User:James Chappell/ Ribozymes

Three possible trans acting ribozymes:
 * RNase P- Found naturally in E.coli and requires a protein subunit for in vivo- means chassis would cross talk with one of the inputs into the NAND gate. Use archeal or euk homologues - problem is require more protein subunits (8 in euk) and so increasing the transcriptional basis of our design (could consiutivly express them?)
 * RNase P has a role in catalysing the processing of primary tRNA transcript by removal of a 5'precusor sequence to form the mature tRNA. Found in three main branches of life; Euk, Arch,Prok.


 * Hammer head - good candidate, one problem is that using the method proposed where RNA self anneals, the hammer head ribozyme has no basis for contact. This could be avoided by having relatively weak binding to the RBS, preceeded by hairpin or structure to block RNA polymerase preceeded by a unpaired loop. The unparied loop provides a basis for interaction which can be cleaved and due to weak interactions on RBS should diassociate.
 * Second approach is to use a small stand of RNA that is comp to our RBS binding RNA so that once cleaved this can compete and bind to cleaved RBS binding RNA.
 * delta - Recognises the 11-mer ribonucleotide substrate. Km of 17.9nM. k2 (second order of 1.89*107 min -1 M -1. Originally fuond in genome of


 * Hair pin-

Papers:

 * PMCID: PMC1369943 - looking at monitoring rate of clevage by FRET

Design-mRNA NAND •	To date most research in synthetic biology has been focused on designing and engineering biological devices that are regulated on the transcriptional level. Numerous projects from the International Genetically Engineered Machines (iGEM) competition have focused on the use of protein transcriptional activators and repressors to construct simple devices such as logic gates. Moreover, research groups have also focused on regulation of transcription, see ….for a review. •	Recently a new class of synthetic biology has emerged looking at use RNA within biological devices. The potential of RNA synthetic biology is that it allows the control of biological devices at the translational level, giving synthetic biologists a whole new category of parts and a new level of control. The potential parts and devices that RNA synthetic biology offer are numerous from riboswitches to allow the detection of small molecules to a variety of RNA interference methods to knock out expression of specific genes. •	Another key reason why these RNA devices are so appealing for synthetic biology is two fold. Firstly, we understanding how RNA sequence can be manipulated to create a diverse away of structures and functions. Furthermore, alternative fields have driven the need for a large variety of computational and RNA specific techniques that synthetic biologists can easily reuse and build upon. We now have an surplus of algorithms and statistical analysis that can accurately predict the structure of an RNA sequence [examples]. •	Limitless designs •	With this in mind our approach was to design a NAND gate based upon a RNA device that would be regulated at the translational level. Inspiration for our design came from previous RNA devices (see review). The key concept behind our design is to have one input that produces an auto-inhibited mRNA. This inhibition is based upon sequence of mRNA that is upstream and complementary to the ribosome binding site (RBS), so that when the mRNA is transcribed the complementary sequence binds to the RBS and inhibits ribosome binding (figure 1.1). The second input to our NAND gate would then provide a way to remove the inhibition of the first input by some how disrupting the binding of the complementary-sequence and RBS to allow the ribosome to bind. •	There are really three key ways to remove the binding of the complementary-sequence to the RBS, these are explained below and summarised in figure 1.1. •	RNase, annealing an RNA, ribozyme. •	The first idea was to include within the complementary-sequence a motif that could be recognised by a RNase synthesised on the second input. There are several advantages to the use of RNase which include the efficiency of the cleavage and the specificity of cleavage. In addition, cleavage of the complementary-sequence into a small single mRNA will reduce its half-life dramatically and should relieve the possibility of re-annealing. However, expression of RNase involved transcription of the mRNA that has to then be translated. This provides an intrinsic delay of the RNase input compared to the auto-inhibited input. Because of this delay we then considered two approaches that removed auto-inhibition via RNA devices.

•	The second idea that has been previously expored (ref) is to use a secondary mRNA that is capable of binds with high efficiency to the complementary-sequence, there by removing the inhibition of the RBS. However, from the research paper it become that there was a key potential problem; the binding of the complementary-sequence to RBS needs to be as strong as possible to prevent translation however the binding of the second annealing mRNA must be sufficiently stronger to enable an efficient efficient switch and NAND gate. The problem being there is a compromise between reducing background translation leakiness and the effectiveness of removal of auto-inhibition.

•	The problem of the annealing strand led us to look at the use of ribozymes to remove the autoinhibition. Ribozymes are RNA enzymes that a capable of catalysing a diverse array of interactions. In particular the use of ribozymes to carry out RNA splicing events in trans seemed a good approach to carry out the cleavage of the auto-inhibited mRNA. These trans RNA ribozymes have been heavily researched in the context of diverse functional applications particularly there use to knock-out gene expression in cell cultures. Three potential ribozymes were explored in more detail and are summarised in table 1.1. In the end the δ ribozyme seemed like a good candidate for our design, firstly because of the specificity and secondly due to its high rate of catalytic activity.

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