Enzymatic Beacons for Specific Sensing of Dilute Nucleic Acid and Potential Utility for SARS-CoV-2 Detection

Enzymatic beacons, or E-beacons, are 1:1 bioconjugates of the nanoluciferase enzyme linked covalently at its C-terminus to hairpin forming DNA oligonucleotides equipped with a dark quencher. We prepared E-beacons biocatalytically using the promiscuous hedgehog protein-cholesterol ligase, HhC. Instead of cholesterol, HhC attached nanoluciferase site-specifically to mono-sterylated hairpin DNA, prepared in high yield by solid phase synthesis. We tested three potential E-beacon dark quenchers: Iowa Black, Onyx-A, and dabcyl. Prototype E-beacon carrying each of those quenchers provided sequence-specific nucleic acid sensing through turn-on bioluminescence. For practical application, we prepared dabcyl-quenched E-beacons for potential use in detecting the COVID-19 virus, SARS-CoV-2. Targeting the E484 codon of the SARS-CoV-2 Spike protein, E-beacons (80 x 10-12 M) reported wild-type SARS-CoV-2 nucleic acid at greater than or equal to 1 x 10-9 M with increased bioluminescence of 8-fold. E-beacon prepared for the E484K variant of SARS-CoV-2 functioned with similar sensitivity. These E-beacons could discriminate their complementary target from nucleic acid encoding the E484Q mutation of the SARS-CoV-2 Kappa variant. Along with specificity, detection sensitivity with E-beacons is two to three orders of magnitude better than synthetic molecular beacons, rivaling the most sensitive nucleic acid detection agents re-ported to date.

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Development of Strategies for Glycopeptide Synthesis: An Overview on the Glycosidic Linkage

Current Organic Chemistry ◽

10.2174/1385272824999200701121037 ◽

2020 ◽

Vol 24 (21) ◽

pp. 2475-2497

Author(s):

Andrea Verónica Rodríguez-Mayor ◽

German Jesid Peralta-Camacho ◽

Karen Johanna Cárdenas-Martínez ◽

Javier Eduardo García-Castañeda

Keyword(s):

Chemical Synthesis ◽

Solid Phase ◽

Building Blocks ◽

Heterogeneous Environments ◽

Phase Synthesis ◽

Low Concentrations ◽

Biological Sources ◽

Synthetic Routes ◽

The Impact ◽

Potential Use

Glycoproteins and glycopeptides are an interesting focus of research, because of their potential use as therapeutic agents, since they are related to carbohydrate-carbohydrate, carbohydrate-protein, and carbohydrate-lipid interactions, which are commonly involved in biological processes. It has been established that natural glycoconjugates could be an important source of templates for the design and development of molecules with therapeutic applications. However, isolating large quantities of glycoconjugates from biological sources with the required purity is extremely complex, because these molecules are found in heterogeneous environments and in very low concentrations. As an alternative to solving this problem, the chemical synthesis of glycoconjugates has been developed. In this context, several methods for the synthesis of glycopeptides in solution and/or solid-phase have been reported. In most of these methods, glycosylated amino acid derivatives are used as building blocks for both solution and solid-phase synthesis. The synthetic viability of glycoconjugates is a critical parameter for allowing their use as drugs to mitigate the impact of microbial resistance and/or cancer. However, the chemical synthesis of glycoconjugates is a challenge, because these molecules possess multiple reaction sites and have a very specific stereochemistry. Therefore, it is necessary to design and implement synthetic routes, which may involve various protection schemes but can be stereoselective, environmentally friendly, and high-yielding. This review focuses on glycopeptide synthesis by recapitulating the progress made over the last 15 years.

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Cholecystokinin Heptapeptide Analogues with Multiple Modification in Peptide Chain

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19932761 ◽

1993 ◽

Vol 58 (11) ◽

pp. 2761-2765 ◽

Cited By ~ 2

Author(s):

Jan Hlaváček ◽

Jana Pírková ◽

Miroslava Žertová ◽

Jan Pospíšek ◽

Lenka Maletínská ◽

...

Keyword(s):

Solid Phase Synthesis ◽

Solid Phase ◽

Peptide Chain ◽

Phase Synthesis ◽

C Terminus

Using solid phase synthesis we prepared the cholecystokinin fragment Boc-CCK-7 (Boc-Tyr(SO3-Na+)-Met-Gly-Trp-Met-Asp-Phe-NH2) Ia and its seven analogues Ib - Ih. In the analogues Ib and Ic the Met residue in the carboxyterminal part of the molecule was substituted for L- or D-Phe Me3. In the analogues Id and Ie with Phe residue substituted by L- or D-Phe Me3 the Neo was inserted in the place of this Met residue and in the analogues If and Ig, an addition to PheMe3 substitution in the carboxyterminus, both Met residues were replaced for Neo. This dual substitution for Met residues was also applied in the analogue Ih with coded Phe residue in the C-terminus.

