Difference between revisions of "Part:BBa K1614019"

Revision as of 13:21, 21 September 2015

ATP Aptamer JAWS1 Spinach 2.1

This part is an improvement of BBa_K1330000, Spinach2.1. We designed it to be ATP-dependent by joining an ATP aptamer to stem 2 using our software JAWS (http://2015.igem.org/Team:Heidelberg/software/jaws). This part was developed as a tool to sense ATP consumption within biochemical reactions. This part is cloned into the RFC 110 and can be transcribed using the T7 RNA Polymerase directly from Plasmid for further information refer to (File:BBF RFC 110.pdf).

Usage and Biology

The Spinach RNA aptamer binds a small molecule that resembles the fluorophore of the green fluorescent protein (GFP)(Jaffrey, 2011). Upon binding of the small molecule to the Spinach a green fluorescence comparable to that of the GFP is emitted. In our project we use variants of these fluorescent aptamer and engineer them to be dependent on small molecules of our interest. The ATP aptamer (Sassanfar, 1993) has been previously published. We used both parts together to obtain an ATP-dependent Spinach. To have a RFC 10 compatible version we used the Spinach2.1 as it was described by DTU Denmark in BBa_K1330000.
We have cloned this part into our new cut-site-independent RFC 110. According to our results, this fusion should contain the same properties as BBa_K1614014 (listed below in "Proporties of the ATP Aptamer JAWS1 Spinach2")This Spinach2.1 is ligand dependent on ATP and should find its use in the detection of the small molecule ATP. Applications such as nucleotide sensing during in vitro transcription are possible.

Proporties of the ATP Aptamer JAWS1 Spinach2

We tested the ATP-dependency of ATP Aptamer JAWS1 Spinach2 by tracking the in vitro transcription of RNA in real-time. During the reaction, the ATP concentration get less and thus the fluorescence we detect reduces over time. The construct is a fusion of an ATP aptamer (Sassanfar 1993) and Spinach2 (Strack 2013), which we will call Spinach2-ATP-Aptamer system. To improve the binding of the ATP aptamer to ATP we apply our own implemented JAWS software (Fig. 1). Using our software, nucleotides which form the stem region of the ATP aptamer can be predicted, which will improve binding properties of this RNA to ATP advanced the stemming behavior of the ATP Aptamer which was then fused to the Spinach2. Measurements with the spectro fluorometer show that the ATP Aptamer JAWS1 Spinach2 has a lower fluorescence than ATP Aptamer JAWS2 Spinach2 (Fig.2) which is caused by a weaker stemming behavior. Therefore ATP Aptamer JAWS1 Spinach2 is a better candidate for sensing ATP changes during biochemical reactions such as the in vitro transcription.
For the in vitro transcription assay the RNA was renatured in 1x Renaturing buffer at 95 °C. 500 nM of the RNA was used for the in vitro transcription for measuring the ATP consumption during transcription. The construct was excited at 460 nm. Emission was measured at 500 nm. Experiments have shown that the detection range of this ATP sensor which correlates to the transcribed RNA is much more sensitive than traditional techniques that require UV-shadowing. The real time fluorescent readout system even allows the study of enzyme kinetics that depends on ATP (Fig.3).
This part was generated by fusion an ATP aptamer to the Spinach Aptamer (BBa_K1330000). This leads to an ATP-dependent fluorescence. The fluorescence of Spiach2 and Spinach2.1 generated by DTU Denmark show similar fluorescence characteristics. Thus we characterized the ATP-dependency in BBa_K1614014. Here we provide a RFC compatible Spinach2.1 that is ATP-dependent.

Fig.1 Fusion of Aptamers to Spinach to generate fluorescent small molecule sensors. (A) For the design of a new small molecule sensor, the second stem of the Spinach2 aptamer can be exchanged by an aptamer that binds specifically to a small molecule. We fused an ATP-binding Aptamer (yellow) to the Spinach. (B) We applied our JAWS software to predict the best ATP-Aptamers, that can form the best stem structure (blue and red highlighted) in presence of ATP. (C) The JAWS predicted stems can be fused to the Spinach Aptamer. The software can be validated by analyzing the fluorescence emission in presence of ATP and DFHBI (Fig.4B and C).

Fig. 2. Establishment of a system to sense small molecule using the Spinach2 Aptamer. (A) Emission spectrum of the original Spinach2 Aptamer, which was applied as an internal control. (B) As another internal control, we reproduce the data for the c-di-GMP Spinach2 system, published by Kellenberger et al.. Indeed, highest fluorescence maximum for the c-di-GMP Spinach2 system was measured in presence of the ligand. (C) Analysis of the fluorescent properties of our ATP Aptamer Spinach2 constructs. The Spinach2 containing the Szostak ATP Aptamer shows the lowest fluorescence of all three ATP Aptamer Spinach2 variations. The JAWS-generated ATP AptamerJAWS1 Spinach2 and the ATP AptamerJAWS2 Spinach2 show higher fluorescence maxima in presence of ATP.

Fig.3.Sensing of ATP using the ATP Aptamer Spinach in real time during in vitro transcription. (A) Assay design of the ATP-Aptamer Spinach2: ATP-AptamerJAWS1 Spinach2 RNA will be applied to a classical in vitro transcription. In presence of ATP, fluorescence emission can be determined. (B) As proof of principle, transcriptions were performed with different concentrations of T7 RNA polymerase. ATP consumption was monitored in real time by measuring the fluorescence of the ATP-AptamerJAWS1 Spinach RNA in regular intervals.(C) To confirm the results of the fluorescence measurements, the in vitro transcription reaction was analyzed using denaturing acrylamide gels.

References:

1.Sassanfar, M. and J.W. Szostak, An Rna Motif That Binds Atp. Nature, 1993. 364(6437): p. 550-553. 2.Strack, R.L. and S.R. Jaffrey, Live-cell imaging of mammalian RNAs with Spinach2. Methods Enzymol, 2015. 550: p. 129-46. 3. Strack, R.L., M.D. Disney, and S.R. Jaffrey, A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat Methods, 2013. 10(12): p. 1219-24.

Sequence and Features