Part:BBa_K738002

Theophyline riboswitch regulated RNA Scaffold (clover vision 2)

RNA scaffold D0 (Bba_K738000) was constructed from a single RNA module d0, which folded into a duplex with PP7 and MS2 aptamer domains that bind PP7 and MS2 fusion proteins. A designed theophylline aptamer was added on the original scaffold D0 in order to produce an interaction with MS2 aptamer in the absence of theophylline, thus disturbing the bind of MS2 aptamer and corresponding protein. (Fig 1)

Fig 1 Secondary structure (left) and tertiary structure (right)

However, when theophylline is added, the fold of the loop is changed and thus the interaction will disappear, leading to the binding of MS2 aptamer and corresponding protein (Fig 2).

Fig 2 The control mechanism of the theophylline aptamer.

Fig 3 Three versions of clovers. Version 2 has adjacent MS2 and theophylline aptamer. It has an interaction between the loop of theophylline aptamer and the stem of MS2 aptamer.

RNA riboscaffold clover 2 can be regulated and controlled through conformational change by theophylline. The interaction is between the loop of the theophylline aptamer and the stem of the MS2 apatamer. And the theophylline aptamer is just beside the MS2 apatamer. When the concentration of Theophylline is in the range from 0mM to 0.5mM, the concentration of Theophylline and the resulting fluorescence intensity are directly proportional (Fig 4). Theophylline concentration beyond certain extent will be hazardous to cells and how it affects cells depends on strain type.

[[Image:Fig 4 tests of fluorescence.jpg]

Fig 4 Tests of fluorescence/ OD change over theophylline concentration. There’s evident positive correlation fluorescence intensity between concentration of theophylline added.

Fig 5 RNA scaffold’s effect on split GFP showing by fluorescence microscopy.

Usage and Biology

This scaffold, by theophylline management, could have a variety of functions, more than accelerate the reaction, but whether to accelerate or not, the degree of acceleration and even reduce the reaction rate.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]