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.
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.
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.
We describe a single-step, single-component, fluorescence-based method of detecting single nucleotide polymorphisms at room temperature without exogenous reagents. The sensor is comprised of a ...single, self-complementary DNA strand forming a triple-stem structure, which undergoes a large conformational change only upon binding to perfectly-matched targets, resulting in a significant increase in fluorescence.
Eine Abweichung genügt: Bei einer einstufigen, nur eine Komponente nutzenden, fluoreszenzbasierten Methode zum Nachweis von Einzelnucleotidpolymorphismen bei Raumtemperatur besteht der Sensor aus ...einem einzigen, selbstkomplementären DNA‐Strang, der eine Dreifachstammstruktur bildet. Die große Konformationsänderung beim Binden an perfekt komplementäre (PM‐)Ziele führt zu einer deutlichen Zunahme der Fluoreszenz (siehe Bild; F=Fluorophor, Q=Quencher).
Eine Abweichung genügt: Bei einer einstufigen, nur eine Komponente nutzenden, fluoreszenzbasierten Methode zum Nachweis von Einzelnucleotidpolymorphismen bei Raumtemperatur besteht der Sensor aus einem einzigen, selbstkomplementären DNA‐Strang, der eine Dreifachstammstruktur bildet. Die große Konformationsänderung beim Binden an perfekt komplementäre (PM‐)Ziele führt zu einer deutlichen Zunahme der Fluoreszenz (siehe Bild; F=Fluorophor, Q=Quencher).