In vitro selection of structure-switching, self-reporting aptamers

Abstract

We describe an innovative selection approach to generate selfreporting 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.G-quadruplex | systematic evolution of ligands by exponential enrichment | sensor | binding induced folding | molecular recognition N ucleic acid-based aptamers (1, 2) can be selected in vitro against a wide range of targets (3-8), chemically modified and synthetically produced, thereby making them a promising class of molecules for many applications including diagnostics (9, 10), in vivo imaging (11,12), and targeted therapeutics (13,14). Unlike most conventional affinity reagents (e.g., antibodies), aptamers can also be engineered to perform complex molecular functions beyond binding. For example, our group has recently demonstrated the use of an aptamer that undergoes target-binding-induced conformational change for continuous, real-time detection of a small molecule in undiluted blood serum (15). However, the selection process required to directly isolate aptamers with the desired function poses a significant technical challenge. One common approach entails selection for an aptamer on the basis of target binding, followed by postmodification and optimization through rational design in order to link the molecular binding capability to the desired functions (16,17). Unfortunately, such approaches rely upon prior knowledge of the threedimensional aptamer structure and require lengthy optimization steps that often compromise aptamers' affinity and specificity for their targets (18)(19)(20).Alternately, one can directly isolate aptamers with the desired function by combining a specifically designed nucleic acid library and a suitable selection process. For example, pioneering work by Nutiu and Li (21) described the isolation of structure-switching aptamers using a DNA library that was designed to incorporate two short randomized stretches sandwiching a central fixedsequence motif, with binding sites for two PCR...

DNA-based advanced logic circuits for nonarithmetic information processing

“…As a 1:2 DEMUX requires two output signals, NMM was selected as another signal reporter, presenting enhanced fluorescence responses on formation of a G-4/NMM complex (G-4: G-quadruplex). 32 Figure S5) are available in the SI. After normalizing the fluorescence intensity of FAM and NMM (Figure 2f), the correspondingly obtained truth table (Figure 2g) indicates that the single input line is reported to the related output, depending on the binary state of the address input (A).…”

Research Progress of the Functional Nucleic Acid Techniques in GMO Detection

“…Functional nucleic acids are a kind of nucleic acid can realized the self-reporting [44][45][46] . We believe that more and more functional nucleic acids which novel structure and principle will be established, and will have broad applicability for rapid, easy, quantitative, and ultrasensitive, analysis of any DNA.…”

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…In this case, ferromagnetic grids are used to capture and retain magnetic beads as they flow into the device, allowing a wider range of flow rates, as well as long and extensive washes to be performed in the channel. 114,115 In a technique utilizing dilutions 116 called Volume Dilution Challenge (VDC), VDC-MSELEX uses the MMS chip to capture and re-concentrate magnetic beads following dilutions of up to one thousandth of the equilibrated solution, which helps to irreversibly dissociate aptamers with poorer off-rates. 117 M-SELEX has also been coupled to highthroughput sequencing (HiTS) in a method called Quantitative Selection of Aptamers through Sequencing (QSAS), to analyze the copy numbers and enrichments of millions of aptamer candidates in multiple pools.…”