Published online 1 September 2004
Nucleic Acids Research, Vol. 32 No. 15 © Oxford University Press 2004; all rights reserved
Synthetic tRNALys,3 as the replication primer for the HIV-1HXB2 and HIV-1Mal genomes
Reverse Transcriptase Biochemistry Section, HIV Drug Resistance Program, National Cancer Institute at Frederick, Frederick, MD 21702, USA and 1 Dharmacon, Inc., Lafayette, CO 80026, USA
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Received June 17, 2004; Revised and Accepted August 19, 2004
In order to determine the contribution of modified bases on the efficiency with which tRNALys,3 is used in vitro as the HIV-1 replication primer, the properties of synthetic derivatives prepared by three independent methods were compared to the natural, i.e. fully modified, tRNA. When prepared directly by in vitro run-off transcription, we show here that the predominant tRNA species is 77 nt, representing a non-templated addition of a single nucleotide. As a consequence, this aberrant tRNA inefficiently primes (–) strand strong stop DNA synthesis from the primer binding site (PBS) on the HIV-1 viral RNA genome to which it must hybridize. In contrast, correctly sized tRNALys,3 can be prepared by (i) total chemical synthesis and ligation of ‘half’ tRNAs, (ii) transcription of a cassette whose DNA template contained strategically placed 2'-O-Methyl-containing ribonucleotides and (iii) processing from a larger precursor by means of targeted cleavage with Escherichia coli RNase H. When each of these 76 nt tRNAs was supplemented into a (–) strand strong stop DNA synthesis reaction utilizing the HXB2 strain of HIV-1, the amount of product obtained was comparable to that from the fully modified counterpart. Parallel assays monitoring early events in (–) strand strong stop DNA synthesis using either the HXB2 or Mal strain of HIV-1 RNA as the template indicated little difference in the pattern or total product amount when primed with either natural or synthetic tRNALys,3. In addition, nuclease mapping of PBS-bound tRNA suggests inter-molecular base pairing between bases of the tRNA anticodon domain and the U-rich U5-IR loop of the viral 5' leader region is less stable on the HIV-1HXB2 genome than the HIV-1Mal isolate.