Highly efficient random mutagenesis in transcription-reverse-transcription cycles by a hydrogen bond ambivalent nucleoside 5'-triphosphate analogue: potential candidates for a selective anti-retroviral therapy.

The nucleoside P can base pair with both A and G. We evaluated the mutation frequency induced by the 5'-triphospbate of the ribonucleoside P (PTP) in an in vitro retroviral replication model. After 4 cycles of replication in the presence of PTP, the mutation frequency was raised to 3.8 x 10(-2) per nucleotide and C-to-U and U-to-C mutations were dominantly observed. These results suggest that ambivalent NTP analogues, like PTP, could induce mutations beyond the error threshold of retroviruses.

Survey and summary: The applications of universal DNA base analogues.

A universal base analogue forms 'base pairs' with each of the natural DNA/RNA bases with little discrimination between them. A number of such analogues have been prepared and their applications as biochemical tools investigated. Most of these analogues are non-hydrogen bonding, hydrophobic, aromatic 'bases' which stabilise duplex DNA by stacking interactions. This review of the literature of universal bases (to 2000) details the analogues investigated, and their uses and limitations are discussed.

Binding specificities of the mismatch binding protein, MutS, to oligonucleotides containing modified bases.

We have studied the effects of DNA mismatch repair on mutagenesis induced by nucleoside analogs. Among them, the mutagenic action of 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one 2'-deoxyriboside (dP) showed high susceptibility to the mismatch repair system, while mutagenesis by N4-aminocytidine and N4-hydroxycytidine was only weakly affected. 2-Aminopurine mutagenesis showed intermediate susceptibility. MutS protein specifically bound to an oligonucleotide duplex containing a dP-dG pair, while the dP-dA pair was bound only weakly. The binding to the dP-dG pair was as strong as binding to a dA-dC mismatch. These specific binding properties can explain the effective avoidance of dP-induced mutagenesis by the mismatch repair system. We have also studied the effects of the repair system on mutagenesis induced by methylating and ethylating agents.

Reverse transcriptional mutagenesis induced by ribonucleoside triphosphate analogue, PTP and its availability for anti-HIV-1 therapy.

The bicyclic pyrimidine analogue, 3,4-dihydro-6H,8H-pyrimido[4,5-c][1,2]oxazin-7-one (P) can base pair with both A and G. The riboside 5'-triphsophate of P (PTP) efficiently induces mutation during in vitro transcription and reverse transcription cycles using a phage promoter. In the present study, we have constructed an in vitro transcription system promoted by the human immunodeficiency virus type 1 (HIV-1) 5'-long terminal repeat (LTR) using HeLa nuclear extract supplemented with HIV-1 Tat protein. Using this system, the effects of mutagenic ribonucleotide analogues were studied.

Synthesis and incorporation of pyrrole carboxamide nucleoside triphosphates by DNA polymerases.

We have synthesised and examined the enzymatic incorporation properties of the 5'-triphosphates of 2'-deoxyribosyl pyrrole 3-monocarboxamide (dMTP) and 2'-deoxyribosyl pyrrole 3,4-dicarboxamide (dDTP). These analogues we had hoped would behave as ambivalent base analogues in that they can present two alternative hydrogen-bonding faces either by rotation about the carboxamide group or about the glycosidic bond. The two pyrrole derivatives, dMTP and dDTP, exhibit a preference for incorporation with Klenow polymerase. They are preferentially incorporated as either A or C.

Retrovirus-specific random mutagenesis by a nucleoside 5'-triphosphate analogue, PTP.

In a retrovirus replication model system, which consists of in vitro transcription and reverse transcription cycles, 6-(beta-D-ribofuranosyl)-3,4- dihydro-8H-pyrimido[4,5-c][1,2]oxazin-7-one-5'-triphosphate (PTP) induced highly efficient random mutations and this was due to the ambiguous incorporation of PTP by RNA polymerases. The types of mutations were mainly C-to-U or U-to-C transition mutations and the frequency was about 4 x 10(-2)/nucleotide during four cycles of the replication. Since a high mutation rate is harmful to species, PTP may be new candidate for anti-retroviral drugs. N4-aminoCTP and N4-hydroxyCTP were also incorporated ambiguously by RNA polymerase. These compounds may have a potential to induce mutation by the same mechanism as PTP.

Disturbance of genetic information by a ribonucleotide analogue.

