## 2 Influence of tRNA Core Modification on tRNA Structure and Stability  
### Modifications and Their Effect on the Physicochemical Properties of the Nucleotides  
The majority of the basic knowledge concerning the impact of core modifications on tRNA structure was mainly discovered towards the end of the last century and has been comprehensively reviewed and discussed elsewhere [16; 17; 45; 46; 47; 48]. We provide here only a concise overview of these fundamentals, which are at the root of all local and global molecular changes associated with the introduction of modifications.  
Pseudouridylation, 2\({}^{\prime}\)-O-methylation and 2-thiolation enhance tRNA structural stability through stabilization of the C3\({}^{\prime}\)-endo conformation of the ribose moiety and improvement of the base stacking properties of these modified nucleotides [49; 50; 51; 52]. This enhances the thermostability of tRNAs containing these modifications. For instance, \(\Psi\)55, \(\Psi\)40 and Gm18 individually increase the melting temperature of _E. coli_ tRNASer [53], and m5\({}^{\prime}\)52U54 increases the melting temperature of thermophilic tRNAs [54; 55]. In addition, 4-thiolation, such as s4U8, also increases tRNA melting temperatures [53], probably by strongly reinforcing the s4U8-A14 reverse Hoogsteen base pair (Figure 1c) [56]. Furthermore, as Gm18 and \(\Psi\)55 are involved in a tertiary interaction between the D- and T-arms of the tRNAs (Figure 1c,d), they are implicated in stabilizing their global tertiary structure and, more particularly, their elbow region [57]. Pseudouridine exhibits a third stabilizing effect by providing an additional hydrogen-bond donor (H-N1) that may form a water-mediated interaction with the phosphate backbone [58; 52; 59]. This additional link increases the rigidity of the local tRNA structure and may contribute to the proper tertiary folding of tRNAs [60].  
Simple methylations such as m5U or m5C also exhibit stabilizing properties due to their increased hydrophobicity, increased base polarizability, increased stacking capabilities and reinforcement of m5U54-m1A58 and G15-m5C48 tertiary interactions (Figure 1d) [60; 61; 62]. This explains why the presence of m5U54 increases the melting temperature of tRNAs [53; 54]. Other methylations such as m1A58 and m7G46 introduce a positive charge at the purine aromatic ring of the base, which may stabilize the tRNA structure by interacting with negatively charged phosphates ions of the tRNA backbone or by stabilizing certain tertiary interactions, such as the C13-G22-m7G46 base triplet (Figure 1c,d) [47; 63]. Other methylations, such as m1A9 and m2G26, disrupt the Watson-Crick base-pairing capacity of these nucleotides and are individually essential for the formation of the proper cloverleaf secondary structure and three-dimensional folding of certain tRNAs [64; 65; 66].  
Only the dihydrouridine (D) modification has the capacity to enhance tRNA structural flexibility. Dihydrouridines effectively promote the C2\({}^{\prime}\)-endo sugar conformation and prevent proper stacking interaction with neighboring bases, thereby allowing for greater conformational flexibility and dynamic motion in the tRNA D-loop (reviewed in [67]).