The effect of hybridization-induced secondary structure alterations on RNA detection using backscattering interferometry.

Backscattering interferometry (BSI) has been used to successfully monitor molecular interactions without labeling and with high sensitivity. These properties suggest that this approach might be useful for detecting biomarkers of infection. In this report, we identify interactions and characteristics of nucleic acid probes that maximize BSI signal upon binding the respiratory syncytial virus nucleocapsid gene RNA biomarker. The number of base pairs formed upon the addition of oligonucleotide probes to a solution containing the viral RNA target correlated with the BSI signal magnitude. Using RNA folding software mfold, we found that the predicted number of unpaired nucleotides in the targeted regions of the RNA sequence generally correlated with BSI sensitivity. We also demonstrated that locked nucleic acid (LNA) probes improved sensitivity approximately 4-fold compared to DNA probes of the same sequence. We attribute this enhancement in BSI performance to the increased A-form character of the LNA:RNA hybrid. A limit of detection of 624 pM, corresponding to ∼10(5) target molecules, was achieved using nine distinct ∼23-mer DNA probes complementary to regions distributed along the RNA target. Our results indicate that BSI has promise as an effective tool for sensitive RNA detection and provides a road map for further improving detection limits.

Toward rapid, high-sensitivity, volume-constrained biomarker quantification and validation using backscattering interferometry.

Realizing personalized medicine, which promises to enable early disease detection, efficient diagnostic staging, and therapeutic efficacy monitoring, hinges on biomarker quantification in patient samples. Yet, the lack of a sensitive technology and assay methodology to rapidly validate biomarker candidates continues to be a bottleneck for clinical translation. In our first direct and quantitative comparison of backscattering interferometry (BSI) to fluorescence sensing by ELISA, we show that BSI could aid in overcoming this limitation. The analytical validation study was performed against ELISA for two biomarkers for lung cancer detection: Cyfra 21-1 and Galectin-7. Spiked serum was used for calibration and comparison of analytical figures of merit, followed by analysis of blinded patient samples. Using the ELISA antibody as the probe chemistry in a mix-and-read assay, BSI provided significantly lower detection limits for spiked serum samples with each of the biomarkers. The limit of quantification (LOQ) for Cyrfa-21-1 was measured to be 230 pg/mL for BSI versus 4000 pg/mL for ELISA, and for Galectin-7, it was 13 pg/mL versus 500 pg/mL. The coefficient of variation for 5 day, triplicate determinations was <15% for BSI and <10% for ELISA. The two techniques correlated well, ranging from 3-29% difference for Cyfra 21-1 in a blinded patient sample analysis. The label-free and free-solution operation of BSI allowed for a significant improvement in analysis speed, with greater ease, improved LOQ values, and excellent day-to-day reproducibility. In this unoptimized format, BSI required 5.5-fold less sample quantity needed for ELISA (a 10 point calibration curve measured in triplicate required 36 µL of serum for BSI vs 200 µL for ELISA). The results indicate that the BSI platform can enable rapid, sensitive analytical validation of serum biomarkers and should significantly impact the validation bottleneck of biomarkers.

Characterizing aptamer small molecule interactions with backscattering interferometry.

Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors, therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins, peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because they can be identified in vitro by SELEX, produced rapidly and relatively economically using oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and because of their unique properties they represent an attractive alternative to antibodies. Current assay methodology for characterizing small molecule-aptamer binding is limited by either mass sensitivity, as in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need for using a fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free and free-solution sensing technique, can be used to effectively characterize SM-aptamer interactions, providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A, tenofovir, and epirubicin showing BSI provided values consistent with those published previously. We then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All measurements produced R(2) values >0.95 and an excellent signal to noise ratio at target concentrations that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed, expediting the assay which is also insensitive to the large relative mass difference between the interacting molecules.

Comparison of free-solution and surface-immobilized molecular interactions using a single platform.

