A second characteristic is tuning of the response curve, the quantitative relationship between ligand concentration and gene expression levels. Designs exhibiting ON behavior (A) or OFF behavior (B), as indicated by the expression level of the target gene in the presence of the ligand. The ligand-regulated behavior of shRNA switches can be programmed by applying the competitive strand and aptamer tuning strategies described above.
Each nucleotide change produced the expected shift in the transfer function corresponding to the relative stabilization (increased KComp) or destabilization (decreased KComp) of the inactive conformation. Relative GFP levels were measured for cells transfected in the presence of theophylline, hypoxanthine, or the combination of the two (Figure 3.5E). When the linkers were cotransfected, high GFP levels only coincided in the presence of both ligands as expected based on the circuit configuration.
Based on the results, shRNA switches allow the construction of fine-tuned genetic networks that can process multiple inputs. For all competing strand alignment strategies, increased stability of the inactive conformation always resulted in an increase in basal expression (Figure 3.3B-G). In the previous design, ligand control of RNAi was achieved through direct coupling of the theophylline aptamer and an shRNA strain.
Based on the proven modularity and configurability of our platform, shRNA switches can be implemented in a variety of applications.
GFP100%GFP
The next step was to correlate the proportion of shRNA switches in the active conformation with the relative expression levels of the target gene. Base pairs were selected such that the same nucleotide in the shRNA stem was part of the selected base pair in both conformations. The main stem for the active conformation extends from the bulge of the shRNA stem to the tip.
Therefore, proper Drosha processing and gene silencing correlate with the size of the bulge in the miRNA basal segments. We developed a design strategy based on this phenomenon and the dependence of Drosha processing on the structure of the basal segments to introduce ligand control of miRNA-mediated gene silencing (Figure 4.2A). The results demonstrate that the observed effect of theophylline on miRNA-mediated gene silencing is specific to the incorporation of the theophylline aptamer into the basal segments of the miRNA.
The binding core of each aptamer was initially integrated next to the lower stem instead of the bulge (Figure 4.3A), and the resulting miRNAs were tested using the cell culture assay in the presence or absence of the corresponding ligand. However, compared to the theophylline aptamer, insertion of the tetracycline aptamer reduced silencing and ligand sensitivity. The altered silencing in the absence of ligand can be attributed to the nature of the unbound aptamer structure, where the tetracycline aptamer folds into a preformed pocket (Muller et al, 2006).
In contrast, insertion of the binding core of the xanthine aptamer (xa1) completely abolished silencing (Figure 4.3C). Most of the synthetic ligand-responsive RNA-based regulatory systems are encoded in the target transcript, providing regulation in cis (Desai and Gallivan, 2004; Drosha processing of the miRNA located in the 3' UTR separates the coding region from the poly( A) tail, thereby inactivating the transcription B) The impact of ligand-responsive miRNA copy number on expression of the transgene by regulation in cis in the presence (grey) or absence (white) of 5 mM theophylline.
DsRed-Express levels of the constructs tested in Figure 4.4C were characterized via identical cell culture assays. -Target miRNAs are located in the 3' UTR of the trans target transcript encoding GFP, so that Drosha cleavage and RISC targeting down-regulate expression. We further examined the effects of miRNA copy number and regulatory mechanism (cis, dual cis/trans) on the dynamics of the ligand-responsive miRNA regulatory response (Figure 4.7D).
Ligand-responsive miRNAs function through ligand-mediated regulation of Drosha processing of a pri-miRNA based on modulation of the structured nature of the basal segment region through aptamer-ligand-binding interactions. Transfected cells could be distinguished from untransfected cells even when four miRNAs were present in the 3'UTR of the DsRed-Express-encoding transcript (data not shown).
2 Error 2
Studies of naturally occurring riboswitches that function through transcriptional termination revealed that the time delay between transcription of the aptamer and the terminator stem can regulate the effective ligand concentration at which a half-. For transcription termination, riboswitches effectively choose between termination (kTA, kTB) and elongation (kMA, kMB) after transcription of the terminator base. While all four parameters affect the dynamic range, kdMA and kdMB also affect the dependence of the dynamic range on the conformational separation (Figure S5.3).
The ratio of the mRNA degradation rate constants in the expression for EC50 accounts for the modified dependence of the dynamic range on K1 for riboswitches that function by mRNA destabilization. Thus, riboswitches that function by either regulatory mechanism show the same trade-off between EC50 and dynamic range. The second regime begins when either of the irreversible rate constants balances the associated reversible rate constant (either γ1 or γ2 is between zero and one).
Regimes are qualitatively labeled for dynamic range and basal and ligand saturation levels according to the ratio of the rate constants for terminator stem formation (kM) and the progression from conformation A to conformation B (k1). This effect reduces the dynamic range (Figure 5.3A) and shifts basal and ligand saturation levels according to the degree of transcriptional folding (Figure 5.3B). Second, biased transcriptional folding can modulate the relationship between irreversible rate constants and the dynamic range (Figure 5.3A).
As a result, influencing transcriptional folding toward conformation B in the kinetically driven regime increases the dynamic range. The competition between terminator stem formation (kM) and the progression from conformation B to A (k1') determines the dynamic range. Our analysis of the kinetically controlled regime revealed that performance can be maintained by directing transcriptional folding toward conformation B and ensuring that k1' exceeds the irreversible rate constant kA.
The dynamic range is greatest when the conformational progression occurs much faster than the formation of the terminator stem (Figure 5.4B), as predicted by our analysis of the kinetically driven regime (Figure 5.3A). An in vitro study of the ribD FMN riboswitch, which acts through transcriptional termination, revealed a reduced dynamic range upon removal. In contrast, the EC50 of the thermodynamically driven riboswitch approaches the dissociation constant of the aptamer as the B conformation stabilizes, resulting in a concomitant decrease in dynamic range (Figure 5.2D).
Limiting L' changes the dependence of the dynamic range (Figure 5.5B) and apparent EC50 (Figure 5.5C) on the model parameters, as shown for riboswitches operating in a thermodynamically driven regime. Mutations made on the aptamer stem of the parent synthetic riboswitch (m1-4) are expected to modulate exclusively conformational partitioning (K1).
