Table I. Antiswitches were designed with an antisense sequence and an aptamer sequence. Each antiswitch had specific characteristics that enable the testing of various variables.

Summary of Results:

Figure 1 a: Figure 1 a shows a schematic illustrating the general mechanism of action an antiswitch in the presence and absence of an effector ligand. In the absence of the effector ligand, as can be seen on the left side of the figure, the antiswitch forms a double-stranded region called the antisense stem which prevents base pairing with the target mRNA molecule. As a result, the antiswitch does not bind to the target mRNA which can then be translated and the GFP encoded by it can thus be produced. In the presence of the effector ligand, as can be seen on the right side of the figure, the affinity of the binding site for the ligand causes a region of the RNA to swing outward and overlap with the previously antisense region of the antiswitch, thereby forming an aptamer stem and leaving a single-stranded region of the antiswitch RNA free for binding. Consequently, the antiswitch binds the target mRNA which cannot be translated, and thus, no GFP is produced.

Figure 1 b: Figure 1 b shows a detailed representation of antiswitch s1 and the changes it undergoes when bound to theophylline. In the absence of theophylline, the antiswitch in is in inactive state where the antisense sequence (red) is bound in a double-stranded region called the antisense stem. This is favored due to the relatively lower value of free energy for this domain (ΔG = -18.3 kcal/mol) versus the free energy of the aptamer domain (ΔG = -10.8 kcal/mol). In the presence of theophylline, however, the aptamer domain binds to it (not shown). Consequently, the aptamer sequence (blue) swings in and is bound into a double-stranded region called the aptamer domain, forcing the antisense sequence to be single stranded and allowing it to bind to the target mRNA strand. The equilibrium of this reaction as indicated by the interconversion arrows lies towards the formation of products. In accordance with Le Chatelier’s principle, since the free energy associated with the formation of the aptamer stem changes by 8.9 kcal/mol when theophylline is added, the aptamer sequence becomes more unstable and is forced into its aptamer stem conformation, thereby leaving the antisense sequence single-stranded.

Figure 1 c: Figure 1 c shows a graph of the relative GFP expression in cells bearing containing antiswitch s1 in the presence and absence of theophylline respectively. Several RNA constructs were created for the production of antiswitches in S. cerivisiae under an inducible promoter (Table II).

Table II. RNA expression constructs were created for the production of antiswitches which contained an antisense domain and an aptamer domain.

In the absence of aptamer domain, the antisense domain is free to bind to the mRNA and therefore, GFP expression is inhibited, remaining at the constant low level of approximately 0.05. In the absence of the antisense domain, the aptamer domain binds theophylline willingly and target mRNA is left free to be translated, thereby resulting in a high value of GFP expression of approximately 1. In the presence of both domains, the aptamer binds the ligand and mRNA to inhibit target mRNA translation and subsequent GFP expression. If caffeine, which is distinct from theophylline only in a single methyl group (Figure I), is used as a ligand, a constant level of GFP expression is seen around 0.75, which indicates the expression seen in the presence of s1 but absence of ligand, since caffeine will not bind to theophylline-specific aptamer domain. When theophylline is used as a ligand, the GFP expression seen with the s1 antiswitch mimics that seen in the presence of caffeine until the concentration of theophylline reaches about 0.75 mM. At this point, the GFP expression experiences a sharp drop and mimics that seen only in the presence of the antisense domain at an intensity of about 0.05.

Caffeine	Theophylline

If the s1 antiswitch were concentration sensitive, the GFP expression would taper off gradually with increasing theophylline concentrations. However, since there is a sudden drop in the GFP expression level, it is possibly because the antiswitch has an all-or-nothing response, like that in the nervous system, to theophylline and that the threshold level beyond which the antiswitch remains active is approximately 0.8 mM of theophylline.