While it is generally accepted that surface immobilization affects the binding properties of proteins, it has been difficult to quantify these effects due to the lack of technology capable of making affinity measurements with species tethered and in free solution on a single platform. Further, quantifying the interaction of binding pairs with widely differing masses has also been challenging, particularly when it is desirable to tether the high molecular weight protein. Here we describe the use of backscattering interferometry (BSI) to quantify the binding affinity of mannose and glucose to concanavalin A (ConA), a 106 KDa homotetramer protein, in free solution using picomoles of the protein. Using the same platform, BSI, we then studied the effect on the binding constants of the ConA-carbohydrate interactions upon chemically immobilizing ConA on the sensor surface. By varying the distances (0, 7.17, and 20.35 nm) of the ConA tether and comparing these results to the free-solution measurements, it has been possible to quantify the effect that protein immobilization has on binding. Our results indicate that the apparent binding affinity of the sugar-lectin pair increases as the distance between ConA and the surface decreases. These observations could lend insight as to why the affinity values reported in the literature sometimes vary significantly from one measurement technique to another.

Measurement of aptamer-protein interactions with back-scattering interferometry.

We report the quantitative measurement of aptamer-protein interactions using backscattering interferometry (BSI) and show that BSI can determine when distinct binding regions are accessed. As a model system, we utilized two DNA aptamers (Tasset and Bock) that bind to distinct sites of a target protein (human α-thrombin). This is the first time BSI has been used to study a multivalent system in free solution wherein more than one ligand binds to a single target. We measured aptamer equilibrum dissociation constants (K(d)) of 3.84 nM (Tasset-thrombin) and 5.96 nM (Bock-thrombin), in close agreement with the literature. Unexpectedly, we observed allosteric effects such that the binding of the first aptamer resulted in a significant change in the binding affinity of the second aptamer. For example, the K(d) of Bock aptamer binding to preformed Tasset-thrombin complexes was 7-fold lower (indicating higher affinity) compared to binding to thrombin alone. Preliminary modeling efforts suggest evidence for allosteric linkage between the two exosites.

Correction to Free Solution Assay Signal Modulation in Variable-Stem-Length Hairpin Aptamers.

[This corrects the article DOI: 10.1021/acsomega.9b04341.].

Free Solution Assay Signal Modulation in Variable-Stem-Length Hairpin Aptamers.

Interferometric measurements of free solution assays (FSAs) quantify changes in molecular conformation and hydration upon binding. Here, we demonstrate that aptamer probes designed to undergo varying levels of conformational change upon binding produce corresponding variations in FSA signals. A series of hairpin aptamers were synthesized for the small molecule (tenofovir) with identical loop regions that contain the binding pocket, with between 2 and 10 self-associating base pairings in the stem region. Aptamers selected for tenofovir showed a decrease in the FSA signal and binding affinity (increase in K D) with increasing stem length. Thermodynamic calculations of the Gibbs free energy (ΔG) reported a decrease in ΔG with respect to a corresponding increase in the aptamer stem length. Collectively, these observations provide an expanded understanding of FSA and demonstrate the potential for the rational design of label-free aptamer beacons using FSA as readout.

A Highly Compensated Interferometer for Biochemical Analysis.

Here we report an improved interferometric sensing approach that facilitates high sensitivity nanovolume refractive index (RI) measurements and molecular interaction assays without a temperature controller. The compensated backscattering interferometer (CBSI) is based on a helium-neon (He-Ne) laser, a microfluidic chip, and a CCD array. The CBSI enables simultaneous differential RI measurements within nanoliter volumes, at a compensation level of ca. 5 × 10-8 RIU in the presence of large thermal perturbations (8 °C). This level of d n/d T compensation is enabled by elongating the laser beam along the central axis of the microfluidic channel and measuring the difference in positional shift of interference patterns from two adjacent regions of the channel. By separating two solutions by an air gap or oil droplet, CBSI can discriminate the difference in RI for the sample and reference at a detection limit of 7 × 10-7 RIU in the absence of electronic filtering. At this level of ΔRI sensitivity, it is possible to perform label-free, free-solution biochemical assays at the 10s of nM level without the typical high-resolution temperature control needed in conventional interferometers. Here we illustrate the effective use of CBSI by quantifying the binding affinities for mannose-concanavalin A and Ca2+-recoverin interactions.