Effect of ionic strength on the hybridization of oligodeoxynucleotides with reduced charge due to methylphosphonate linkages to unmodified oligodeoxynucleotides containing the complementary sequence.
A 12-mer oligodeoxynucleotide containing 10 methylphosphonate bonds and 1 phosphodiester bond was shown to bind specifically to the restriction endonuclease fragment containing complementary DNA in a Southern blot. This 12-mer as well as 14-mer oligodeoxynucleotides containing 3 methylphosphonate and 10 phosphodiester bonds was used to examine the effect of reduced charge on the thermodynamics of binding to complementary DNA or complementary oligodeoxynucleotides with additional nucleotides overlapping both the 3' and 5' ends. The 14-mer oligodeoxynucleotides were synthesized with one methylphosphonamidite (A, C, G, or T). Melting profiles were examined by spectrophotometry for the 14-mers and by a gel-shift assay for the 12-mer. Nearest-neighbor free energy values were compiled for predicting concentration-dependent melting temperatures for all oligodeoxynucleotide hybridizations, including those involving adjacent dG residues. The free energy contribution to duplex formation from the dangling ends was about 1 kcal/mol. The free energy decrement due to introduction of each methylphosphonate linkage was -0.75 kcal/mol in high salt independent of the methylphosphonamidite used for synthesis of the oligodeoxynucleotide. However, the change in charge per nearest-neighbor base pair decreased from 0.26 to 0.0 when the nearest-neighbor base pair contained one methylphosphonate. Thus at very low salt, methylphosphonate-substituted oligodeoxynucleotides form more stable hybrids than analogous phosphodiester sequences. The 12-mer with 10 methylphosphonate bonds outcompetes the analogous phosphodiester 12-mer below 0.01 M NaCl. The temperature of 50% dissociation of bound oligodeoxynucleotide after being washed for 30 min was measured with a dot-blot assay. These results, together with the thermodynamic results, indicate that the substitution of methylphosphonate linkages at high salt only affects the reverse rate constant.
Quartin
R S
RS
Department of Microbiology, Mount Sinai School of Medicine, New York 10029.
Wetmur
J G
JG
D001482:Base Composition; D015139:Blotting, Southern; D004247:DNA; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D009994:Osmolar Concentration; D010957:Plasmids; D013816:Thermodynamics
D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; C032627:methylphosphonic acid; D004247:DNA