The synthetic base analogue, 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one (P), can efficiently base pair with A and G. We have previously demonstrated that the deoxyribonucleoside of P (dP) is highly mutagenic and that this is due to the ambiguous base pairing ability of P. In this work, we have shown that the ribonucleoside triphosphate of P (rPTP) induces C to U mutation on an in vitro model of retroviral genomic RNA replication pathway. This mutation induction by rPTP may be specific to retroviruses, since host genomic DNA should not be affected by such a ribonucleotide analogue, although temporary transcription-translation errors may occur.

The use of tailed octamer primers for cycle sequencing.

Studies have been carried out on the use of octamer oligonucleotides tailed with different base analogues as primers in cycle sequencing reactions. 5-Nitroindole tails improved the performance as primers of a number of octamers. A tail length of three or four 5-nitroindole residues significantly increased the sequencing signal intensity for almost all primers. The use of incomplete libraries of tailed octamer primers for primer walking is discussed.

A thymine-like base analogue forms wobble pairs with adenine in a Z-DNA duplex.

The DNA hexamer d(CACGPG), in which dP is the ambivalent pyrimidine nucleoside analogue 2'-deoxy-beta-d-ribofuranosyl-(6H,8H-3, 4-dihydropyrimido[4,5-c][1,2]oxazin-7-one), crystallises as a left-handed Z-DNA duplex. X-ray analysis at 1.5 A shows that both P. A base-pairs are of the wobble type. This result appears inconsistent with other evidence from hybridisation and NMR studies of P-containing oligonucleotides, which suggests that, while P can form stable base-pairs with either A or G, thymine-like properties are more pronounced. Thermal denaturation experiments over a range of solution pH values indicate that protonation of the P.A base-pairs is unlikely to be responsible for the anomalous behaviour. No specific crystal packing effects can be identfied as an explanation, and it is concluded that base stacking and other interactions between nucleotide residues in Z-DNA are responsible.

Use of 5-nitroindole-2'-deoxyribose-5'-triphosphate for labelling and detection of oligonucleotides.

The 5'-triphosphate of 5-nitroindole-2'-deoxyriboside has been shown to be a good substrate for terminal deoxynucleotidyl transferase (TdT). An antibody has been prepared for the detection of 5-nitroindole and has been used for the detection of 5-nitroindole tailed DNA both in single-stranded form and after hybridisation to a template. This is therefore a new method for the detection of nucleic acid probes.

Synthesis and RNA polymerase incorporation of the degenerate ribonucleotide analogue rPTP.

The synthesis and enzymatic incorporation into RNA of the hydrogen bond degenerate nucleoside analogue 6-(beta-d-ribofuranosyl)-3, 4-dihydro-8H-pyrimido[4,5-c]-[1,2]oxazin-7-one (P) is described. The 5'-triphosphate of this analogue is readily incorporated by T3, T7 and SP6 RNA polymerases into RNA transcripts, being best incorporated in place of UTP, but also in place of CTP. When all the uridine residues in an HIV-1 TAR RNA transcript are replaced by P the transcript has similar characteristics to the wild-type TAR RNA, as demonstrated by similar melting temperatures and CD spectra. The P-substituted TAR transcript binds to the Tat peptide ADP-1 with only 4-fold lowered efficiency compared with wild-type TAR.

Triple helices formed at oligopyrimidine*oligopurine sequences with base pair inversions: effect of a triplex-specific ligand on stability and selectivity.

Oligonucleotide-directed triple helix formation is mostly restricted to oligopyrimidine*oligopurine sequences of double helical DNA. An interruption of one or two pyrimidines in the oligopurine target strand leads to a strong triplex destabilisation. We have investigated the effect of nucleotide analogues introduced in the third strand at the site opposite the base pair inversion(s). We show that a 3-nitropyrrole derivative (M) discriminates G*C from C*G, A*T and T*A in the presence of a triplex-specific ligand (a benzo[e]pyridoindole derivative, BePI). N6-methoxy-2,6-diaminopurine (K) binds to an A*T base pair better than a T*A, G*C or C*G base pair. Some discrimination is still observed in the presence of BePI and triplex stability is markedly increased. These findings should help in designing BePI-oligonucleotide conjugates to extend the range of DNA sequences available for triplex formation.

Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases.

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazin-7-one and the purine base N6-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by Taq polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of Caenorhabditis elegans, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and Taq polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of C. elegans. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.

Comparative mutagenicities of N6-methoxy-2,6-diaminopurine and N6-methoxyaminopurine 2'-deoxyribonucleosides and their 5'-triphosphates.

