Structure, Function and Utility in Medicine
Structure of RNA
Ribonucleic acid (RNA) is a biopolymer, consisting of individual nucleotides. Like DNA, RNA nucleotides are very similar in that there are four nitrogen bases that make up its sequence. Three of the nitrogenous bases are absolutely identical to DNA: adenine, guanine, and cytosine. However, instead of a thymine base as in DNA, there is a structurally close pyrimidine in RNA called uracil. Also, unlike DNA, RNA consists of a single strand, not a duplex. RNA is utilized in many ways in the cell, either as messenger RNA (mRNA), transfer RNA (tRNA) or ribosomal RNA (rRNA). In contrast to the deoxyribose sugars in DNA, ribose sugars that form RNA are less stable and easily degrade. Also, RNA is typically single-stranded, shaped as a hairpin, or double-stranded as in viruses.
In contrast to DNA, RNA is not a single long strand of genetic material that contains all the genes in an organism’s genome. Each gene is broken up into many introns and exons, with each gene having its own functional properties. The exon regions of DNA, which are the parts of sequence that contain useful instructional sequences, are read by RNA polymerases, and the resultant exon fragments are pieced together to form a single mRNA transcript whose sequence is completely dependent on the genetic code of the template DNA sequence.
Function of RNA
RNA is transcribed from DNA when a particular gene is needed by the cell. Unlike DNA, which is maintained at a constant level (i.e. 23 pairs of chromosomes in humans), the amount of mRNA in a cell is continually changing. Depending on the type of cell and the function of a particular gene in response to certain stimuli, the cell may require a massive production of one gene (i.e. upregulation) and zero transcription of another gene (i.e. downregulation).
Messenger RNA transcripts are translocated out of the nucleus and into the cytoplasm where protein synthesis occurs. At this point, tRNA molecules bring amino acids to ribosomes where rRNA links the amino acids together to form full length proteins.
Ribosomal RNA makes up a majority of all cellular RNA (up to 80%). This amount of rRNA demands an intensive transcription of genes which code for its production. The genome compensates for this need by providing copies of the genes that code for rRNA. In eukaryotes, this consists from several hundred (~200 in yeast) to tens of thousands of organized genes. Genes coding rRNAs in a human being are also organized into groups and located in central regions of chromosomes 13, 14, 15, 21, and 22.
RNA is manufactured in the cell during transcription, where RNA polymerases use DNA as a template to produce complementary RNA. Ribosomes in the cell use the RNA as a template to produce proteins, in a process called translation. Three types of RNA are involved in this unique process: messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA). In addition to transcription and translation, RNA is also involved in the activation and deactivation of genes.
Transfer RNA is involved with decoding the messenger RNA sequence into amino acids that form various proteins. tRNAs function at specific locations inside the ribosomes during the translation process — the A (aminoacyl), P (peptidyl), and E (exit) sites. tRNAs are folded structures that contain three hairpin loops, where one loop contains the complementary RNA sequence (anticodon) to the mRNA codons. Each tRNA has its own anticodon on its end, and when it recognizes a codon on the mRNA, it will transfer the corresponding amino acid to the growing amino acid chain. Emerging research has also found some evidence suggesting that tRNAs participate in stress responses as signaling molecules (https://www.nature.com/articles/nrg3861).
Utility of RNA in Medicine
RNA enables extensive benefits to the laboratory and to novel therapeutic medicines. Following well established RNA isolation protocols, RNA can be isolated from tissues of study animals, plants, bacteria, RNA-containing viruses, and tumors. Upon isolation, the concentration of RNA can be measured since the nucleotide bases of RNA absorb ultraviolet light at a wavelength near 260 nm. The isolated RNA enables researchers to determine individual gene expression levels of a test sample (i.e. treated or tumor sample) relative to a control (i.e. non-treated or normal tissue sample).
After gathering a full understanding of differential gene expression via qRT-PCR or RNA-Seq, scientists then attempt to genetically correct any cellular alterations by intracellular delivery of RNA molecules via transfection in which RNA is reintroduced into cells by electroporation or using liposomes that fuse with the cell membrane to deliver the RNA payload to the interior of the cell. The resultant delivery and resultant downregulation of the target RNA molecules is due to RNA interference (RNAi) in which innate mechanisms find and then regulate or degrade the complementary mRNA sequence of the transfected RNA (i.e. miRNA, siRNA, piRNA). RNAi effects have been induced in both mammalian cells and in tissues for endogenous and alien genes. The resultant loss of expression can be partial or complete, allowing the response to be tailored to the needs of a given experiment. A crucial consideration in RNAi products is the length of the double-stranded RNA (dsRNA), as any strand longer than thirty base-pairs may trigger an interferon response leading to cleavage of mRNA and possibly apoptosis. Care must also be taken in the design of RNAi therapeutics such that they do not induce innate immune responses or normal cell functions in context of foreign particles.
Double-stranded RNA can be delivered into cells through several specialized transfection techniques:
- Viral induction
- Electroporation of siRNA
- Plasmid producing shRNA
- miRNA introduction via nanoparticles
RNA has been harnessed for its ability to silence genes or lower gene expression, where a small complementary fragment of RNA is used by cellular mechanisms to limit expression of a gene. In vivo transfection (mRNA or siRNA transfection) is known to be difficult due to short RNA being easily degradable. Some companies have developed specialized RNA transfection reagents (see transfection kits) to help researchers achieve high transfection efficiency both in vitro and in vivo.
RNA has also been the basis of many therapeutic techniques over the past years. Antisense RNAs and siRNAs are being used in drugs that are being implemented in clinics, and RNA aptamer and mRNA has been the topic of active research for new drugs. RNA therapy has the ability to target all genes in the cells, which provides it with advantages over other small molecule-based therapies. Givosiran is one example of an siRNA drug that recently got approved by the FDA in 2019. The drug is aimed to treat acute hepatic porphyria, which is a rare, inherited genetic disease by suppressing the translation of an mRNA, reducing neurotoxic intermediates (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7250668/).