Guanine quadruplexes and disease: Insight into mechanisms and therapeutic potential Katie Manas and Celena Hoeve Abstract Understanding Guanine Quadruplexes (G4s) provides valuable insight into a wide array of diseases at a genetic level and their potential therapy. These unusual architectures can form in DNA and RNA, composed of planar, stacked tetrads of four hydrogen bonded guanines. G4s are widely implicated in a range of disease categories, including parasites, viral infections, cancer, and neurodegenerative diseases, with varied roles within each. Of current relevance, a connection to the coronavirus family may be an intriguing avenue of investigation for recent outbreaks. The planar surface of the G4 tetrad also provides an attractive target for small molecule binders that can provide therapeutic value. Overall, this review aims to address the exciting relevance of G4s in these felds, a few major mechanisms of action, and their potential as therapeutic targets. 1. Introduction arise from the orientation of the four guanine segments with Though most are familiar with DNA as a double stranded respect to the 3’ and 5’ ends. Parallel G4s have all strands helical structure composed of four nucleobases (ACTG), it oriented in the same direction, while antiparallel G4s have also has the ability to form other higher-order architectures, the four strands in alternating directions. Hybrid G4s have such as the well-known guanine quadruplex (G4). G4s were three strands oriented in one direction and the fourth strand frst proposed in 1962 (Gellert et al. 1962), as three-dimen- in the other direction. G4s can also be described based on sional architectures that form in guanine-rich environments. the loops, which create a unique environment surrounding Four guanine nucleobases self-assemble via hydrogen bond- the stacked guanine tetrads of the G4. The structural features ing into a planar guanine tetrad; these tetrads then stack on of G4s have previously been reviewed (Keniry 2001). These one another to form the three-dimensional G4 architecture structures are often determined by techniques such as solu- in the presence of stabilizing monovalent cations (Figure 1) tion-state NMR, which has been used with the c-Myc G4, (Pinnavaia et al. 1978). found in the promoter region preceding the gene (Figure 2) (Ambrus et al. 2005). A B Figure 1. A) Chemical structure of a guanine tetrad, showing the hydrogen bonding interactions of four guanine nucleobas- es and stabilized by a central cation (M+). Figure 2. Solution-state NMR structure of the c-Myc pro- B) G4 formed by stacked tetrads (stabilizing cations are omit- moter G4 with stabilizing cations (PDB ID: 1XAV) (Ambrus ted). et al. 2005). G4s can adopt parallel, anti-parallel, and hybrid topologies, G4s can assemble when guanine-rich regions of nucleic acid which can be useful for classifcation purposes. The topologies are in single-stranded forms. This can occur during activi- 40 40 ties such as replication and transcription, where the typically helicase, which is suggested to have the ability to unwind double stranded DNA is temporarily separated into single G4s. Therefore, P. falciparum can use the RecQ helicase to turn strands to allow for processing. It can also occur in RNA on transcription by unwinding the G4 structures upstream of molecules, which are intrinsically more diverse in shape, and the var genes (Smargiasso et al. 2009). often exist as single strands (Ji et al. 2011). G4 structures play important roles in nucleic acid processing in both humans and other organisms, which has made G4s an active area of research in recent years. We aim to provide a review that demonstrates the relevance of G4s by describing their im- portance in parasitic infection, viral infection, cancer, and neurodegeneration. We will cover G4 mechanisms of action in these areas, as well as the potential of G4s as targets for therapeutic treatments. 2. Parasites 2.1. History of G4s in parasites Although G4s are now better known for their roles in can- cer and viral activity, they were originally characterized in protozoa such as Oxytricha and Tetrahymena (Williamson et al. 1989). Early crystal structures were also determined in Oxytri- cha (Kang et al. 1992). Despite this early interest in protozoan G4s, research in this area decreased when the focus shifted Figure 3. Trypanosoma parasites to G4s in cancer and infectious disease. In recent years, new A) Transcription-on state in mediated by the formation of research into the roles of G4s in parasites has started to re- RNA G4s. B) Replication-on state mediated by the formation surface. The new research has provided further insight into of DNA/RNA hybrid G4s. the action mechanisms of G4s in protozoa, and the potential to exploit these structures in treatments of parasitic protozoa. There is also evidence suggesting that G4s play