Mild Detritylation of Nucleic Acid Hydroxyl Groups by Warming Up

It is challenging to effectively deprotect hydroxyl groups of acid-or-base sensitive bio-macromolecules without causing even minor defects and compromising high quality of final products. We report here a mild detritylation strategy in mildly acidic buffers to remove the DMTr protection from the 5′-hydroxyl groups of synthetic nucleic acids. The DMTr-groups can be easily and effectively removed at pH 4.5 or 5.0 with slight warming up (40°C), offering virtually quantitative deprotection. This warming-up strategy is particularly useful for deprotection of the modified nucleic acids that are sensitive to the conventional acid deprotection. As a first step towards our long-term goal of synthesizing defect-free nucleic acids, our novel and simple strategy further increases the quality of synthetic nucleic acids.     

Design and synthesis of dinucleotide 5'-triphosphates with expanded functionality

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.     

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se‐Met) strategy). This selenium derivatization strategy via MAD (multi‐wavelength anomalous dispersion) phasing has revolutionized protein X‐ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X‐ray crystal‐structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O‐atom at the positions 2′, 4′, 5′, and in nucleobases and non‐bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid‐phase chemical synthesis and enzymatic methods, and the Se‐derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein–nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2′‐position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom‐specific substitution of the nucleotide O‐atom, better stability under X‐ray radiation, and structure isomorphism. Therefore, our Se‐derivatization strategy has great potentials to provide rational solutions for both phase determination and high‐quality crystal growth in nucleic‐acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site‐specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se‐derivatization strategy in nucleic acid and protein research are also described in this review.     

Functional lactide monomers: methodology and polymerization

Side-chain-functionalized lactide analogues have been synthesized from commercially available amino acids and polymerized using stannous octoate as a catalyst. The synthetic strategy presented allows for the incorporation of any protected amino acid for the preparation of functionalized diastereomerically pure lactide monomers. The resulting functionalized cyclic monomers can be homopolymerized and copolymerized with lactides and then quantitatively deprotected forming new functional poly(lactide)-based materials. This strategy allows for the introduction of functional groups along a poly(lactide) (PLA) backbone that after deprotection can be viewed as chemical handles for further functionalization of PLA, yielding improved biomaterials for a variety of applications.     

Synthesis of Polyamide Nucleic Acids (Pnas), Pna /Dna-Chimeras and Phosphonic Ester Nucleic Acids (Phonas)

Abstract

The synthesis of polyamide nucleic acids (PNAs) and derivatives thereof by different synthetic routes is described. The first strategy makes use of 9-Fluorenylmethoxycarbonyl (Fmoc)/monomethoxytrityl (Mmt) protected building blocks, whereas the second approach involves the use of Mmt/acyl protected monomers, which allows the preparation of PNADNA chimera. Additionally, a block coupling strategy is presented for the synthesis of novel phosphonic ester nucleic acids (PHONAs).     

Synthesis of novel MMT/acyl-protected nucleo alanine monomers for the preparation of DNA/alanyl-PNA chimeras

Alanyl-peptide nucleic acid (alanyl-PNA)/DNA chimeras are oligomers envisaged to be beneficial in efficient DNA diagnostics based on an improved molecular beacon concept. A synthesis of alanyl-PNA/DNA chimera can be based on the solid phase assembly of the oligomer with mixed oligonucleotide/peptide backbone under DNA synthesis conditions, in which the nucleotides are introduced as phosphoramidites, whereas the nucleo amino acids make use of the acid labile monomethoxytrityl (MMT) group for temporary protection of the α-amino groups and acyl protecting groups for the exocyclic amino functions of the nucleobases. In this work, we realized for the first time the synthesis of all four MMT/acyl-protected nucleo alanines, achieved by deprotection/reprotection of the newly synthesized Boc/acyl intermediates, useful monomers for the obtainment of (alanyl-PNA)/DNA chimeras by conditions fully compatible with the standard phosphoramidite DNA synthesis strategy.     

