PROBING CONTACTS TO THE DNA BACKBONE IN THE TRP REPRESSOR-OPERATOR SEQUENCE-SPECIFIC PROTEIN-NUCLEIC ACID COMPLEX USING DIASTEREOMERIC METHYLPHOSPHONATE ANALOGS

Fourteen analogue DNA sequences containing the trp operator sequence a nd a single diastereomeric methylphosphonate linkage are each prepared from the stereochemically pure nucleoside methylphosphonate dimer bui lding block, prepared as a phosphoramidite. The analogue sequences are shown to be single diastereomers on the basis of HPLC analysis of the digestion mixture; in each case, only a single diastereomeric dimer i s present. These analogue sequences can be used effectively to probe f or interactions to either of the prochiral phosphate oxygens as illust rated by their use to identify critical interactions in the trp repres sor-operator complex. In a number of cases, the pairs of diastereomeri c analogue sequences exhibit variable binding affinities that can be u sed to identify one of the prochiral phosphate oxygens as a critical s ite for complex-stabilizing interactions. Upon the basis of dissociati on constants, apparent incremental binding energies are assigned to sp ecific interactions. In all but one example, these identified sites fo r interactions to the phosphate backbone can be correlated with contac ts implicated by the crystal structure analysis of the trp repressor-o perator complex.