Uncovering Molecular Quencher Effects on FRET Phenomena in Microsphere-Immobilized Probe Systems.

Double-stranded (ds) oligonucleotide probes composed of quencher-dye sequence pairs outperform analogous single-stranded (ss) probes due to their superior target sequence specificity without any prerequisite target labeling. Optimizing sequence combinations for dsprobe design requires promoting a fast, accurate response to a specific target sequence while minimizing spontaneous dsprobe dissociation events. Here, flow cytometry is used to rapidly interrogate the stability and selective responsiveness of 20 candidate LNA and DNA dsprobes to a 24 base-long segment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA and ∼243 degenerate RNA sequences serving as model variants. Importantly, in contrast to quantifying binding events of dye-labeled targets via flow cytometry, the current work employs the Förster resonance energy transfer (FRET)-based detection of unlabeled RNA targets. One DNA dsprobe with a 15-base-long hybridization partner containing a central abasic site emerged as very stable yet responsive only to the SARS-CoV-2 RNA segment. Separate displacement experiments, however, indicated that ∼12% of these quencher-capped hybridization partners remain bound, even in the presence of an excess SARS-CoV-2 RNA target. To examine their quenching range, additional titration studies varied the ratios and spatial placement of nonquencher and quencher-capped hybridization partners in the dsprobes. These titration studies indicate that these residual, bound quencher-capped partners, even at low percentages, act as nodes, enabling both static quenching effects within each residual dsprobe as well as longer-range quenching effects on neighboring FAM moieties. Overall, these studies provide insight into practical implications for rapid dsprobe screening and target detection by combining flow cytometry with FRET-based detection.

Quantitative Analysis of In Situ Locked Nucleic Acid and DNA Competitive Displacement Events on Microspheres.

Synthetic analogues of natural oligonucleotides known as locked nucleic acids (LNAs) offer superior nuclease resistance and cytocompatibility for numerous scenarios ranging from in vitro detection to intracellular imaging of nucleic acids. While recognized as stronger hybridization partners than equivalent DNA residues, quantitative analysis of LNA hybridization activity is lacking, especially with respect to competitive displacement of the original hybridization partner by another oligonucleotide. In the current study, we perform in situ measurements of toehold-mediated competitive displacement of soluble, fluorescently labeled primary targets from probe strands immobilized on microspheres using high throughput flow cytometry. Both LNA-DNA hybrid sequences and pure DNA sequences are employed as the immobilized strands, as soluble, fluorescently labeled 9-base-long primary targets, and as unlabeled 15-base-long secondary or competitive targets. In addition to comparing chemically substituted and unsubstituted sequences, we explore the effects of mismatched primary targets and the location of the toehold segment within the primary duplexes on the resulting displacement profiles. The primary duplex or double-stranded probe (dsprobe) systems implemented here exhibited varying responses to unlabeled secondary targets ranging from surprisingly modest primary target displacement activity despite the presence of a six base-long nucleotide toehold segment at the dsprobe free end to distinctive displacement profiles sensitive to LNA substitutions and the placement of the toehold segment closer to the microsphere surface.

Competition-Enhanced Ligand Selection to Screen for DNA Aptamers for Spherical Gold Nanoparticles.

The Competition-Enhanced Ligand Selection (CompELS) approach was used to identify aptamer candidates for spherical gold nanoparticles (AuNPs). This approach differs from conventional Systematic Evolution of Ligands by EXponential enrichment (SELEX)-based aptamer screening by eliminating repeated elution and polymerase chain reaction (PCR) amplification steps of bound candidate sequences between each selection round to continually enrich the candidate aptamer pool with oligonucleotides remaining from an earlier SELEX selection round. Instead, a new pool of unenriched oligonucleotides is added during each CompELS selection round to compete with existing target-bound oligonucleotides species for target binding sites. In this study, 24 aptamer candidates for AuNPs were identified using the CompELS approach and then compared to reveal similarities in their primary structures and their predicted secondary structures. No strong patterns in individual base identities (position-dependent) nor in segments of consecutive bases (independent of position) prevailed among the identified sequences. Motifs in predicted secondary structures, on the other hand, were shared among otherwise unrelated aptamer sequences. These motifs were revealed using a systematic classification and enumeration of distinct secondary structure elements, namely, hairpins, duplexes, single-stranded segments, interior loops, bulges, and multibranched loops.

Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides.

In the last three decades, oligonucleotides have been extensively investigated as probes, molecular ligands and even catalysts within therapeutic and diagnostic applications. The narrow chemical repertoire of natural nucleic acids, however, imposes restrictions on the functional scope of oligonucleotides. Initial efforts to overcome this deficiency in chemical diversity included conservative modifications to the sugar-phosphate backbone or the pendant base groups and resulted in enhanced in vivo performance. More importantly, later work involving other modifications led to the realization of new functional characteristics beyond initial intended therapeutic and diagnostic prospects. These results have inspired the exploration of increasingly exotic chemistries highly divergent from the canonical nucleic acid chemical structure that possess unnatural physiochemical properties. In this review, the authors highlight recent developments in modified oligonucleotides and the thrust towards designing novel nucleic acid-based ligands and catalysts with specifically engineered functions inaccessible to natural oligonucleotides.

Analyzing Secondary Structure Patterns in DNA Aptamers Identified via CompELS.

In contrast to sophisticated high-throughput sequencing tools for genomic DNA, analytical tools for comparing secondary structure features between multiple single-stranded DNA sequences are less developed. For single-stranded nucleic acid ligands called aptamers, secondary structure is widely thought to play a pivotal role in driving recognition-based binding activity between an aptamer sequence and its specific target. Here, we employ a competition-based aptamer screening platform called CompELS to identify DNA aptamers for a colloidal target. We then analyze predicted secondary structures of the aptamers and a large population of random sequences to identify sequence features and patterns. Our secondary structure analysis identifies patterns ranging from position-dependent score matrixes of individual structural elements to position-independent consensus domains resulting from global alignment.

Competition-Enhanced Ligand Selection to Identify DNA Aptamers.

Competition-enhanced ligand screening (CompELS) was employed to rapidly screen through large DNA libraries to identify single-stranded, oligonucleotide-based ligands called aptamers that bind to a nonbiological target. This previously unreported aptamer screening approach involves the repeated introduction of unenriched random sequence populations during the biopanning process, but avoids iterative elution and polymerase chain reaction (PCR) amplification steps inherent to traditional SELEX (systematic evolution of ligands by exponential enrichment) screening. In this study, 25 aptamers were identified against a gold surface via CompELS and evaluated to identify patterns in primary structures and predicted secondary structures. Following a final one-round competition experiment with the 25 identified aptamers, one particular aptamer sequence (1N) emerged as the most competitive adsorbate species for the gold substrate. Binding analysis indicated at least an order of magnitude difference in the binding affinity of 1N ( Kd = 5.6 × 10-10 M) compared to five other high affinity aptamer candidates ( Kd = 10-8-10-9 M) from identical secondary structure families. Collectively, these studies introduce a rapid, reliable screening and ranking platform along with a classification scheme well-suited for identifying and characterizing aptamers for nonbiological as well as biological targets.

Analysis of in Situ LNA and DNA Hybridization Events on Microspheres.

The hybridization activity of single-stranded DNA and locked nucleic acid (LNA) sequences on microspheres is quantified in situ using flow cytometry. In contrast to conventional sample preparation for flow cytometry that involves several wash steps for posthybridization analysis, the current work entails directly monitoring hybridization events as they occur between oligonucleotide-functionalized microspheres and fluorescently tagged 9 or 15 base-long targets. We find that the extent of hybridization between single-stranded, immobilized probes and soluble targets generally increases with target sequence length or with the incorporation of LNA nucleotides in one or both oligonucleotide strands involved in duplex formation. The rate constants for duplex formation, on the other hand, remain nearly identical for all but one probe-target sequence combination. The exception to this trend involves the LNA probe and shortest perfectly matched DNA target, which exhibit a rate constant that is an order of magnitude lower than any other probe-target pair, including a mismatched duplex case. Separate studies entailing brief heat treatments to suspensions generally do not consistently yield appreciable differences in associated target densities to probe-functionalized microspheres.