The peptide nucleic acids (PNAs): introduction to a new class of probes for chromosomal investigation

Peptide nucleic acids (PNAs) are synthetic DNA mimics in which the sugar phosphate backbone is replaced by repeating N-(2-aminoethyl) glycine units linked by an amine bond and to which the nucleobases are fixed. Peptide nucleic acids hybridize with complementary nucleic acids with remarkably high affinity and specificity, essentially because of their uncharged and flexible polyamide backbone. The unique physicochemical properties of PNAs have led to the development of a large variety of biological research assays, and, over the last few years, PNAs have proved their powerful usefulness in genetic and cytogenetic diagnostic procedures. Several sensitive and robust PNA-dependent methods have been designed for modulating polymerase chain reactions, detecting genomic mutation or capturing nucleic acids. The more recent applications of PNA involve their use as molecular hybridization probes. Thus, the in situ detection of several human chromosomes has been reported in various types of tissues.Communicated by E.A. Nigg     

Assessing site-isolation of amine groups on aminopropyl-functionalized SBA-15 silica materials via spectroscopic and reactivity probes

The average degree of separation and the accessibility of aminopropyl groups on SBA-15 silica materials prepared using different silane grafting approaches are compared. Three specific synthetic approaches are used: (1) the traditional grafting of 3-aminopropyltrimethoxysilane in toluene, (2) a protection/deprotection method using benzyl- or trityl-spacer groups, and (3) a cooperative dilution method where 3-aminopropyltrimethoxysilane and methyltrimethoxysilane are co-condensed on the silica surface as a silane mixture. The site-isolation and accessibility of the amine groups are probed via three methods: (a) evaluation of pyrene groups adsorbed onto the solids using fluorescence spectroscopy, (b) the reactions of chlorodimethyl(2,3,4,5-tetramethyl-2,4-cyclopentadien-1-yl)silane (Cp′Si(Me)₂Cl) and chloro(cyclopenta-2,4-dienyl)dimethylsilane (CpSi(Me)₂Cl) with the tethered amine sites, and (c) comparison of the reactivity of zirconium constrained-geometry-inspired catalysts (CGCs) prepared using the Cp′Si(Me)₂-modified aminosilicas in the catalytic polymerization of ethylene to produce poly(ethylene). The spectroscopic probe of site-isolation suggests that both the protection/deprotection method and the cooperative dilution method yield similarly isolated amine sites that are markedly more isolated than sites on traditional aminosilica. In contrast, both reactivity probes show that the protection/deprotection strategy leads to more uniformly accessible amine groups. It is proposed that the reactivity probes are more sensitive tests for accessibility and site-isolation in this case.     

Shuffling of amino acid sequence: an important control in synthetic peptide studies of nucleic acid-binding domains. Binding properties of fragments of a conserved eukaryotic RNA binding motif.

We have used synthetic peptides to study a conserved RNA binding motif in yeast poly(A)-binding protein. Two peptides, 45 and 44 amino acids in length, corresponding to amino and carboxyl halves of a 90-amino acid RNA-binding domain in the protein were synthesized. While the amino-terminal peptide had no significant affinity for nucleic acids, the carboxyl-terminal peptide-bound nucleic acids with similar characteristics to that for the entire 577 residue yeast poly(A)-binding protein. In 100 mM NaCl, the latter peptide retained over 50% of the intrinsic binding free energy of the protein, as well as, similar RNA versus DNA binding specificity. However, shuffling of the sequence of this 44 residue peptide had surprisingly little effect on its nucleic acid binding properties suggesting the overriding importance of amino acid composition as opposed to primary sequence. Deletion studies on the 44 residue peptide with the "correct" sequence succeeded in identifying amino acids important for conferring RNA specificity and for increasing our understanding of the molecular basis for nucleic acid binding by synthetic peptides. The shuffled peptide study, however, clearly indicates that considerable caution must be exercised before extrapolating results of structure/function studies on synthetic peptide analogues to the parent protein.     

Forks and combs and DNA: the synthesis of branched oligodeoxyribonucleotides.

Nucleoside phosphoramidite derivatives containing two protected primary hydroxyl functions have been incorporated into synthetic oligonucleotides as 'branching monomers'. With selective deprotection, multiple identical copies of an additional oligonucleotide can be incorporated to form fork- or comb-like structures for use as signal amplification materials in nucleic acid hybridization assays.     

