Small RNA Biosensor Design Strategy To Mitigate Off-Analyte Response.

Several bottlenecks in the design of current sensor technologies for small noncoding RNA must be addressed. The small size of the sensors and the large number of other nucleotides that may have sequence similarity makes selectivity a real concern. Many of the current sensors have one strand with an exposed region called a toehold. The toehold serves as a place for the analyte nucleic acid strand to bind and initiate competitive displacement of sensors' secondary strands. Since the toehold region is not protected, any endogenous oligonucleotide sequences that are similar or only different by a few nucleic acids will interact with the toehold and cause false signals. To address sensor selectivity, we investigated how the toehold location in the sensor impacts the sensitivity and selectivity for the analyte of interest. We will discuss the differences in sensitivity and selectivity for a miR-146a-5p biosensor in the presence of different naturally occurring mismatch sequences. We found that altering the toehold location lowered the rate of the false signal from off-analyte microRNA by upward of 20 percentage points. Detection limits as low as 56 pM were observed when the sensor concentration was 5 nM. The findings herein are broadly applicable to other small and large RNAs as well as other types of sensing platforms.

Molecular Approaches To Address the Challenges of RNA Analysis in Complex Matrices.

We present on a design change and addition of an internal polyethylene glycol (PEG) spacer to an existing biosensor. There were two reasons for changing the sensor design. The first was to increase the stability of the biosensor to avoid binding off-analytes with single nucleotide polymorphisms. The second was to prevent sensor degradation by nucleases. The biosensor, designed for detection of short noncoding RNA strands, is composed of Reporter and Probe nucleic acid strands that form a partially complementary duplex. The internal PEG was added to the Reporter, and subsequently diminished false negatives that resulted from off-oligonucleotide binding. Furthermore, the PEG eliminated degradation of the sensor by DNase1 endonuclease. Currently, in situ and crude cell lysate RNA analysis is hindered by nonspecific interactions and degradation by endogenous nucleases. Together, the design changes presented here mitigate these matrix effects and allow for robust RNA analysis in complex media.

Performance of nano-assembly logic gates with a DNA multi-hairpin motif.

DNA nano-assemblies have far-reaching implications for molecular computers. Boolean logic gates made from DNA respond to specific combinations of chemical or molecular inputs. In complex samples an assortment of other chemicals and molecules may interfere with the gate's recognition and response mechanisms. For logic gates to accept an increasing number of inputs, while maintaining selectivity, their design must only respond when specific input combinations are available simultaneously. Here we present proof-of-principle for a fluorescent-based nano-assembly logic gate for three inputs. Central to the gate's design is a multi-hairpin motif that distinguishes it from other works in this area. The multi-hairpin motif facilitates a larger and increasing number of inputs and a place to generate FRET-based signal enhancement. We will show the nano-assembly logic gate worked in aqueous buffer and in crude MCF-7 cell lysate. We will demonstrate the gate's selectivity against off-analyte cocktails. Finally, multi-hairpin motifs with different chemical and physical properties were evaluated to test their logic capabilities. Future work will demonstrate the gate's ability to visually identify specific combinations of oligonucleotides called small non-coding RNAs (ncRNAs) in cells. This nano-assembly logic gate for small ncRNA has far reaching cellular computation and single-cell analysis applicability. The gate can be used for basic cellular analysis, computing and observing the unique molecular expression patterns in tumor microenvironments, and advancing the field of therapeutics.

Förster resonance energy transfer to impart signal-on and -off capabilities in a single microRNA biosensor.

Many studies have found that over- or under-expression of biomolecules called microRNAs (miRNA) regulates several diseases. Biosensors are in need to visually identify the relative expression level of miRNA to determine the direction these miRNA change in cells and tissues. Our established reporter+probe miRNA biosensor design requires that miRNA outcompete and displace the reporter from the probe. Once displaced, the reporter folds into a hairpin structure to force together a pair of Förster Resonance Energy Transfer (FRET) dyes. The donor and acceptor signal changes can be used to indicate the over-/under-expression of miRNA. The bright signal from the donor will indicate miRNA under-expression; the bright acceptor signal will indicate miRNA over-expression. Since close proximity of the dyes to each other and nucleic acids often quench fluorescence, polyethylene glycol spacers were added in-between the dyes and nucleic acids. We compared reporter designs with and without spacers to investigate the effects on the following analytical metrics: (1) extent of signal change, (2) limits of detection and quantitation, and (3) sensitivity. Systematic errors and amount of reporter+probe biosensor formed were evaluated for one of the biosensors. Cy3|Cy5 and 6-carboxyfluorescein (6-FAM)|ATTO 633 dye pairs on reporters containing spacers showed an increase in the acceptor signal change by ∼190 and ∼484%, respectively, compared to no spacers. Transduction mechanisms that enhance and quench the signal both showed LODs that ranged from 3-17 nanomolar (nM) with 100 nM of the biosensor.