Figure 1 d: Figure 1 d shows a graph of the expression intensity of GFP in the cells containing antiswitch s1 in the presence and absence of theophylline over a period of time in vivo. In the absence of theophylline (blue), the antisense stem is formed and GFP is expressed. However, with time, GFP expression increases since there is no inhibition of GFP expression at all and this construct thus serves as a negative control. In the presence of theophylline at a concentration of 2mM, the antiswitch should inhibit GFP expression altogether as can be seen from the data in Figure 1 c where GRP expression falls to 0.05 after 0.8 mM concentration of theophylline is reached, indicating that the antiswitch s1 is "off." However, this inhibition is not immediate, since there is a time lag between the transcription of DNA to make GFP mRNA and the translation of mRNA into GFP. Thus, at time point 0 hours, translation of GFP mRNA begins, just as gradual inhibition of GFP translation begins. However, a certain amount of GFP is already present before the inhibition occurs and this GFP, which has a half-life of 0.5-1 hours, persists in intensity until its lifetime is exhausted at approximately 3 hours. Beyond this point, due to lack of further translation, no more GFP is produced and the intensity of GFP tapers off.

Figure 1 e: Figure 1 e shows the a radiograph of a gel containing radiolabeled antiswitch s1 in varying concentrations of theophylline. While 0.2 µM and 2 µM concentrations of theophylline show a lower MW for s1, 10µM, 20 µM and 200 µM show the same higher MW band. This indicates that at the two lower concentrations, s1 does not bind to theophylline since the antisense stem is probably still more stable than the aptamer stem conformation of the molecule. The higher concentrations of theophylline, however, provide the adequate energy to overcome the activation energy barrier to make the switch from the antisense stem or "off" conformation to the aptamer stem or "on" conformation. Therefore, the threshold concentration of theophylline required to just induce the switch from "off" to "on" must lie between 2 µM and 10 µM.

Figure 2 a: Figure 2 a shows the four variations of the theophylline-specific antiswitch - s1, s2, s3 and s4. The free energy change, ΔG = -10.8 kcal/mol, required for the aptamer sequence to form the aptamer stem in each case is the same. The difference lies, however, in the free energy of the antisense stem which determines the intrinsic stability of the antisense stem and its subsequent affinity to bind to the target mRNA. The lower the magnitude of the ΔG i.e. the more negative the value, the less stable the antisense stem, leading to a higher propensity to bind to theophylline and move towards a lower energy conformation. Consequently, the higher the magnitude of ΔG, the greater the amount of theophylline ligand needed to produce GFP inhibition.

Figure 2 b: Figure 2 b shows the effect of increasing concentrations of theophylline on the relative GFP expression for different identity and length of the antisense stems as in each of the theophylline-specific antiswitches - s1, s2, s3 & s4. s1 (blue) is the control which is shows its drastic drop in GFP expression at approximately 0.8 mM of theophylline as seen before. s3 (orange), having a higher magnitude of free energy change associated with the formation of its antisense stem, requires a higher concentration of theophylline before it can be turned "on" and can serve to inhibit GFP expression. In accordance with this hypothesis, s3 does not inhibit GFP expression until a theophylline concentration of 1.25 mM. In contrast to this, s2 (red) and s4 (green), having lower free energy change associated with their antisense stems, tend to require a lower concentration of theophylline before they can change to their aptamer stem or "on" conformation and thus, inhibit GFP expression at low theophylline concentration of approximately 0.4 mM.

Figure 2 c: Figure 2 c shows the effect of increasing theophylline concentrations on the relative GFP expression for different aptamer domains in two additional theophylline-specific antiswitches with lower theophylline affinity than s1 - s5 & s6. s1 (blue) is the control, once again showing the drastic drop in GFP expression at approximately 0.8 mM of theophylline as seen before. s5 (green) and s6 (red) are similar and they require ten times the concentration of theophylline for the same inhibition of GFP expression as s1. Since the Kd of the aptamer domain of s5 and s6 is approximately ten times higher than that of s1 for theophylline, there is a likely correlation between the Kd and the concentration of effector ligand required for antiswitches.

