No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Recent Advances in Oligonucleotide Synthesis and Their Applications
Abstract
Short synthetic oligonucleotides are finding wide variety of applications in area of genomics and medicinal chemistry. Since the isolation of nucleic acids to the mapping of human genome, chemical synthesis of nucleic acids has undergone tremendous advancements. Further improvements in this area such as, introduction of high throughput synthesizers, better coupling reagents, improved polymer supports, newer sets of protecting groups for exocyclic amino groups of nucleic bases and introduction of universal polymer supports have completely revolutionized the entire field of nucleic acids chemistry. Most of these developments have been targeted to assemble these molecules more efficiently in a cost-effective manner and rapidly. Preparation of oligonucleotide conjugates has further helped in identifying the newer areas of their applications. A number of conjugates with biological and abiological ligands have been discussed in this article along with their possible wide spectrum of applications. Recently developed microarray technology, which refers to attachment of short oligonucleotides on a solid/polymeric surface, has proved to be useful for screening of genetic mutations, study of polymorphism, as diagnostics, etc. The major developments in these areas are presented in the review.
... Direct syntheses (in situ) of oligonucleotides on the chip surface following conventional or photolithographic methods and immobilization of presynthesized oligonucleotides on selected substrates (the deposition method) are two predominant methods for fabrication of oligonucleotide microarrays (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). The in situ synthesis protocol, which allows the preparation of high-density oligonucleotide microarrays, suffers from certain drawbacks (6)(7)(8)17). ...
... Direct syntheses (in situ) of oligonucleotides on the chip surface following conventional or photolithographic methods and immobilization of presynthesized oligonucleotides on selected substrates (the deposition method) are two predominant methods for fabrication of oligonucleotide microarrays (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). The in situ synthesis protocol, which allows the preparation of high-density oligonucleotide microarrays, suffers from certain drawbacks (6)(7)(8)17). The deposition method, on the other hand, offers flexibility, in the sense that a variety of biomolecules can be immobilized on a surface of choice but is limited to generation of low-to-medium-density arrays. ...
Microarray technology has become an important tool for detection and analysis of nucleic acid targets. Immobilization of modified and unmodified oligonucleotides on epoxy-functionalized glass surfaces is often used in microarray fabrication. Here, we demonstrate a protocol that employs coating of SU-8 (glycidyl ether of bisphenol A) onto glass microslides to obtain high density of epoxy functions for efficient immobilization of aminoalkyl-, thiophosphoryl-, and phosphorylated oligonucleotides with uniform spot morphology. The resulting microarrays exhibited high immobilization (∼65%) and hybridization efficiency (30-36%) and were sufficiently stable over a range of temperature and pH conditions. The prominent feature of the protocol is that spots can be visualized distinctly at 0.05 μM probe (a 20-mer oligonucleotide) concentration. The constructed microarrays were subsequently used for detection of base mismatches and bacterial diseases (meningitis and typhoid).
... nate Cas9 nucleases, species, or CRISPR-Cas systems (Cong et al., 2013; Kleinstiver et al., 2015a; Kleinstiver et al., 2015b; Ran et al., 2015; Kleinstiver et al., 2016). It is important to note that with traditional chemistries, such as TBDMS or 2 -TOM (Pitsch et al., 2001; Sproat, 2005 ), it can be challenging to accurately and efficiently synthesize RNA greater than ∼70 bases. For S. pyogenes, the crRNA, tracrRNA and sgRNA are approximately 40, 70 and 100 nucleotides in length, respectively (Jinek et al., 2012), which would make synthesis of the tracrRNA or an sgRNA challenging for most chemistries. ...
The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events.
... These approaches are relatively simple and published protocols are available. Recently, technologies and applications of DNA synthesis have been reviewed [2,11,12]. However, while these approaches generally succeed in producing usable oligos, they each have limitations than can lead to errors in assembly [13]. ...
Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.
... 7 Therefore, 3′-modified oligonucleotides bearing reactive functionalities such as aminoalkyl, carboxyl, mercaptoalkyl, aminooxyalkyl, and phosphoryl are finding important biomedical applications. 8 Recently, microarrays have extensively been used for various applications such as the detection of nucleic acid sequences, gene expression analysis, mutation detection, and so forth. 9,10 The technology is based on hybridization of targets with the immobilized probes, either single-or double-stranded nucleic acids, followed by detection through a scanning process. ...
An engineered polymer support 5 has been prepared for the solid-phase assembly of 3'-carboxyalkyl-modified oligonucleotides using commonly available reagents. A two-step deprotection procedure resulted in the quantitative cleavage of oligonucleotides from the support and removal of the protecting groups from phosphodiesters and exocyclic amino groups of the nucleic bases. The fully deprotected oligomers, obtained in high yield, were desalted and analyzed on RP-HPLC. After characterization by MALDI-TOF, these carboxyalkylated oligonucleotides were immobilized onto the epoxy-functionalized glass microslides to prepare biochips. The performance of these biochips was evaluated under different sets of conditions and then successfully validated by the detection of base mismatches and human infectious disease, bacterial meningitis, caused by N. meningitidis.