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Synthesis of [1-β-mercaptopropionic acid, 8-D-arginine]vasopressin (DDAVP) in solid phase. Simple optimalization

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19791173 ◽

1979 ◽

Vol 44 (4) ◽

pp. 1173-1178 ◽

Cited By ~ 4

Author(s):

Viktor Krchňák ◽

Milan Zaoral

Keyword(s):

Arginine Vasopressin ◽

Solid Phase Synthesis ◽

Solid Phase ◽

Experimental Conditions ◽

Phase Synthesis ◽

Mercaptopropionic Acid ◽

Better Than

A series of solid-phase syntheses of the protected precursor II of DDAVP was carried out. Experimental conditions were developed under which practically pure II can reproducibly be obtained in yields better than 60%. The protected precursors of DDAVP obtained by liquid- and solid-phase synthesis and DDAVP samples obtained from these precursors were undistinguishable by conventional analytical or pharmacological assays.

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Solid-phase synthesis of benzodiazepinediones mimicking the C-terminus of the H-Ras protein

Tetrahedron Letters ◽

10.1016/j.tetlet.2009.01.016 ◽

2009 ◽

Vol 50 (26) ◽

pp. 3148-3150 ◽

Cited By ~ 3

Author(s):

Björn Ludolph ◽

Herbert Waldmann

Keyword(s):

Solid Phase Synthesis ◽

Solid Phase ◽

Phase Synthesis ◽

Ras Protein ◽

C Terminus

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Microwave-assisted solid-phase synthesis of antisense acpP peptide nucleic acid-peptide conjugates active against colistin- and tigecycline-resistant E. coli and K. pneumoniae

European Journal of Medicinal Chemistry ◽

10.1016/j.ejmech.2019.02.024 ◽

2019 ◽

Vol 168 ◽

pp. 134-145 ◽

Cited By ~ 2

Author(s):

Anna Mette Hansen ◽

Gitte Bonke ◽

Wouter Frederik Johan Hogendorf ◽

Fredrik Björkling ◽

John Nielsen ◽

...

Keyword(s):

Nucleic Acid ◽

Peptide Nucleic Acid ◽

Solid Phase Synthesis ◽

Solid Phase ◽

Peptide Conjugates ◽

Phase Synthesis ◽

E Coli ◽

Microwave Assisted

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Rapid, High-Yield, Solid-Phase Synthesis of the Antitumor Antibiotic Sansalvamide A Using a Side-Chain-Tethered Phenylalanine Building Block

Organic Letters ◽

10.1021/ol0002830 ◽

2000 ◽

Vol 2 (23) ◽

pp. 3743-3746 ◽

Cited By ~ 34

Author(s):

Younghee Lee ◽

Richard B. Silverman

Keyword(s):

Building Block ◽

Solid Phase Synthesis ◽

Solid Phase ◽

High Yield ◽

Side Chain ◽

Antitumor Antibiotic ◽

Phase Synthesis ◽

Sansalvamide A

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Combinatorial synthesis using nucleic acid-based (NAB™) scaffold: parallel solid-phase synthesis of nucleotide libraries

Tetrahedron Letters ◽

10.1016/s0040-4039(99)02089-4 ◽

2000 ◽

Vol 41 (4) ◽

pp. 441-445 ◽

Cited By ~ 6

Author(s):

Wenqiang Zhou ◽

Arlène Roland ◽

Yi Jin ◽

Radhakrishnan P Iyer

Keyword(s):

Nucleic Acid ◽

Solid Phase Synthesis ◽

Solid Phase ◽

Combinatorial Synthesis ◽

Phase Synthesis

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Solid-Phase Synthesis of DNA Chemical Sensor

Advanced Materials Research ◽

10.4028/www.scientific.net/amr.815.305 ◽

2013 ◽

Vol 815 ◽

pp. 305-311 ◽

Cited By ~ 1

Author(s):

Hui Yong Zhang

Keyword(s):

Nucleic Acid ◽

Chemical Sensor ◽

Solid Phase Synthesis ◽

Solid Phase ◽

Acid Synthesis ◽

Synthetic Approach ◽

Biological Interactions ◽

Oligonucleotide Synthesis ◽

Phase Synthesis ◽

Essential Components

Oligonucleotides are essential components of many applications in molecular biology. The synthesis chemistry is robust and commercial oligonucleotide synthesizers have taken advantage of the chemistry to provide oligonucleotides of high quality and purity. This paper established nucleic acid synthesis platform to carry out the synthesis of the labeled nucleic acid probes based on the DNA synthesizer and solid-phase synthesis technology. We chose to study the automated synthesis starting from DMT protected FAM labeled amidite attached to controlled pore glass (CPG) support and the standard trityl-off oligonucleotide synthesis cycle was performed, yielding the solid-supported oligonucleotide. The reported automated solid-phase oligonucleotide synthesis procedure successfully employs the common iterative synthesis, deblocking, activation, coupling, capping, oxidation, and isolation steps in standard oligonucleotide synthesis. The automated synthetic approach can also be applied to oligonucleotides of different length, composition of nucleotide, demonstrating the universality of the method. Moreover, the synthesis involved the use of commercially available, safe, stable, and inexpensive reagents, particularly advantageous and attractive for their use in automated solid-phase synthesis. The synthesis allows custom tailoring of their structure to the requirements of biological assays within hours, as opposed to traditional approaches that require weeks or months of work in the laboratory. Therefore it will become much easier to investigate biological interactions and optimize for objectives such as the receptor mediated targeting of oligonucleotides.

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