Three triple-helical hairpin DNAs with substitution of 5-bromocytosine for cytosine in different strands have been investigated by molecular mechanics and Raman spectroscopy. The stability of the three substituted triplexes were compared with the corresponding unsubstituted triplex DNA by the molecular mechanics method. Base stacking interactions and strand]strand interactions of each triplex were analyzed in detail. Sugar conformations in these triplexes have been determined by both vibrational spectroscopy and molecular dynamics simulation. The hairpin triplexes with substitution occurring in strand I or both in strands I and III have the main sugar conformation of C39-endo, while the triplex with substitution occurring in strand III is the combination of C39-endo and C29-endo sugar conformation. Theoretical results are basically in agreement with experiments. Q 1999 Elsevier Science B.V. All rights reserved.