Dendrimers and dendrons are classes of highly branched ?tree-shaped? nanosized molecules. This chapter deals with dendrimers in connection with biology and medicine, and begins with a discussion on their properties. In a divergent approach of synthesis, the dendrimer is essentially synthesized beginning at the core and building outward, whereas in the convergent approach the dendrimer is built starting from the periphery and going toward the core. Polyamidoamine (PAMAM) dendrimers, are synthesized by Michael addition of methylacrylate to polyamines followed by amidation by treatment with an excess of 1,2-ethanediamine. The chapter highlights toxicity of dendrimers, and then goes on to discuss the use of dendrimers in diagnostics and dendrimer-based reagents in imaging. Other topics that are discussed include anti-inflammatory activities of dendrimers, DNA transfection, and small interfering RNA (siRNA).
Polypyrrole (PPy)-bile salt composite was used for sensing ethanol vapor. PPy was synthesized by interface polymerization for subsequent fabrication of thin film of its composite with bile salt, by in-situ co-dispersion method and then exposed to ethanol vapour. Sensing was visualized through changes in morphological, structural and optical characterizations. The ethanol exposed film showed larger agglomeration as revealed in its surface morphology on scanning electron microscope (SEM) and greater crystallinity as seen through X-Ray diffraction (XRD). Fourier transform infra red (FTIR) and nuclear magnetic resonance spectroscopy (NMR) of the ethanol incorporated film also gave signature of the presence of bile salt and alcohol. Alcohol incorporation pattern resulted in increase in electrical conductance from 7.08539 x 10(-5) mA/V to 8.0356 x 10(-5) mA/V, as determined from current voltage characterizations. Average molecular weight (M(n)) obtained from gel permeation chromatography changed from 6160 to 10300 on ethanol intake. Photoluminescence (PL) intensity was quenched and the PL peak shifted from 430 to 409 on ethanol exposure. Changes in morphological, structural, optical and electrical properties of the composite on ethanol exposure showed its prospective application for sensing ethanol.
DNA microarray technology offers potential for future high-throughput variation genotyping. Allele-specific primer extension procedure on microarray has been considered as an efficient method for single nucleotide polymorphism(SNP) genotyping. However, the high cost of the fluorescent-labeled dNTP used for signal detection in this method limits its application. In the present study, we evaluated the characteristics of solid-phase allele-specific primer extension, in terms of specificity and efficiency and demonstrated that compared to liquid-phase reaction, it requires lower annealing temperature, and higher template and Mg2+ concentrations. The extension efficiency and specificity were though linked, behave diametric during the gradient change of template and Mg2+ concentrations or annealing temperature. To obtain both optimal signal intensity and specificity, we introduced an artificial mismatched base at the third position from the primer 3'end, which enhanced the specificity significantly. The PicoGreen staining method, which could decrease the cost greatly, was then introduced to replace the fluorescent-labeled dNTP for signal detection.
A novel approach for the construction of oligonucleotide microarrays under the influence of light and microwaves is described. For that purpose, a new heterobifunctional crosslinking reagent, 1-N-(maleimidohexanoyl)-6-N-(anthraquinon-2-oyl) hexanediamine (MHAHD), possessing a photoactive anthraquinone moiety on one terminus and an electrophilic maleimide group on the other one, was developed. The immobilization of oligonucleotides using the reagent, MHAHD, was realized via two routes (A and B). In order to speed up the immobilization procedure, the reaction between 3′- or 5′-mercaptoalkylated oligonucleotides and maleimide moiety of MHAHD was carried out under microwaves in just 15 min. The oligonucleotide arrays produced by both the routes were analyzed by hybridization experiments (hybridization efficiency 30.13%) and subsequently used for the discrimination of base mismatches. The constructed microarrays were found to possess good thermal stability (only ∼4.5% loss of fluorescence intensity observed after ten cycles).
The development of oligonucleotide-based microarrays (biochips) is a major thrust area in the rapidly growing biotechnology industry, which encompasses a diverse range of research areas including genomics, proteomics, computational biology, and pharmaceuticals, among other activities. Microarray experiments have proved to be unique in offering cost-effective and efficient analysis at the genomic level. In the last few years, biochips have gained increasing acceptance in the study of genetic and cellular processes. As the increase in experimental throughput has posed many challenges to the research community, considerable progress has been made in the advancement of microarray technology. In this review, chemical strategies for immobilization of oligonucleotides have been highlighted with special emphasis on post-synthetic immobilization of oligonucleotides on glass surface. The major objective of this article is to make the researchers acquainted with some most recent advances in this area.
We propose the new approach to the synthesis of 5'-triphosphate derivatives of natural and modified dinucleotides with expanded functionality. Our strategy includes the combination of the solution phase synthesis of necessary dimers using the wide range of nucleic acids chemistry methods and the subsequent introduction of the triphosphate residue. A number of the new potential substrates for the template dependent synthesis of nucleic acids with expanded functionality are obtained, namely, 5'-triphosphates of dinucleotides containing the functionally active groups in heterocyclic bases, in carbohydrate-phosphate backbone, and the groups mimicking the residues of natural amino acids. The abilities of the proposed synthetic route are also demonstrated by the synthesis of 5'-triphosphates of dinucleotides with modified carbohydrate-phosphate backbone.
A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5'- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5'-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.
This review covers the research findings related to almost all aspects of the peptide-oligonucleotide conjugates (POCs). First, a brief introduction into the need for conjugation of peptides to oligonucletides and their potential applications in diverse fields is given. Different POCs known so far are presented in a systematic manner to have a clear understanding on the possible modes of conjugation of peptides to oligonucleotides. Second, different types of chemical linkages and the means to achieve the same are summarized. There are two major synthetic protocols: solid-phase divergent and solid- or liquid-phase convergent methods. For synthesis of oligonucleotides or peptides, the solid-phase divergent method is the primary method of preparation but for preparation of conjugates, the solid-phase sequential method fails.
