Search

The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT I (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHII (5'-G_CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHII. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco52I (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQBI PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by DraI (3'-TTT_AAA-3') was not inhibited by 5 micro M conjugate 14. That ImImPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBI PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as was required to protect supercoiled DNA from hydrolysis.

The T:G mismatched base pair is associated with many genetic mutations. Understanding its biological consequences may be aided by studying the structural perturbation of DNA caused by a T:G base pair and by specific probing of the mismatch using small molecular ligands. We have shown previously that AR-1-144, a tri-imidazole (Im-Im-Im) minor groove binder, recognizes the sequence CCGG. NMR structural analysis of the symmetric 2:1 complex of AR-1-144 and GAACCGGTTC revealed that each AR-1-144 binds to four base pairs with the guanine N2 amino group forming a bifurcated hydrogen bond to a side-by-side Im/Im pair. We predicted that the free G-N2 amino group in a T:G wobble base pair can form two individual hydrogen bonds to a side-by-side Im/Im pair. Thus an Im/Im pair may be a good recognition motif for a T:G base pair in DNA. Cooperative and tight binding of an AR-1-144 homodimer to GAACTGGTTC permits a detailed structural analysis by 2D NOE NMR refinement and the refined structure confirms our prediction. Surprisingly, AR-1-144 does not bind to GAATCGGTTC. We further show that both the Im-Im-Im/Im-Py-Im heterodimer and the Im-Im-Im/Im-Im-Im homodimer bind strongly to the CACGGGTC + GACTCGTG duplex. These results together suggest that an Im/Im pair can specifically recognize a single T:G mismatch. Our results may be useful in future design of molecules (e.g. linked dimers) that can recognize a single T:G mismatch with specificity.

The new solid phase synthesis of sequence-specific DNA alkylating polyamides containing segment A of Du86 (Duo), N-methylimidazole (Im) and N-methylpyrrole (Py) amino acids is described. New monomer building block N-carboxylmethyl Py (Pyc) was synthesized from 2-methylpyrrolecarboxylate by eight steps. After normal coupling of FMOC-protected-Im and -Py monomer, the deprotection of silyl group generates free carboxylic acid. Introduction of various types of functional groups on solid support will be presented.

Thyroid nodularity following continuous low-dose radiation exposure in China was determined in 1,001 women aged 50-65 years who resided in areas of high background radiation (330 mR/yr) their entire lives, and in 1,005 comparison subjects exposed to normal levels of radiation (114 mR/yr). Cumulative doses to the thyroid were estimated to be of the order of 14 cGy and 5 cGy, respectively. Personal interviews and physical examinations were conducted, and measurements were made of serum thyroid hormone levels, urinary iodine concentrations, and chromosome aberrations in circulating lymphocytes. For all nodular disease, the prevalences in the high background and control areas were 9.5% and 9.3%, respectively. For single nodules, the prevalences were 7.4% in the high background area and 6.6% in the control area (prevalence ratio = 1.13; 95% confidence interval = 0.82-1.55). There were no differences found in serum levels of thyroid hormones. Women in the high background region, however, had significantly lower concentrations of urinary iodine and significantly higher frequencies of stable and unstable chromosome aberrations. Increased intake of allium vegetables such as garlic and onions was associated with a decreased risk of nodular disease, which seems consistent with experimental studies suggesting that allium compounds can inhibit tumor growth and proliferation. The prevalence of mild diffuse goiter was higher in the high background radiation region, perhaps related to a low dietary intake of iodine. These data suggest that continuous exposure to low-level radiation throughout life is unlikely to appreciably increase the risk of thyroid cancer. However, such exposure may cause chromosomal damage.