Solid-phase Chemical Synthesis Research Articles

Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET). The Python tool uses thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.

Development of an in vitro aggregation assay for long synthetic polypeptide, amyloidogenic gelsolin fragment AGelD187N 173-242.

Aggregation of the gelsolin protein fragment is the hallmark of the hereditary systemic disease gelsolin amyloidosis. As with other protein misfolding diseases, there is an urgent need for efficient disease-modifying treatment for gelsolin amyloidosis. The formation of amyloids can be reproduced by incubating the disease-causing amyloidogenic 8 kDa polypeptide, 70-residue gelsolin protein fragment, AGelD187N 173-242, in vitro and monitoring the process by thioflavin T dye. However, for screening of potential aggregation inhibitors, the required protein amounts are large and the biotechnological production of amyloidogenic proteins has many challenges. Conversely, use of shorter synthetic regions of AGelD187N 173-242 does not mimic the in vivo aggregation kinetics of full-length fragment as they have different aggregation propensity. In this study, we present an in vitro aggregation assay for full-length AGelD187N 173-242 that has been produced by solid-phase chemical synthesis and after that monomerized carefully. Chemical synthesis allows us to produce high quantities of full-length fragment efficiently and at low cost. We demonstrate that the generated aggregates are fibrillar in nature and how the purity, terminal modification, initial aggregates and seeding affect the aggregation kinetics of a synthetic gelsolin fragment. We also present sufficient quality criteria for the initial monomerized synthetic polypeptide.

Practical Synthesis of N-Formylmethionylated Peptidyl-tRNA Mimics.

Hydrolysis-resistant RNA-peptide conjugates that mimic peptidyl-tRNAs are frequently needed for structural and functional studies of protein synthesis in the ribosome. Such conjugates are accessible by chemical solid-phase synthesis, allowing for the utmost flexibility of both the peptide and the RNA sequence. Commonly used protection group strategies, however, have severe limitations with respect to generating the characteristic Nα-formylmethionyl terminus because the formyl group of the conjugate synthesized at the solid support is easily cleaved during the final basic deprotection/release step. In this study, we demonstrate a simple solution to the problem by coupling appropriately activated Nα-formyl methionine to the fully deprotected conjugate. The structural integrity of the obtained Nα-formylmethionyl conjugate─and hence the chemoselectivity of the reaction─were verified by Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry sequence analysis. Additionally, we confirmed the applicability of our procedure for structural studies by obtaining two structures of the ribosome in complex with either fMAI-nh-ACCA or fMFI-nh-ACCA in the P site and ACC-PMN in the A site of the bacterial ribosome at 2.65 and 2.60 Å resolution, respectively. In summary, our approach for hydrolysis-resistant Nα-formylated RNA-peptide conjugates is synthetically straightforward and opens up new avenues to explore ribosomal translation with high-precision substrate mimics.

O1-conotoxin Tx6.7 cloned from the genomic DNA of Conus textile that inhibits calcium currents.

Conotoxins exhibit great potential as neuropharmacology tools and therapeutic candidates due to their high affinity and specificity for ion channels, neurotransmitter receptors or transporters. The traditional methods to discover new conotoxins are peptide purification from the crude venom or gene amplification from the venom duct. In this study, a novel O1 superfamily conotoxin Tx6.7 was directly cloned from the genomic DNA of Conus textile using primers corresponding to the conserved intronic sequence and 3' UTR elements. The mature peptide of Tx6.7 (DCHERWDWCPASLLGVIYCCEGLICFIAFCI) was synthesized by solid-phase chemical synthesis and confirmed by mass spectrometry. Patch clamp experiments on rat DRG neurons showed that Tx6.7 inhibited peak calcium currents by 59.29 ± 2.34% and peak potassium currents by 22.33 ± 7.81%. In addition, patch clamp on the ion channel subtypes showed that 10 μM Tx6.7 inhibited 56.61 ± 3.20% of the hCaV1.2 currents, 24.67 ± 0.91% of the hCaV2.2 currents and 7.30 ± 3.38% of the hNaV1.8 currents. Tx6.7 had no significant toxicity to ND7/23 cells and increased the pain threshold from 0.5 to 4 hours in the mouse hot plate assay. Our results suggested that direct cloning of conotoxin sequences from the genomic DNA of cone snails would be an alternative approach to obtaining novel conotoxins. Tx6.7 could be used as a probe tool for ion channel research or a therapeutic candidate for novel drug development.