Isolation of a non-genomic origin fluoroquinolone responsive regulatory element using a combinatorial bioengineering approach

Advances in chemical biology have led to selection of synthetic functional nucleic acids for in vivo applications. Discovery of synthetic nucleic acid regulatory elements has been a long-standing goal of chemical biologists. Availability of vast genome level genetic resources has motivated efforts for discovery and understanding of inducible synthetic genetic regulatory elements. Such elements can lead to custom-design of switches and sensors, oscillators, digital logic evaluators and cell–cell communicators. Here, we describe a simple, robust and universally applicable module for discovery of inducible gene regulatory elements. The distinguishing feature is the use of a toxic peptide as a reporter to suppress the background of unwanted bacterial recombinants. Using this strategy, we show that it is possible to isolate genetic elements of non-genomic origin which specifically get activated in the presence of DNA gyrase A inhibitors belonging to fluoroquinolone (FQ) group of chemicals. Further, using a system level genetic resource, we prove that the genetic regulation is exerted through histone-like nucleoid structuring (H-NS) repressor protein. Till date, there are no reports of in vivo selection of non-genomic origin inducible regulatory promoter like elements. Our strategy opens an uncharted route to discover inducible synthetic regulatory elements from biologically-inspired nucleic acid sequences.     

Real-time monitoring of nucleic acid ligation in homogenous solutions using molecular beacons

Nucleic acids ligation is a vital process in the repair, replication and recombination of nucleic acids. Traditionally, it is assayed by denatured gel electrophoresis and autoradiography, which are not sensitive, and are complex and discontinuous. Here we report a new approach for ligation monitoring using molecular beacon DNA probes. The molecular beacon, designed in such a way that its sequence is complementary with the product of the ligation process, is used to monitor the nucleic acid ligation in a homogeneous solution and in real-time. Our method is fast and simple. We are able to study nucleic acids ligation kinetics conveniently and to determine the activity of DNA ligase accurately. We have studied different factors that influence DNA ligation catalyzed by T4 DNA ligase. The major advantages of our method are its ultrasensitivity, excellent specificity, convenience and real-time monitoring in homogeneous solution. This method will be widely useful for studying nucleic acids ligation process and other nucleic acid interactions.     

High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis

The use of capillary electrophoresis with fluorescently labeled nucleic acids revolutionized DNA sequencing, effectively fueling the genomic revolution. We present an application of this technology for the high-throughput structural analysis of nucleic acids by chemical and enzymatic mapping ('footprinting'). We achieve the throughput and data quality necessary for genomic-scale structural analysis by combining fluorophore labeling of nucleic acids with novel quantitation algorithms. We implemented these algorithms in the CAFA (capillary automated footprinting analysis) open-source software that is downloadable gratis from https://simtk.org/home/cafa. The accuracy, throughput and reproducibility of CAFA analysis are demonstrated using hydroxyl radical footprinting of RNA. The versatility of CAFA is illustrated by dimethyl sulfate mapping of RNA secondary structure and DNase I mapping of a protein binding to a specific sequence of DNA. Our experimental and computational approach facilitates the acquisition of high-throughput chemical probing data for solution structural analysis of nucleic acids.     

The enzymatic synthesis of antiviral agents.

The majority of potential antiviral agents which are currently undergoing clinical trials are inhibitors of the replication of nucleic acids. The most common class of these inhibitors are nucleoside analogues and the elucidation of synthetic routes to these compounds has been of interest for many years as many are anticancer agents. One synthetic development has been the application of bio-transformations to nucleoside syntheses. This topic has been reviewed recently (Shirae et al., 1991) but this review is not widely available. In the present review, the application of biotechnology to the synthesis of antiviral agents including those which are not nucleoside analogues will be discussed. Enzymatic syntheses of nucleosides can be simpler and quicker than syntheses carried out by chemical methods. The most useful enzymes are those found in catabolic pathways. Nucleoside phosphorylases and N-deoxyribosyltransferases have both been widely used for nucleoside synthesis catalysing the transfer of sugar residues from a donor nucleoside to a heterocyclic base. Enzymatic methods have also been applied to the resolution of racemic mixtures and adenosine deaminase is a convenient catalyst for the hydrolysis of amino groups on purines and purine analogues. Regioselective deprotection of nucleoside esters has been achieved with lipases and these enzymes have also been applied to the synthesis of esters of sugar-like alkaloids. The latter have potential as inhibitors of the replication of HIV. Esterases have also been used in combined chemical and enzymatic syntheses of organophosphorus antiviral agents.     

