Lighting Up the Thrombin-Binding Aptamer G-Quadruplex with an Internal Cyanine-Indole-Quinolinium Nucleobase Surrogate. Direct Fluorescent Intensity Readout for Thrombin Binding without Topology Switching

Gray, Micaela D.; Deore, Prashant S.; Chung, Andrew; Riesen, Abigail J Van; Manderville, Richard A.; Prabhakar, Preethi Seelam; Wetmore, Stacey D.

doi:10.1021/acs.bioconjchem.0c00530

Cited by 12 publications

(24 citation statements)

References 70 publications

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“…The UV excitation and emission of 2AP suffer from strong light scattering and absorption in real samples. Environment‐sensitive fluorophores in the visible region are more suitable for biosensors [43,44] . In the original paper where this aptamer was selected, a field‐effect transistor based biosensor was fabricated and it reached a middle point of response below 1 mM glucose in diluted whole blood [17] …”

Section: Resultsmentioning

confidence: 99%

2‐Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

Huang

Zheng

et al. 2022

ChemBioChem

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Glucose is the most important analyte for biosensors. Recently a DNA aptamer was reported allowing binding-based detection. However, due to a relatively weak binding affinity, it is difficult to perform binding assays to understand the property of this aptamer. In this work, we replaced the only adenine base in the aptamer binding pocket with a 2-aminopurine (2AP) and used fluorescence spectroscopy to study glucose binding. In the selection buffer, glucose increased the 2AP fluorescence with a K d of 15.0 mM glucose, which was comparable with the 10 mM K d previously reported using the strand displacement assay. The binding required two Na + ions or one Mg 2 + that cannot be replaced by Li + or K + . The binding was weaker at higher temperature and its van't Hoff plot indicated enthalpy-driven binding. While other monosaccharides failed to achieve saturated binding even at high concentrations, two glucosecontaining disaccharides, namely trehalose and sucrose, reached a similar fluorescence level as glucose although with over 10-fold higher K d values. Detection limits in both the selection buffer (0.9 mM) and in artificial interstitial fluids (6.0 mM) were measured.

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Section: Resultsmentioning

confidence: 99%

2‐Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

Huang

Zheng

et al. 2022

ChemBioChem

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“…To incorporate the 4QI surrogate into DNA oligonucleotides, our previous work utilized solid-phase DNA synthesis with a fully synthesized 4QI–phosphoramidite . However, the reactivity of the dye to ammonium hydroxide (NH 4 OH) required the use of ultramild conditions with an extended coupling time of 2 h for optimal insertion.…”

Section: Resultsmentioning

confidence: 99%

“…To incorporate the 4QI surrogate into DNA oligonucleotides, our previous work utilized solid-phase DNA synthesis with a fully synthesized 4QI−phosphoramidite. 24 However, the reactivity of the dye to ammonium hydroxide (NH 4 OH) required the use of ultramild conditions with an extended coupling time of 2 h for optimal insertion. This tedious protocol suggested the use of an on-strand approach through aldol-type condensations between heterocyclic quaternary salts bearing an active methyl group with an oligonucleotide containing an indole−aldehyde modification.…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…23 Recently, we incorporated a molecular rotor cyanine− indole−quinolinium (4QI) hemicyanine dye tethered to an acyclic 1,2-propanediol linker into the T3-loop position of TBA to monitor probe response to thrombin binding by the antiparallel GQ motif. 24 Although the 4QI surrogate provided a turn-off response to thrombin binding, it displayed impressive fluorescence brightness (ε.Φ fl ∼ 5400 cm −1 M −1 ) within TBA compared to free dye binding to the antiparallel GQ (∼11-fold increase in intensity for an internal label versus free dye) and had a minimal impact on the stability and structure of the antiparallel GQ bound by K + . These attractive features suggested the potential utility of internal hemicyanine nucleobase surrogates as loop residue replacements to monitor antiparallel GQ-folding given the turn-on fluorescence response of free cyanine−styryl dyes to molecular rigidity.…”

Section: ■ Introductionmentioning

confidence: 99%

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On-Strand Knoevenagel Insertion of a Hemicyanine Molecular Rotor Loop Residue for Turn-On Fluorescence Detection of Pb-Induced G-Quadruplex Rigidity

