Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis

Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified ‘ultra-mild’ DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of β-mercaptoethanol) designed to remove protecting groups of commercially available ‘ultra-mild’ phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.     

Porphyrin substituted phosphoramidites: new building blocks for porphyrin-oligonucleotide syntheses

Thymidine phosphoramidites containing trispyridylphenyl and tetraphenylporphyrin chromophores attached via a short amide linker in the 3'-position have been synthesized and used as building blocks in solid-phase synthesis of self-complementary 8-mer oligonucleotides 3'-T-5'-GCGCGCA-3' and 5'-ACGCGCGT-3'. To our knowledge, these are the first porphyrin-oligonucleotide conjugates carrying the porphyrin chromophores in the 3'-position. Chain assembly was achieved by automated solid-phase synthesis and by inexpensive straightforward 'in flask' modification of commercially available solid supported oligonucleotides. This approach allows the synthesis of modified oligonucleotides without using costly instrumentation for automated DNA synthesis. Porphyrin-containing self-complementary oligonucleotides are expected to be a valuable model for drug binding studies and determination of conformational changes in DNA sequences using circular dichroism.     

Solid phase-supported thymine dimers for the construction of dimer-containing DNA by combined chemical and enzymatic synthesis: a potentially general method for the efficient incorporation of modified nucleotides into DNA.

The ability to study the structure-activity relationships of the cis-syn thymine dimer, the major photoproduct of DNA, has been greatly aided by the availability of a building block suitable for its sequence-specific incorporation into oligonucleotides by standard automated DNA synthesis. Unfortunately, its usefulness is compromised by the fact that it takes six steps to synthesize in low overall yield and, as with all phosphoramidite building blocks, has to be used in great excess over the support in standard automated synthesis. To extend the usefulness of this building block, we have directly coupled it to standard A, C, G and T long chain alkylamine-linked controlled pore glass supports to yield a solid phase-supported dimer. We then demonstrate that 13mers containing a 3'-terminal d(T[cis-syn]TN) group synthesized with this support at 0.2 micromol scale can be efficiently incorporated into longer oligonucleotides by both primer extension with 3'-->5'exonuclease-deficient Klenow fragment or T4 polymerase and dNTPs or by enzymatic ligation with T4 DNA ligase to another oligonucleotide opposite a complementary template. The site specificity and integrity of the cis-syn thymine dimer after both primer extension and ligation was confirmed by cis-syn dimer-specific cleavage with T4 denV endonuclease V. This general approach should be applicable to the synthesis of many types of site-specific nucleic acid modifications and would be of particular use for those for which the required building blocks are expensive or difficult to make.     

Evaluation of oligonucleotides containing two novel 2'-O-methyl modified nucleotide monomers: a 3'-C-allyl and a 2'-O,3'-C-linked bicyclic derivative.

The two ribo-configured nucleosides 1-(3-C-allyl-2-O-methyl-beta-D-ribo-pentofuranosyl)thymine 3 and (1S,5R,6R,8R)-5-hydroxy-6-(hydroxymethyl)-1-methoxy-8-(thymin-1-yl )- 2,7-dioxabicyclo[3.3.0]octane 6 have been transformed into their corresponding phosphoramidites, 5 and 8 respectively, and used as building blocks for the synthesis of modified oligonucleotides. The oligonucleotides were shown to hybridize with decreased binding affinity towards complementary single stranded DNA and RNA.     

Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation

We report the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazides. The reaction of these hydrazide oligonucleotides with active esters and aldehydes is demonstrated for solution conjugation and immobilization. Compared with the established amino modified oligonucleotides, hydrazides show enhanced reactivity at neutral and acidic buffer conditions. One method to introduce hydrazides is using amidites with preformed, protected hydrazides. A completely novel approach is the generation of the hydrazide functionality during the oligonucleotide cleavage and deprotection with hydrazine. Therefore, building blocks for the introduction of esters as hydrazide precursors are described. For the enhanced attachment on surfaces branched modifier amidites, which introduce up to four reactive groups to the oligonucleotide, are applied. The efficiency of branched hydrazide oligonucleotides compared with standard amino modified oligonucleotides for the immobilization of DNA on active electronic Nanogen chips is demonstrated.     

DNA containing non-nucleosidic phenanthrene building blocks with asymmetrical linkers

The synthesis and hybridization properties of oligonucleotides containing phenanthrene building blocks with non-nucleosidic linkers of different length are described. It was found that the length of the linkers, as well as the combination of unequal linkers can have a substantial influence on the thermal stability of the modified DNA.     

