Synthesis and properties of oligonucleotides containing aminodeoxythymidine units

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120

Synthetic oligonucleotides and their analogs have attracted considerable interest recently. These compounds may lead to highly specific therapeutic agents, as well as to powerful diagnostic tools. Here, we present the synthesis of uniformly modified oligodeoxyribonucleotide N3' -> P5' phosphoramidates containing 3'-NHP(O)(0-)0-5' internucleoside linkages and the study of Synthetic oligonucleotides have a great potential to become a new type of rationally designed therapeutic agent. These compounds could interfere with the expression of selected genes through a specific interaction with RNA or DNA regions of interest (1). Several modifications of the natural phosphodiester internucleoside bond have been introduced to improve hybridization properties of oligonucleotides as well as their cellular membrane penetration and stability and distribution inside cells (2-4). Unfortunately, the vast majority of these more hydrolytically stable analogs show reduced binding with RNA or DNA via duplex or triplex formation (5).In the present report we describe the synthesis and hybridization properties of the new analogs of nucleic acidsuniformly modified oligonucleotide N3' -* P5' phosphoramidates (Fig. 1). These compounds possess some very attractive features for therapeutic and diagnostic applications, such as a negatively charged and achiral phosphorus, good solubility in water, and phosphodiesterase digestion resistance, buttressed by a high affinity for natural RNA and DNA. MATERIALS AND METHODSOligonucleotide Synthesis. Oligodeoxyribonucleotides and oligoribonucleotides were prepared on an ABI 380B synthesizer using standard DNA or RNA assembly protocols via the phosphoramidite method. Oligonucleotide N3' -> P5' phosphoramidates were synthesized on 1 ,umol scale using an ABI 394 synthesizer and 5'-4,4'-dimethoxytrityl (DMT)-N-acyl-3'-amino-2',3'-dideoxynucleosides, as described (6). Oligonucleotides were purified by ion exchange HPLC on a Pharmacia Mono Q 10/10 column at pH 12 (10 mM NaOH) with a 1%The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. per min gradient of 1.5 M NaCl in 10 mM NaOH and a flow rate of 2 ml/min. Oligonucleotides were desalted immediately after purification on Pharmacia NAP-5 gel filtration columns and then lyophilized in vacuo.Thermal Dissociation Experiments. A Cary-Varian 1E spectrophotometer equipped with a temperature programmer and data processor from Varian was used according to the reported procedure for thermal dissociation experiments (7). Buffer concentrations and compositions are listed in the legends to Tables 1-3. Absorbance values at 260 nm were obtained at 1-min intervals with a temperature increase of 1.0°C/min. The reported tm (°C) is the temperature at the midpoint of the slope of a plot of absorbance vs. temperature.Thermodynamic Parameters Determination. Thermodynamic parameters for the dodecamers 11-13 (see T...

Section: Methodsmentioning

confidence: 99%

Oligonucleotide N3'-->P5' phosphoramidates.

Gryaznov¹,

Lloyd²,

Chen³

et al. 1995

162

120

“…Much of this binding enhancement by tertiary interactions (BETI) can be retained when these hexamers are modified to be resistant to nuclease degradation (11). In particular, d(AnTnGnAnCn)rU with N3Ј 3 P5Ј phosphoramidate (19) linkages ( Fig. 1) binds 2,000-fold more tightly than expected for base pairing.…”

mentioning

confidence: 99%

In vitro suicide inhibition of self-splicing of a group I intron from Pneumocystis carinii by an N 3′ → P 5′ phosphoramidate hexanucleotide

Testa

Gryaznov

Turner

1999

Self Cite

Binding enhancement by tertiary interactions is a strategy that takes advantage of the higher order folding of functionally important RNAs to bind short nucleic acid-based compounds tightly and more specifically than possible by simple base pairing. For example, tertiary interactions enhance binding of specific hexamers to a group I intron ribozyme from the opportunistic pathogen Pneumocystis carinii by 1,000-to 100,000-fold relative to binding by only base pairing. One such hexamer, d(AnTnGnAnCn)rU, contains an N3 3 P5 phosphoramidate deoxysugar-phosphate backbone (n) that is resistant to chemical and enzymatic decay. Here, it is shown that this hexamer is also a suicide inhibitor of the intron's self-splicing reaction in vitro. The hexamer is ligated in trans to the 3 exon of the precursor, producing dead-end products. At 4 mM Mg 2؉ , the fraction of trans-spliced product is greater than normally spliced product at hexamer concentrations as low as 200 nM. This provides an additional level of specificity for compounds that can exploit the catalytic potential of complexes with RNA targets.Most human therapeutics have been discovered by screening natural products. Synthetic organic chemistry has made it possible to synthesize such natural products and derivatives thereof in large quantities, thus broadening the range of compounds that can be used clinically (1-3). Synthetic methodology coupled with the outpouring of protein structural information has also allowed rational design of completely new therapeutic compounds (4,5). Similarly, the recent explosion in nucleic acid sequence information is providing a knowledge base for structure-based targeting of RNA. The first generation of such therapeutics consists of antisense nucleic acids that bind mRNA through Watson-Crick base pairing and thereby regulate translation (6, 7). Because of the long sequences employed, typically 15-20 nucleotides, potential disadvantages include high cost of synthesis (8) and lack of specificity (9). Cost of synthesis can be reduced and specificity increased by designing short antisense agents whose binding to RNA targets is enhanced by tertiary interactions (10, 11). Here we show that one such hexanucleotide can also be a suicide inhibitor (12, 13) of RNA function. This provides an additional design principle for increasing specificity of compounds that can exploit the catalytic potential of complexes with RNA targets.Our model system is a large-subunit ribosomal RNA (rRNA) precursor from the opportunistic pathogen Pneumocystis carinii, which is a common cause of death in immunocompromised patients (14, 15). The rRNA precursor contains a group I self-splicing intron (10, 16) that provides a potential therapeutic target (16, 17) because self-splicing is required for assembly of active ribosomes (18). Hexamers that mimic this intron's 5Ј exon can bind to the catalytic core of a ribozyme derived from the intron as much as 100,000-fold more tightly than expected if the hexamers bound by simple base pairing (10). Much of this bindi...