Spectroscopic studies of nucleic acid additions during seed-mediated growth of gold nanoparticles.

The effect of adding nucleic acids to gold seeds during the growth stage of either nanospheres or nanorods was investigated using UV-Vis spectroscopy to reveal any oligonucleotide base or structure-specific effects on nanoparticle growth kinetics or plasmonic signatures. Spectral data indicate that the presence of DNA duplexes during seed ageing drastically accelerated nanosphere growth while the addition of single-stranded polyadenine at any point during seed ageing induces nanosphere aggregation. For seeds added to a gold nanorod growth solution, single-stranded polythymine induces a modest blue-shift in the longitudinal peak wavelength. Moreover, a particular sequence comprised of 50% thymine bases was found to induce a faster, more dramatic blue-shift in the longitudinal peak wavelength compared to any of the homopolymer incubation cases. Monomeric forms of the nucleic acids, however, do not yield discernable spectral differences in any of the gold suspensions studied.

Evaluating the dual target binding capabilities of immobilized aptamers using flow cytometry.

In the current study, the authors quantify the binding activity of particle-immobilized DNA aptamers to their nucleotide and non-nucleotide targets. For the purposes of this work, DNA and vascular endothelial growth factor (VEGF) binding analysis was carried out for VEGF-binding aptamers and compared to that of an ampicillin-binding aptamer as well as a non-aptamer DNA probe. Binding analysis followed incubation of one target type, coincubation of both DNA and VEGF targets, and serial incubations of each target type. Moreover, recovery of aptamer binding activity following displacement of the DNA target from aptamer:DNA duplexes was also explored. Flow cytometry served as the quantitative tool to directly monitor binding events of both the DNA target and protein target to the various aptamer and non-aptamer functionalized particles. The current work demonstrates how processing steps such as annealing and binding history of particle-immobilized aptamers can affect subsequent binding activity. To this end, the authors demonstrate the ability to fully recover DNA target binding activity capabilities and to partially recover protein target binding activity.

Altering colloidal surface functionalization using DNA encapsulated inside monodisperse gelatin microsphere templates.

Soluble oligonucleotides are typically introduced to bulk solution to promote hybridization activity on DNA-functionalized surfaces. Here, an alternative approach is explored by encapsulating secondary target strands inside semipermeable colloidal satellite assemblies, then triggering their release at 37 °C for subsequent surface hybridization activity. To prepare DNA-loaded satellite assemblies, uniform gelatin microspheres are fabricated using microfluidics, loaded with 15 base-long secondary DNA targets, capped with a polyelectrolyte bilayer, and finally coated with a monolayer of polystyrene microspheres functionalized with duplexes comprised of immobilized probes and soluble, 13 base-long hybridization partner strands. Once warmed to 37 °C, secondary DNA targets are released from the gelatin template and then competitively displace the shorter, original hybridization partners on the polystyrene microspheres.

Measuring in situ primary and competitive DNA hybridization activity on microspheres.

Microspheres serve as convenient substrates for studying DNA activity on surfaces. Here, in addition to employing conventional sample preparation involving multiple wash and resuspension steps prior to flow cytometry measurements, we also directly sampled the reaction volume to acquire in situ measurements of primary and competitive hybridization events. Even in the absence of post hybridization wash steps, nonspecific binding events were negligible and thus allowed for direct, quantitative assessment of hybridization events as they occurred on colloidal surfaces. The in situ results indicate that primary duplex formation between immobilized probes and soluble targets on microsphere surfaces is less favorable than predicted by solution models. The kinetics of competitive displacement of primary hybridization partners by secondary targets measured in situ or post washing also deviate from expectations based on theoretical solution thermodynamics, but are consistent with predicted kinetic trends stemming from differences in either the toehold base length or branch migration.

Using double-stranded DNA probes to promote specificity in target capture.