Figure 2 d: Figure 2 d shows the effect of different increasing concentrations of different effector ligands on different ligand-specific antiswitches. s1 shows the effect of increasing concentrations of theophylline on GFP expression showing inhibition at approximately 0.8 mM. s7 shows the effect of increasing the concentrations of another ligand, tetracycline, on GFP expression, showing inhibition at approximately 1 mM. This shows that the "on" and "off" mechanism or all-or-nothing response is observed not only with theophylline-specific antiswitches, but also with other ligand-specific antiswitches.

Figure 3 a: Figure 3 a shows a detailed representation of the antiswitch s8 and the changes it undergoes when it binds to theophylline. In the absence of the theophylline ligand, the antiswitch s8 is in its aptamer stem conformation which is its "on" conformation. In the presence of theophylline, s8 is in its antisense conformation which is its "off" conformation. Thus, in contrast to s1 in Figure 1 b, s8 is on while s1 is off in the absence of theophylline and s8 is off while s1 is on in the presence of theophylline. It is thus, the opposite of the antiswitch s1. The aptamer ΔG differs for s1(-10.8 kcal/mol) and s8 (-8.9 kcal/mol) while their antisense ΔG has the same value (-18.3 kcal/mol). Since antisense ΔG < aptamer ΔG for s8, s8 is found in its aptamer stem conformation in the absence of theophylline and since antisense ΔG > aptamer ΔG for s1, s1 is found in its antisense stem conformation in the absence of theophylline.

Figure 3 b: Figure 3 b further illustrates the nature of the opposite antiswitches with regards to relative GFP expression versus increasing concentrations of the theophylline ligand. As concentrations increase till approximately 0.8 mM, s1 (blue) remains in its "off" conformation while s8 (red) remains in its "on" conformation, with its antisense sequence bound to target mRNA thereby inhibiting GFP expression. At this concentration, the two curves cross and take on opposite natures. s1 is now turned "on" and inhibits GFP expression by binding to its mRNA while s8 turns "off" into its antisense stem conformation.

Figure 4 a: Figure 4 a shows a schematic of the antiswitches s1 and s9 and their mechanism of action respectively. s1 is turned "on" by binding to theophylline as shown in Figure 1 a. s9 is turned on by the same mechanism by binding instead to tetracycline as it is tetracycline-specific. Furthermore, while s1 regulates the expression of Green Fluorescent Protein (GFP), s9 regulates the expression of Yellow Fluorescent Protein (YFP). In this case, the binding of s1 and s9 to theophylline and tetracycline respectively leads to inhibition of GFP and YFP expression respectively.

Figure 4 b: Figure 4 b shows a graph relative expression of the target protein when the antiswitches s1 and s9 are stimulated either in turn or simultaneously by the addition of their respective effector ligands. The 0mM theophylline and 0mM tetracycline serves as a negative control to demonstrate GFP and YFP expression levels in the absence of effector ligands. These expression levels are relatively high since neither antiswitch is turned "on." With 5 mM theophylline and no tetracycline, only s1 is turned "on" and consequently GFP levels are negligible while YFP levels are still high. With 5 mM tetracycline, only s9 is turned "on" and consequently YFP levels are negligible while GFP levels are still high. When both tetracycline and theophyline are added as 5 mM concentrations, expression of both YFP and GFP is inihibited.

Critique:

The study of the design and effectiveness of RNA molecules controlled by ligands in the the regulation of gene expression is a novel approach to gene regulation. Bayer and Smolke's presentation of the results is very thorough and the conclusions they draw are consistent with their data.

Bayer and Smolke show detailed images of the antiswitches they designed, yet fail to show the specific binding pocket into which the theophylline molecule fits. Figure 1 a makes an attempt to show where the theophylline molecule binds in relation to the aptamer stem but a detailed view would also help the reader understand why the antiswitch s1 is specific to theophylline and does not even bind caffeine.