The primary literature concerning the combinatorial synthesis of organophosphorus compounds is reviewed and discussed. The subject matter is divided into three main sections describing the solid phase, solution phase and solvent-free synthesis of phosphorus containing organic molecules. The review covers the synthesis of compounds in which the final products contain phosphorus-carbon bonds, primarily phosphonates, phosphinates, phosphine oxides and phosphines.
Solid-phase chemistry, photolabile protecting groups, and photolithography have been combined to achieve light-directed, spatially
addressable parallel chemical synthesis to yield a highly diverse set of chemical products. Binary masking, one of many possible
combinatorial synthesis strategies, yields 2n compounds in n chemical steps. An array of 1024 peptides was synthesized in
ten steps, and its interaction with a monoclonal antibody was assayed by epifluorescence microscopy. High-density arrays formed
by light-directed synthesis are potentially rich sources of chemical diversity for discovering new ligands that bind to biological
receptors and for elucidating principles governing molecular interactions. The generality of this approach is illustrated
by the light-directed synthesis of a dinucleotide. Spatially directed synthesis of complex compounds could also be used for
microfabrication of devices.
Coupling efficiencies for the covalent attachment of oligonucleotides (17–29 bases in length) to solid supports derivatized
with alkyl-amino and -carboxylic functionalities have been determined. Attachment efficiencies of 60–80% were obtained for
coated long-chain alkylamino controlled pore glass (CPG) supports. Similar efficiencies of immobilization were observed for
carbox-yl-bearing supports, which additionally exhibited lower levels of non-covalent binding. The extent of terminally linked
oligonu-cleotide was determined to be 50–55% of the overall attachment in the carbodiimide-mediated coupling reaction of a
5′-aminohexyl phosphoramidate derivative of a 29-mer to Sephacryl carboxyl support. While lower overall efficiencies of attachment
were obtained in the reaction with Sephacryl N-hydroxysuccinimide-activated carboxyl support, greater than 80% of this coupling
results in end-attached oligonucleotides.
Fluorescent dye-labeled DNA primers have been developed that exploit fluorescence energy transfer (ET) to optimize the absorption and emission properties of the label. These primers carry a fluorescein derivative at the 5' end as a common donor and other fluorescein and rhodamine derivatives attached to a modified thymidine residue within the primer sequence as acceptors. Adjustment of the donor-acceptor spacing through the placement of the modified thymidine in the primer sequence allowed generation of four primers, all having strong absorption at a common excitation wavelength (488 nm) and fluorescence emission maxima of 525, 555, 580, and 605 nm. The ET efficiency of these primers ranges from 65% to 97%, and they exhibit similar electrophoretic mobilities by gel electrophoresis. With argon-ion laser excitation, the fluorescence of the ET primers and of the DNA sequencing fragments generated with ET primers is 2- to 6-fold greater than that of the corresponding primers or fragments labeled with single dyes. The higher fluorescence intensity of the ET primers allows DNA sequencing with one-fourth of the DNA template typically required when using T7 DNA polymerase. With single-stranded M13mp18 DNA as the template, a typical sequencing reaction with ET primers on a commercial sequencer provided DNA sequences with 99.8% accuracy in the first 500 bases. ET primers should be generally useful in the development of other multiplex DNA sequencing and analysis methods.
A method is described for the detection of DNA hybrids formed on a solid support, based upon the pairing of oligonucleotide chemistry and the technologies of electronic microdevice design. Surface matrices have been created in which oligonucleotide probes are covalently linked to a thin SiO2 film. 32P labeled target nucleic acid is then hybridized to this probe matrix under conditions of high stringency. The salient feature of the method is that to achieve the highest possible collection efficiency, the hybridization matrix is placed directly on the surface of a charge coupled device (CCD), which is used to detect 32P decay from hybridized target molecules (1, Eggers, M.D., Hogan, M.E., Reich, R.K., Lamture, J.B., Beattie, K.L., Hollis, M.A., Ehrilich, D.J., Kosicki, B.B., Shumaker, J.M., Varma, R.S., Burke, B.E., Murphy, A., and Rathman, D.D., (1993), Advances in DNA Sequencing Technology, Proc. SPIE, 1891, 13-26). Two implementations of the technology have been employed. The first involves direct attachment of the matrix to the surface of a CCD. The second involves attachment of the matrix to a disposible SiO2 coated chip, which is then placed face to face upon the CCD surface. As can be predicted from this favorable collection geometry and the known characteristics of a CCD, it is found that as measured by the time required to obtain equivalent signal to noise ratios, 32P detection speed by the direct CCD approach is at least 10 fold greater than can be obtained with a commercial gas phase array detector, and at least 100 fold greater than when X-ray film is used for 32P detection. Thus, it is shown that excellent quality hybridization signals can be obtained from a standard hybridization reaction, after only 1 second of CCD data acquisition.
