RNA duplex with
tandem dimethylguanosine: adenosine pairs (m2G: A pairs)
Created by Ramzi Shaykh
RNA molecules are typically single-stranded, but it is common to have double stranded regions that form complementary sequences within the chain to join via intra-strand base pairing 1. RNA-RNA duplexes can, however, occur elsewhere. Double stranded RNA is often a sign of trouble because many viruses form long strands of double-stranded RNA as they replicate their genome. RNA interference is a process that occurs in plant and animal cells that attacks the viral RNA, and short sequences of double stranded RNA called small interfering RNA (siRNA) are involved in this process2. Similarly, microRNAs (miRNA) are a family of small, non-coding RNAs also around 21-25 nucleotides long that have been found to regulate gene expression in a post-transcriptional level3.
RNA duplexes are being studied intensively in order to discover what other functions they can have. Additionally, non-natural interfering RNA can now be used as a fast and easy way to determine the function of a gene. This is done by putting the synthetic RNA duplex into a cell and destroying certain mRNA that we want to target for destruction. This destruction of the mRNA prevents the translation of the protein; then, one can determine the initial function of the gene by seeing what is wrong with the cell. 2 The RNA duplex in this assignment is an RNA duplex with tandem dimethylguanosine: adenosine pairs (m2G: A pairs) (PDB ID: 3CJZ) and can be made synthetically and thus could very well be used for this purpose. Further research hopes to discover further functions of these RNA duplexes as well as more ways to utilize them in medicine.
In biochemistry, a common theme is the importance of the association between structure and function of a particular molecule. This paper will examine the structure of an RNA duplex with tandem dimethylguanosine: adenosine pairs (m2G: A pairs) as well as how that structure affects the properties of the nucleic acid. This is a nucleic acid sequence is a double stranded RNA with two complimentary strands (strand A and strand B). The RNA duplex is 13 nucleotides long, has a structure weight of 8401.24 daltons, and has a %GC of 69.23% [4]. The two methyl groups added to the exocyclic nitrogen of guanosine lead to significant changes in properties compared to an RNA molecule that does not have the extra methyl groups on its guanine. The dual methylation on the guanosine eliminates the ability of the exocyclic nitrogen to donate a hydrogen and create a hydrogen bond between that hydrogen and the N7 nitrogen atom on the next nucleotide, adenine. This, in turn, alters its pairing behavior with cytosine. The less noticeable consequence of the dimethylation is its role in controlling the pairing modes between G and the adjacent A4 .
Four varieties of G:A base pairs have been observed in the past: imino hydrogen-bonded both with and without a bridging water molecule, the sheared type in which the major groove face of adenine interacts with the minor-groove face of guanine, and the unusual G(syn):A(anti) base pairing. In the case of tandem G·A pairs, the type of pairing has been shown to depend on both the order (G·A/A·G vs. A·G/G·A) and the surrounding base pairs.5
The methylation at the guanine nucleobase can affect the two most common G:A mismatch pairing modes, which are the sheared and the imino-hydrogen bonded G:A conformations. In the native state (with no methylation), the G:A pair can adopt either conformation. Singly methylated guanine can only pair with A in the sheared conformation. The presence of two methyl groups on N2 limits the conformation to only the imino- hydrogen bonded type. The sheared conformation often seen in tandem G:A pairs is avoided due to a possible steric clash between N2-methyl groups and the major groove edge of A4.
Many RNA duplexes have modifications that can alter their function. Another example of this is an RNA duplex in which there is a single bulge motif. The PDB ID number of such an RNA duplex is 2JXQ 6. A bulge, otherwise known as an internal loop, occurs where the RNA strand is forced into a short single-stranded loop because one or more base pairs along one strand in an RNA double helix finds no base pairing partners1. In the nucleic acid examined here, the bulge is due to only one base pair that does not have a partner. They are common motifs in folded RNA molecules, providing structural flexibility required for RNA folding 3.
The structures of these two RNA duplexes do have some commonalities. Both of the nucleic acids are arranged in a right-handed helix, and the lengths of the nucleotide sequences were very similar (13 vs 10). The helix of each has characteristics similar to that of the Watson and Crick model for nucleic acids pairing in a helix. The RNA duplex with tandem m22 G:A pairs has a %GC of 69.23% and the RNA duplex with a single bulge motif that was also looked at had a %GC of 70%[4], [6].. The modifications on each of the RNA duplexes plays a role in its function. Both of the RNA duplexes have two instances of G: A adjacent pairing modes in their sequences. Although the two have different modifications, it is likely that both play a role in controlling secondary and tertiary structure.
It is also significant to look at changes in secondary structure as a result of methylation. Methylation can affect the equilibrium of the duplex-hairpin conversion with RNA nucleotides. One study looks at the self-complementary sequence rCGCGAAUUCGCGA similar to that of the nucleic acid being looked at in this paper, which forms a stable Watson and Crick base-paired duplex. It is shown that the sequence is forced to adopt a hairpin conformation if one of the central 6 nucleotides is replaced by the corresponding methylated nucleotide N2,N2-dimethylguanosine. This is almost exactly the same as the RNA duplex examined in this paper in that it is 13 nucleobases long, has tandem G:A base pairs, and considers the dimethylation of the guanosine. Nucleotides that are methylated at the Watson–Crick base pairing site can greatly affect RNA structure by controlling folding into different secondary structure motifs. This can be shown by a comparison of selected sequences that exist in duplex conformations but change into hairpin conformations if single nucleobases are replaced by the corresponding methylated ones. The methyl groups on the guanosine are located at sites that usually involve H-bonding within a normal A-form duplex. Methylation of a nucleobase reduces the possibilities for hydrogen bonding and leads to a large amount of steric interference. This restricts the number of energetically favorable mismatches between a methylated nucleobase and the former pairing partner. Subsequently, the probability of retaining the duplex structure after methylation is decreased compared to after the introduction of a mismatch.7
The geometry of m2 2G:A pairs differs significantly from that of G:A base pairs. In normal 13 nucleotide RNA duplexes with tandem G:A base pairs that are not methylated, there is little propeller twisting. Also the lengths of the hydrogen bonds between (G)N1-H and N1(A) and (G)O6and H-N6(A) are almost equivalent. On the other hand, the lengths of the same hydrogen bonds in the duplex with m2 2G:A differ from one another by .49 Å. In addition, some propeller twisting exists. The m22G6:A20 pair displays an opening of −35°, and the A7:m2 2G19 pair also shows a significant opening of 23°. These changes in geometry help to minimize the steric hindrance that is created by the additional methyl groups. The increase in length of the hydrogen bonds does, however, decrease the thermodynamic stability of the molecule. There is also a reduction of hydration around the around m2 2G:A pairs that also contributes to the decrease in thermodynamic stability. This reduction of hydration is a result of the increase in hydrophobicity as a result of the methyl groups. The major groove has a normal hydration pattern due to more space, but the presence of methyl groups interferes with the water structure in the center of the minor groove, leading to an overall decrease in the amount of hydration. The major and minor groove thus have differences in hyrophobicity due to the difference free space. The decrease in thermodynamic stability can be seen by its lower values for Tm in comparison with the same RNA duplex that is not dimethylated.4 Dimethylation may seem like a simple modification to a molecule, bus as seen in this paper, it can lead to significant changes in the secondary structure as well as other properties of this 13 nucleotide RNA duplex.