In Figure 1 e, a negative control lane could be included to show the MW of s1 only in 0 µM theophylline. If the conclusions drawn from this figure are that the lower MW represents the unbound s1 antiswitch and the higher MW band represents s1 + theophylline, then the negative control lane should show the same band of lower MW. Also, the range of theophylline used for this experiment was very large, from 0.2µM to 200µM. If this was an initial screen to detect the concentration at which the switch is turned "on" or "off," another screen of closer concentration range of theophylline, for instance between 2µM and 10µM, should have been performed to demonstrate a narrower range for the critical concentration of theophylline required. This narrow range, however, is identified from the graph in Figure 1 c to be around 0.8 mM.

Figure 4 a is unnecessary as simply stating the difference between s1 and s9 with respect to their ligands and target proteins is sufficient to gain an understanding of the contructs created.

On the whole, Bayer and Smolke present a convincing argument of the feasibility of the use of ligand-dependent RNA molecules for the regulation of gene expression. The range of theophylline at which the GFP is inhibited, although not effectively illustrated in their gel-shift experiment, is possibly accurately described since the proximity of the data points on the graphs in the 0.8-1 mM range of theophylline is great showing the relative accuracy of the data. Moreover, the use of positive and negative controls such as those in Figure 2 b, make it possible to compare the actual effect of theophylline on constructs that are not drastically different but have subtle similarities, such as s1, s2, s3 and s4. The color coding of the aptamer and antisense sequences enables easier reading of the images and the values of free energy provided enables one to understand the reason for interconversion between the antisense stem and aptamer stem conformations of these antiswitches. They also show that gene expression can not only be inhibited, as with s1, but also induced, as with s8. Moreover, using the dual construct of GFP and YFP, Bayer and Smolke also demonstrate that two riboregulators can be simultaneously or individually used in a single cell using a single promoter.

Future Studies:

An idea that could be explored in the future, could be examination of the regulation of gene expression using riboregulators in higher organisms since Bayer and Smolke conduct studies only in S. cerivisiae. This could be done by genetically engineering mice to produce such riboregulators and then examining their effects on gene expression by administering effector ligands, such as theophylline, to these mice. Expression levels could be tested on tissue samples from these mice to measure the intensity of GFP expression in much the same way as in S. cerivisiae.

Moreover, the importance of examining the ability of these riboregulators to regulate the expression of genes other than those encoding GFP should be tested to ensure that the use of this technique can be generalized to other proteins.

Riboregulators could be of importance in a disease such as diabetes. An antiswitch sensitive to glucose could be designed such that once the glucose levels exceed a certain level in the tissues, the antiswitch begins to act by either downregulating glucagon which converts glycogen to glucose or by inducing expression of insulin which converts glucose into insoluble glycogen, thereby serving to reduce the blood sugar level (BSL) of diabetic patients and alleviating the symptoms of the disease. The concern, however, would be the introduction of these riboregulators into humans.

Another application of riboregulators could be in combatting the influence of retroviruses. Retroviruses use RNA as their genetic material rather than DNA and use the cellular replication machinery of their host cells to replicate this RNA. If riboregulators that could bind the RNA characteristic of such viruses at its aptamer domain, and could, subsequently, inhibit the expression of genes responsible for replication i.e. transcription and translation factors, the virus would be unable to replicate and infect the host cell. This, however, may not be very effective, since retroviruses such as HIV, mutate at a very high rate compared to DNA viruses (Drake 1993). The high mutation rate of approximately 1 per replication cycle and only one order of magnitude lower for retroviruses, would involve a ridiculously high rate of making new riboregulators.

References:

Chemfinder. <http://www.chemfinder.com> Accessed April 29th, 2005.

Drake, J. W. (1993). "Rates of spontaneous mutation among RNA viruses." Proceedings of the National Academy of Sciences. 90: 4171-4175.

Bayer, T. S. and C. D. Smolke. (2005). "Programmable ligand-controlled riboregulators of eukaryotic gene expression." Nature Biotechnology. 23 (3) March: 337-343.

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