National Cancer Institute Formulary: A Public-Private Partnership Providing Investigators Access to Investigational Anticancer Agents.

As part of the White House Cancer Moonshot Initiative, the National Cancer Institute (NCI) has developed a drug formulary to provide investigational anticancer agents to the extramural research community. This article describes how the NCI Formulary functions, how researchers may apply for access to drugs in the formulary, and the NCI's initial goals for formulary participation. Approved investigators may apply for access to formulary agents at: https://nciformulary.cancer.gov.

Physical mapping of HIV reverse transcriptase to the 5' end of RNA primers.

Enzymatic analysis of RNA cleavage products has suggested that human immunodeficiency virus (HIV) reverse transcriptase (RT) binds to the 5' end of RNAs that are recessed on a longer DNA template (RNA primers) yet binds to the 3' end of DNA primers. One concern is that RT molecules bound at the 3' end of RNA would not be easily detected because RT may not catalyze substantial RNA extension or cleavage when bound to the 3' end. We used physical mapping to show that RT binds preferentially to the 5' end of RNA primers. An HIV-RT that lacked RNase H activity (HIV-RT(E478Q)) was incubated with the RNA-DNA hybrid followed by the addition of Escherichia coli RNase H. RT protected a approximately 23-base region at the 5' end of the RNA and 4 additional bases on the DNA strand. This footprint correlated well with the crystal structure of HIV-RT. No protection of the RNA 3' end was observed, although when dNTPs were included, low levels of extension occurred, indicating that RT can bind this end. Wild-type HIV-RT cleaved the RNA and then extended a small portion of the cleaved fragments, suggesting that very small RNAs may be bound similar to DNA primers.

In vitro strand transfer from broken RNAs results in mismatch but not frameshift mutations.

An in vitro system to compare the fidelity of strand transfers from truncated vs full-length RNAs was constructed. A donor RNA, on which reverse transcriptase (RT)-directed DNA synthesis was initiated, shared homology with an acceptor RNA, to which DNAs initiated on the donor could transfer. All RNAs were derived from the N-terminal portion of the alpha-lac gene. On full-length donors, transfers occurred when DNAs migrated to the acceptor prior to being completed on the donor. On donors that were truncated, most transfers occurred after DNAs reached the end of the donor. Transfer products were amplified by PCR and used to replace the corresponding region in a vector containing the alpha-lac gene. Transformed Escherichia coli were screened for alpha-complementation by blue-white phenotype analysis, with white colonies scored as those with errors in alpha-lac. These errors were derived from RT synthesis and strand transfer. The mutant colony frequency approximately doubled for transfer products derived from truncated donors (0.026+/-0.005 vs. 0.053+/-0.011 (three experiments +/- SD), for full-length vs. truncated, respectively). The increases resulted from additional non-template-directed bases (mostly thymidines) added to the DNAs before transfer. Sequence analysis of DNAs synthesized on truncated donors showed that about 60% had additions (20/34); however, those without additions transferred at a much higher rate than those with. Transfer of the DNAs with additions always resulted in substitutions; no frameshifts were observed. Results are consistent with RT adding nontemplated nucleotides at template termini. Transfer and subsequent extension of these products is severely inhibited relative to products without additions. The potential relevance of these findings to retrovirus replication is discussed.

Human immunodeficiency virus reverse transcriptase base misincorporations can promote strand transfer.

A system to determine if HIV-reverse transcriptase (RT) base misincorporations can promote strand transfer was constructed. A donor RNA, on which RT-directed DNA synthesis was initiated, shared homology over a 119 base internal region with an acceptor RNA, to which DNAs initiated on the donor could transfer. Products completed on the donor in the presence or absence of acceptor were isolated and PCR was used to amplify these DNAs. PCR products were ligated into a vector which had this same region (near the N-terminus of the alpha-lac gene) removed. Transformed E. coli were screened in an alpha-complementation assay by blue-white phenotype analysis with white colonies scored as those with errors in plasmid-derived alpha-lac. The frequence of white colonies +/- standard deviations was 0.031 +/- 0.006 and 0.0037 +/- 0.009, for plasmids with inserts derived from donor-directed products synthesized with 100 microM dNTPs in the presence and absence of acceptor template, respectively. Statistical analysis indicated a lower white colony frequency in the presence of acceptor (p = 0.0025). The lower frequency with acceptor implies that a portion of the errors made on the donor are transferred to the acceptor suggesting that base misincorporations can induce strand transfer.