In order to increase the efficiency of use of automated DNA synthesisers (i.e. the number of oligomers prepared per day),
we have devised and prepared novel phosphoramidite reagents that contain a linking group which, while stable under the normal
synthesis conditions, is cleaved under basic conditions. When one of these linkers is introduced at the desired position in
the synthesis of an oligonucleotide, subsequent detritylation enables the synthesis of a second oligonucleotide sequence upon
the first. During deprotection of the oligonucleotide with ammonium hydroxide, the chain is cleaved at either side of the
points of introduction of the novel reagent, generating two oligonucleotides free in solution. These reagents are of particular
use in applications where oligomers are used in pairs (such as PCR, chemical synthesis of genes etc.) and means that an automated
synthesis facility can be used more efficiently, without the need for operator intervention, after the working day is over.
In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition.
Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3′ end) of short
oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino
or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in
the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino
gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are
based on an acrylamide gel activated with glutaral-dehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride.
The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization
methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated
support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed
for preparing oligonucleotide microchips, they may be used for other purposes as well.
A simple, rapid, and efficient method for the covalent binding of oligonucleotides to solid glass supports was developed. Glass slides were derivatized with aminophenyl or aminopropyl silanes and 5'-succinylated target oligonucleotides were attached by carbodiimide-mediated coupling. Approximately 40 to 50% of the applied target oligonucleotides covalently bound to the derivatized glass. Hybridizations with radioactively labeled oligonucleotide probes showed that up to 90% of the attached oligonucleotides were available for hybridization. This system can conveniently be applied for studies on hybridization and detection of nucleic acids.
Different chemical methods used to attach oligonucleotides by their 5′-end on a glass surface were tested in the framework
of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first
evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5′-specific linkage
that satisfyingly withstands PCR conditions and leaves the 3′-ends available for DNA polymerase activity. The best results
were obtained with 5′-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional
cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can
be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers.
Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The
newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface
amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5′-end on a solid support
and can be used as an alternative route for producing DNA chips for genomic studies.
The chemical processes involved in the preparation of oligonucleotide-intercalator conjugates are reviewed. Two strategies are used to covalently attach an intercalating agent to oligonucleotides; it can be either directly incorporated during the oligonucleotide chain assembly or linked to the unblocked oligonucleotide using a specific coupling reaction. The covalent attachment of different intercalating agents to various positions of an oligonucleotide chain: 5′-, 3′-, both 5′- and 3′-ends, internucleotidic phosphate, 1′- or 2′-position of a sugar residue or nucleic base is reported. Studies of the binding of these modified oligonucleotides to their targets are also presented together with some of the unique properties which are conferred upon the oligonuleotides by attachment of an intercalating agent.
Facile modification of oligodeoxyribonucleotides is required for efficient immobilization to a pre-activated glass surface.
This report presents an oligodeoxyribonucleotide which contains a hairpin stem–loop structure with multiple phosphorothioate
moieties in the loop. These moieties are used to anchor the oligo to glass slides that are pre-activated with bromoacetamidopropylsilane.
The efficiency of the attachment reaction was improved by increasing the number of phosphorothioates in the loop, as shown
in the remarkable enhancement of template hybridization and single base extension through catalysis by DNA polymerase. The
loop and stem presumably serve as lateral spacers between neighboring oligodeoxyribonucleotides and as a linker arm between
the glass surface and the single-stranded sequence of interest. The oligodeoxyribonucleotides of this hairpin stem–loop architecture
with multiple phosphorothioate moieties have broad application in DNA chip-based gene analysis.
With the advent of oligonucleotides as potential therapeutics, the search for modified analogs of nucleic acids having improved properties, such as increased stability against nucleases, improved cellular uptake and enhanced binding affinity to complementary nucleic acids with high specificity, has accelerated rapidly and has attracted considerable attention. Specific binding through natural base pairing has become increasingly important for the development of DNA based diagnostics as well as therapeutics in the form of antisense and antigene oligonucleotides. Modified DNA based oligonucleotides can affect the gene expression at the level of translation as well as transcription. The automation of oligonucleotide synthesis, the development of better coupling reagents as well as support system and efficient scale-ups have expanded the application of these molecules to diverse areas of fundamental as well as applied biological research. The modified molecules are expected to find answers to understanding, unveiling and utilizing the intricate methods of cell functioning at the molecular level and would provide applications in genetic engineering, gene therapy and cellular regulations in an array of conditions. In this review, the synthesis or the assembling of oligonucleotides modified at the backbone, or at the base, or at the sugar residues, has been brought into sharp focus.
A simple protocol based on polymerization reactions has been developed for the preparation of high-loading polymer supports, useful for large-scale synthesis of oligonucleotides. Polymer supports of different pore sizes have been employed in the present investigation to improve the functional-group density on them. A ten- to twelvefold increase in the loading of the functional groups, after the polymerization reaction, has been observed. The support was then used in the subsequent reaction to attach the leader nucleoside to obtain fully functionalized supports 6a–6c by oligonucleotide synthesis in an automated DNA synthesizer. The aminoalkylated-supports 5a–5c were directly employed for the synthesis of oligonucleotide 3′-phosphates. The oligonucleotides and oligonucleotide 3′-phosphates synthesized on these supports were compared with the corresponding standard oligomers with respect to their retention time on HPLC. These were further characterized on MALDI-TOF mass spectrometry.
A hybrid comprising an oligodeoxyribonucleotide linked the 3′-hydroxyl to the amino terminus of a peptide has been prepared using a combination of solid-phase FMOC peptide synthesis methodology and phosphoramidite chemistry.
