Tools for Studying RNA Structure and Function

By affecting cellular process mechanisms, RNA is believed to play a central role in transcription initiation, elongation, termination, mRNA splicing, as well as retroviral infection of eukaryotic cells.

RNA molecules, typically comprised of single- and double-stranded regions, assemble in complex, three-dimensional structures. It is these structures that go on to affect RNA’s interactions with other nucleic acids, proteins, and small molecules in the aforementioned cellular processes. 

To further understand RNA’s structure and function, we have developed an extensive portfolio of products for RNA synthesis and modification.

Used to study base-pairing, ribonuclease (RNase) cleaves at either single- or double-stranded RNA regions. The below RNase can be used for RNA structure studies, RNA sequencing, protein footprinting, and boundary experiments.

• RNase A: Cleaves single stranded RNA (ssRNA) at phosphodiester bonds between the 3’-phosphate groups of pyrimidine nucleotides (C and U).
• RNase T1: When applied to ssRNA,cleaves guanine’s 3’-phosphate group’s phosphodiester bond.

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RNA structure and function can be determined by the use of RNA polymerases, which are enzymes that produce different types of RNA. Common RNA polymerases are listed below.

• Poly(A) polymerase: Catalyzes the addition of adenosine to the 3´ end of RNA in a sequence-independent fashion.
• T7 RNA polymerase: Catalyzes the 5´→3´ synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter.
• SP6 RNA polymerase: Catalyzes the 5´→3´ synthesis of RNA on either single-stranded DNA or double-stranded DNA downstream from its promoter and incorporates modified nucleotides.

See RNA polymerase promoter sequences
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Both RNA structure and function studies use enzymes, such as ligases and transferases, to distinguish sections of RNA that are important to its natural function.

• Anza T4 polynucleotide kinase: Transfers the terminal phosphate of ATP to the 5´ hydroxyl terminus of DNA or RNA. Used for 5´ end-labeling of oligonucleotides and polynucleotides.
• T4 RNA ligase: Catalyzes the formation of a phosphodiester linkage between a 5´-phosphoryl-terminated ribonucleic acid and a 3´-hydroxyl-terminated ribonucleic acid.
• Terminal transferase: Catalyzes the addition of deoxynucleotides to the 3´ hydroxyl terminus of DNA.

Phosphorylation, or the addition of a phosphoryl group to a molecule, is a modification that can be done on nucleic acids to signal to downstream processes. Phosphatases have been used in studies of RNA function.

• CIP (calf intestinal phosphatase): Phosphomonoesterase that removes 3´ and 5´ phosphates from DNA and RNA.
• BAP (bacterial alkaline phosphatase): Removes 3´ and 5´ phosphates from DNA and RNA. BAP is active at 65°C for at least 1 hour and is inactivated by phenol extraction.

Running the same assays with multiple samples? These transcription kits efficiently isolates total RNA from a variety of samples with high yields.

• MEGAshortscript High Yield Transcription Kit: Efficiently transcribes large quantities of RNA from short templates, <300 bases.
• MEGAscript SP6, T7, and T3 kits: Synthesize ultra-high yields of RNA, 10 to 50 times as much as produced by conventional transcription reactions.

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Visualization of nucleic acid fragments is important for studying RNA structure and function.

• KinaseMax 5' End Labeling Kit: For end-labeling DNA, RNA, and oligonucleotides using T4 polynucleotide kinase and [γ-³²P]ATP.

RNA function is studied through the use of caps and modified UTPs, which allow RNA to either get tagged for degradation or translation.

• Cap analog and cap variants: m⁷G(5´)ppp(5´)G (cap analog) and cap variants. The cap analog is used for synthesis of 5´-capped RNA molecules.
• Modified UTPs: Incorporate these nucleotides to confer unique characteristics and test for particular reactive groups.

1. Moine H, Ehresmann B, Ehresmann C, and Romby P. (1998) Probing RNA structure and function in solution. In: Simons RW and Grunberg-Manago M (editors). RNA Structure and Function, Cold Spring Harbor Laboratory Press. p. 77–115. 
2. Knapp G. (1989) Enzymatic approaches to probing of RNA secondary and tertiary structure. Methods Enzymol 180:192–212. 
3. Krol A and Carbon P. (1989) A guide for probing native small nuclear RNA and ribonucleoprotein structures. Methods Enzymol 180:212–227. 
4. Tinoco I Jr, and Bustamante C. (1999) How RNA folds. J Mol Biol 293(2):271–281. 
5. Doudna J A. (2000) Structural genomics of RNA. Nature Struct Biol 7 Suppl:954–956.