The hypermodified dinucleoside monophosphates, uridylyl(3'-5')-N-(9-(..beta..-D-ribofuranosyl)purin-6-ylcarbamoyl)threonine (Upt/sup 6/A), adenylyl(3'-5')-N/sup 6/-(..delta../sup 2/-isopentenyl)adenosine (Api/sup 6/A), adenylyl(3'-5')-N/sup 6/-(..delta../sup 2/-isopentenyl)-2-methylthioadenosine (Apms/sup 2/i/sup 6/A), and adenylyl(3'-5')-l,N/sup 6/-ethenoadenosine (Ap epsilon A,, a synthetic model for adenylyl(3'-5')wybutosine, ApyW), which represent the most common sequences found as the third letter of the anticodon triplet and its adjacent 3' neighbor, have been isolated. Their solution properties have been investigated using ultraviolet absorption, circular dichroism (CD), and high resolution proton magnetic resonance. The properties of these molecules have been compared with those of their unmodified counterparts, uridylyl(3'-5')adenosine (UpA) and adenylyl(3'-5')adenosine (ApA). These properties measured as a function of temperature have been analyzed employing a two-state intramolecular stacking model. All of the properties show that the stacking of Upt/sup 6/A is stabilized relative to UpA, while Api/sup 6/A, Apms/sup 2/i/sup 6/A, and Ap epsilon A are slightly destabilized relative to ApA. Thus, Upt/sup 6/A, Api/sup 6/A, Apms/sup 2/i/sup 6/A, and Ap epsilon A have comparable stacking equilibria, indicating that the modifications remove the large difference in stacking between UpA and ApA. Furthermore, cytidylyl(3'-5')adenosine (CpA), which is the most common unmodified sequence in this particular anticodon region,exhibits a stability similar to those of the hypermodified dinucleoside phosphates. Hypermodification therefore seems to keep themore » flexibility of this crucial part of the tRNA constant. It is proposed that this may result in a more smoothly regulated translation step. Also, it is proposed that the enhanced stacking of Upt/sup 6/A relative to UpA prevents the incorrect wobble base pairing of this U residue in the tRNA during translation.« less