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Albany 2001

category image Biomolecular
Stereodynamics
SUNY at Albany
June 19-23, 2001

Triplex Formation by Psoralen-Conjugated Chimeric Oligonucleoside Methylphosphonates

Anti-gene oligonucleotides have been proposed as reagents for modulating gene expression. This can be achieved by utilizing triplex forming oligonucleotides (TFOs), that are capable of binding to purine sequences of duplex DNA. We have used electrophoretic mobility shift assays to investigate the binding interactions between nuclease resistant, 5Õ-psoralen-conjugated, chimeric methylphosphonate oligodeoxyribo- or oligo-2Õ-O-methylribo-triplex-forming oligonucleotides (TFOs), and a purine sequence found in the envelope gene of HIV proviral DNA (env-DNA). {1,2} These chimeric TFOs have backbones that are mixtures of methylphosphonate and phosphodiester internucleotide bonds. For example, 1mp, a pyrimidine chimeric TFO composed of thymidine and 5-methyl-2'-deoxycytidine (C), d-PS-TpCpTpCpTpCpTpTpTpTpTpTpCpTpC , (where PS is trimethylpsoralen, and p is a methylphosphonate internucleotide linkage), forms a stable triplex with env-DNA, whose dissociation constant is 1.3 mM at 22 oC and pH 7.0. TFO 2mp, d-PS-UpCpTpCpTpCpTpUpTpUpTpUpCpTpC, contains 5-propynyl-2'-deoxyuridines (U), and has a dissociation constant of 400nM. This increase in binding affinity is consistent with the increased stacking interactions and hydrophobic nature of 5-propynyl-U. A chimeric oligo-2'-O-methylribo pyrimidine TFO, mr-PS- UpCpUpCpUpCpUpUpUpUpUpUpCpUpC (4mp) also forms a triplex, whose stability is comparable to that of 1mp (Kd = 1.5 mM). In addition to maintaining strong binding affinity, the 2Õ-O-methylribo backbone, in combination with methylphosphonate linkages, is very resistant to nuclease hydrolysis. The binding affinities of 1mp, 2mp, and 4mp were less than those of their all phosphodiester counterparts (Kd 470-50nM). This reduced binding affinity may be due in part to the racemic nature of the methylphosphonate linkages. We are currently investigating the effect of methylphosphonate configuration on TFO binding affinity.

References and Footnotes
  1. Cassidy, R.A.; Kondo, N.S.; Miller, P.S. "Triplex Formation by Psoralen-Conjugated Chimeric Oligonucleoside Methylphosphonates". Biochemistry, 2000, 39, 8683-8691.
  2. Miller, P.S.; Cassidy, R.A.; Hamma, T.H.; Kondo, N.S. "Studies on anti-human immunodeficiency virus oligonucleotides that have alternating methylphosphonate/phosphodiester linkages". Pharmacology & Therapeutics, 2000, 85, 159-163.

Rachel A. Cassidy* and Paul S. Miller

Department of Biochemistry and Molecular Biology, School of Hygiene and Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205
*Phone 410-955-3171; Fax 410-955-2926; cassidy@jhu.edu