Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes

Abstract

Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes.

Solid-Phase Nucleic Acid Sequence-Based Amplification and Length-Scale Effects during RNA Amplification

“…Despite the fact that there is an excess of complementary target, a significant fraction of MBs tethered to the 80 and 100 wt % PEG-B microgels do not hybridize. This result is consistent with our previous measurements exploring crowding effects on MB performance in a system involving only tethered MBs with no tethered primers or bridges . The reduced hybridization efficiency was previously attributed to the increased role of steric factors on either target access to the tethered MBs or the ability of the target to sufficiently hybridize to a tethered MB.…”

“…In addition to microgels consisting of 20 wt % PEG-B, we explored the SP-NASBA reaction for microgels consisting of 40 and 60 wt % PEG-B (Figure ). The fact that the microgel intensities for both the positive and negative control specimens increase linearly with increasing PEG-B content is consistent with previously published experiments on microgels made from PEG-OH/PEG-B blends and indicates that the PEG-B content controls the density of tethering sites. However, while the SP-NASBA reaction is very efficient at low tethering densities (20 wt % PEG-B), it becomes extremely inefficient as the tethering density is increased.…”

“…We create poly­(ethylene glycol) (PEG) microgels produced by electron-beam cross-linking PEG thin films cast on solid substrates. , Microgels made from PEG with biotin end groups can be activated with streptavidin, and biotinylated oligonucleotides can then be tethered to the highly hydrated outermost portions of the microgels. The tethering density can be controlled using precursor thin films comprising miscible homopolymer blends of biotin-terminated PEG (PEG-B) and hydroxyl-terminated PEG (PEG-OH) . Precursor films made from pure PEG-OH present no tethering sites and resist nonspecific protein adsorption. , There is a linear increase in tethering density as PEG-B is blended into PEG-OH reaching a maximum density of just under 50 000/μm 2 for pure PEG-B (5 kDa M w precursor) .…”

Surface‐Bound Microgels for Separation, Sensing, and Biomedical Applications

“…The team discovered that the surface density of the conjugated linear DNA probes increased with the ratio of biotin/hydroxylterminated PEG, while the binding capacity rapidly plateaued when binding hairpin DNA due to steric hinderance. [44] Besides peptides, oligonucleotide aptamers furnish highly selective binding moieties that enable sensitive detection of target analytes and signal amplification. For example, Ma et al developed a solid-phase oligonucleotide amplifier by integrating RNA primers on the surface of biotinylated-PEG microgels combined with reverse transcriptase and RNase H, which promoted the formation of double-stranded DNA on the microgel surface.…”

“…Specifically, controlled swelling has been leveraged to release physically bound molecules, [20,[34][35][36] adjust the surface stiffness, [5,25,[37][38][39] and enable sensing activity. [9,40,41] Furthermore, the monomers used to construct the microgels are often amenable to post-synthetic modification with (bio)macromolecules, [42][43][44][45][46] nanoparticles, [47][48][49] and small molecules, [50][51][52][53] thus expanding their technological potential.…”