Synthesis of New Substituted Lactones by "Click" Chemistry.
Decreases in oligonucleotide density over multiple dehybridization cycles could also result from the loss or leaching of small numbers of oligonucleotide-functionalized polymer chains that are physically entrapped (as opposed to covalently crosslinked) in the topmost layers of a film. We note, however, that images of these rehybridized films did not reveal changes in the individual features of these arrays (e.g., feature broadening or blurred edges) that would also be expected to result from the lateral diffusion of free polymer chains (data not shown). We also did not observe large-scale physical delamination of films during any of these experiments (e.g., by optical or fluorescence microscopy), and such delamination would be more likely to result in complete, rather than gradual, loss of signal as observed here. Finally, as outlined above, the decreases in fluorescence intensity shown in could also result from changes in the extent to which surface-bound oligonucleotides are physically accessible after multiple treatment cycles. For example, repeated chemical and physical manipulation of these films could result in the exposure of segments of PEI (present in underlying layers of the films) that could interact with and sequester negatively charged oligonucleotides through ionic interactions. Additional characterization will be necessary to understand the extent to which such changes could occur in these experiments. In the context of this current study, however, we conclude that arrays fabricated on these film-coated substrates are stable and robust, and that they can be reused for at least three hybridization/rehybridization cycles without significant deterioration of signal.
Modular Synthesis of ABC Type Block Copolymers by "Click" Chemistry.
Oligonucleotide arrays were in situ synthesized on hydroxyl-functionalized films in a base-by-base manner using 3’-nitrophenylpropyloxycarbonyl (NPPOC)-protected nucleosides and a Maskless Array Synthesizer (see Materials and Methods and reference 18 for additional details). shows the sequences of the two different oligonucleotides (Probes 1 and 2) used in this study. Control arrays were also synthesized on hydroxyl-terminated (uncoated) glass substrates, the conventional substrate used for this application, to provide a direct comparison to established methods and materials. We note here that each iterative cycle of MAS (during which a single new nucleoside is added) exposes the solid-phase substrate to multiple different chemical processing steps (including flowing and static exposure to different organic and drying media, exposure to activator or base pair solutions, photo-irradiation steps, and oxidation procedures). The complete multi-step synthesis of the oligonucleotide arrays used in the studies described below thus requires the exposure of a surface to ~450 individual (albeit iterative) chemical processing steps. The fabricated arrays were then hybridized with fluorescently labeled complementary oligonucleotides and imaged using a fluorescence scanner to characterize both the fidelity of patterning and the ability of the immobilized sequences to pair with complementary oligonucleotide sequences.
The flexible nature of the underlying PET films did not have an apparent influence on the fluorescence intensities or the average signal-to-noise ratios of the hybridized oligonucleotide features. We performed two experiments to investigate the possible influence of substrate flexing either before or after hybridization. In the first experiment, a PET array was hybridized, a fluorescence image was obtained, and the curvature of the hybridized array was temporarily altered by manual bending of the substrate for one minute (each substrate was flexed such that the opposite ends of the surface were brought in contact, but a crease was not formed). No difference in the average fluorescence intensity or signal-to-noise ratio was observed as a result of bending. In a second set of experiments, we compared properties of PET arrays that were not bent prior to hybridization to those of arrays that were bent prior to use. The hybridization densities, average fluorescence intensities, and signal-to-noise ratios for arrays subjected to these treatments were statistically indistinguishable (data not shown). While we did not characterize the effects of flexing these array substrates surface during hybridization, several reports suggest that the curvature of an array surface can influence the density of hybridization., The ability to deposit PEI/PVDMA multilayers on a variety of different soft and flexible substrates could thus provide new tools to investigate the effects of curvature and dynamic flexing on hybridization.
Oligonucleotide Array Sequence Analysis
N2 - This contribution presents a brief overall look of the methods for the preparation of various types of DNA microarrays and a thorough examination of the methods for in situ synthesis of oligonucleotide microarrays.
In Situ Synthesis of Oligonucleotide Microarrays
The types of chemical reactions and environments used during in situ synthesis often restrict the types of substrates that can be used for array fabrication. In many approaches to the synthesis of oligonucleotide arrays, for example, substrates are exposed repeatedly to organic solvents, ultraviolet light, oxidizing agents, etc. An additional design requirement specific to the development of substrates for use with phosphoramidite chemistry is the need for free hydroxyl groups on the surface of a substrate to enable coupling of the first nucleoside phosphoramidite. Finally, in addition to stability during synthesis, substrates should also, ideally, be able to withstand exposure to the range of solvents, reagents, and other physical/mechanical challenges associated with downstream use of the arrays (e.g., in subsequent biochemical/screening assays, etc.). A number of approaches have been developed to functionalize the surfaces of glass, nanocrystalline diamond,, and amorphous carbon, substrates with terminal hydroxyl groups to permit in situ synthesis, improve array performance, and address other issues that can arise during subsequent studies.
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The work reported here was motivated by our interests in the design of reactive, polymer-based interfaces– and expanding the range of substrates that are compatible with in situ oligonucleotide synthesis.,– Polymer-based substrates could present attractive alternatives to glass and carbon-based substrates used in past studies because they are low cost, durable, and easily processed. Approaches based on polymer thin films could also be attractive because they can often be used to functionalize the surfaces of non-planar (i.e., curved) objects and porous/flexible substrates that could provide practical advantages during synthesis or subsequent screening. One drawback common to many conventional polymer-based materials, however, is that they can exhibit poor resistance to organic solvents commonly used during synthetic reactions (e.g., they either dissolve or swell upon prolonged exposure). Conversely, many of the more chemically- and mechanically stable polymer-based materials are, inherently, more resistant to facile chemical functionalization. Here, we report a step toward the design of polymer-based thin films as substrates for the in situ synthesis of oligonucleotide arrays. Our approach is based on methods developed for the ‘layer-by-layer’ fabrication of polymer thin films on surfaces.