Characterizing aptamer small molecule interactions with backscattering interferometry.

Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors, therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins, peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because they can be identified in vitro by SELEX, produced rapidly and relatively economically using oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and because of their unique properties they represent an attractive alternative to antibodies. Current assay methodology for characterizing small molecule-aptamer binding is limited by either mass sensitivity, as in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need for using a fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free and free-solution sensing technique, can be used to effectively characterize SM-aptamer interactions, providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A, tenofovir, and epirubicin showing BSI provided values consistent with those published previously. We then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All measurements produced R(2) values >0.95 and an excellent signal to noise ratio at target concentrations that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed, expediting the assay which is also insensitive to the large relative mass difference between the interacting molecules.

The porphyrin TmPyP4 unfolds the extremely stable G-quadruplex in MT3-MMP mRNA and alleviates its repressive effect to enhance translation in eukaryotic cells.

We report that the cationic porphyrin TmPyP4, which is known mainly as a DNA G-quadruplex stabilizer, unfolds an unusually stable all purine RNA G-quadruplex (M3Q) that is located in the 5'-UTR of MT3-MMP mRNA. When the interaction between TmPyP4 and M3Q was monitored by UV spectroscopy a 22-nm bathochromic shift and 75% hypochromicity of the porphin major Soret band was observed indicating direct binding of the two molecules. TmPyP4 disrupts folded M3Q in a concentration-dependent fashion as was observed by circular dichroism (CD), 1D (1)H NMR and native gel electrophoresis. Additionally, when TmPyP4 is present during the folding process it inhibits the M3Q RNA from adopting a G-quadruplex structure. Using a dual reporter gene construct that contained the M3Q sequence alone or the entire 5'-UTR of MT3-MMP mRNA, we report here that TmPyP4 can relieve the inhibitory effect of the M3Q G-quadruplex. However, the same concentrations of TmPyP4 failed to affect translation of a mutated construct. Thus, TmPyP4 has the ability to unfold an RNA G-quadruplex of extreme stability and modulate activity of a reporter gene presumably via the disruption of the G-quadruplex.

An RNA G-quadruplex is essential for cap-independent translation initiation in human VEGF IRES.

RNA G-quadruplexes located within the 5'-UTR of mRNA are almost always known to be associated with repression of cap-dependent translation. However, in this report we present functional as well as structural evidence that sequence redundancy in a G-rich segment within the 5'-UTR of human VEGF mRNA supports a 'switchable' RNA G-quadruplex structure that is essential for IRES-mediated translation initiation. Additionally, utilization of a specific combination of G-tracts within this segment allows for the conformational switch that implies a tunable regulatory role of the quadruplex structure in translation initiation. A sequence engineered from a functionally handicapped mutant moderately rescued the activity, further indicating the importance of G-quadruplex structure for VEGF IRES-A function. This to our knowledge is the first report of a conformationally flexible RNA G-quadruplex which is essential for IRES-mediated translation initiation.

An unusually stable G-quadruplex within the 5'-UTR of the MT3 matrix metalloproteinase mRNA represses translation in eukaryotic cells.

MT3-MMP is a matrix metalloproteinase involved in the regulation of cancer cell invasion and metastasis. The MT3-MMP mRNA contains a 20-nucleotide G-rich region (M3Q) upstream of the initiation codon. Herein, we report that the M3Q purine-only sequence forms an extremely stable intramolecular G-quadruplex structure and has an inhibitory role on translation of a reporter gene in eukaryotic cells. The formation of the G-quadruplex structure was indicated by circular dichroism (CD) spectroscopy and enzymatic footprinting with RNase T1. The unusual stability of the G-quadruplex was evidenced when addition of only 1 mM KCl resulted in about a 30 degrees C increase in the melting temperature (T(m)), as compared to that obtained in the absence of added salt. The T(m) was independent of the RNA concentration, suggesting an intramolecular G-quadruplex structure. Additionally, in a dual luciferase reporter assay performed in eukaryotic cells, the M3Q motif present in the context of the entire 5'-UTR of MT3-MMP repressed activity of its downstream gene by more than half. To the best of our knowledge, the naturally occurring M3Q sequence forms one of the most stable, intramolecular RNA G-quadruplexes reported. This report is the first to establish a functional role of a G-quadruplex forming sequence within the MT3-MMP 5'-UTR in the regulation of translation in eukaryotic cells.

Engineered domain swapping indicates context dependent functional role of RNA G-quadruplexes.

RNA domain swapping typically demonstrates conservation of the native function of the domain in a non-native context. In contrast, we employed RNA engineering to demonstrate deviation of G-quadruplex (GQ) function that is contingent upon its context dependent location, which is opposite to their native functional role. Known translation repressing RNA GQs were engineered into human VEGF IRES A replacing the endogenous GQ domain essential for translation. Alternatively, the translation inhibitory GQ motif within the 5'-UTR of MT3-MMP mRNA was replaced with two known GQ motifs that are essential for translation. The results indicate that the engineered GQ domains can adopt GQ structures in a foreign environment with a functional role reversal to accommodate the need of the endogenous swapped motifs. The observations establish the functionality and context dependent modularity of RNA GQ structures.

A library screening approach identifies naturally occurring RNA sequences for a G-quadruplex binding ligand.

An RNA G-quadruplex library was synthesised and screened against kanamycin A as the ligand. Naturally occurring G-quadruplex forming sequences that differentially bind to kanamycin A were identified and characterized. This provides a simple and effective strategy for identification of potential intracellular G-quadruplex targets for a ligand.

Analysis of catalytic RNA structure and function by nucleotide analog interference mapping.

Nucleotide analog interference mapping (NAIM) is a quick and efficient method to define concurrently, yet singly, the importance of specific functional groups at particular nucleotide residues to the structure and function of an RNA. NAIM can be utilized on virtually any RNA with an assayable function. The method hinges on the ability to successfully incorporate, within an RNA transcript, various 5'-O-(1-thio)nucleoside analogs randomly via in vitro transcription. This could be achieved by using wild-type or Y639F mutant T7 RNA polymerase, thereby creating a pool of analog doped RNAs. The pool when subjected to a selection step to separate the active transcripts from the inactive ones leads to the identification of functional groups that are crucial for RNA activity. The technique can be used to study ribozyme structure and function via monitoring of cleavage or ligation reactions, define functional groups critical for RNA folding, RNA-RNA interactions, and RNA interactions with proteins, metals, or other small molecules. All major classes of catalytic RNAs have been probed by NAIM. This is a generalized approach that should provide the scientific community with the tools to better understand RNA structure-activity relationships.