2021

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We demonstrate the ability to distinguish Pb 2+ from K + within the central cavity of the antiparallel G-quadruplex (GQ) DNA produced by the thrombin binding aptamer (TBA) using an internal molecular rotor fluorescent probe. An indole−aldehyde containing an acyclic N-glycol group was first employed in the on-strand Knoevenagel condensation with five different heterocyclic quaternary cationic acceptors to assess the molecular rotor character of the resulting cyanine−styryl dyes within duplex DNA. An indole−pyridinium (4PI) nucleobase surrogate displayed the greatest turn-on emission response to duplex formation and was thus inserted into the loop residues of TBA to monitor GQ-folding in the presence of Pb 2+ versus K + . TBA-4PI exhibits turn-on emission upon Pb 2+ -binding with a brightness (ε•Φ fl ) of 9000 cm −1 M −1 compared to K +binding (ε•Φ fl ∼ 2000 cm −1 M −1 ) due to Pb 2+ -induced GQ rigidity with 4PI-Gtetrad stacking interactions. The Pb 2+ -bound TBA-4PI GQ also provides energytransfer (ET) fluorescence with a diagnostic excitation at 310 nm for distinguishing Pb 2+ from K + within the antiparallel GQ. The TBA-4PI GQ affords the desired turn-on fluorescence response for detecting Pb 2+ ions with an apparent dissociation constant (K d ) of 63 nM and a limit of detection (LOD) of 19 nM in an aqueous buffer. It can also distinguish Pb 2+ (230 nM) from K + (1.5 mM, 6500-fold excess) in an antiparallel GQ recognition motif without topology twitching.

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“…For these studies, [COOTh]An was incorporated into the TT loop positions of the 15-mer thrombin binding aptamer (TBA, 5′-GGT 3 TGG-TGTGGTTGG-3′), which folds into an antiparallel GQ with the TT loops interacting directly with exosite I of thrombin . We have previously incorporated internal fluorescent labels as T replacements within TBA to study thrombin binding, and the best fluorescent responses have been with the label occupying the T3 position, , which binds thrombin through hydrophobic interactions . For the present study, on-strand Suzuki–Miyaura coupling was used to insert the negatively charged [COOTh]An surrogate into T3, T4 and between T3 and T4 (T3XT4) to create a bulged loop, given its impressive emission response within a −1 bulged duplex when flanked by Ts (see the SI for Table S3 summarizing the MS analysis of modified TBA samples including yields from the on-strand reactions, MS spectra, and representative HPLC chromatograms).…”

Section: Results and Discussionmentioning

confidence: 99%

Screening Internal Donor–Acceptor Biaryl Nucleobase Surrogates for Turn-On Fluorescence Affords an Aniline–Carboxythiophene Probe for Protein Detection by G-Quadruplex DNA

2021

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Donor−acceptor biaryls serve as microenvironment fluorescent sensors with highly quenched intramolecular charge transfer (ICT) emission in polar protic solvents that turns on in aprotic media. In DNA, canonical donor−acceptor fluorescent base analogs can be prepared through on-strand Suzuki−Miyaura cross-coupling reactions involving 8-bromo-2′-deoxyguanosine (8-Br-dG) with an acceptor aryboronic acid. Herein, we demonstrate that replacement of 8-Br-dG with N-methyl-4-bromoaniline (4-Br-An) containing an acyclic N-glycol group can be employed in the on-strand Suzuki−Miyaura reaction to afford new donor−acceptor biaryl nucleobase surrogates with a 40-fold increase in emission intensity for fluorescent readout within singlestrand oligonucleotides. Screening the best acceptor for turn-on fluorescence upon duplex formation afforded the carboxythiophene derivative [COOTh]An with a 7.4-fold emission intensity increase upon formation of a single-bulged duplex (−1) with the surrogate occupying a pyrimidine-flanked bulge. Insertion of the [COOTh]An surrogate into the lateral TT loops produced by the antiparallel G-quadruplex (GQ) of the thrombin binding aptamer (TBA) afforded a 4.1-fold increase in probe fluorescence that was accompanied by a 20 nm wavelength shift to the blue upon thrombin binding. The modified TBA afforded a limit of detection of 129 nM for thrombin and displayed virtually no emission response to off-target proteins. The fluorescence response of [COOTh]An to thrombin binding highlights the utility of the thienyl-aniline moiety for monitoring DNA−protein interactions.

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Lighting Up the Thrombin-Binding Aptamer G-Quadruplex with an Internal Cyanine-Indole-Quinolinium Nucleobase Surrogate. Direct Fluorescent Intensity Readout for Thrombin Binding without Topology Switching

Cited by 12 publications

References 70 publications

2‐Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

2‐Aminopurine Fluorescence Spectroscopy for Probing a Glucose Binding Aptamer

On-Strand Knoevenagel Insertion of a Hemicyanine Molecular Rotor Loop Residue for Turn-On Fluorescence Detection of Pb-Induced G-Quadruplex Rigidity

Screening Internal Donor–Acceptor Biaryl Nucleobase Surrogates for Turn-On Fluorescence Affords an Aniline–Carboxythiophene Probe for Protein Detection by G-Quadruplex DNA

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