Iminodiacetic-phosphoramidates as metabolic prototypes for diversifying nucleic acid polymerization in vivo

Abstract

Previous studies in our laboratory proved that certain functional groups are able to mimic the pyrophosphate moiety and act as leaving groups in the enzymatic polymerization of deoxyribonucleic acids by HIV-1 reverse transcriptase. When the potential leaving group possesses two carboxylic acid moieties linked to the nucleoside via a phosphoramidate bond, it is efficiently recognized by this error-prone enzyme, resulting in nucleotide incorporation into DNA. Here, we present a new efficient alternative leaving group, iminodiacetic acid, which displays enhanced kinetics and an enhanced elongation capacity compared to previous results obtained with amino acid deoxyadenosine phosphoramidates. Iminodiacetic acid phosphoramidate of deoxyadenosine monophosphate (IDA-dAMP) is processed by HIV-1 RT as a substrate for single nucleotide incorporation and displays a typical Michaelis-Menten kinetic profile. This novel substrate also proved to be successful in primer strand elongation of a seven-base template overhang. Modelling of this new substrate in the active site of the enzyme revealed that the interactions formed between the triphosphate moiety, magnesium ions and enzyme's residues could be different from those of the natural triphosphate substrate and is likely to involve additional amino acid residues. Preliminary testing for a potential metabolic accessibility lets us to envision its possible use in an orthogonal system for nucleic acid synthesis that would not influence or be influenced by genetic information from the outside.

Figure 1.

Chemical structure of (a) deoxyadenosine triphosphate (b) l-Asp-dAMP and (c) IDA-dAMP. The bond to be cleaved during enzymatic polymerisation is coloured in grey.

Scheme 1.

Obtention of IDA-dAMP 1.

Figure 2.

Incorporation of IDA-dAMP and L-Asp-dAMP into P1:T1 by HIV-1 reverse transcriptase. Aliquots were taken at 5, 10, 20, 30 and 60 min.

Figure 3.

Elongation of P1:T2 with IDA-dAMP by HIV-1 reverse transcriptase. Aliquots were taken at 15, 30, 60, 90 and 120 min. Blank: no triphosphate analogue was added in the reaction.

Figure 4.

Elongation of P1:T3 with IDA-dAMP by HIV-1 reverse transcriptase. Aliquots were taken at 15, 30, 60, 90 and 120 min. Blank: no triphosphate analogue was added in the reaction.

Figure 5.

(a) The thymidine triphosphate (TTP) in the RT dNTP pocket [X-ray structure from Huang 1998 (18)]. The primer strand is shown as a green ribbon; the template strand is in blue. The stabilizing interactions of the TTP within the complex are shown: hydrogen bond distances between the bases are indicated in angstroms. The ligation of the 2 Mg²⁺ ions to the complex is also visualized by dashes and distances in angstroms. (b) Average structure of IDA-dAMP in the RT dNTP binding pocket. This figure was generated using Bobscript, Molscript and Raster3d (37–39).

Scheme 2.

Hypothesized metabolic pathway towards iminodiacetic acid deoxyadenosine phosphoramidate.