A simple solid-phase scheme for deprotection and enzymatic purification of oligodeoxyribonucleotides synthesized by a phosphoramidite method using hydrazine-sensitive protection is presented.
A procedure for the preparation of oligonucleotide-peptide hybrid molecules by means of automated synthesis is described. The conjugates have been assembled on silica supports including CPG (Controlled Pore Glass) and Fractosil supports. The novel Nε-lysine protecting group, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene) ethyl (Dde) was used.
A dithymidine dinucleotide (V) has been synthesised by condensing 3′-O-acetylthymidine with thymidine 3′-(benzyl phosphorochloridate) 5′-(dibenzyl phosphate) and subsequently removing the protecting groups. This represents the first preparation of a dinucleotide by chemical means and since the synthetic material behaves towards enzymes exactly as the dinucleotidic fragments obtained by degrading deoxyribonucleic acids the postulate of a 3′:5′-internucleotidic linkage in the latter is further confirmed. Thymidine-3′ thymidine-5′ phosphate has also been prepared and by-products isolated include a dinucleoside pyrophosphate and a dinucleotide pyrophosphate.
Incorporation of an azobenzene-4,4′-diamide group via a linker arm into the 3′-hydroxyl function of one oligonucleotide segment and the 5′-OH of other oligonucleotide has been described. The binding of the oligonucleotides containing the azobenzene linker was investigated by UV melting behaviors. The azobenzene linker has been shown to be useful as an effective bridge for stabilizing hairpin duplex and triplex.
Conjugates of pyrene and perylene with oligodeoxynucleotides were synthesized and tested as hybridisation probes. A 13-mer containing a 3-peryleneacetic acid residue attached to the 5′ end through a hexamethylenediamine linker showed no response in the fluorescent spectrum upon hybridisation to the complementary sequence. At the same time, pyrene-labelled probes are sensitive to duplex formation. A pyrene pseudonucleotide unit based on 4-(1-pyrenyl)-1,3-butanediol can be introduced into any predetermined position(s) of the oligonucleotide chain. The probes polylabelled in this fashion displayed considerable changes in the excimer-to-monomer fluorescence intensity ratio after duplex formation. The internal location of two pyrene residues in the probe provides a drastic enhancement of excimer fluorescence (∼470 nm) upon hybridisation. When two pyrene units were brought into close proximity to two pyrenes in the complementary strand upon duplex formation, strong excimer emission at ∼450 nm was detected. This effect provides a basis for a sensor construction designed to detect nucleic acid hybridisation.
Binary non-covalent systems for site-selective RNA scission are prepared from Lu(III) ion and a DNA bearing an acridine in the internal position. On the formation of DNA/RNA hetero-duplex, the target phosphodiester linkage is activated and preferentially hydrolyzed.
Phenylazonaphthalene was tethered to the 5′-end of oligo(T) as photo-responsive moiety for the photo-regulation of DNA-triplex formation. The melting temperature of DNA-triplex changed as greatly as 40.5°C on trans↔cis isomerization of phenylazonaphthalene. This change was much greater than that achieved by azobenzene.
meta-Aminoazobenzene has been introduced in the side chain of oligonucleotides as a photo-responsive molecule. Compared with the para-aminoazobenzene which was previously used, the thermal cis→trans isomerization was much slower: the half-lives of the cis-isomers of m- and p-aminoazobenzene were 13.2 h and 20 min at 50°C, respectively. By using the present oligonucleotides, duplex formation and dissociation was efficiently regulated on photo-irradiation without being disturbed by the thermal isomerization.
We describe a general methodology for the solid phase synthesis of directly linked peptide-oligonucleotide hybrids using a sarcosine-modified controlled pore glass (CPG) support and employing phosphoramidite chemistry for the oligonucleotide synthesis and Fmoc strategy for the peptide assembly. Using this approach a special linker is not needed and the resultant peptide-oligonucleotide hybrid can be further elongated with an oligonucleotide sequence.
Nucleopeptides incorporating, besides the linking residue, other trifunctional amino acids such as lysine, arginine, tryptophan, serine, threonine and tyrosine have been obtained following stepwise solid-phase assembly. The best alternatives for the protection of the side chains of these trifunctional amino acids are discussed. The use of different solid supports (polystyrene-co-1%-divinylbenzene, controlled pore glass and polyethylene glycol-polystyrene) and nucleopeptide-resin linkers has also been evaluated.
synthesis of a photoisomerizable linker containing an azobenzene unit for connecting oligonucleotide segments has been described. 4,4′-Azobenzene dicarbonyl chloride was converted to the fully protected diol by treatment with the O-silyl protected aminopentanol. Partial deprotection of the silyl group afforded the monoprotected diol, which was protected by a dimethoxytrityl group. Then the remaining silyl protecting group was removed to afford the alcohol. The hydroxyl function was converted to the phosphoramidite. The amidite can be used for joining two oligonucleotides via the 5′-terminal hydroxyl group of one oligomer and the 3′-terminal oxygen of the other.
A simple protocol for the preparation of high-loading supports, suitable for large-scale synthesis of oligonucleotides, has been developed. The method involves the use of inexpensive reagents and is amenable to large-scale preparation of supports. The derivatized supports were successfully employed in an automated DNA synthesizer without any difficulty. The quality of the synthesized oligonucleotides was found to be comparable to that of the corresponding oligomers prepared with commercially available standard supports.
