Synthesis, cyclopolymerization and cyclo-copolymerization of 9-(2-diallylaminoethyl)adenine and its hydrochloride salt.

We report herein the synthesis and characterization of 9-(2-diallylaminoethyl) adenine. We evaluated two different synthetic routes starting with adenine where the optimal route was achieved through coupling of 9-(2-chloroethyl)adenine with diallylamine. The cyclopolymerization and cyclo-copolymerization of 9-(2-diallylaminoethyl)adenine hydrochloride salt resulted in low molecular weight oligomers in low yields. In contrast, 9-(2-diallylaminoethyl)adenine failed to cyclopolymerize, however, it formed a copolymer with SO2 in relatively good yields. The molecular weights of the cyclopolymers were around 1,700-6,000 g/mol, as estimated by SEC. The cyclo-copolymer was stable up to 226 °C. To the best of our knowledge, this is the first example of a free-radical cyclo-copolymerization of a neutral alkyldiallylamine derivative with SO2. These polymers represent a novel class of carbocyclic polynucleotides.

Rapid construction and characterization of synthetic antibody libraries without DNA amplification.

We report on a simple method to rapidly generate very large libraries of genes encoding mutant proteins without the use of DNA amplification, and the application of this methodology in the construction of synthetic immunoglobulin variable heavy (V(H)) and light (V(kappa)) libraries. Four high quality, chemically synthesized polynucleotides (90-140 bases) were annealed and extended using T4 DNA polymerase. Following electroporation, >10(9) transformants could be synthesized within 1 day. Fusion to beta-lactamase and selection on ampicillin resulted in 3.7 x 10(8) V(H) and 6.9 x 10(8) V(kappa) clones highly enriched for full-length, in-frame genes. High-throughput 454 DNA sequencing of >250,000 V(H) and V(kappa) genes from the pre- and post-selection libraries revealed that, in addition to the expected reduction in reading-frame shifts and stop codons, selection for functional expression also resulted in a statistical decrease in the cysteine content. Apart from these differences, there was a good agreement between the expected and actual diversity, indicating that neither oligonucleotide synthesis nor biological constrains due to protein synthesis of V(H)/V(kappa)-beta-lactamase fusions introduce biases in the amino acid composition of the randomized regions. This methodology can be employed for the rapid construction of highly diverse libraries with the near elimination of PCR errors in invariant regions.

Synthesis of polynucleotide analogs containing a polyvinyl alcohol backbone.

Water soluble homo-base polynucleotide analogues were synthesized in which polyvinyl alcohol and partially phosphonated polyvinyl alcohol constituted the backbones,onto which were grafted uracil or adenine via 1,3-dioxane spacers formed by acetal formation with the 1,3-diol moieties in PVA. The resulting adenine-PVA polynucleotide analogs exhibited hyperchromic effects, which was not the case for the corresponding uracil compounds. Mixtures of the adenine- and aracil PVA-phosphate polynucleotide analogs in solutions exhibited characteristic S-shaped UV-absorbance vs temperature and melting curves with melting points at approximately 40 degrees C.

Oligo- and poly-nucleotides: 50 years of chemical synthesis.

It is fifty years since the first chemical synthesis of a dinucleoside phosphate and a dinucleotide with natural 3'-->5'-internucleotide linkages was reported. The main developments in the methodology of oligo- and poly-nucleotide synthesis that have taken place since are described.

In vitro replication and repair of DNA containing a C2'-oxidized abasic site.

Abasic lesions are unable to form Watson-Crick hydrogen bonds with nucleotides. Nonetheless, polymerase and repair enzymes distinguish between various oxidized abasic lesions, as well as from nonoxidized abasic sites (AP). The C2-AP lesion is produced when DNA is exposed to gamma-radiolysis. Its effects on polymerases and repair enzymes are unknown. A recently reported method for the chemical synthesis of oligonucleotides containing C2-AP at a defined site was utilized for studying the activity of Klenow exo(-) and repair enzymes on templates containing the lesion. The C2-AP lesion has a similar effect on Klenow exo(-) as do AP and C4-AP sites. Deoxyadenosine is preferentially incorporated opposite C2-AP, but extension of the primer past the lesion is strongly blocked. C2-AP is incised less efficiently by exonuclease III and endonuclease IV than are other abasic lesions. Furthermore, although a Schiff base between C2-AP and endonuclease III can be chemically trapped, the location of the 3'-phosphate alpha with respect to the aldehyde prevents beta-elimination associated with the lyase activity of type I base excision repair enzymes. The interactions of the C2'-oxidized abasic site with Klenow exo(-) and repair enzymes suggest that the lesion will be mutagenic and that it will be removed by strand displacement synthesis and flap endonuclease processing via a long patch repair mechanism.