A universal platform for sensitive and selective colorimetric DNA detection based on Exo III assisted signal amplification

Rapid growth of available sequence data has made the detection of nucleic acids critical to the development of modern life sciences. Many amplification methods based on gold nanoparticles and endonuclease for sensitive DNA detection have been developed. However, these approaches require specific target sequence for endonuclease recognition, which cannot be fulfilled in all systems. Replacing the restriction enzyme with a nuclease that does not require any specific recognition sequence may offer a universally adaptable system. Here we have developed a novel homogeneous, colorimetric DNA detection method, which consists of Exo III, a linker DNA, and two DNA-modified gold nanoparticles. This system is simple, low-cost, sensitive and selective. By coupling cyclic enzymatic cleavage and gold nanoparticle for signal amplification, our system provides a colorimetric detection limit of 15 pM, which is 3 orders of magnitude more sensitive than that of a general three-component sandwich assay format. Due to the intrinsic property of Exo III, our method shows excellent detection selectivity for single-base discrimination. More importantly, superior to other methods based on nicking and FokI endonuclease, our target sequence-independent platform is generally applicable for DNA sensing. This new approach could be widely applied to sensitive nucleic acids detection.     

A convergent liquid-phase synthesis of salmon calcitonin.

Salmon calcitonin (sCT) was prepared in good yield and high purity by the condensation of Nalpha-Boc-cyclic decapeptide, Boc-C1SNLSTC7VLG-OH (1,7-disulfide), with protected docosapeptide (Psc)LSQE(OPse)LHK(Psc)LQTYPRTNTGSGTP-NH2 x 3TFA, followed by deprotection of Boc with trifluoroacetic acid and Psc/Pse with piperidine. The 2-(phenylsulfonyl)ethoxycarbonyl (Psc) and 2-(phenylsulfonyl)ethyl (Pse) protecting groups were recently developed. The two peptides were built up by stepwise and fragment condensation using appropriate Nalpha-Boc-amino acids and subsequent deprotection in solution. The synthetic sCT exhibited hypocalcemic potency of more than 4000 IU/mg in rats.     

Sensitivity of PCR assays for murine gammaretroviruses and mouse contamination in human blood samples

Gammaretroviruses related to murine leukemia virus (MLV) have variously been reported to be present or absent in blood from chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) patients and healthy controls. Using subjects from New York State, we have investigated by PCR methods whether MLV-related sequences can be identified in nucleic acids isolated from whole blood or from peripheral blood mononuclear cells (PBMCs) or following PBMC culture. We have also passaged the prostate cancer cell line LNCaP following incubation with plasma from patients and controls and assayed nucleic acids for viral sequences. We have used 15 sets of primers that can effectively amplify conserved regions of murine endogenous and exogenous retrovirus sequences. We demonstrate that our PCR assays for MLV-related gag sequences and for mouse DNA contamination are extremely sensitive. While we have identified MLV-like gag sequences following PCR on human DNA preparations, we are unable to conclude that these sequences originated in the blood samples.     

Real time monitoring of acylations during solid phase peptide synthesis: a method based on electrochemical detection

Monitoring of acylation reactions during solid phase peptide synthesis is important to ensure high coupling yields in all steps of the synthesis. We describe in this paper a simple and reliable method for monitoring the time course of the acylation steps as well as the washing and deprotection steps during computer-controlled solid phase peptide synthesis. The method is based on the continuous measurement of electrical conductivity in the reaction vessel. It is shown that there is a close correspondence between the degree of acylation (as determined from the amount of 9-fluorenylmethoxycarbonyl- (Fmoc) groups released during deprotection) and the conductivity profile obtained during coupling of the amino acids to the growing peptide chain. The measurements are fed back to the computer providing data for software control of the duration of the acylation, deprotection and washing steps. The method is demonstrated with pentafluorophenol esters, but is equally applicable to dihydroxybenzotriazole esters and symmetric anhydrides using the Fmoc-polyamide strategy in a continuous flow set-up with dimethylformamide (DMF) as the general solvent.     

Recent advances in oligonucleotide synthesis and their applications

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.