The most prevalent nucleic acid detection schemes employ single-stranded sequences as probes for capture and detection of oligonucleotide targets in solution. In these systems elevated temperature conditions are generally used to enhance specificity and limit false positives from occurring with mismatched targets. In contrast, the current study uses a strand displacement approach between soluble targets and double-stranded DNA probes (dsProbes) immobilized on microspheres. In our approach the displacement of reporter strands from the dsProbes by the target of interest is promoted by the affinity differences between the reporter strand and the soluble DNA or RNA targets for the immobilized sequences. While displacement activity occurred readily in center mismatched dsProbes with a weaker intrinsic affinity, incorporating a two base-long single-stranded segment at the free end of the immobilized dsProbes resulted in target discrimination not observed for dsProbes possessing only a center mismatch.

Hybridization kinetics between immobilized double-stranded DNA probes and targets containing embedded recognition segments.

We have investigated the time-dependent strand displacement activity of several targets with double-stranded DNA probes (dsProbes) of varying affinity. Here, the relative affinity of various dsProbes is altered through choices in hybridization length (11-15 bases) and the selective inclusion of center mismatches in the duplexes. While the dsProbes are immobilized on microspheres, the soluble, 15 base-long complementary sequence is presented either alone as a short target strand or as a recognition segment embedded within a longer target strand. Compared to the short target, strand displacement activity of the longer targets is slower, but still successful. Additionally, the longer targets exhibit modest differences in the observed displacement rates, depending on the location of recognition segment within the long target. Overall, our study demonstrates that the kinetics of strand displacement activity can be tuned through dsProbe sequence design parameters and is only modestly affected by the location of the complementary segment within a longer target strand.

DNA density-dependent assembly behavior of colloidal micelles.

A key advantage of DNA-mediated colloidal assembly is the ability to tune the strength of adhesion between particles based on sequence characteristics. In the current study, we have investigated DNA-mediated assembly of polystyrene colloidal particles as a function of sequence length, sequence fidelity, and probe density for DNA sequences patterned from the Salmonella genome. The results of our work indicate that the density of DNA probe strands heavily influences the ability of immobilized sequences to hybridize between surfaces of bidisperse colloidal particles. Incubating suspensions at higher temperatures (to minimize secondary structures that might otherwise compromise duplex formation) was also found to have less effect than duplex density on DNA-mediated particle assembly. We believe these results may add to the understanding and design considerations of directed particle assembly using DNA hybridization, especially in the submicrometer and micrometer size regime.

Manipulating DNA probe presentation via enzymatic cleavage of diluent strands.

We previously reported a system for the controlled redispersion of DNA-linked aggregates using secondary, competitive hybridization events and found that complete redispersion is contingent upon dilution of the active 20 base-long probe strands with 20 base-long nonhybridizing strands. Here, to reduce the steric interference of nonhybridizing or diluent strands on probe activity, we investigate the effect of shorter diluent strands on the hybridization activity of immobilized probes using the following two approaches: (1) simultaneously coupling shorter diluent strands and longer probe strands to microspheres and (2) simultaneously coupling diluent and probe strands of the same base length to microspheres and then clipping diluent strands with the restriction endonuclease AluI. Results indicate that one can reduce the duplex density down by 50-70% of its initial value, depending on the location of the recognition motif along the hybridization segment. In addition, tighter control over the number of probe-target duplexes is achieved with the enzyme-based approach.

Quantitative measurement of nanoparticle halo formation around colloidal microspheres in binary mixtures.

A new colloidal stabilization mechanism, known as nanoparticle "haloing" (Tohver, V.; Smay, J. E.; Braem, A.; Braun, P. V.; Lewis, J. A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, (16), 8950-8954), has been predicted theoretically and inferred experimentally in microsphere-nanoparticle mixtures that possess high charge and size asymmetry. The term "halo" implies the existence of a nonzero separation distance between the highly charged nanoparticles and the negligibly charged microspheres that they surround. By means of ultrasmall-angle X-ray scattering, we have quantified the microsphere-nanoparticle separation distance as well as the number of nanoparticles and their lateral separation distance within the self-organized halos that form in these binary mixtures.

Reversing DNA-mediated adhesion at a fixed temperature.