Towards the use of an amino acid cleavable linker for solid-phase chemical synthesis of peptides and proteins.

The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from the paucity of linkers that can be cleaved under mild conditions. Here, we deployed a spontaneous nickel-assisted cleavage (SNAC) tag, known to undergo spontaneous cleavage in the presence of nickel(II), as a linker for C-to-N SPCL.

Rapid and reliable RNA resonance assignment by combining chemical and enzymatic stable isotope labeling

In this work a rapid RNA assignment approach by combining chemical and enzymatic 13C and 15N stable isotope labeling is introduced. We exemplify the assignment strategy for imino N1H1 purine and N3H3 pyrimidine and aromatic C6H6 pyrimidine, C8H8 purine and C2H2 adenine resonances for a non-coding RNA comprising 66 nucleotides. The assignment strategy is based on position specific labeling by chemical solid phase synthesis and dilute stable isotope 13C/15N-labeling by mixing labeled and commercially available unlabeled RNA phosphoramidites. The assignment process is further facilitated by nucleotide specific labeling using T7 RNA polymerase in vitro transcription with in house produced atom specific 13C labeled ribonucleotide triphosphates. The approach is fast with a total NMR measurement time of only 22 h and also competitive in terms of costs as compared to the standard methodology relying on in vitro transcription using 2H, 15N and 13C/15N uniformly labeled ribonucleotide triphosphates. Furthermore, the assignment procedure revealed a slow exchange process on the NMR chemical shift time scale in the 66 nt non-coding RNA with possible biological implications in the regulation of bacterial competence.

Rapid identification of short oligonucleotide impurities using lithium adduct consolidated MALDI-TOF mass spectrometry

Nucleic acids are an increasingly popular platform for the development of pharmaceuticals to treat and prevent multiple diseases including those where traditional small molecule and protein-based drug development efforts have failed. Short oligonucleotide therapeutics, which consist of antisense oligonucleotides (ASOs) and short interfering ribonucleic acids (siRNAs), are prepared by solid phase chemical synthesis, which can generate various impuriies that have the potential to lower drug safety and efficacy. Drug substance-related impurities can be especially difficult to identify and characterize. To address this short coming, here we apply lithium adduct consolidation with matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) to afford a simplified, quick, and facile method for identification of a known drug substance impurity, isobutyryl (iBu) groups arising from incomplete deprotection during solid phase synthesis. We further employ high-resolution nuclear magnetic resonance (NMR) spectroscopy to confirm assessment of the iBu impurity. This lithium adduction consolidation method should find general applicability for routine quality assessment of synthetic oligonucleotides in both academic and industrial laboratories.

Quantification of synthetic errors during chemical synthesis of DNA and its suppression by non-canonical nucleosides

Substitutions, insertions, and deletions derived from synthetic oligonucleotides are the hurdles for the synthesis of long DNA such as genomes. We quantified these synthetic errors by next-generation sequencing and revealed that the quality of the enzymatically amplified final combined product depends on the conditions of the preceding solid phase chemical synthesis, which generates the initial pre-amplified fragments. Among all possible substitutions, the G-to-A substitution was the most prominently observed substitution followed by G-to-T, C-to-T, T-to-C, and A-to-G substitutions. The observed error rate for G-to-A substitution was influenced by capping conditions, suggesting that the capping step played a major role in the generation of G-to-A substitution. Because substitutions observed in long DNA were derived from the generation of non-canonical nucleosides during chemical synthesis, non-canonical nucleosides resistant to side reactions could be used as error-proof nucleosides. As an example of such error-proof nucleosides, we evaluated 7-deaza-2´-deoxyguanosine and 8-aza-7-deaza-2´-deoxyguanosine and showed 50-fold decrease in the error rate of G-to-A substitution when phenoxyacetic anhydride was used as capping reagents. This result is the first example that improves the quality of synthesized sequences by using non-canonical nucleosides as error-proof nucleosides. Our results would contribute to the development of highly accurate template DNA synthesis technologies.

