Biology subjects

Soh, H. T.

Publications and source records attributed to Soh, H. T..

42 records · Page 3Linked to original sources

bioRxiv2020

Continuous detection of glucose and insulin in live animals

Real-time biosensors that can continuously measure circulating biomolecules in vivo would provide valuable insights into a patients health status and their response to therapeutics even when there is considerable variability in pharmacokinetics and pharmacodynamics across patient populations. Unfortunately, current real-time biosensors are limited to a handful of analytes (e.g. glucose and blood oxygen) and are limited in sensitivity (high nanomolar). In this work, we describe a general approach for continuously and simultaneously measuring multiple analytes with picomolar sensitivity and sub-second temporal resolution. As exemplars, we report the simultaneous detection of glucose and insulin at picomolar concentrations in live diabetic rats. Using our system, we demonstrate the capacity to resolve inter-individual differences in the pharmacokinetic responses to insulin and discriminate profiles from different insulin formulations at a high temporal resolution. Critically, our approach is general and could be readily modified to continuously and simultaneously measure other circulating analytes in vivo by swapping the affinity reagents, thus making it a versatile tool for biomedical research.

bioengineering↗

bioRxiv2019

A Dual-sensing DNA Nanostructure with an Ultra-broad Detection Range

Despite considerable interest in the development of biosensors that can measure analyte concentrations with a dynamic range spanning many orders of magnitude, this goal has proven difficult to achieve. We describe here a modular biosensor architecture that integrates two different readout mechanisms into a single-molecule construct that can achieve target detection across an extraordinarily broad dynamic range. Our dual-mode readout DNA biosensor (DMRD) combines an aptamer and a DNAzyme to quantify ATP with two different mechanisms, which respond to low (micromolar) and high (millimolar) concentrations by generating distinct readouts based on changes in fluorescence and absorbance, respectively. Importantly, we have also devised regulatory strategies to finely tune the target detection range of each sensor module by controlling the target-sensitivity of each readout mechanism. Using this strategy, we report the detection of ATP at a dynamic range spanning 1-500,000 M--more than five orders of magnitude, representing the largest dynamic range reported to date with a single biosensor construct.

bioengineering↗

bioRxiv2019

Independent Control of the Thermodynamic and Kinetic Properties of Aptamer Switches

Molecular switches that change their conformation upon target binding offer powerful capabilities for biotechnology and synthetic biology. In particular, aptamers have proven useful as molecular switches because they offer excellent binding properties, undergo reversible folding, and can be readily engineered into a wide range of nanostructures. Unfortunately, the thermodynamic and kinetic properties of the aptamer switches developed to date are intrinsically coupled, such that high temporal resolution (i.e., switching time) can only be achieved at the cost of lower sensitivity or high background. Here, we describe a general design strategy that decouples the thermodynamic and kinetic behavior of aptamer switches to achieve independent control of sensitivity and temporal resolution. We used this strategy to generate an array of aptamer switches with effective dissociation constants (KD) ranging from 10 M to 40 mM and binding kinetics ranging from 170 ms to 3 s--all generated from the same parent ATP aptamer. Our strategy is broadly applicable to other aptamers, enabling the efficient development of switches with characteristics suitable for diverse range of biotechnology applications.

bioengineering↗

bioRxiv2019

Click-PD: A Quantitative Method for Base-Modified Aptamer Discovery

Base-modified aptamers that incorporate non-natural chemical moieties can achieve greatly improved affinity and specificity relative to natural DNA or RNA aptamers. However, conventional methods for generating base-modified aptamers require considerable expertise and resources. In this work, we have accelerated and generalized the process of generating base-modified aptamers by combining a click-chemistry strategy with a fluorescence-activated cell sorting (FACS)-based screening methodology that measures the affinity and specificity of individual aptamers at a throughput of [~]10^7 per hour. Our \"click-PD\" strategy offers many advantages. First, almost any chemical modification can be introduced with a commercially-available polymerase. Second, click-PD can screen vast numbers of individual aptamers based on quantitative on- and off-target binding measurements to simultaneously achieve high affinity and specificity. Finally, it requires minimal specialized equipment or reagents besides a FACS instrument which is now widely-available. Using click-PD, we generated a boronic acid-modified aptamer with [~]1 M affinity for epinephrine, a target for which no aptamer has been reported to date. We subsequently generated a mannose-modified aptamer with nanomolar affinity for the lectin concanavalin A (ConA). The strong affinity of both aptamers is fundamentally dependent upon the presence of chemical modifications, and we show that their removal essentially eliminates aptamer binding. Importantly, our ConA aptamer exhibited exceptional specificity, with minimal binding to other structurally-similar lectins. Finally, we show that our aptamer remarkable biological activity. Indeed, to the best of our knowledge, this aptamer is the most potent inhibitor of Con A-mediated hemagglutination reported to date.

bioengineering↗

bioRxiv2019

High-Fidelity Nanopore Sequencing of Ultra-Short DNA Sequences

Nanopore sequencing offers a portable and affordable alternative to sequencing-by-synthesis methods but suffers from lower accuracy and cannot sequence ultra-short DNA. This puts applications such as molecular diagnostics based on the analysis of cell-free DNA or single-nucleotide variants (SNV) out of reach. To overcome these limitations, we report a nanopore-based sequencing strategy in which short target sequences are first circularized and then amplified via rolling-circle amplification to produce long stretches of concatemeric repeats. These can be sequenced on the Oxford Nanopore Technologys (ONT) MinION platform, and the resulting repeat sequences aligned to produce a highly-accurate consensus that reduces the high error-rate present in the individual repeats. Using this approach, we demonstrate for the first time the ability to obtain unbiased and accurate nanopore data for target DNA sequences of < 100 bp. Critically, this approach is sensitive enough to achieve SNV discrimination in mixtures of sequences and even enables quantitative detection of specific variants present at ratios of < 10%. Our method is simple, cost-effective, and only requires well-established processes. It therefore expands the utility of nanopore sequencing for molecular diagnostics and other applications, especially in resource-limited settings. One Sentence SummaryWe introduce a simple method of accurately sequencing ultra-short (<100bp) target DNA on a nanopore sequencing platform.

bioengineering↗

bioRxiv2018

A direct selection strategy for isolating aptamers with pH-sensitive binding activity

An aptamer reagent that can switch its binding affinity in a pH-responsive manner would be highly valuable for many biomedical applications including imaging and drug delivery. Unfortunately, the discovery of such aptamers is difficult and only a few have been reported to date. Here we report the first experimental strategy for generating pH-responsive aptamers through direct selection. As an exemplar, we report streptavidin-binding aptamers that retain nanomolar affinity at pH 7.4 but exhibit a ~100-fold decrease in affinity at pH 5.2. These aptamers were generated by incorporating a known streptavidin-binding DNA motif into an aptamer library and performing FACS-based screening at multiple pH conditions. Upon structural analysis, we found that one aptamers affinity-switching behavior is driven by a non-canonical G-A base-pair that controls its folding in a highly pH-dependent manner. We believe our strategy could be readily extended to other aptamer-target systems because it does not require a priori structural knowledge of the aptamer or the target.

synthetic biology↗