Molecular structure and thermodynamic predictions to create highly sensitive microRNA biosensors.

Many studies have established microRNAs (miRNAs) as post-transcriptional regulators in a variety of intracellular molecular processes. Abnormal changes in miRNA have been associated with several diseases. However, these changes are sometimes subtle and occur at nanomolar levels or lower. Several biosensing hurdles for in situ cellular/tissue analysis of miRNA limit detection of small amounts of miRNA. Of these limitations the most challenging are selectivity and sensor degradation creating high background signals and false signals. Recently we developed a reporter+probe biosensor for let-7a that showed potential to mitigate false signal from sensor degradation. Here we designed reporter+probe biosensors for miR-26a-2-3p and miR-27a-5p to better understand the effect of thermodynamics and molecular structures of the biosensor constituents on the analytical performance. Signal changes from interactions between Cy3 and Cy5 on the reporters were used to understand structural aspects of the reporter designs. Theoretical thermodynamic values, single stranded conformations, hetero- and homodimerization structures, and equilibrium concentrations of the reporters and probes were used to interpret the experimental observations. Studies of the sensitivity and selectivity revealed 5-9 nM detection limits in the presence and absence of interfering off-analyte miRNAs. These studies will aid in determining how to rationally design reporter+probe biosensors to overcome hurdles associated with highly sensitive miRNA biosensing.

Dye-Specific Wavelength Offsets to Resolve Spectrally Overlapping and Co-Localized Two-Photon Induced Fluorescence.

The fundamentally important fluorescent imaging has one major limitation, resolution of over three dyes. This limitation is in part due to the overlap of the broad emission profile of each of the emitters used in fluorescence detection. The overlapping emission contaminates each emitter's detection channel, referred to as cross-talk. To reduce fluorescence cross-talk for two photon applications, we present an innovative Two-Photon-Dye-Specific Excitation-Emission Offset (TP-DSO) method. TP-DSO selectively detects each dye by synchronously scanning the excitation and emission wavelengths at defined wavelength offsets. This technique advances multicolor analysis significantly by resolving dyes with highly overlapping spectral profiles. We identified three benefits: reduced excitation spectral bandwidth, reduced emission cross-talk between colocalized emitters with closely overlapping fluorescence, and validated use of thin-film variable optical emission filters for tuning the bandwidth and center wavelength. TP-DSO will advance multicolor analysis for many applications.

Detection of miRNA using a double-strand displacement biosensor with a self-complementary fluorescent reporter.

Design of rapid, selective, and sensitive DNA and ribonucleic acid (RNA) biosensors capable of minimizing false positives from nuclease degradation is crucial for translational research and clinical diagnostics. We present proof-of-principle studies of an innovative micro-ribonucleic acid (miRNA) reporter-probe biosensor that displaces a self-complementary reporter, while target miRNA binds to the probe. The freed reporter folds into a hairpin structure to induce a decrease in the fluorescent signal. The self-complementarity of the reporter facilitates the reduction of false positives from nuclease degradation. Nanomolar limits of detection and quantitation were capable with this proof-of-principle design. Detection of miRNA occurs within 10 min and does not require any additional hybridization, labeling, or rinsing steps. The potential for medical applications of the reporter-probe biosensor is demonstrated by selective detection of a cancer regulating microRNA, Lethal-7 (Let-7a). Mechanisms for transporting the biosensor across the cell membrane will be the focus of future work.

Mapping vortex-like hydrodynamic flow in microfluidic networks using fluorescence correlation spectroscopy.

