Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species.

RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC-Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30-tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

Determination of In Vitro and Cellular Turn-on Kinetics for Fluorogenic RNA Aptamers.

Fluorogenic RNA aptamers have been applied in live cells to tag and visualize RNAs, report on gene expression, and activate fluorescent biosensors that detect levels of metabolites and signaling molecules. In order to study dynamic changes in each of these systems, it is desirable to obtain real-time measurements, but the accuracy of the measurements depends on the kinetics of the fluorogenic reaction being faster than the sampling frequency. Here, we describe methods to determine the in vitro and cellular turn-on kinetics for fluorogenic RNA aptamers using a plate reader equipped with a sample injector and a flow cytometer, respectively. We show that the in vitro kinetics for the fluorescence activation of the Spinach2 and Broccoli aptamers can be modeled as two-phase association reactions and have differing fast phase rate constants of 0.56 s-1 and 0.35 s-1, respectively. In addition, we show that the cellular kinetics for the fluorescence activation of Spinach2 in Escherichia coli, which is further limited by dye diffusion into the Gram-negative bacteria, is still sufficiently rapid to enable accurate sampling frequency on the minute timescale. These methods to analyze fluorescence activation kinetics are applicable to other fluorogenic RNA aptamers that have been developed.

TLR8 activation and inhibition by guanosine analogs in RNA: Importance of functional groups and chain length.

Toll-like receptor 8 (TLR8) is an important component of the human innate immune system that recognizes single stranded RNA (ssRNA). Recent X-ray crystal structures of TLR8 bound to ssRNA revealed a previously unrecognized binding site for a 5'-UpG-3' dinucleotide. Here we use an atomic mutagenesis strategy coupled with a cellular TLR8 activation assay to probe the importance of specific functional groups present on the guanine base in RNA-mediated receptor agonism and antagonism. Results from RNA analogs containing 7-deazaguanosine, 2-aminopurine and inosine confirm the importance of guanine N7, O6 and N2, respectively, in TLR8 activation. Nevertheless, these RNAs each retained TLR8 antagonism activity. RNA containing 7-deaza-8-azainosine (7d8aI) was prepared from a novel phosphoramidite and found to be a weaker TLR8 activator than guanosine-containing RNA. However, 7d8aI-containing RNA also retained TLR8 antagonism activity indicating that removal of multiple TLR8 H-bonding sites on guanine is insufficient for blocking TLR8 antagonism by guanine-containing RNA. We also identified an oligoribonucleotide length dependence on both TLR8 activation and antagonism. These studies extend our understanding of the effects of nucleobase modification on immune stimulation and will inform the design of novel RNA-based therapeutics.

Controlling miRNA-like off-target effects of an siRNA with nucleobase modifications.

SiRNAs can cause unintended gene silencing due to miRNA-like effects because of the similarity in function of an siRNA guide strand and a miRNA. Here we evaluate the effect on miRNA-like off targeting of introducing the adenosine derivative 7-EAA and triazoles prepared from 7-EAA at different positions in an siRNA guide strand. We find that a sterically demanding triazole placed in the RNA duplex major groove at position six of the guide strand dramatically reduces miRNA-like off targeting potency. A high-resolution structure of an RNA duplex bearing a novel, major-groove localized triazole is reported, which suggests that modified triazoles could be disrupting the hAgo2-guide-target RNA ternary complex. Five different triazole modifications were tested at the guide strand 6-position for effects on on-target and miRNA-like off target knockdown potency. A 7-EAA triazole bearing a benzylamine substituent displayed on-target knockdown activity as potent as the native siRNA, while having an IC50 against a miRNA-like off target >100-fold higher. Melting temperature studies revealed no obvious correlation between potency in knockdown assays and a modification's effect on duplex stability. These results, along with known structures of hAgo2-guide-target ternary complexes, are used to rationalize the effect of 7-EAA triazoles on miRNA-like off target effects.