Aptamers are single-stranded nucleic acids with defined tertiary structures for selective binding to target molecules. Aptamers are also able to bind a complementary DNA sequence to form a duplex structure. In this report, we describe a strategy for designing aptamer-based fluorescent reporters that function by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and a small oligonucleotide modified with a quenching moiety (denoted QDNA). When the target is absent, the aptamer binds to QDNA, bringing the fluorophore and the quencher into close proximity for maximum fluorescence quenching. When the target is introduced, the aptamer prefers to form the aptamer-target complex. The switch of the binding partners for the aptamer occurs in conjunction with the generation of a strong fluorescence signal owing to the dissociation of QDNA. Herein, we report on the preparation of several structure-switching reporters from two existing DNA aptamers. Our design strategy is easy to generalize for any aptamer without prior knowledge of its secondary or tertiary structure, and should be suited for the development of aptamer-based reporters for real-time sensing applications.

SUMMARY Messenger RNAs (mRNAs) can fold into complex structures that regulate gene expression. Resolving such structures de novo has remained challenging and has limited understanding of the prevalence and functions of mRNA structure. We use SHAPE-MaP experiments in living E. coli cells to derive quantitative, nucleotide-resolution structure models for 194 endogenous transcripts encompassing approximately 400 genes. Individual mRNAs have exceptionally diverse architectures, and most contain well-defined structures. Active translation destabilizes mRNA structure in cells. Nevertheless, mRNA structure remains similar between in-cell and cell-free environments, indicating broad potential for structure-mediated gene regulation. We find that translation efficiency of endogenous genes is regulated by unfolding kinetics of structures overlapping the ribosome binding site. We discover conserved structured elements in 35% of untranslated regions, several of which we validate as novel protein binding motifs. RNA structure regulates every gene studied here in a meaningful way, implying that most functional structures remain to be discovered.

Picking out probes: A novel approach permits isolation of standard DNA aptamers by in vitro selection and conversion into fluorescence signaling probes. This method comprises the isolation of DNA aptamers capable of duplex‐to‐complex structure switching and labeling the derived aptamers with a pair of short DNA strands with a fluorophore F and a quencher Q to create a reporter system for real‐time sensing (see picture).

Sequence-specific DNA binding by transcription factors is central to gene expression regulation. While a number of methods for characterizing DNA-protein interactions are currently available1-6, none have demonstrated both quantitative measurement of affinity and high throughput. To address this challenge, we developed HiTS-FLIP, a technique that couples high-throughput sequencing with direct visualization of in vitro binding to provide quantitative protein-DNA binding affinity measurements at unprecedented depth. HiTS-FLIP analysis of GCN4, the master regulator of the yeast amino acid starvation response7, yielded 440 million binding measurements, enabling determination of context-averaged dissociation constants for all 12mer sequences having submicromolar affinity. These data revealed complex interdependency between motif positions, yielded improved discrimination of in vivo GCN4 binding sites and regulatory targets relative to previous models, and identified sets of genes with distinct GCN4 affinity levels which had distinct functions and expression kinetics. This approach promises to deepen understanding of the interactions that drive transcription.

The development of aptamer technology considerably broadens the utility of nucleic acids as molecular recognition elements, because it allows the creation of DNA or RNA molecules for binding a wide variety of analytes (targets) with high affinity and specificity. Several recent studies have focused on developing rational design strategies for transducing aptamer-target recognition events into easily detectable signals, so that aptamers can be widely exploited for detection directed applications. We have devised a generalizable strategy for designing nonfluorescent aptamers that can be turned into fluorescence-signaling reporters. The resultant signaling probes are denoted "structure-switching signaling aptamers" as they report target binding by switching structures from DNA/DNA duplex to DNA/target complex. The duplex is formed between a fluorophore-labeled DNA aptamer and an antisense DNA oligonucleotide modified with a quencher (denoted QDNA). In the absence of the target, the aptamer hybridizes with QDNA, bringing the fluorophore into close proximity of the quencher for efficient fluorescence quenching. When this system is exposed to the target, the aptamer switches its binding partner from QDNA to the target. This structure-switching event is coupled to the generation of a fluorescent signal through the departure of QDNA, permitting the real-time monitoring of the aptamer-target recognition. In this article, we discuss the conceptual framework of the structure-switching approach, the essential features of structure-switching signaling aptamers as well as remaining challenges and possible solutions associated with this new methodology.

Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of the G-run class of SREs is ∼4-fold higher when adjacent to intermediate strength 5'ss relative to weak 5'ss, and ∼1.3-fold higher relative to strong 5'ss. This dependence on 5'ss strength was observed in splicing reporters and in global microarray and mRNA-Seq analyses of splicing changes following RNAi against heterogeneous nuclear ribonucleoprotein (hnRNP) H, which crosslinked to G-runs adjacent to many regulated exons. An exon’s responsiveness to changes in hnRNP H levels therefore depends in a complex way on G-run abundance and 5'ss strength, and other splicing factors may function similarly. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting the frequency of 5'ss polymorphism and the evolution of new splicing patterns.

We propose a new method that allows the use of low-affinity aptamers as affinity probes in quantitative analyses of proteins. The method is based on nonequilibrium capillary electrophoresis of the equilibrium mixture (NECEEM) of a protein with its fluorescently labeled aptamer. In general, NECEEM of a protein with a fluorescently labeled aptamer generates an electropherogram with three characteristic features: two peaks and an exponential curve. Two peaks correspond to (i) the equilibrium amount of free aptamer in the equilibrium mixture and (ii) the amount of the protein-aptamer complex that remains intact at the time of detection. The exponential part is ascribed to the complex decaying during separation under nonequilibrium conditions. Simple analysis of the three features in experiments with known concentrations of the protein can be used for the determination of the equilibrium dissociation constant, Kd, of the aptamer-protein complex. Similar analysis of the three features in the experiment with unknown concentration of the protein and known Kd value allows the determination of the protein concentration. In this proof-of-principle work, the NECEEM method was applied to the analysis of thrombin using a fluorescein-labeled aptamer under the conditions at which the protein-aptamer complex completely decayed during the separation. We demonstrated that, despite the decay, as few as 4 x 10(6) molecules of the protein could be detected with NECEEM without sacrificing the accuracy. This sensitivity is comparable with that reported by others for the aptamer-based equilibrium method. Thus, the proposed NECEEM-based method allows the use of aptamers for highly sensitive affinity analysis of proteins even when protein-aptamer complexes are unstable.

No abstract