Starting from 2,3:5,6-di-O-isopropylidene-α-D-mannofuranose, benzyl 5,6-ditrifluoroacetamido-5,6-dideoxy-2-O-(4,4′-dimethoxytrityl)-α-D-mannofuranose was prepared and attached to a controlled pore glass to generate a new universal solid support for oligonucleotide synthesis. This support is applicable for the preparation of all types of oligonucleotides as well as oligomers with unusual baselabile nucleosides.
A review with 411 refs. on antisense oligonucleotides including synthesis, properties, control of gene expression, toxicity, and applications. [on SciFinder (R)]
A new method for attaching oligodeoxyribonucleotides to glass involving monoalkoxylated and dialkoxylated silanes and bromoacetamide/phosphorothioate linking chemistry has been developed. Three novel bromoacetamide silanes were synthesized for derivatization of glass microscope slides by traditional dipping methods. A thin film method that conserves silane and provides a consistent protocol for test experiments was also used. Oligonucleotides bearing 5‘-phosphorothioates were synthesized by literature methods. Immobilization conditions were initially established by treatment of bromoacetamidosilyl slides with fluoresceinated oligonucleotides, which were imaged by confocal fluorescence microscopy. Spotting can be accomplished in water at oligonucleotide concentrations down to 0.1 mM. Oligonucleotides immobilized using this method can serve as primers for templated, polymerase-based extension reactions with a fluoresceinated dideoxynucleotide terminator. When such primers are formatted into small arrays, specific extension is observed only in the presence of complementary template, with the amount of immobilized primer reflected in the fluorescence signal.
A novel surface modification procedure for the creation of sulfhydryl-terminated alkanethiol monolayers that can be used for the attachment of biomolecules onto gold surfaces is described. A self-assembled monolayer of the amine-terminated alkanethiol 11-mercaptoundecylamine (MUAM) is reacted with the heterobifunctional cross-linker N-succinimidyl S-acetylthiopropionate (SATP) in order to create a protected sulfhydryl-terminated monolayer. This monolayer can then be deprotected in an alkaline solution to create an active sulfhydryl surface. Compounds that have been modified to contain a maleimide moiety can be easily attached onto the sulfhydryl-derivatized gold surface. In a second attachment strategy, the sulfhydryl-terminated monolayer is reacted with 2,2'-dipyridyl disulfide to form disulfide bonds on the surface. These disulfide bonds are then used in a thiol-disulfide exchange reaction with free sulfhydryls in order to attach biomolecules, such as thiol-modified DNA or cysteine-containing polypeptides, onto the surface. In contrast to the maleimide-attached monolayers, the disulfide-immobilized species can be cleaved in the presence of dithiothreitol (DTT) in order to regenerate the free sulfhydryl surface. Polarization modulation FTIR reflection-absorption spectroscopy (PM-FTIRRAS) has been used to characterize these surface reactions, and fluorescence "wash off" measurements provided an estimate of 1.5 x 10(12) molecules/cm(2) for the surface coverage of DNA immobilized using a thiol-disulfide exchange reaction. Surface plasmon resonance (SPR) imaging measurements were employed to monitor in situ hybridization onto DNA arrays fabricated using this surface immobilization reaction.
The enzymatic manipulation of sets of short, 16-base oligonucleotides, or DNA "words", is demonstrated with applications toward DNA computing on surfaces. The enzyme T4 DNA ligase is used to ligate (join) DNA words on a chemically modified gold thin film. The efficiency of this surface ligation reaction is 80%, as determined by removal of the ligated molecules from the surface followed by gel electrophoresis, This surface ligation reaction is used in two new operations for DNA computing on surfaces. In a "Surface Word Append" operation, the complexity and information density of DNA word strands attached to gold surfaces are increased by appending additional words onto these word strands. The ligation reaction is also utilized as part of a "Two-Word Mark and Destroy" operation in which singly marked two-word DNA strands are selectively removed from the surface in the presence of doubly marked two-word strands. These new operations are essential for manipulation of the large combinatorial sets of linked DNA word strands required for DNA computing.
Rapid and novel deprotection conditions have been developed for the cleavage of oligodeoxyribonucleotides from cis-diol group based universal polymer supports and simultaneous removal of protecting groups from exocyclic amino functionalities of nucleic bases and internucleotide phosphates. The fully deprotected oligodeoxyribonucleotides were compared with the corresponding oligomers prepared and deblocked using standard protocols. They were found to be identical with respect to their retention times on HPLC and biological activity.
Porous glass containing 2′(3′)--benzoyluridine 5′-succinyl ligands serves as a universal support for construction of oligodeoxyribonucleotides of any sequence, using either the phosphotriester or the phosphite approach. At the conclusion of a synthesis, the released and deprotected oligomer bears a terminal uridine residue which can be readily removed as its 2′,3′-cyclic phosphate by treatment with Pb++ ion.
DNA microarray is a powerful tool allowing simultaneous detection of many different target molecules present in a sample. The efficiency of the array depends mainly on the sequence of the capture probes and the way they are attached to the support. The coupling procedure must be quick, covalent, and reproducible in order to be compatible with automatic spotting devices dispensing tiny drops of liquids on the surface. We compared several coupling strategies currently used to covalently graft DNA onto a glass surface. The results indicate that fixation of aminated DNA to an aldehyde-modified surface is a choice method to build DNA microarrays. Both the coupling procedure and the hybridization efficiency have been optimized. The detection limit of human cytomegalovirus target DNA amplicons on such DNA microarrays has been estimated to be 0.01 nM by fluorescent detection.
