Figure 2.12 Catalytic activity of deoxyribozyme.
(2.19)
If quencher is absent, then the fluorophore again returns to the ground state by the emission of light which causes the fluorescence.
(2.20)
In presence of quencher, the excited fluorophore nonradiatively, i.e., without emitting light, transfers its energy to the quencher. This causes the quencher to enter into excited state, on the other hand, the fluorophore returns to its ground state.
(2.21)
In the next step, the quencher returns to its ground state by releasing the absorbed energy. The release of energy can occur through emissive decay which causes fluorescence; or by dark quenching in which the energy releases non-radiatively, i.e., through molecular vibrations (heat).
(2.22)
The fluorescence intensity increases proportionally as the distance between fluorophore and quencher increases.
In Figure 2.12, if substrate is cleaved, then the two short strands go apart which allow the fluorescence of the output signal. Otherwise, the fluorescence is suppressed by the quencher.
2.5.1.2 Controlling Deoxyribozyme Logic Gate
Deoxyribozyme can be either in active or in inactive state. This enzyme can be made switch sensitive to an input DNA strand by adding a stem-loop structure to the molecule. In the stem-loop structure, the two substrate binding regions are complementary to each other, thus these regions hybridize to each other to form the stem component. These formed structures inhibit the substrates to bind to its recognition site in deoxyribozyme.
Figure 2.13 Mechanism to switch on deoxyribozyme logic gate.
The mechanism of controlling the catalytic activity of deoxyribozyme is shown in Figure 2.13. Deoxyribozyme logic gate can be switched on by addition of a single stranded short input DNA strand. This input oligonucleotide binds to the complementary single stranded loop region of deoxyribozyme. The hybridization leads to the destabilization of the stem-loop structure of deoxyribozyme. As a result, the stem-loop module opens up which permits the substrate to bind with deoxyribozyme. Thus, it can be concluded that the addition of the input oligonucleotides leads to the conformation change of the stemloop region of the enzyme which causes it is the catalytic activity. Following this method, the DNA logic gate can be switched on. This mechanism can also be applied for large DNA logic circuit. In this case, many different DNA oligonucleotides selectively bind to the specific corresponding stem-loop regions of deoxyribozyme. Thus, the activation of the logic circuit can be controlled by adding all inputs and enzymes in the same solution.
Now, in the next subsections, we will discuss the basic logic gates that can be constructed by using deoxyribozyme [6].
2.5.1.3 YES Gate
Deoxyribozyme logic gate works as YES gate if it is modified by including a single stemloop region which regulates the binding of substrate, as explained in the previous subsection. The substrate cannot bind to deoxyribozyme if the stem-loop is in closed form. As a result, the enzyme is inactivated as no output can be produced. Addition of input single stranded DNA sequence causes the hybridization of it to the loop region and breakage of stem-loop structure. This conformational change activates the enzyme. Now, the substrate binding regions are free to hybridize which causes the cleavage of the substrate. Now, the output DNA signal can be produced. This mechanism is shown in Figure 2.14, where a single input ix activates the YESx logic gate. Table 2.4 represents the truth table for YES gate.
Figure 2.14 Mechanism of YES gate.
Table 2.4 Truth table for YES gate.
| Input (ix) | Output (iy) |
| 0 | 0 |
| 1 | 1 |
2.5.1.4 NOT Gate
Deoxyribozyme logic gate works as NOT gate if the inclusion of single stem-loop region modifies the catalytic core of the enzyme. It controls the enzyme activity by controlling the binding of substrate to the specific region of the enzyme. The mechanism of NOT gate is pictorially explained in Figure 2.15. The NOTz gate as shown in the figure is first in its active mode. If a single stranded input sequence iz is added to deoxyribosome, then it hybridizes to the input binding region (red) of the enzyme which leads to the deformation of the catalytic core. This conformational alteration of the enzyme structure inhibits the enzyme activity, i.e., inactivates the DNA logic gate. Thus, if the input iz is bound to the enzyme, then the substrate cannot be cleaved. Table 2.5 represents the truth table for NOT gate.
2.5.1.5 AND Gate
Deoxyribozyme logic gate works as AND gate if it is modified by including two stemloop modules. This region regulates the activity of the enzyme by controlling the binding of substrate. The substrate cannot bind to deoxyribozyme if any of the stem-loops is in closed form. As a result, the enzyme is inactivated as no output can be generated. The mechanism is shown in Figure 2.16. If two specific single stranded input DNA sequences, i.e., ix and iy, are added, then these oligonucleotides hybridize to the two loop regions and break the stem-loop structures. Then, the substrate can anneal to the substrate binding regions which causes the substrate cleavage. Hence, the output DNA signal is produced. Thus, the xANDy logic gate is activated only when both the inputs, i.e., ix and iy are present. The addition of any one of the two inputs cannot open both substrate binding regions. The truth table for AND gate is presented is Table 2.1.