nucleic Acid–Nanomaterial Interaction and Performance Improvement of Peptide Nucleic Acid-Based Nucleic Acid Detection by Using Electrostatic Effects

Faikhruea

Choopara

Somboonna

et al. 2022

ACS Appl. Bio Mater.

Self Cite

View full text Add to dashboard Cite

Single-stranded peptide nucleic acid (PNA) probes interact strongly with several nanomaterials, and the interaction was diminished in the presence of complementary nucleic acid targets which forms the basis of many nucleic acid sensing platforms. As opposed to the negatively charged DNA probes, the charges on the PNA probes may be fine-tuned by incorporating amino acids with charged side chains. The contribution of electrostatic effects to the interaction between PNA probes and nanomaterials has been largely overlooked. This work reveals that electrostatic effects substantially enhanced the quenching of dyelabeled conformationally constrained pyrrolidinyl PNA probes by several nanomaterials including graphene oxide (GO), reduced graphene oxide, gold nanoparticles (AuNPs), and silver nanoparticles. The fluorescence quenching and the color change from red to purple in the case of AuNPs because of aggregation were inhibited in the presence of complementary nucleic acid targets. Thus, fluorescence and colorimetric assays for DNA and RNA that can distinguish even single-base-mismatched nucleic acids with improved sensitivity over conventional DNA probes were established. Both the GO-and AuNP-based sensing platforms have been successfully applied for the detection of real DNA and RNA samples in vitro and in living cells. This study emphasizes the active roles of electrostatic effects in the PNA−nanomaterial interactions, which paves the way toward improving the performance of PNA−nanomaterial based assays of nucleic acids.

show abstract

Section: Methodsmentioning

confidence: 99%

Enhancing Peptide Nucleic Acid–Nanomaterial Interaction and Performance Improvement of Peptide Nucleic Acid-Based Nucleic Acid Detection by Using Electrostatic Effects

Faikhruea

Choopara

Somboonna

et al. 2022

ACS Appl. Bio Mater.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Fluorescence images were obtained on BLooK LED transilluminator (GeneDireX, UK) with the emission maximum at 470 nm. The Fmoc-protected pyrrolidinyl PNA monomers (A, T, C, and G) and the ACPC spacers were synthesized according to a previously published protocol …”

Section: Methodsmentioning

confidence: 99%

“…The Fmoc-protected pyrrolidinyl PNA monomers (A, T, C, and G) and the ACPC spacers were synthesized according to a previously published protocol. 46 RPA Primer and Flu-acpcPNA Probe Designs. The 16S rRNA gene sequences of C. l. familiariz (Clu) and related tickborne bacteria including E. canis (Eca), E. chaffeensis (Ech), E. ewingii (Eew), A. platys (Apl), and A. phagocytophilum (Aph) available in the GenBank nucleotide sequence database were downloaded and aligned by Multalin sequence alignment (http://multalin.toulouse.inra.fr/multalin/).…”

Section: ■ Conclusionmentioning

confidence: 99%

“…The acpcPNA probe bearing a 5(6)-carboxyfluorescein label at the N-terminus was synthesized via solid-phase peptide synthesis according to the previously developed protocol using the Fmoc-SPPS strategy employing the pentafluorophenyl activated PNA monomers (base = A, T, C), and ACPC spacer. 46 Briefly, the SPPS was performed at the 1.5 μmol scale. The Fmoc group was deprotected by treatment with 100 μL of 20% v/v piperidine, 2% v/v DBU in DMF for 10 min followed by an exhaustive wash with DMF.…”

Section: ■ Conclusionmentioning

confidence: 99%

“…The Flu-acpcPNA probe was successfully synthesized by following the previously published method 46 to give the PNA product in 25% isolated yields with >90% purity by HPLC. The labeled PNA probe appeared as two peaks in the HPLC chromatogram due to the 5-and 6-carboxyfluorescein isomers.…”

