A chimeric RNA/DNA ribozyme (HRz) was synthesized in pure form. The 30 nucleotide long sequences was such that it could be targeted to cleave the HIV-1 gag RNA. Named HRz-M, it a had a uridine in the invariable region replaced by a guanine. The ribozyme is demonstrated to possess a compact base paired secondary structure with a stem region and two loops ( Sarma et al FEBS Leters 357, 317-323, 1995). By NMR studies at 500 MHz in 90% water and 10% deuterium oxide, and following the imino protons it is demonstrated that this ribozyme forms a stable abortive complex with its DNA substrate. It is further demonstrated that in the ribozyme-DNA complex, the nucleotidyl units adopt the C3~-endo A type conformation. A detailed introduction to our anti-HIV ribozyme project was provided in our 1994 NIH Annual Report. Of the two ribozymes synthesized, HRz-W and HRz-M, the study in 1995 was limited to HRz-W. It was demonstrated earlier that the ribozymes themselves exist as a mixture of several conformations. And from these mixtures we have been able to identify their stable secondary structure, and this was reported in FEBS Letters 357, 317-323 (1995). The secondary structure had a stem region consisting of AT and GT pairs along with two loop regions. In 1995 our main concern has been to investigate whether the ribozyme forms a stable complex with the corresponding DNA substrate. A 17-mer DNA substrate TTGTTGGTCCAAAATGC was synthesized, and was titrated with HRz-M, and the complex formation was followed by monitoring the imino protons. At a concentration ratio of 1:1 ribozyme and substrate, clearly distiguishable resonances from the complex was present. We did not observe any resonance doublings, and in the temperature range of 5 to 30 degrees, the complex was stable. The lack of resonance doublings in the ribozyme-DNA complex in the temperature range of 5 to 30 degrees suggests the absence of detectable multiple conformations. Hence it should be possible to perform a reasonable assignment of the imino proton resonances. We have also measured NOESY and P.E.COSY spectra of the complex at 500 MHz in the temperature range of 15-30 degrees. A detailed analysis and assignment of the complex spectra is impossible without 13C and 15N labelled ribozyme. Nevertheless, by following base-base cross peaks, and cross peaks from the methyl groups of the thymines in the DNA part and the ribozyme part in the NOESY we are attempting to unearth certain tertiary interactions. The H1~ vs H2~H2~ cross section in the P.E.COSY spectrum clearly revealed no detectable coupling constants, clearly suggesting that the sugar conformation of all the nucleotidyl units belong to the A family, viz C(3~)-endo. If they belonged to the B-family (C2~-endo) we would have detected couplings of the magnitude of 8 to 10 hertz. Conditions under which P.E.COSY was performed were such that in the H6 vs H5 cross section of the cytosines we could cleraly see the H5-H6 couplings. This suggests that the lack of observation of couplings in the H1~ vs H2~H2~ region was not due to excessive line widths or lack of resolution. We are in the process of making 13C and 15N labelled ribozymes so that the above system can be investigated in detail. The current study reveals the ribozyme-DNA complex system that we have investigated to be a fairly clean stable system, unlike the ribozyme itself, and it is worth undertaking the task of preparing uniformly 13C and 15N labelled materials.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR000995-21
Application #
5221950
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
21
Fiscal Year
1996
Total Cost
Indirect Cost
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