Functionalization of universal polymer supports is described for the synthesis of oligonucleotides bearing different functionalities (mercaptoalkyl or aminoalkyl) at 3′-end during final deprotection.A functionalization process for universal polymer supports is described for the synthesis of oligonucleotides bearing mercaptoalkyl or aminoalkyl at 3′-end during final deprotection.
Hydrogen-terminated Si(111) surfaces are modified by attachment of oligodeoxynucleotides and characterized with respect to DNA surface density, chemical stability, and DNA hybridization binding specificity. Surface functionalization employs the reaction of omega-unsaturated alkyl esters with the Si(111) surface using UV irradiation. Cleavage of the ester using potassium tert-butoxide yields a carboxyl-modified surface, which serves as a substrate for the attachment of DNA by means of an electrostatically adsorbed layer of polylysine and attachment of thiol-modified DNA using a heterobifunctional cross-linker. The resultant DNA-modified surfaces are shown to exhibit excellent specificity and chemical stability under the conditions of DNA hybridization. This work provides an avenue for the development of devices in which the exquisite binding specificity of biomolecular recognition is directly coupled to semiconductor devices.
Oligonucleotide-peptide conjugates have several applications, including their potential use as therapeutic agents. We developed a strategy for the chemical ligation of unprotected peptides to oligonucleotides in aqueous solution. The two compounds are joined via a stable amide bond in a template-directed reaction.
Peptides, ending in a carboxy-terminal thioester, were converted to thioester-linked oligonucleotide-peptide intermediates. The oligonucleotide portion of the intermediate binds to a complementary oligonucleotide template, placing the peptide in close proximity to an adjacent template-bound oligonucleotide that terminates in a 3' amine. The ensuing reaction results in the efficient formation of an amide-linked oligonucleotide-peptide conjugate.
An oligonucleotide template can be used to direct the ligation of peptides to oligonucleotides via a highly stable amide linkage. The ligation reaction is sequence-specific, allowing the simultaneous ligation of multiple oligonucleotide-peptide pairs.
A number of phosphoramldite monomers have been prepared and used in the synthesis of antisense phosphorothioate oligonucleotides
bearing 5′-polyalkyl and cholesterol moieties. Similar groups have also been attached to the 3′-end of oligonucleotides by
means of functionalised CPG. Melting temperatures of duplexes formed between phosphorothioate oligonucleotides with lipophilic
end-groups and complementary DNA strands were found to be identical to those formed by the equivalent unmodified phosphorothioates.
Conjugates consisting of staphylococcal nuclease crosslinked to oligonucleotides hybridize to supercoiled duplex DNA by Watson--Crick base-pairing. Here we describe this strand invasion. Affinity cleavage by these conjugates provides a probe for the local topology of the DNA duplex and is most efficient at a target DNA sequence known to form a cruciform. Additional supercoiling of the substrate DNA increases selective cleavage at other sequences. Hybridization of the conjugate to duplex DNA is temperature dependent and is stable over time. Affinity cleavage is not substantially inhibited by a 200-fold excess of the analogous unmodified oligonucleotide, demonstrating that hybridization of the unmodified oligonucleotide must be less favored and that the nuclease is involved in substrate binding. Surprisingly, affinity cleavage is also not effectively inhibited by complementary oligonucleotides unless they contain an extended 5'-sequence capable of separate interactions with the nuclease domain of the conjugate. These results suggest that the oligonucleotide-nuclease conjugate prefers to hybridize to target sequences which will allow interactions with both the oligonucleotide and the nuclease domains. Affinity cleavage by oligonucleotide-nuclease conjugates provides general insights for the design of oligonucleotides and their conjugates for strand invasion and affords a convenient competition assay for their hybridization.
Conjugates consisting of oligoarginine peptides linked to oligodeoxynucleotides have been synthesized, including a new type of conjugate, in which a pair of oligonucleotides is bridged by a cationic peptide. Two different 9-mer oligonucleotides were conjugated to the terminal cysteine residues of the peptide series H-Cys-(Arg)n-Cys-NH2 (n = 3, 5, 7). Different thiol protecting groups were utilized on the amino- and carboxy-terminal cysteine residues of the peptide to allow selective attachment to the 3'- or 5'-terminus of each specific oligonucleotide. The conjugates containing oligoarginine peptides were purified by anion-exchange chromatography, and their structures were confirmed by polyacrylamide gel electrophoresis and amino acid analysis.
The ability of compounds to adsorb passively to hydrophobic polymer surfaces composed of, e.g., polystyrene generally is restricted to limited types of molecules such as proteins. Some proteins, many peptides, polysaccharides, oligonucleotides, and small molecules as well as pro- and eucaryotic cells cannot adsorb directly to such surfaces. Also, solid phase adsorbed antigens, antibodies, or gene probes may not be recognized by its corresponding ligand due to denaturation or steric hindrance of the molecular tertiary structure. Covalent binding, on the other hand, orientates all immobilized compounds in a defined way on the solid phase, thereby exposing the interacting sites on the enzymes, antibodies, gene probes, etc. Here we describe a method for modifying a polymer surface by contacting the polymer with derivatives of psoralen under irradiation with long-wavelength UV light. The psoralen derivatives were immobilized covalently on the polymer surface by this process. The psoralen molecules was conjugated to appropriate chemical linkers, incubated in aqueous solutions, and irradiated with UV light. This resulted in solid phase introduction of functional groups such as, e.g., amino groups on the polystyrene surface. The functional groups could subsequently be used for immobilization of biomolecules using conventional cross-linker technology. The method only involved premodification of the psoralens to be immobilized whereas no pretreatment of the polymer was required. Psoralen modified microtiter plates seems to have future application for the development of solid phase hybridization and immunoassays.
