Chemoselective Covalent Coupling of Oligonucleotide Probes to Self-Assembled Monolayers

Abstract

Surface arrays of single-stranded DNA are at the center of some of the most active areas in biological research. These include conventional applications in genome sequencing and disease diagnostics, as well as more novel emerging examples, such as combinatorial drug and reaction discovery. 1 Most methods of oligonucleotide immobilization rely on traditional nucleophilicelectrophilic reactions to achieve coupling of the oligonucleotide to the surface. 2 Unfortunately, this strategy is susceptible to side reactions, for instance, with amino groups on the nucleotides or the small molecule contaminants inherent to oligonucleotide synthesis. 3 Additionally, popular reactive electrophiles, such as N-hydroxysuccinimide esters, are prone to hydrolysis before and during the coupling reaction, which both reduce coupling yields and can make the yields irreproducible. 4 Accurate and reproducible detection of target oligonucleotides depends on the accuracy and reproducibility with which the surface can be functionalized. In this paper, we report a chemoselective approach to the formation of oligonucleotide probe surfaces using copper(I) tris(benzyltriazolylmethyl)amine (TBTA)-catalyzed triazole formation between a controlled density of azide groups on densely packed selfassembled monolayers (SAMs) and acetylene groups on the oligonucleotide probe to be immobilized. We have found this strategy to be highly predictable, very fast, and resistant to side reactions, unaffected even by the presence of excess nucleophilic or electrophilic impurities.Recently, we have demonstrated that Sharpless "click" chemistry can be used to covalently attach acetylene-bearing molecules to azide-terminated SAMs. 5,6 The surface reaction is quantitative and regioselective, exclusively yielding a single product at a single orientation. The chemistry is orthogonal to most typical organic transformations and thus is chemoselective. 7 Recent studies have demonstrated that the chemistry is well suited for the coupling of biomolecules to surfaces. 8 Although application of this chemistry to the attachment of oligonucleotides may appear straightforward, the majority of past work used free Cu(I), typically generated and maintained in aqueous solution by an excess of reducing agent. Unfortunately, in the presence of dioxygen, Cu(I) rapidly damages DNA via the generation of reactive oxygen species. 9 In order for a surface array of oligonucleotides to be useful as a sensor, the structure of the oligonucleotides must be preserved. Recently, the Sharpless group has introduced a triazolylamine copper ligand, tris-(benzyltriazolylmethyl)amine, that can accelerate the cycloaddition reaction. 10 At the same time, this ligand was found to significantly deter the redox chemistry of Cu(I) with oxygen, which is essential for preventing damage to the oligonucleotides. Encouraged by the highly desirable features of the Cu(I)TBTA-catalyzed azide-alkyne cycloaddition, we studied its applicability to the attachment of oligonucleotide probes onto well-defined SAMs.Oligode...

Preparation of Carbodiimide-terminated Dithiolane Self-Assembly Monolayers as a New DNA-Immobilization Method

“…According to previous reports, DNA can be immobilized to the density of a 1.0 × 10 12 -10 14 molecules/cm 2 (treatment of thiolated DNA on a bare gold surface), [16][17][18][19] 1.0 -4.0 × 10 12 molecules/cm 2 (reaction of 5′-aminated DNA with the surface of active ester-covered SAMs), 20 and 1.0 -4.0 × 10 12 molecules/cm 2 (reaction of acetylated DNA with the surface of azide-covered SAMs). 22 The amount estimated for 30 meric ODN3(-) is in agreement with those literature values. The 100-times smaller amount of immobilization observed for poly(dT) may also be reasonable, given that its size is an order of magnitude larger than that of the oligonucleotides.…”

“…[10][11][12][13][14][15] To construct such a system, it is important to develop a reproducible method to immobilize a capture DNA probe on the gold surface, and many kinds of immobilization methods have been reported. [16][17][18][19][20][21][22] An immobilization method through the Au-S linkage is one of the conventional methods. It is known that thiolated DNA can bind to a gold surface, and detailed studies, such as an estimation of the density of DNA immobilization, were carried out.…”

“…20,21 Recently, Childsey et al reported a novel immobilization method for acetylated DNA on a SAM surface having an azide group. 22 Such a SAM system can avoid nonspecific absorption by introducing an additional hydroxyl alkanethiol, such as 6-mercaptohexanol (6MH). An immobilization method based on biotin-avidin interaction, where biotinyl DNA was immobilized on an avidin-covered gold electrode, was also reported.…”

Rate of Interfacial Electron Transfer through the 1,2,3-Triazole Linkage

“…34 This catalyst has the advantage that it is free of side reactions with O 2 that form partially reduced oxygen species and so can be safely used under aerobic conditions in the presence of oxidatively sensitive compounds, such as the coupling of single-stranded DNA to an azide-terminated mixed monolayer. 36 Most recently, we have used this chemistry to selectively modify an array of independently addressable microelectrodes by electrochemically localizing the copper(I) catalyst. 37 The work reported here demonstrates that monolayer assemblies coupled to a redox species by Cu(I)-catalyzed triazole formation are well suited for studies where the electron-transfer rate between a given surface redox species and the electrode needs to be reproducibly and predictably controlled but where assembly of the redox-active mixed monolayer in one step is prohibited by the reactivity of free thiols and/or the overall complexity of the redox species.…”

Cellulose Chemistry Meets Click Chemistry: Syntheses and Properties of Cellulose-Based Glycoclusters with High Structural Homogeneity

“…Exclusive chemoselectivity and perfect conversion yield are therefore highly required for the further modification reaction. To satisfy these criteria, we applied click chemistry for the further modification on Cel-N 3 [29][30][31][32][33].…”