Section: ■ Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Isothermal Detection of Canine Blood Parasite (Ehrlichia canis) Utilizing Recombinase Polymerase Amplification Coupled with Graphene Oxide Quenching-Based Pyrrolidinyl Peptide Nucleic Acid

et al. 2021

Self Cite

View full text Add to dashboard Cite

Canine monocytic ehrlichiosis (CME), caused by transmitted Ehrlichia canis infection, is a major disease in dogs with worldwide distribution. Herein, a nucleic acid assay was established for the identification of E. canis infection employing a fluorescently labeled conformationally constrained pyrrolidinyl PNA probe (Flu-acpcPNA) designed to sequence-specifically target the 16S rRNA gene. The sensing principle is based on the excellent quenching ability of graphene oxide (GO) of the free PNA probe, that was diminished upon binding to the DNA target. The addition of DNase I improved the performance of the detection system by eliminating the nonspecific quenching capability of long-chain dsDNA and thus enhancing the fluorescence signaling. The assay was coupled with a recombinase polymerase amplification (RPA) technique, which could be performed under isothermal conditions (37 °C) without DNA denaturation and purification steps. The established method is simple to set up and execute, proving a rapid, specific, and sensitive detection of 16S rRNA gene of E. canis with a limit of detection at least 11.1 pM. This technique shows good potential for the visual detection of double-stranded DNA targets without the need for PCR or complicated instruments, which shows great promise for practical usage in resource limited areas.

show abstract

Pyrrolidinyl Peptide Nucleic Acid Probes Capable of Crosslinking with DNA: Effects of Terminal and Internal Modifications on Crosslink Efficiency

Muangkaew

Vilaivan

2020

ChemBioChem

Self Cite

View full text Add to dashboard Cite

In this study, we describe a furan-modified acpcPNA as a probe that can form an interstrand crosslink (ICL) with its DNA target upon activation with N-bromosuccinimide (NBS). To overcome the problem of furan instability under acidic conditions, a simple and versatile post-synthetic methodology for the attachment of the furan group to the PNA probe was developed. Unlike in other designs, the furan was placed at the end of the PNA molecule or tethered to the PNA backbone with all the base pairs in the PNA • DNA duplexes fully preserved. Hence, the true reactivity of each nucleobase towards the crosslinking could be compared. We show that all DNA bases except T could participate in the crosslinking reaction when the furan was placed at the end of the PNA strand. The crosslinking process was sensitive to mispairing, and lower crosslinking efficiency was observed in the presence of a base-mismatch in the PNA • DNA duplex. In contrast, when the furan was placed at internal positions of the acpcPNA • DNA duplex, no ICL was observed; this was explained by the inability of a hydrogenbonded nucleobase to participate in the crosslinking reaction. The crosslinking efficiency was considerably improved, despite lower duplex stability, when an unpaired base (in the form of Cinsertion) was present in the complementary DNA strand close to the furan modification site.

show abstract

Synthesis of Pyrrolidinyl PNA and Its Site-Specific Labeling at Internal Positions by Click Chemistry

Cited by 7 publications

References 28 publications

Enhancing Peptide Nucleic Acid–Nanomaterial Interaction and Performance Improvement of Peptide Nucleic Acid-Based Nucleic Acid Detection by Using Electrostatic Effects

Enhancing Peptide Nucleic Acid–Nanomaterial Interaction and Performance Improvement of Peptide Nucleic Acid-Based Nucleic Acid Detection by Using Electrostatic Effects

Isothermal Detection of Canine Blood Parasite (Ehrlichia canis) Utilizing Recombinase Polymerase Amplification Coupled with Graphene Oxide Quenching-Based Pyrrolidinyl Peptide Nucleic Acid

Pyrrolidinyl Peptide Nucleic Acid Probes Capable of Crosslinking with DNA: Effects of Terminal and Internal Modifications on Crosslink Efficiency

Contact Info

Product

Resources

About