We report here an electrochemical approach that offers, for the first time, single-step, room-temperature single nucleotide polymorphism (SNP) detection directly in complex samples (such as blood ...serum) without the need for target modification, postwashing, or the addition of exogenous reagents. This sensor, which is sensitive, stable, and reusable, is comprised of a single, self-complementary, methylene blue-labeled DNA probe possessing a triple-stem structure. This probe takes advantage of the large thermodynamic changes in enthalpy and entropy that result from major conformational rearrangements that occur upon binding a perfectly matched target, resulting in a large-scale change in the faradaic current. As a result, the discrimination capabilities of this sensor greatly exceed those of earlier single- and double-stem electrochemical sensors and support rapid (minutes), single-step, reagentless, room-temperature detection of single nucleotide substitutions. To elucidate the theoretical basis of the sensor’s selectivity, we present a comparative thermodynamic analysis among single-, double-, and triple-stem probes.
Identifying the interactions of small molecules with biomolecules in complex cellular environments is a significant challenge. As one important example, despite being widely used for decades, much is ...still not understood regarding the cellular targets of Pt(II)-based anticancer drugs. In this study we introduce a novel method for isolation of Pt(II)-bound biomolecules using a DNA hybridization pull-down approach. Using a modified Pt reagent, click-ligation of a DNA oligonucleotide to both a Pt(II)-bound DNA hairpin and bovine serum albumin (BSA) are demonstrated. Subsequent hybridization to a biotin-labeled oligonucleotide allows for efficient isolation of Pt(II)-bound species by streptavidin pulldown. We also find that platinated bovine serum albumin readily crosslinks to DNA in the absence of click ligation, and that a fraction of BSA-bound Pt(II) can transfer to DNA over time. Interestingly, in in vitro studies, fragmented mammalian DNA that is crosslinked to BSA through Pt(II) exhibits significantly increased protection from degradation by serum nucleases.
This work presents a novel click-based method to enrich Pt(II)-bound biomolecules. Pt(II)-bound DNA and proteins are readily isolated using an oligonucleotide hybridization strategy. Interestingly, Pt(II)-dependent and click-independent crosslinking of a DNA oligonucleotide to serum albumin was observed, and DNA crosslinked to protein is dramatically protected from serum nucleases. Display omitted
•New method for isolating Pt-bound biomolecules by DNA click-ligation and hybridization•Successful click-ligation of DNA to Pt-bound biomolecules•Surprising DNA-protein crosslinking with non-click Pt(II) reagents•Some Pt(II) transfer from protein to DNA•Significant increase in serum half-life for DNA crosslinked to proteins
Singled out for its singularity: In a single-step, single-component, fluorescence-based method for the detection of single-nucleotide polymorphisms at room temperature, the sensor is comprised of a ...single, self-complementary DNA strand that forms a triple-stem structure. The large conformational change that occurs upon binding to perfectly matched (PM) targets results in a significant increase in fluorescence (see picture; F=fluorophore, Q=quencher).
The catalysis of nucleic acid, as well as nucleobases, metals and other cofactors, is examined, focusing on metal-ion-assisted catalysis in ribozymes. Characteristics of RNA are also addressed.
We describe an innovative selection approach to generate self-reporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional ...modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye, N-methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.
Methods to map small-molecule binding sites on cellular RNAs are important for understanding interactions with both endogenous and exogenous compounds. Pt(
ii
) reagents are well-known DNA and RNA ...crosslinking agents, but sequence-specific and genome-wide identification of Pt targets following in-cell treatment is challenging. Here we describe application of high-throughput 'Pt-Seq' to identify Pt-rRNA adducts following treatment of
S. cerevisiae
with cisplatin.
Methods to map small-molecule binding sites on cellular RNAs are important for understanding interactions with both endogenous and exogenous compounds. Here, 'Pt-Seq' is presented as a high-throughput method to identify Pt adducts on RNA resulting from cisplatin treatment.
Methods to map small-molecule binding sites on cellular RNAs are important for understanding interactions with both endogenous and exogenous compounds. Pt(ii) reagents are well-known DNA and RNA ...crosslinking agents, but sequence-specific and genome-wide identification of Pt targets following in-cell treatment is challenging. Here we describe application of high-throughput 'Pt-Seq' to identify Pt-rRNA adducts following treatment of S. cerevisiae with cisplatin.
Many biological systems employ allosteric regulatory mechanisms, which offer a powerful means of directly linking a specific binding event to a wide spectrum of molecular functionalities. There is ...considerable interest in generating synthetic allosteric regulators that can perform useful molecular functions for applications in diagnostics, imaging and targeted therapies, but generating such molecules through either rational design or directed evolution has proven exceptionally challenging. To address this need, we present an in vitro selection strategy for generating conformation-switching DNA nanostructures that selectively release a small-molecule payload in response to binding of a specific trigger molecule. As an exemplar, we have generated a DNA nanostructure that hybridizes with a separate ‘cargo strand’ containing an abasic site. This abasic site stably sequesters a fluorescent cargo molecule in an inactive state until the DNA nanostructure encounters an ATP trigger molecule. This ATP trigger causes the nanostructure to release the cargo strand, thereby liberating the fluorescent payload and generating a detectable fluorescent readout. Our DNA nanostructure is highly sensitive, with an EC50 of 30 μM, and highly specific, releasing its payload in response to ATP but not to other chemically similar nucleotide triphosphates. We believe that this selection approach could be generalized to generate synthetic nanostructures capable of selective and controlled release of other small-molecule cargos in response to a variety of triggers, for both research and clinical applications.
Methods to map small-molecule interactions with cellular RNAs are important for understanding endogenous activation, such as in riboswitches, as well as the potential for exogenous compounds to ...target RNA. Cisplatin is one of the most widely used of the platinum anticancer drugs that are prescribed in approximately 40-50% of all chemotherapy treatments (Dyson and Sava, 2006; Harper et al., 2010). Despite nearly 40 years of experience with this class of drugs, we still lack a comprehensive understanding of the targets of Pt compounds and their effects on cells. Pt(II) compounds are well-known DNA and RNA crosslinking agents, but the latter area is under-studied. In order to better understand the impacts of cisplatin and other platinum(II)-derived small molecules on cellular RNA, we have developed a technique we call “Platinum-seq,” which couples reverse transcription mapping of platinated RNAs to high-throughput sequencing. Chapter 1 is a study of cisplatin and a novel click-functionalized platinum compound (2-ADAP Pt) binding to the HDV ribozyme, a small catalytic RNA. Chapter 2 moves our platinum mapping approaches from low-throughput, sequencing gel based methods into next-generation sequencing for high-throughput analysis of all platinum sites in cellular RNA, a method we have named “Platinum-seq.” Chapter 3 is a study of differential gene expression of Saccharomyces cerevisiae treated with cisplatin and a second novel platinum(II) compound (azaplatin), using data acquired from the work in Chapter 2. Chapter 4 describes recent efforts to implement pre-enrichment of sequencing targets using click chemistry followed by DNA hybridization, in order to enrich for platinated fragments before sequencing library construction. Together, this work represents a significant step forward in advancing analysis of Pt(II) binding to cellular RNA, a potentially important target for this widely used class of anticancer compounds. Methods developed here are broadly applicable to genome-wide identification of platinum accumulation on DNA as well, which has not been pursued despite the extensive use of these compounds.
PDF