Binding mode of cationic monomer and dimer porphyrin with native and synthetic polynucleotides studied by polarized light spectroscopy.

Binding properties of the tricationic porphyrin monomer with a phenolic substituent at the periphery and the porphyrin dimer conjugated with hydrophilic triethylene glycol were investigated in this study using absorption and polarized spectroscopy, namely, circular dichroism (CD) and linear dichroism (LD). The spectral properties of the porphyrin monomer, when complexed with polynucleotides, were essentially the same as that of the well-known meso-tetrakis(N-methylpyridiniumyl)porphyrin, indicating that the substitution at one peripheral pyridiniumyl ring did not affect the binding mode. When the porphyrin dimer formed a complex with poly[d(G-C)(2)], a negative CD band and a negative LD(r) spectrum were apparent in the Soret absorption region, with its LD(r) magnitude significantly smaller than that in the DNA absorption region. As the complex was stabilized over time, the intensity of the negative CD band and the negative LD(r) increased. These observations indicated that one of the porphyrin moieties of the dimer intercalated initially and than the other one also intercalated consecutively within a few hours. In the porphyrin dimer-poly[d(A-T)(2)] complex case, a bisignate CD was apparent and remained for at least 12 h, indicating that the porphyrins are stacked along the polynucleotide stem even at a very low [porphyrin]/[DNA base] ratio. A wavelength-dependent and time-dependent LD(r) of this complex suggests that the porphyrin molecular plane tilts strongly relative to the polynucleotide helix axis. The spectral properties of the porphyrin dimer-DNA complex are similar to those of the porphyrin dimer-poly[d(G-C)(2)] complex. However, some of the porphyrin moieties were located at the groove, which was evident by some positive characters in the CD and LD(r) spectra at the short wavelength in the Soret band.

Enzyme screening with synthetic multifunctional pores: focus on biopolymers.

This report demonstrates that a single set of identical synthetic multifunctional pores can detect the activity of many different enzymes. Enzymes catalyzing either synthesis or degradation of DNA (exonuclease III or polymerase I), RNA (RNase A), polysaccharides (heparinase I, hyaluronidase, and galactosyltransferase), and proteins (papain, ficin, elastase, subtilisin, and pronase) are selected to exemplify this key characteristic of synthetic multifunctional pore sensors. Because anionic, cationic, and neutral substrates can gain access to the interior of complementarily functionalized pores, such pores can be the basis for very user-friendly screening of a broad range of enzymes.

Antiviral amphipathic oligo- and polyribonucleotides: analogue development and biological studies.

A series of novel N1 alkylated purine nucleic acids were polymerized either enzymatically or by automated synthesis to further establish the SAR requirements for HIV, RT, and HCMV activity. Out of the series, two constructs, 2'-O-methyl-1-allylinosinic acid phosphorothioate 33-mer (16) and an oligomer incorporating 1-propyl-6-thioinosinic acid residues (20), were found to be highly active under all three assay conditions. SAR studies indicate that sulfur incorporation, high molecular weight, and low steric bulk at N1 all can be important for activity.

Classification of CD and absorption spectra in the Soret band of H(2)TMPyP bound to various synthetic polynucleotides.

The binding mode of porphyrins, namely meso-tetrakis(N-methyl pyridinium-4-yl)porphyrin (H(2)TMPyP), was classified in this work by absorption and circular dichroism(CD) spectroscopy. The three binding modes of intercalation, minor groove binding and external stacking exhibit their own characteristic absorption and CD spectra. Intercalation occurs for this porphyrin when bound to GC-rich polynucleotides at a low mixing ratio, as expected. This binding mode produces hypochromism and a red shift in the absorption band and a negative CD band in the Soret absorption region. When it is complexed with AT-rich polynucleotides at a low mixing ratio, hypochromism and a red shift in the absorption band and a positive CD peak is apparent, and this species can easily be assigned to the minor groove-binding mode. For both AT- and GC-rich polynucleotides at a high binding ratio, an excitonic CD was apparent. The sign of excitonic CD depends on the order of the DNA bases; the CD spectra of H(2)TMPyP complexed with non-alternating homopolymer (disregarding the nature of base pairs, i.e. AT or GC) are characterized by a positive band at short wavelengths followed by a negative band at long wavelengths. In contrast, those complexed with alternating polynucleotide were opposite to those of non-alternating homopolymers.