Porphyrin-DNA: a supramolecular scaffold for functional molecules on the nanometre scale

We are pursuing the aim to use DNA as a supramolecular scaffold for the creation of electronically functional molecules on the nanometre scale. Here, we give a review on our results on porphyrin modified nucleotides used for this purpose. A general synthetic route to porphyrin-nucleotides has been devised, and the building blocks can be incorporated into oligonucleotides using standard solid phase synthesis methods. Up to 11 porphyrins were incorporated into DNA, reaching a length of approximately 4 nm in the array. The spectroscopic data are consistent with a porphyrin induced secondary structure stabilisation in the single strands.     

Solid-phase synthesis of oligonucleotides labeled with luminescent lanthanide(III) chelates

The synthesis of phosphoramidite building blocks that allow introduction of luminescent europium(III), terbium(III), dysprosium(III), and samarium(III) chelates to oligonucleotides on the solid phase is described. Several labeled oligonucleotides using these building blocks were prepared, and the photophysical properties of these bioconjugates were investigated.     

Synthesis and hybridization properties of inverse oligonucleotides.

The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and DNA is significantly reduced.     

Recursive construction of perfect DNA molecules from imperfect oligonucleotides

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error‐free DNA molecules and their libraries from error‐prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem‐solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error‐prone oligonucleotides are recursively combined in vitro, forming error‐prone DNA molecules; error‐free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error‐free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.     

一种新的等温单向生长基因合成法初步探索

基因合成正日益成为基因获取的一种有效手段。两种常用的基因合成方法是基于PCR的基因合成（P法）和基于连接酶的基因合成(L法)。这两种方法都不是独立于所合成基因的序列内容的。文章首次提出了一种新的基因合成策略—等温单向生长法(Isothermal Unidirectional Elongation Method, IUEM), 在3种酶的作用下, DNA链在等温状态下单向串行延伸, 直到获得目标基因。由于引物设计成特殊的发夹结构, 该方法可以做到合成过程独立于或部分独立于目标基因的序列内容。本文探索了该策略的实验可行性, 检测了影响基因合成的各种因素, 并利用该方法成功地合成了一段254 bp和一段300 bp的DNA序列。     

Alpha-hydroxybenzylphosphonate modified oligonucleotides: synthesis, properties, and a novel route via monomer building blocks

In general, alpha-hydroxybenzylphosphonate modified 2'-deoxyadenosine-thymidine dimer building blocks 1, 2 are utilized for the incorporation into alpha-hydroxybenzylphosphonate pro-oligonucleotides. For a universal application of our pro-oligonucleotide concept on biologically relevant oligonucleotides a route for the synthesis of modified monomer building blocks 3 was developed and is presented herein.     

Evaluation of Oligonucleotides Containing Two Novel 2′-O-Methyl Modified Nucleotide Monomers: A 3′-C-Allyl and a 2′-O-3′-C-Linked Bicyclic Derivative

Abstract

The two ribo-configured nucleosides 1-(3-C-allyl-2–0-methyl-β-D-ribo-pentofuranosyl)thymine 3 and (1S,5R,6R,8R)-5-hydroxy-6-(hydroxymethyl)-1-methoxy-8-(thymin-1-yl)-2,7-dioxabicyclo[3.3.0]octane 6 have been transformed into their corresponding phosphoramidites, 5 and 8 respectively, and used as building blocks for the synthesis of modified oligonucleotides. The oligonucleotides were shown to hybridize with decreased binding affinity towards complementary single stranded DNA and RNA.     

Benzimidazolium triflate-activated synthesis of (6-4) photoproduct-containing oligonucleotides and its application.

In the solid-phase synthesis of oligonucleotides containing the pyrimidine(6-4)pyrimidone photoproduct using a dinucleotide building block, considerable amounts of by-products were found as the chain length increased. The by-products were the major product when a 49mer was synthesized on a 40 nmol scale. It was assumed that these by-products were formed by the coupling of phosphoramidites with the N3 imino function of the 5' component of the (6-4) photoproduct. We examined imidazolium triflate and benzimidazolium triflate to find an alternative activator for DNA synthesis. Imidazolium triflate prevented by-product formation to some extent, but the coupling yields were low. Benzimidazolium triflate was comparable to tetrazole in coupling efficiency and reduced by-product formation to a great extent, without modification of the synthesizer program. The obtained 49mer was used to detect proteins that recognize UV-damaged DNA in HeLa cell extracts. Two major protein-DNA complexes were found when a 49mer duplex was used as probe, while a 30mer duplex failed to detect one of them. This application showed the usefulness of long chain 'damaged' oligonucleotides in biochemical studies.     

