Triple-helical structures involving the interaction of an oligonucleotide third strand with a duplex nucleic acid sequence have recently gained attention as a therapeutic strategy in the "antigene" approach [cf. Helene, C. (1991) Eur. J. Cancer 27, 1466-1471]. This method utilizes the triple helix formed from the cellular duplex and an added third strand to directly regulate the activity of a selected gene. The limited stability of nucleic acid triple-helical interactions, particularly if the third strand has backbone modifications such as methylphosphonate or phosphorothioate substitutions, is a limiting condition for the use of this approach. We have designed and synthesized compounds, on the basis of the following three criteria, that we feel should provide selective interactions and significant stabilization of triplexes: appropriate aromatic surface area for stacking with triplex bases in an intercalation complex, positive charge, and limited torsional freedom in the aromatic system to match the propeller twist of the triple-base interactions in the triplex. A series of quinoline derivatives with an alkylamine side chain at the 4-position and with different aryl substituents at the 2-position has been synthesized as our first compounds. A 2-naphthyl derivative provides significant and selective stabilization of the triplex. In a 0.2 M NaCl buffer, the naphthyl derivative increased the Tm for the triplex (triplex to duplex and third strand transition) by approximately 30 degrees C more than the Tm increase for the duplex (duplex to single strands transition). Spectral changes and energy-transfer results indicate that the naphthyl compound and related derivatives bind to the triplex by intercalation.(ABSTRACT TRUNCATED AT 250 WORDS)
Pasteurella multocida is a mucosal pathogen that colonizes the respiratory system of susceptible hosts. Most isolates of P. multocida produce sialidase activity, which may contribute to colonization of the respiratory tract or the production of lesions in an active infection. We have cloned and sequenced a sialidase gene, nanH, from a fowl cholera isolate of P. multocida. Sequence analysis of NanH revealed that it exhibited significant amino acid sequence homology with many microbial sialidases. Insertional inactivation of nanH resulted in a mutant strain that was not deficient in sialidase production. However, this mutant exhibited reduced enzyme activity and growth rate on 2-3 sialyl lactose compared to the wild type. Subsequently, we demonstrated the presence of two sialidases by cloning another sialidase gene that differed from nanH in DNA sequence and substrate specificity. NanB demonstrated activity on both 2-3 and 2-6 sialyl lactose, while NanH demonstrated activity only on 2-3 sialyl lactose. Neither enzyme liberated sialic acid from colominic acid (2-8 sialyl lactose). Recombinant E. coli containing the sialidase genes were able to utilize several sialoconjugants when they were provided as sole carbon sources in minimal medium. These data suggest that sialidases have a nutritional function and may contribute to the ability of P. multocida to colonize and persist on vertebrate mucosal surfaces.Pasteurella multocida is a gram-negative coccobacillus of the family Pasteurellaceae and is a normal inhabitant of the upper respiratory system of many animals (24). The organism has a broad host range and is commonly a secondary pathogen in upper respiratory infections. Serotype D virulent isolates are toxigenic, but all serotypes produce capsules which confer serum resistance and resistance to phagocytosis (42). However, it is unusual to isolate a P. multocida strain that does not produce sialidase activity (40). Sialidases (neuraminidases; EC 3.2.1.18) are enzymes that liberate sialic acid from sialylconjugated glycoproteins, glycolipids, or colominic acids by cleaving alphaketosidic linkages. It is hypothesized that sialidase contributes to the virulence of some pathogenic organisms, especially those that inhabit and invade mucosal surfaces (7). Drzeniek (14) found sialidase activity in bacterial isolates that belong to the orders Pseudomonadales and Eubacteriales, and sialidases have been cloned from Clostridium species (35,36,37), Vibrio cholerae (48), Streptococcus pneumoniae (4, 5), Micromonospora viridifaciens (38), and Salmonella enterica serotype Typhimurium (21). Many of these bacterial sialidases have about 20% similarity at the amino acid level (21).Sialidases have been implicated as bacterial virulence factors (7, 34). It has been shown that a sialidase-deficient mutant of S. pneumoniae was less able to colonize and persist on mucosal surfaces than the wild type (46). In addition, a Bacteroides fragilis sialidase-deficient mutant was attenuated in the rat abscess model (18). The role of sialidase in ...
Deoxyribooligonucleotides containing 19 repeating bases of A, T or U were prepared with normal phosphodiester (dA19, dT19, dU19) or methylphosphonate (dA*19, dT*19, dU*19) linkages. Complexes of these strands have been investigated at 1:1 and 1:2 molar ratios (purine:pyrimidine) by thermal melting and gel electrophoresis. There are dramatic sequence dependent differences in stabilities of complexes containing methylphosphonate strands. Duplexes of dA*19 with dT19 or dU19 have sharp melting curves, increased Tm values, and slopes of Tm versus log (sodium ion activity) plots reduced by about one half relative to their unmodified 'parent' duplexes. Duplexes of dA19 with either dT*19 or dU*19, however, have broader melting curves, reduced Tm values at most salt concentrations and slopes of less than one tenth the values for the unmodified duplexes. Duplex stabilization due to reduced phosphate charge repulsion is offset in the pyrimidine methylphosphonate complexes by steric and other substituent effects. Triple helical complexes with dA19 + 2dT19 and dA19 + 2dU19, which can be detected by biphasic melting curves and gel electrophoresis, are stable at increased Na+ or Mg+2 concentrations. Surprisingly, however, no triple helix forms, even at very high salt concentrations, when any normal strand(s) is replaced by a methylphosphonate strand. Since triple helical complexes with methylphosphonates have been reported for shorter oligomers, inhibition with larger oligomers may vary due to their length and extent of substitution.
The effects of ligand structure and properties, DNA backbone modifications and DNA sequence on the interaction of a variety of well-known groove-binding agents and intercalators with DNA duplexes and triplexes have been evaluated by thermal melting experiments and molecular modeling. Both methylphosphonate and phosphorothioate substitutions generally destabilize DNA duplexes and triplexes. Modified duplexes can be strongly stabilized by both groove-binding agents and intercalators whereas triplexes are primarily stabilized by intercalators. Of the compounds tested, the intercalators coralyne and quinacrine provide the largest stabilization of the triplex dT19.dA19.dT19. Molecular modeling studies suggest that the large intercalating ring system of coralyne stacks well with the triplex bases whereas the alkylamino side chain of quinacrine fits snugly into the remaining space of the major groove of dT19.dA19.dT19 triplex and forms extensive van der Waals contacts with the thymine methyl groups that line the groove. Converting some of the T.A.T base triples to C+.G.C (e.g. dT19.dA19.dT19 to d(T4C+)3T4.d(A4G)3A4.(T4C)3T4) causes very significant decreases in observed Tm increases for compounds such as quinacrine and coralyne. Although removal of thymine methyl groups and addition of positive charge on substitution of C+.G.C for T.A.T should reduce binding of cationic intercalators, the large difference observed between the pure AT and the mixed sequence triplexes suggest that they may also have differences in structure and properties.
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