Synthesis of N‐Acetylated Deoxyribonucleoside 5′‐Triphosphates and their Utilization in Enzymatic Formation of Single‐Stranded Polydeoxyribonucleotides

Abstract

The N-acetylated deoxyadenosine and deoxyguanosine 5'4riphosphates (dAAcTP and dGAcTP) have been synthesized and found competent as substrates for enzymatic chain lengthening of polydeoxyribonucleotides without concomitant loss of the acetyl group. The enzyme used was terminal deoxyribonucleotidyltransferase. The addition of dGAc units proceeds without aggregation that self-limits addition of nonacetylated dG units. The resulting polymer 3'-ended with dGAC units is an efficient initiator for subsequent chain lengthening. Hydrogen bonding is not destroyed by N-acetylation of the dA-dT Watson-Crick pair, although it is considerably weakened. As a practical result of this, a polymer 3'-ended with dAAC units is a much more efficient initiator for enzymatic chain lengthening by dT units than is a corresponding polymer with dA units. Venom phosphodiesterase does not hydrolyze a polymer 3'-ended with dAAc U n i t s .Synthesis of polydeoxyribonucleotides with desired sequences can be a key contributor to successful, designed cellular transformation. According to current knowledge about the chemistry of the cell genome, the arrangement of nucleotide units is a linear mixture of hetero-and homopolymer sequences (CBN) ; overlining of a subscript means that the number is the average of a distribution; the superscripts Ac or Ac, following a capital letter show that the nucleotide is acetylated or diacetylated; monomer units in a polymer are referred to by dA, dG*c, etc.; labeling in compounds is sparingly designated, usually just a t the start of an experiment ; the mono-ymole is a micromole of monomer units contained in a polymer. 33.tion of dG units onto an initiator but also very inefficient utilization as an initiator of an oligodeoxyribonucleotide 3'-terminated with a run of dG units. However, when dG units were enzymatically copolymerized with any other nucleotide or combination of nucleotides, aggregation was absent [20]. Aggregation of dC units interferes with use of dC oligomers longer than decamer as initiators for chain lengthening [21]. Hydrogen bonding slows the rate of dT unit addition onto poly dA initiators [22].It has been shown that N-acetylated dG oligomers do not have the property of aggregation [17], and it may readily be inferred that this same chemical modification on other amino-bearing nucleotides could eliminate the other difficulties mentioned above. Therefore, we have synthesized the N-acetylated 5'-triphosphates dGAcTP and dAACTP in order to determine whether these can function as substrates for enzymatic chain lengthening, whether the products retain the N-acetyl group and are devoid of the aggregation and hydrogen-bonding difficulties, and whether the N-acetyl group is Gnally removable with no damage to the polydeoxyribonucleotide. METHODS AND N A T E R

Induction of stable linkage between the deoxyribonucleic acid dependent ribonucleic acid polymerase and d(A-T)n·d(A-T)n by ultraviolet light

“…The reaction was then extracted with phenol as previously described and dialyzed exhaustively against 0.01 M NaCl. The labeled polymer was then eluted from a calibrated Bio-Gel A-50m column (Bio-Rad Laboratories) according to procedures outlined elsewhere (Hayes et al, 1968;Hayes and Mitchell, 1969). The preparation of [1JC]d(A-T)"-d(A-T)" had an average monomer unit length of 456.…”

O4-Methyldeoxythymidine replacing deoxythymidine in poly[d(A—T)] renders the polymer resistant to the 3′ → 5′ exonuclease activity of the Klenow and T4DNA polymerases

“…0.5 ml fractions were collected. The polymer peak fractions (#6-9) were well separated from monomeric material (# [13][14][15][16][17][18][19][20][21][22][23]. No labeled nucleoside monoor triphosphate was detectable in polymer peak fractions after thin layer chromatography on Eastman #6065 cellulose developed in 75 ethanol/30 ammonium acetate (pH 7).…”

Polydeoxythymidylate as template for complementary enzymatic synthesis

“…The reactions employed dAZ4,, , dTlo , and dTTP-2-14 C and were allowed to proceed until no further synthesis was measurable using the filter paper disc method [9]. By varying the amount of polymerase for each reaction the equivalence point was determined ( fig.…”

“…1) A series of reactions using dTr re , dTr,e, or dT&,, were run for a minimum of 24 hr at 37" in 2.8 ml total volume containing 5 PM template, a 5-fold excess of dATP-8-14C over that required for l-fold synthesis, 8 mM MgClz , 1 mM 2-mercaptoethanol, 40 mM potassium phosphate buffer (pH 7.0), and calf thymus DNA polymerase in equivalence ratios to template (R) of 4, 2, 1, 0.5,0.3 and 0.2. The reactions were monitored for completion of synthesis by filter paper disc method [9]. Protein was removed by phenol extraction [ 1 l] , and agarose column chromatography in 5 mM NaCl [9] separated all polynucleotide material from low molecular weight substances.…”

“…The reactions were monitored for completion of synthesis by filter paper disc method [9]. Protein was removed by phenol extraction [ 1 l] , and agarose column chromatography in 5 mM NaCl [9] separated all polynucleotide material from low molecular weight substances. Each isolated polymer was analyzed for phosphorus content [ 121, UV absorption, radioactivity, and thermal hyperchromicity [ 131, fig.…”