Oligonucleotide-peptide hybrids have potential for use as antisense inhibitors of gene expression, with the peptide helping to increase the intracellular concentration of the active oligonucleotide. The preparation of such hybrids can be achieved by the coupling of thiol-derivatized oligonucleotides with maleimido-peptides. We have developed reliable methods for preparing 5'-thiol oligonucleotides in good yields using phosphoramidite chemistry and coupling 6-(tritylthio)hexyl phosphoramidite as the 5'-terminal residue. The use of highly pure thiol phosphoramidite as well as a manual iodine treatment after this coupling were found to be important. Oligonucleotide-peptide hybrids were prepared in high yield (85%) by reacting freshly purified 5'-thiol oligonucleotides with peptides derivatized at their N-terminus with a maleimido functionality.
On the basis of the reported enhanced antisense activity of polylysine-oligonucleotide conjugates, a synthetic 12-mer oligodeoxyribonucleotide has been coupled at its 5' terminus to a series of positively charged (delta-ornithine)n cysteine peptides. Binding between the nucleic acid-peptide conjugate and its complementary DNA target sequence was detected by the impact of complexation on the melting temperature (Tm). It was found that the Tm for the nucleic acid-peptide gradually increased with increasing net charge on the conjugated peptide. Site-directed cleavage with RNase H demonstrates that the peptide-modified oligomer also hybridizes with its RNA target sequence. Increased affinity for target mRNA with net charge was shown by a cell-free translation arrest assay.
We have designed and synthesized fluorescent oligonucleotide primers having improved fluorescence and electrophoretic properties by exploiting the concept of resonance fluorescence energy transfer (ET). These primers carry a fluorescein derivative at the 5' end as a common fluorescence donor and other fluorescein and rhodamine derivatives attached to a modified thymidine within the primer sequence as acceptors. These primers all have strong absorption at a common excitation wavelength (448 nm) and fluorescence emission maxima of 525, 555, and 605 nm. The fluorescence emission intensity of the ET primers increases as the spacing between the donor and acceptors is increased, and of the spacings studied the strongest fluorescence was observed when the number of nucleotides between the donor and acceptors is 10. The electrophoretic mobilities of the primers were also found to be a function of the spacing between the donor and the acceptors, and mobilities of the single base extension DNA fragments generated with primers (F10F, F10J, F10T, and F10R) is 2- to 14-fold greater than that of the corresponding primers labeled with only one dye. The increased fluorescence intensity of the ET primers and the substantially similar mobilities of the DNA fragments generated with the four ET primers allow four-color DNA sequencing on a capillary electrophoresis DNA sequencer using a single laser line at 488 nm for excitation and without applying mobility shift adjustments. With single-stranded M13mp18 DNA as the template, a typical run with the ET primers on a commercial sequencer provided DNA sequences with 99-100% accuracy in the first 500 bases using 8-fold less DNA template than that typically required using T7 DNA polymerase.
High-density arrays of oligonucleotide probes are proving to be powerful new tools for large-scale DNA and RNA sequence analysis. A method for constructing these arrays, using light-directed DNA synthesis with photo-activatable monomers, can currently achieve densities on the order of 10(6) sequences/cm2. One of the challenges facing this technology is to further increase the volume, complexity, and density of sequence information encoded in these arrays. Here we demonstrate a new approach for synthesizing DNA probe arrays that combines standard solid-phase oligonucleotide synthesis with polymeric photoresist films serving as the photoimageable component. This opens the way to exploiting high-resolution imaging materials and processes from the microelectronics industry for the fabrication of DNA probe arrays with substantially higher densities than are currently available.
Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression. In order to direct oligonucleotides to specific compartments within the cell, we have investigated the possibility of coupling them to a signal peptide Lys-Asp-Glu-Leu (KDEL). This sequence should be able to convey oligonucleotides to the endoplasmic reticulum and from there to the cytosol and the nucleus where their targets are located. On this basis we prepared peptide-oligonucleotide conjugates by coupling, in a single step, a Nalpha-bromoacetyl peptide with an oligonucleotide bearing a thiol group, through a thioether bond. This paper deals with the definition of the optimal pH and temperature conditions leading to an efficient synthesis of peptide-oligonucleotide conjugates: the reaction was quantitative at pH 7.5 within few hours. This method was first set up using a 5',3'-modified dodecanucleotide and a (bromoacetyl)pentapeptide as a conjugation model. Then a 5',3'-modified pentacosanucleotide, complementary to the translation initiation region of the gag mRNA of HIV, was coupled to a (bromoacetyl)dodecapeptide containing a KDEL signal sequence. The anti-HIV activity of the pentacosanucleotide was compared with that of pentacosanucleotide-dodecapeptide conjugates linked through either a thioether bond or a disulfide bridge. The conjugate with a thioether bond has a higher antiviral activity than the peptide-free oligonucleotide and the conjugate linked via a disulfide bond.