The ability to quickly measure flow parameters in microfluidic devices is critical for micro total analysis system (microTAS) applications. Macrofluidic methods to assess flow suffer from limitations that have made conventional methods unsuitable for the flow behavior profiling. Single molecule fluorescence correlation spectroscopy (FCS) has been employed in our study to characterize the fluidic vortex generating at a T-shape junction of microscale channels. Due to its high spatial and temporal resolution, the corresponding magnitudes relative to different flow rates in the main channel can be quantitatively differentiated using flow time (tau(F)) measurements of dye molecules traversing the detection volume in buffer solution. Despite the parabolic flow in the channel upstream, a heterogeneous distribution of flow has been detected across the channel intersection. In addition, our current observations also confirmed the aspect of vortex-shaped flow in low-shear design that was developed previously for cell culture. This approach not only overcomes many technical barriers for examining hydrodynamic vortices and movements in miniature structures without physically integrating any probes, but it is also especially useful for the hydrodynamic studies in polymer-glass based micro-reactor and -mixer.

Comparison of methods to classify and quantify free and bound states of complexes using single molecule fluorescence anisotropy.

Steady-state single molecule fluorescence anisotropy (SMFA) is described to quantify free and bound probe molecules from a Biotin-Neutravidin complexation reaction. By formulating a ratio of bound to the total number of molecules sampled (N(b)/N(t) ratio) we quantified the extent of binding. We report on a comparison of three methods to extract fluorescent bursts from single molecules from a ten-minute time trace. The impact on the N(b)/N(t) ratio using either anisotropy values alone or anisotropy values combined with the difference in detector counts (Deltan) were investigated. The data analysis methods reduced the random error due to scatter. Biotin-Rhodamine 110 (BR110) was used as the labeled probe for these studies. Neutravidin was used as the target protein. A competitive reaction between labeled BR110 probe and unlabeled Biotin was also investigated. The use of steady-state SMFA as a tool to probe molecular complexation will be useful in performing sensitive immunoassays, in drug discovery to investigate and enhance the binding of drugs to their substrates, and to study other molecular interactions.

Light tolerance of R-phycoerythrin and a tandem conjugate observed by single molecule recrossing events.

The recrossing of single molecules in a probe volume was used to investigate light harvesting and energy transfer between R-phycoerythrin (R-PE) and a tandem conjugate dye. The normalized recrossing ratio, Nr/Nt, was defined as the number of molecules that reenter the probe volume (Nr) to the total number of molecules detected (Nt). The energy transfer process in phycobiliproteins was studied as a function of excitation irradiation and irradiation time. This was achieved by investigating the average baseline-subtracted fluorescence intensity, normalized molecular recrossing ratio (Nr/Nt), and the number of molecules detected per second. The photon saturation irradiance of the R-PE and the tandem conjugate were compared with each other, showing that energy transfer to the tandem dye significantly improves photostability and light tolerance of the phycobiliprotein. The Nr/Nt ratio was used to study the photophysical properties of R-phycoerythrin and the tandem conjugate Streptavidin R-Phycoerythrin-AlexaFluor-647 (PE-647). Normalized molecular recrossings showed that energy transfer to a tandem conjugate could reduce the formation of triplet states in R-phycoerythrin and extend the light tolerance of certain phycobiliproteins.

Measuring complexation by single-molecule fluorescence anisotropy.

The complexation of a fluorescent probe by a target protein was observed by single-molecule fluorescence anisotropy. Free and bound states, heterogeneities, and rare binding events can all be observed by this approach. Fluorophore-conjugated biotin was used to bind to NeutrAvidin as a proof-of-concept case. Molecular interactions were observed that could not be elucidated with conventional (ensemble) measurements.

Investigation of photobleaching and saturation of single molecules by fluorophore recrossing events.

A method for investigation of photobleaching and saturation of single molecules by fluorophore recrossing events in a laser beam is described. The diffraction-limited probe volumes encountered in single-molecule detection (SMD) produce high excitation irradiance, which can decrease available signal. The single molecules of several dyes were detected and the data was used to extract interpeak times above a defined threshold value. The interpeak times revealed the number of fluorophore recrossing events. The number of molecules detected that were within 2 ms of each other represented a molecular recrossing for this work. Calcein, fluorescein and R-phycoerythrin were analyzed and the saturation irradiance and photobleaching effects were determined as a function of irradiance. This approach is simple and it serves as a method of optimizing experimental conditions for single-molecule detection.