The structure of the deoxyribonucleoside derived from N 6-methoxy-2, 6-diaminopurine (dK) was examined by NMR. The methoxyamino residue was found predominantly in the imino rather than the amino tautomer (ratio: 9:1 in DMSO). The nucleoside proved to be a potent transition mutagen in Escherichia coli , in contrast to the closely related nucleoside derived from the analogue N6-methoxyaminopurine (dZ), which was only weakly mutagenic. The 5'-triphosphate derivatives, dKTP and dZTP, were synthesized; Taq polymerase incorporated dKTP opposite both T and, less well, opposite dC in template DNA. Both analogue triphosphates produced transition mutations when added to PCR reactions. In each case, there was a large excess of AT-->GC compared to GC-->AT mutations (ratios were 15:1 for dKTP and 10:1 for dZTP). Polymerase extension times in each cycle had to be extended, consistent with a decreased rate of DNA synthesis in the presence of the analogues. This and the mutagenic ratios are discussed in terms of syn-anti inversion of the methoxyl group.

Stability and structure of DNA oligonucleotides containing non-specific base analogues.

The nature of DNA containing the deoxyribosyl derivative of 5-nitroindole has been investigated. 5-Nitroindole has been shown to give good stability when present in duplexes opposite natural bases, with only slightly reduced melting temperatures. However, enhanced stability occurs when it is incorporated as an additional bulged base in duplexes. It also markedly enhances the stability of duplexes when it is present as a pendant base at either the 5' or 3'-ends of the two strands. The stabilisation is presumed to be due to enhanced stacking interactions for the nitroindole base. Oligomers containing a number of consecutive 5-nitroindole residues form stable, stacked secondary structures. An oligomer containing 21 such substitutions is presumed to exist as a hairpin structure. This was further investigated by circular dichroism melting experiments, which demonstrated that the single-stranded oligomer contains significant secondary structure in the region of the 5-nitroindole tract, which appears to contain a double-stranded stem. X-ray analysis of 5-nitroindole deoxyriboside provides some indication of how the mode of stacking observed in crystals of the nucleoside may also be responsible for stabilising secondary structures of oligonucleotides.

An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside.

Acyclic nucleoside analogues with carboxamido- or nitro-substituted heterocyclic bases have been evaluated for their possible use as universal bases in oligodeoxynucleotides. The acyclic moiety endows the constructs with enough flexibility to allow good base stacking. The 5-nitroindazole analogue afforded the most stable duplexes among the acyclic derivatives with the least spread in Tm versus the four natural bases. In spite of the acyclic moiety, stabilities are comparable with those of duplexes incorporating the recently described 5-nitroindole nucleoside analogue, but considerably exceed those for the 3-nitropyrrole analogue.

3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR.

3-Nitropyrrole and 5-nitroindole have been assessed as universal bases in primers for dideoxy DNA sequencing and in the polymerase chain reaction (PCR). In contrast to a previous report, we have found that the introduction of more than one 3-nitropyrrole residue at dispersed positions into primers significantly reduced their efficiency in PCR and sequencing reactions. Primers containing 5-nitroindole at multiple dispersed positions were similarly affected; for both bases only a small number of substitutions were tolerated. In PCR experiments neither base, when incorporated into primers in codon third positions, was as effective as hypoxanthine, which was incorporated in six codon third positions in a 20mer oligomer. However, primers containing up to four consecutive 5-nitroindole substitutions performed well in both PCR and sequencing reactions. Consecutive 3-nitropyrrole substitutions were tolerated, but less well in comparable reactions.

5-Nitroindole as an universal base analogue.

4-, 5- and 6-Nitroindole have been investigated and compared with 3-nitropyrrole as universal bases in oligodeoxynucleotides. Of these the 5-nitroindole derivative was found to be superior giving higher duplex stability, and behaving indiscriminately towards each of the four natural bases in duplex melting experiments. 3-Nitropyrrole, whilst not discriminating between the natural bases, was found to lead to considerable destabilisation of the duplexes, particularly when multiple substitutions were made, in contrast to the 5-nitroindole nucleoside.

Isolation of antirhinoviral sesquiterpenes from ginger (Zingiber officinale).

The dried rhizomes of Indonesian ginger, Zingiber officinale, were investigated for antirhinoviral activity in the plaque reduction test. Fractionation by solvent extraction, solvent partition, and repeated chromatography guided by bioassay, allowed the isolation of several sesquiterpenes with antirhinoviral activity. The most active of these was beta-sesquiphellandrene [2] with an IC50 of 0.44 microM vs. rhinovirus IB in vitro.