The role of self-assembled monolayers of the purine and pyrimidine bases in the emergence of life.

The experimental evidence for the spontaneous formation and structure determination of two-dimensional monolayers of the purine and pyrimidine bases is examined. The plausibility of such structures forming spontaneously at the solid-liquid interface following their prebiotic synthesis suggests a functional role for them in the emergence of life. It is proposed that prebiotic interactions of enantiomorphic monolayers of mixed base composition with racemic amino acids might be implicated in a simultaneous origin of a primitive genetic coding mechanism and biomolecular homochirality. The interactions of these monolayers with carbohydrates and other derivatives is also discussed.

Error analysis of chemically synthesized polynucleotides.

Two single-stranded polynucleotide constructs, 123 and 126 nucleotides in length, were chemically synthesized using standard phosphoramidite chemistry. Clonable, double-stranded DNA fragments about 100-bp long were prepared from the polynucleotides by primer extension with a DNA polymerase and end-trimming with two restriction endonucleases, then the fragments were ligated into separate plasmids. Errors in individual insert copies were determined by dideoxy sequencing after in vivo amplification of plasmids. Five of the ten inserts sequenced contained errors, including seven single-base-pair deletions, one four-base-pair deletion and one G-->C transversion. The origins of the latter two errors are unclear, but single-base deletions are inconsistent with errors of polymerases; thus, the most common sequence errors of chemical synthesis are deletion mutations. Deletions are most likely to result from incomplete capping or de-tritylation. The observed error rate can became a significant limiting factor in applications that depend on the correctness of a polynucleotide sequence in individual insert clones.

Spermine conjugated peptide nucleic acids (spPNA): UV and fluorescence studies of PNA-DNA hybrids with improved stability.

Peptide Nucleic Acids (PNAs), the achiral DNA mimics with amide backbone, are emerging as attractive leads for drug development by antisense approach. Two major limitations of PNAs from an application perspective are their limited solubility in aqueous systems and pronounced self-organization. In this paper, it is shown that covalent conjugation of spermine at C-terminus of PNA (spPNA) improves its solubility and binds to complementary DNA 20 times stronger than the corresponding binding of PNA. Fluorescence kinetics shows a 2 fold acceleration of the bimolecular association process in spPNA:DNA hybrids, due to electrostatic interaction cationic spermine tagged to PNA with anionic DNA. This modification is easy to incorporate into PNA synthetic protocols to make them more effective in biological applications and may improve the poor cell uptake of PNA.

Synthetic polynucleotide templates for characterizing sequence-selective small molecule/nucleic acid interactions.

A synthetic polynucleotide sequence was developed and cloned to serve as a target in footprinting and other assays designed to characterize the sequence-selective binding of drugs and other small molecules to various forms of nucleic acids. The target sequence comprehensively represents all base quartet recognition sites in a minimal length sequence. Minimal length target sequences were found to be 144 nt long. One such target sequence was divided into two parts. One strand of each part was chemically synthesized and the complementary strands were generated using a DNA polymerase. Double-stranded sequences were then cloned into pGEM-3Zf(+/-) vectors (Promega, Inc.). The cloned target sequence can be used directly in double-stranded DNA form. Alternatively, features of the plasmid vector allow expression of the target sequences as single-stranded DNA or RNA or as RNA/DNA or RNA/RNA duplexes. These cloned target sequences designed for high information content overcome limitations to the use of natural DNA sequences for footprinting and related experiments arising from the unequal representation of base quartets and the potential for secondary structure formation in single-stranded forms.

Synthesis of oligopeptides as polynucleotide analogs.

