The need for fluorescently tracking

RNA Ribonucleic acid (RNA) is a polymeric molecule essential in various biological roles in coding, decoding, regulation and expression of genes. RNA and deoxyribonucleic acid ( DNA) are nucleic acids. Along with lipids, proteins, and carbohydra ...

rose as its roles in complex cellular functions has grown to not only include

mRNA In molecular biology, messenger ribonucleic acid (mRNA) is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is read by a ribosome in the process of Protein biosynthesis, synthesizing a protein. mRNA is ...

rRNA Ribosomal ribonucleic acid (rRNA) is a type of non-coding RNA which is the primary component of ribosomes, essential to all cells. rRNA is a ribozyme which carries out protein synthesis in ribosomes. Ribosomal RNA is transcribed from ribosoma ...

, and

tRNA Transfer RNA (abbreviated tRNA and formerly referred to as sRNA, for soluble RNA) is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length (in eukaryotes), that serves as the physical link between the mRNA and the amino ac ...

, but also RNAi, siRNA,

snoRNA In molecular biology, Small nucleolar RNAs (snoRNAs) are a class of small RNA molecules that primarily guide chemical modifications of other RNAs, mainly ribosomal RNAs, transfer RNAs and small nuclear RNAs. There are two main classes of snoRNA, ...

, and lncRNA, among others. Spinach is a synthetically derived RNA aptamer born out of the need for a way of studying the role of RNAs at the cellular level. This aptamer was created using Systematic Evolution for Ligands by EXponential enrichment, or SELEX, which is also known as in vitro evolution. Selex1

The aptamer was designed to be an RNA mimic of

green fluorescent protein The green fluorescent protein (GFP) is a protein that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea ...

(GFP); similar to GFP for proteins, Spinach can be used for the fluorescently labeling RNA and tracking it in vivo. A method for inserting the Spinach sequence after an RNA sequence of interest is readily available. GFP’s

fluorophore A fluorophore (or fluorochrome, similarly to a chromophore) is a fluorescent chemical compound that can re-emit light upon light excitation. Fluorophores typically contain several combined aromatic groups, or planar or cyclic molecules with se ...

is made up of three cyclized amino acids within the beta-barrel structure: Serine65-Tyrosine66-Glycine67. This structure, 4-hydroxybenzylidene imidazolinone (HBI) was the basis for the synthetic analogue used in the SELEX studies. Many derivatives of this structure were screened using SELEX, but the chosen fluorophore, 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI), showed the best selective fluorescence with high quantum yield (0.72) when bound to the RNA sequence 24-2, deemed Spinach. Spinach (RNA)

] It was determined that DFHBI only binds Spinach in the phenolate form. At pH < 6.0, both the phenolic and phenolate forms are detected. At pH = 6.0, only the phenolate peak is detected. DFHBI is also incredibly robust and resists

photobleaching In optics, photobleaching (sometimes termed fading) is the photochemical alteration of a dye or a fluorophore molecule such that it is permanently unable to fluoresce. This is caused by cleaving of covalent bonds or non-specific reactions between t ...

over a long period of time as compared to HBI and EGFP. It is believed that the free exchange of bound and unbound ligand allows for this persistence. As the fluorophore of GFP and its derivatives are covalently bound to/a part of the protein, free exchange cannot happen and thus photobleaching results. Spinach is an 84-nucleotide-long structure with two helical strands and an internal bulge with a

G-quadruplex In molecular biology, G-quadruplex secondary structures (G4) are formed in nucleic acids by sequences that are rich in guanine. They are helical in shape and contain guanine tetrads that can form from one, two or four strands. The unimolecular ...

motif. It is at this G-quadruplex that the fluorophore binds. Based on crystallographic data, massive rearrangement of the adjacent bases occurs once the fluorophore binds to accommodate the molecule. This binding is favorable, however, as it promotes base-stacking in a normally unstable region, i.e. the internal bulge. Similar to GFP, the DFHBI is also dehydrated, which would help with its high quantum yield. Spinach has also been adapted for sensing proteins or molecules in vivo. An adapted structure, which includes two binding sites, limits fluorescence of the aptamer to (1) the fluorophore and (2) the protein or small molecule.

References

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External links

SPINACH PLASMIDS
Cell imaging Bioluminescence RNA