Rapid synthesis of oligodeoxyribonucleotides VI. Efficient, mechanised synthesis of heptadecadeoxyribonucleotides by an improved solid phase phosphotriester route.

Efficient mechanised synthesis of heptadecadeoxyribonucleotides has been achieved on an economically small scale by an improved solid phase phosphotriester method on a polydimethylacrylamide resin. Improvements were made in the preparation of dinucleotide building blocks, reaction conditions for oligonucleotide assembly and in purification of deprotected oligonucleotides by h.p.l.c. Several milligrams of pure heptadecamers were obtained. Two of the heptadecamers were designed for sequencing in opposite directions of DNA cloned in phage M13mp2.     

Synthesis and evaluation of RNA transesterification efficiency using stereospecific serinol-terpyridine conjugates

Six novel artificial ribonucleases were synthesized employing a stereochemically pure abasic serinol backbone residue for attachment of the RNA transesterification agent copper(II) terpyridine. These stereochemically pure abasic residues were synthesized as phosphoramidite building blocks from the parent L-serine and D-serine starting building blocks and incorporated into oligonucleotides via solid-phase DNA synthesis. These artificial ribonucleases were constructed to determine if the stereochemistry of the alpha carbon of an abasic serinol residue has influence over RNA transesterification through selective placement of a pendant transesterification agent in either the major or minor groove. The novel artificial ribonucleases and previously synthesized artificial ribonucleases were challenged with a 28-mer and 159-mer RNA substrate. It was determined that the stereochemistry of the carbon atom derived from the alpha-carbon of serine did not influence the extent of cleavage in these studies using copper(II) terpyridine conjugated artificial ribonucleases.     

Triple-helix mediated excimer and exciplex formation

The synthesis and properties of triple-helical hybrids containing non-nucleosidic polyaromatic building blocks are described. Clamp-type oligonucleotides containing a non-nucleosidic pyrene linker form stable triple helices with a polypurine target strand containing a terminal pyrene or phenanthrene moiety. Stacking interactions between the unnatural building blocks enhance triplex stability and lead to strong excimer or exciplex formation, which is monitored by fluorescence spectroscopy.     

Manipulating the salt and thermal stability of DNA multilayer films via oligonucleotide length

DNA films are promising materials for diverse applications, including sensing, diagnostics, and drug/gene delivery. However, the ability to tune the stability of DNA films remains a crucial aspect for such applications. Herein, we examine the role of oligonucleotide length on the formation, and salt and thermal stability, of DNA multilayer films using oligonucleotides of homopolymeric diblocks (polyAG and polyTC), with each block (A, G, T, or C) ranging from 5 to 30 bases (10-, 20-, 30-, 40-, and 60-mer). Using a combination of quartz crystal microgravimetry, dual polarization interferometry, and flow cytometry, we demonstrate that at least 10 bases per hybridizing block in the DNA diblocks (that is, 20-mer) are required for successful hybridization and, hence, DNA multilayer film formation. Films assembled using longer oligonucleotide blocks were more stable in low salt conditions, with the DNA multilayer films assembled from the 60-mer oligonucleotides remaining intact in solutions of about 25 mM NaCl. A systematic increase in film melting temperature ( T m) was observed for the DNA multilayer films (assembled on colloids) with increasing oligonucleotide length, ranging from 38.5 degrees C for the 20-mer films to 53 degrees C for the 60-mer films. Further, an alternating trend in T m of the DNA multilayer films was observed with layer number (AG or TC); DNA multilayer films terminated with an AG layer exhibited a higher T m (44-49 degrees C) than films with an outermost TC layer (ca. 38 degrees C), suggesting a rearrangement of the film structure upon hybridization of the outermost layer. This work shows that the stability of DNA multilayer films can be tuned by varying the length of the oligonucleotide building blocks, thus providing a versatile means to tailor the salt and thermal stability of DNA films, which is necessary for the application of such films.     

Synthesis of encoded modified oligonucleotide libraries. Part 1: orthogonal chemistry

The basis for further development of combinatorial libraries of modified oligonucleotides tagged by a codifying sequence is discussed. The chemistry involved in the orthogonal synthesis of both strands and some representative examples of building blocks are presented.     

Synthesis of oligonucleotide building blocks of 2'-deoxyguanosine bearing a C8-arylamine modification

C8-Arylamine-dG adducts were synthesized by palladium-catalyzed cross-coupling reactions. The corresponding 5'-O-DMTr-3'-O-phosphoramidite-C8-arylamine-dG adducts were synthesized as potential building blocks for the automated synthesis of site-specifically modified oligonucleotides.