Several dipeptides which have a nucleic acid base in their side chains were designed as water-soluble nucleotide analogs. Amino acids having a uracil or adenine moiety in the gamma-position were synthesized and were connected with another amino acid (spacer) to afford dipeptides. The oligopeptides were prepared from the dipeptides as monomer units. Although all the oligopeptides having uracil moieties examined were water-soluble as expected, these exhibited no hypochromic effect with poly A or poly dA. Contrary, the oligopeptides with adenines exhibited large hypochromicity (ca. 30%) base-specifically with poly dT or poly U.

Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation.

The crystal structure of a MyoD basic-helix-loop-helix (bHLH) domain-DNA complex has been solved and refined at 2.8 A resolution. This structure proves that bHLH and bHLH-leucine zipper (bHLH-ZIP) proteins are remarkably similar; it helps us understand subtle differences in binding preferences for these proteins; and it has surprising implications for our understanding of transcription. Specifically, Ala-114 and Thr-115, which are required for positive control in the myogenic proteins, are buried at the protein-DNA interface. These residues are not available for direct protein-protein contacts, but they may determine the conformation of Arg-111. Comparisons with Max suggest that the conformation of this arginine, which is different in the two structures, may play an important role in myogenic transcription.

Expression of inactive stage anti-dsDNA idiotypes on anti-ssDNA antibodies in a lupus patient during active stage of lupus cerebritis.

The possibility that idiotypes (Ids) defined on anti-double stranded DNA (dsDNA) antibodies during active and inactive stages of lupus (1/84 Id and 4/90 Id, respectively) were expressed on anti-DNA antibodies during a subsequent active period (9/90) of the disease was investigated in a lupus patient with lupus cerebritis. Using rabbit (R)-anti-Ids specific to 1/84 Id and 4/90 Id in inhibition assays, the 4/90 Id was shown to be expressed on the framework regions of anti-single stranded DNA (ssDNA) but poorly on co-existing anti-dsDNA antibodies of active (9/90) stage. The 1/84 Id was poorly expressed on both types of 9/90 anti-DNA antibodies. While the 9/90 anti-ssDNA significantly bound to immobilized ssDNA and several single-stranded polynucleotides, only ssDNA inhibited the binding of the anti-ssDNA to ssDNA, suggesting its monospecificity toward ssDNA. Western blot analysis following isoelectric focusing showed that a spectrotype pattern of 4/90 Id-positive 9/90 anti-ssDNA IgG was similar to that of the 4/90 anti-dsDNA, suggesting that they are of related clonal origin. The present study suggests the idiotypic heterogeneity of anti-DNA antibodies and the shift of antigen specificity within an idiotypically related anti-DNA population during exacerbation of the disease.

Structure-activity relationships and mode of action of 5-mercapto-substituted oligo- and polynucleotides as antitemplates inhibiting replication of human immunodeficiency virus type 1.

Introduction of a reactive 5-mercapto group into some of the cytosine and/or uracil bases of various oligo- and polynucleotides by partial thiolation resulted in several potent inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) in primary human lymphocytes. These compounds exhibited little if any toxicity against uninfected peripheral blood mononuclear cells and showed 15 to 75 times higher antitemplate activity against a p66/p51 HIV-1 recombinant reverse transcriptase (RT) than against the DNA polymerase alpha from human lymphocytes. In contrast, the unthiolated oligo- and polynucleotides are void of antitemplate activity, and their apparent inhibitory effect on HIV-1 closely paralleled their toxicity for the cells. Partially thiolated poly(dC) (MPdC) was the most potent of all the compounds tested against HIV-1 in peripheral blood mononuclear cells (50% effective concentration, 1.8 micrograms/ml or 0.019 microM), while showing low cytotoxicity (greater than 100 micrograms/ml). The corresponding unmodified poly(dC) showed no anti-HIV-1 activity at 50 micrograms/ml but had pronounced cytotoxicity. MPdC was also a potent inhibitor of HIV-1 RT (50% inhibitory concentration, 0.30 micrograms/ml). The inhibitory activities of thiolated homooligo(dCs) against both HIV-1 replication and HIV-1 RT increased with increasing chain length. The heterooligonucleotides included in this study were designed as structural analogs of portions of the natural primer of HIV-1 RT, i.e., tRNA(3Lys). An 18-mer analog of the 3' terminus, complementary (antisense) to the primer-binding site of the HIV-1 genome, was attached to an oligo(dC) tail and 5-thiolated; this increased its activity and decreased its toxicity. This compound will serve as a new lead in the development of more effective antitemplates against HIV-1.