Part:BBa_K779116

Short RNA reporter bottom strand (with ROX fluorophore) MammoBlock

Modifications: 3' ROX fluorophore

IDT DNA Oligo Order: mUmGmAmGmAmUmGmUmGmAmUmUmGmUmGmUmUmAmUmG/3ROX_N/

Usage and Biology

Can be annealed to Part:BBa_K779120 (quencher with Alexa 488 fluorophore) or Part:BBa_K779117 (just quencher) to make a reporter for strand displacement as described in [http://www.sciencemag.org/content/332/6034/1196.abstract Qian et al. 2011].

Excitation and emission spectra for the Alexa 488 and ROX fluorophore. When Part:BBa K779116 is annealed to Part:BBa_K779120, there are two fluorophores in the complex, Alexa and ROX. They are ortohgonal fluorophores, which allows the tracking of a quenched complex using the Alexa flurophore, why a strand displacement reaction is indicated by increase from the fluorescence of ROX. Furthermore, Alexa is outside the quenching-zone of the RQ quencher (according to [http://idt.com IDT]).

Can also be annealed to Part:BBa_K779118 to make a double-stranded complex with a fluorophore (dsROX).

Dilutions of Part:BBa_K779116 as a free strand. Measurements carried out on a Tecan Safire II plate reader (see [http://2012.igem.org/Team:MIT/MaterialsAndMethods#iv2bio protocol]).

Effect of carrier RNA on fluorescence levels. Previously ([http://www.sciencemag.org/content/318/5853/1121.abstract Winfree et al. 2007]) it has been shown that RNA and DNA stick to plastics in a non-specific way. This includes pipette tips as well as wells in a plate reader plate. Thus, for accurate measurements, a carrier of some sort is needed, otherwise a decrease in fluorescent signal is observed as the nucleic acids sticks to the sides of a well.
Here, Part:BBa_K779119 is used as an excess carrier (>10 uM whereas dsROX was at 40 nM). Measurements carried out on a Tecan Safire II plate reader (see [http://2012.igem.org/Team:MIT/MaterialsAndMethods#iv2bio protocol]) using a 384-well plate. At timepoint 1, the wells are mixed using a pipette, disturbing the RNA bound to the well. At timepoint 2, carrier RNA is added, which now outcompetes dsROX for binding to the plate. Almost all of the fluorescence is recovered.

Strand displacement in vitro. The input strand (Part:BBa_K779118) can attach via the toehold T to the reporter complex (Part:BBa_K779117:Part:BBa_K779116) and go through toehold-mediated strand displacement as described by [http://www.sciencemag.org/content/332/6034/1196.abstract Qian et al. 2011]. This results in a dsROX complex (see above) and a free quencher strand (Part:BBa_K779117). The dsROX complex is fluorescent, whereas the original reporter is not. Negative control is reporter, but with a scramled-sequence strand (Part:BBa_K779119) as an input. After 2 hours, a roughly 10-fold increase in fluorescence is observed. Measurements carried out on a Tecan Safire II plate reader (see [http://2012.igem.org/Team:MIT/MaterialsAndMethods#iv2bio protocol]).

Reporter response to various input strand levels. Here, a strand displacement reaction is shown with the reporter (Part:BBa_K779117:Part:BBa_K779116) and input (Part:BBa_K779118). Measurements carried out on a Tecan Safire II plate reader (see [http://2012.igem.org/Team:MIT/MaterialsAndMethods#iv2bio protocol], note deviation for amount of input). We expect to see faster kinetics and a higher output level from a higher concentration of input. These data suggest that this expectation is sound.

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]