a role in recombination events in P. falciparum. A strong relationship 2.2. G4 mechanisms of action in parasites has been established between breakpoints and PQSs. A possi- One interesting aspect of protozoan genomes is that some ble mechanism could be that breaks occur when the replica- of them contain a high proportion of A and T nucleotides tion fork is stalled by a G4, and recombination occurs when compared to C and G nucleotides. For example, Plasmodium repair is carried out (Stanton et al. 2016). Parasites of the falciparum, the most severe of the malaria-causing parasites, Trypanosoma genus, implicated in conditions such as sleeping has a genome with an A+T composition of 80.6% (Gardner et sickness (T. brucei) and Chagas disease (T. cruzi), also use G4s al. 2002). However, despite such AT-rich genomes, G4-form- in their genome. Here, G4s are used as a switch to mediate ing sequences have been preserved in P. falciparum as well as transcription and replication. As RNA polymerase synthesiz- several other species (Smargiasso et al. 2009), suggesting that es pre-mRNA, there are two options for G4 formation. The they play an important role in the genome. frst is G4 formation using exclusively the pre-mRNA. This In P. falciparum, 16 putative G-quadruplex sequences (PQSs) leads to RNA editing and continued transcription. The sec- have been identifed upstream of the group B var genes ond is the formation of a DNA/RNA hybrid G4. This blocks (Smargiasso et al. 2009). The var genes encode P. falciparum transcription, allowing replication to occur instead. Making erythrocyte membrane protein 1 (PfEMP1), which is a sur- transcription and replication mutually exclusive allows these face antigen. At any given time, only a single var gene is two processes to be carried out on the circular DNA without being expressed, and switching this expression is a method interfering with each other (Figure 3) (Leeder et al. 2016). used by P. falciparum to evade immunity (Kyes et al. 2007). The Another species in which G4 activity has been investigated presence of PQSs upstream of the group B var genes suggests is Leishmania tarentolae, a parasite that causes leishmaniasis. In that these structures may play a role in regulating the var L. tarentolae, as well as in several other kinetoplastid protists, genes by blocking transcription. P. falciparum encodes a RecQ some T nucleobases in the genome are altered through glu- cosylation and hydroxylation to give base J. The role of base 41 41 J is as a transcription terminator; it has been suggested that CoV) (Kusov et al. 2015). The link with SARS-CoV could be of the absence of base J would cause transcription to continue particular interest because of the recent outbreak of corona- beyond the points where it should be stopped, killing L. taren- virus disease (COVID-19), which is caused by viral infection tolae (van Luenen et al. 2012). The presence of G4s has been with SARS-CoV-2. Considering that both diseases are of the correlated with J insertion, suggesting that G4s may serve as coronavirus family, it may be worth investigating potential a landmark for this process (Genest et al. 2015). Therefore, G4 mechanisms in SARS-CoV-2. targeting G4s in L. tarentolae could be used to kill the parasite by preventing proper transcriptional termination. 3.1. G4s and antigenic variation G4s in viruses have been shown to play a role in antigenic 2.3. G4s as therapeutic targets in parasites variation (Cahoon and Seifert 2009; Kuryavyi et al. 2012), a It can be clearly seen that G4s are implicated in a variety process by which pathogens switch out the antigen-encoding of important processes for several parasitic species, which genes that are being expressed to alter recognizable charac- makes them a potential target for therapeutic purposes. Of- teristics and thus avoid being detected by the immune sys- ten, G4s are targeted with binders composed of a fat, aromat- tem (Domingo et al. 2016). G4-mediated antigenic variation ic core which can stack on top of the terminal guanine tetrad is well modelled by Neisseria gonorrhoeae, where the antigenic in the G4 to provide a stabilizing efect. Several known G4 variation of pilin proteins, which are found on the surface of ligands were tested to determine their stabilization of P. falci- the bacteria, has been linked to a G4 structure (Cahoon and parum G4s. Compounds such as Phen-DC3, Phen-DC6, and Seifert 2009). A G4 motif near the pilE gene forms a parallel telomestatin were shown to have strong stabilization of G4s G4, the structure of which seems to be crucial, as replace- in P. falciparum, which is promising for the use of these com- ment with other G4s resulted in the loss of pilin antigenic pounds as therapeutic tools. However, the G4 binders did not variation (Cahoon and Seifert 2009). This G4 recruits the show strong selectivity for P.