“…However, the nonenzymatic synthesis of 3′-NP-DNA by copying RNA or LNA templates may provide an alternative route to the synthesis of 3′-NP-DNA oligonucleotides if this can be carried out with sufficient fidelity and on an appropriate scale. A potential barrier to 3′-NP-DNA replication is the difficulty of strand separation due to the high T m of duplex 3′-NP-DNA (24)(25)(26), and the additional duplex stabilization expected from 2-thio-T substitution. This problem may be ameliorated by moderate concentrations of formamide or urea (27), or alternatively, a heterogeneous backbone of N 3′ -P 5′ and N 2′ -P 5′ linkages could be used (6).…”

Section: Discussionmentioning

confidence: 99%

Fast and accurate nonenzymatic copying of an RNA-like synthetic genetic polymer

Zhang

Blain

Zielińska

et al. 2013

Self Cite

Recent advances suggest that it may be possible to construct simple artificial cells from two subsystems: a self-replicating cell membrane and a self-replicating genetic polymer. Although multiple pathways for the growth and division of model protocell membranes have been characterized, no self-replicating genetic material is yet available. Nonenzymatic template-directed synthesis of RNA with activated ribonucleotide monomers has led to the copying of short RNA templates; however, these reactions are generally slow (taking days to weeks) and highly error prone. N 3′ -P 5′ -linked phosphoramidate DNA (3′-NP-DNA) is similar to RNA in its overall duplex structure, and is attractive as an alternative to RNA because the high reactivity of its corresponding monomers allows rapid and efficient copying of all four nucleobases on homopolymeric RNA and DNA templates. Here we show that both homopolymeric and mixed-sequence 3′-NP-DNA templates can be copied into complementary 3′-NP-DNA sequences. G:T and A:C wobble pairing leads to a high error rate, but the modified nucleoside 2-thiothymidine suppresses wobble pairing. We show that the 2-thiothymidine modification increases both polymerization rate and fidelity in the copying of a 3′-NP-DNA template into a complementary strand of 3′-NP-DNA. Our results suggest that 3′-NP-DNA has the potential to serve as the genetic material of artificial biological systems.origin of life | nonenzymatic primer extension | artificial genetic systems | nucleotide modifications | mismatch T he phosphoramidate nucleic acids are of particular interest as potential genetic materials for artificial life-forms because of their potential for replication by the nonenzymatic polymerization of amino-sugar nucleotides. Because of the greater nucleophilicity of the amino group relative to the 3′-hydroxyl group of ribo-and deoxyribo-nucleotides, amino-sugar nucleotides exhibit more rapid spontaneous polymerization. Obviating the requirement for a polymerase greatly simplifies the task of creating and assembling the components of an artificial cell, and thus of constructing simple living systems from inanimate materials. We and others have therefore explored the synthesis of a variety of phosphoramidatelinked nucleic acids, their corresponding amino-sugar monomers, and the characterization of nonenzymatic template-directed primer extension reactions in these systems (1-11). Among these systems, we have examined 2′-amino versions of the acyclic glycerol nucleic acid (5), 2′-amino-2′,3′-dideoxyribonucleic acid (4, 6), and 3′-amino-2′,3′-dideoxyribonucleic acid (7).The structural simplicity of the acyclic sugar-phosphate nucleic acid backbones has made them attractive targets for study. Indeed, an acyclic nucleotide consisting of a glycerol-phosphate backbone linked to a formylated nucleobase (12) was among the first of such nucleic acids to be chemically synthesized, but incorporation of this nucleotide into oligomers caused a severe loss of duplex stability. Much later, the glycerol nucleic acids, in which...