Recognition-based assembly of micron- to nano-sized colloidal particles functionalized with DNA has generated great interest in the past decade; however, reversing the assembly process is typically achieved by thermal denaturation of the oligonucleotide duplexes. Here, we report an alternative disassembly approach at a fixed temperature using competitive hybridization events between immobilized and soluble oligonucleotide strands. Microspheres are first aggregated via primary hybridization events between immobilized DNA strands with a weak, but sufficient, affinity for partner strands to link complementary surfaces together. To reverse the aggregation process, soluble oligonucleotides are then added to competitively displace the original hybridization partners through secondary hybridization events. Using flow cytometry to quantify hybridization events and microscopy to examine DNA-mediated aggregation and redispersion, we found that the efficiency of competitive displacement is based upon (1) the difference in base pair matches between the primary and secondary target for the same probe sequence and (2) the concentration of hybridizing oligonucleotides participating in microsphere aggregation. To the best of our knowledge, this study is the first to employ DNA hybridization events to mediate reversible adhesion between colloidal particles at a fixed temperature.

DNA-mediated phase behavior of microsphere suspensions.

We have constructed a phase diagram for DNA-modified microsphere suspensions based on experimental and theoretical studies. The system is comprised of 1 microm red fluorescent colloids functionalized with strands of an identical oligonucleotide sequence and 1 microm green fluorescent colloids functionalized with the complementary sequence. Keeping the suspension composition and temperature fixed, the phase behavior of colloidal mixtures was studied as a function of salt and oligonucleotide concentration. We observed a colloidal fluid phase of dispersed, single particles at low salt concentrations and low DNA densities. We attribute this colloidal fluid phase to unfavorable hybridization conditions. With increasing salt or hybridizing oligonucleotide concentrations, we observed phase transitions of fluid --> fluid + aggregates --> aggregates due to an increase in duplex affinity, duplex number, or both. Computational analysis assigns a 4 kBT attraction between pairs of complementary microspheres at the destabilizing fluid --> fluid + aggregates transition.

Ultrafast electronic relaxation and charge-carrier localization in CdS/CdSe/CdS quantum-dot quantum-well heterostructures.

The relaxation and localization times of excited electrons in CdS/CdSe/CdS colloidal quantum wells were measured using subpicosecond spectroscopy. HRTEM analysis and steady-state PL demonstrate a narrow size distribution of 5-6 nm epitaxial crystallites. By monitoring the rise time of the stimulated emission as a function of pump intensity, the relaxation times of the electron from the CdS core into the CdSe well are determined and assigned. Two-component rise times in the stimulated emission are attributed to intraband relaxation of carriers generated directly within the CdSe well (fast component) and charge transfer of core-localized carriers across the CdS/CdSe interface (slow component). This is the first reported observation of simultaneous photon absorption in the core and well of a quantum-dot heterostructure. With increasing pump intensity, the charge-transfer channel between the CdS core CdSe well contributes less to the stimulated emission signal because of filling and saturation of the CdSe well state, making the interfacial charge-transfer component less efficient. The interfacial charge-transfer time of the excited electron was determined from the slow component of the stimulated emission build-up time and is found to have a value of 1.2 ps.

Differential adhesion of microspheres mediated by DNA hybridization I: experiment.

We have developed a novel method to study collective behavior of multiple hybridized DNA chains by measuring the adhesion of DNA-coated micron-scale beads under hydrodynamic flow. Beads coated with single-stranded DNA probes are linked to surfaces coated with single target strands through DNA hybridization, and hydrodynamic shear forces are used to discriminate between strongly and weakly bound beads. The adhesiveness of microspheres depends on the strength of interaction between DNA chains on the bead and substrate surfaces, which is a function of the degree of DNA chain overlap, the fidelity of the match between hybridizing pairs, and other factors that affect the hybridization energy, such as the salt concentration in the hybridization buffer. The force for bead detachment is linearly proportional to the degree of chain overlap. There is a detectable drop in adhesion strength when there is a single base mismatch in one of the hybridizing chains. The effect of single nucleotide mismatch was tested with two different strand chemistries, with mutations placed at several different locations. All mutations were detectable, but there was no comprehensive rule relating the drop in adhesive strength to the location of the defect. Since adhesiveness can be coupled to the strength of overlap, the method holds promise to be a novel methodology for oligonucleotide detection.