Completely Chemically Synthesized Long DNA Can be Transcribed in Human Cells.

Chemical ligation reaction of DNA is useful for the construction of long functional DNA using oligonucleotide fragments that are prepared by solid phase chemical synthesis. However, the unnatural linkage structure formed by the ligation reaction generally impairs the biological function of the resulting ligated DNA. We achieved the complete chemical synthesis of 78 and 258 bp synthetic DNAs via multiple chemical ligation reactions with phosphorothioate and haloacyl-modified DNA fragments. The latter synthetic DNA, coding shRNA for luciferase genes with a designed truncated SV promoter sequence, successfully induced the expected gene silencing effect in HeLa cells.

Site-specific covalent labeling of large RNAs with nanoparticles empowered by expanded genetic alphabet transcription

Conjugation of RNAs with nanoparticles (NPs) is of significant importance because of numerous applications in biology and medicine, which, however, remains challenging especially for large ones. So far, the majority of RNA labeling relies on solid-phase chemical synthesis, which is generally limited to RNAs smaller than 100 nucleotides (nts). We, here, present an efficient and generally applicable labeling strategy for site-specific covalent conjugation of large RNAs with a gold nanoparticle (Nanogold) empowered by transcription of an expanded genetic alphabet containing the A-T/U and G-C natural base pairs (bps) and the TPT3-NaM unnatural base pair (UBP). We synthesize an amine-derivatized TPT3 (TPT3A), which is site specifically incorporated into a 97-nt 3'SL RNA and a 719-nt minigenomic RNA (DENV-mini) from Dengue virus serotype 2 (DENV2) by in vitro T7 transcription. The TPT3A-modified RNAs are covalently conjugated with mono-Sulfo-N-hydroxysuccinimidyl (NHS)-Nanogold NPs via an amine and NHS ester reaction and further purified under nondenaturing conditions. TPT3 modification and Nanogold labeling cause minimal structural perturbations to the RNAs by circular dichroism, small angle X-ray scattering (SAXS), and binding activity assay. We demonstrate the application of the Nanogold-RNA conjugates in large RNA structural biology by an emerging molecular ruler, X-ray scattering interferometry (XSI). The internanoparticle distance distributions in the 3'SL and DENV-mini RNAs derived from XSI measurements support the hypothetical model of flavivirus genome circularization, thus, validate the applicability of this labeling strategy. The presented strategy overcomes the size constraints in conventional RNA labeling strategies and is expected to have wide applications in large RNA structural biology and RNA nanotechnology.

Optimization and characterization of position-selective labelling of RNA (PLOR) for diverse RNA and DNA sequences

ABSTRACT Modifications of short RNAs at specific sites can be achieved commercially by solid-phase chemical synthesis method. However, labelling long RNAs is still challenging for the routine methods. Position-selective Labelling of RNA (PLOR) is a hybrid phase transcription method that allows to label RNAs at desired sites with great flexibility and decent efficiency. In principle, PLOR is a promising method for synthesis of long modified RNAs that are unable to be generated by solid-phase chemical synthesis and other methods. However, as a recently developed method, PLOR has been only applied to label a 71nt and a 104nt RNA, and the limited sequence applications of PLOR may hinder its potential usages. To extend PLOR to more RNAs, we tested the PLOR performances for various RNA sequences. Considering that the controlled transcriptional pauses at the initiation stage in PLOR may lead to different preferences on RNA sequences from in vitro transcription method, we here focused on identifying the effects of the 5ʹ-end and initiated lengths of RNA on PLOR. In addition, our work demonstrated that PLOR efficiencies also varied with linker sizes of DNA templates. This work can facilitate PLOR to be the choice of synthesizing long modified RNAs for more users in the near future.

The Crucial Role of Methodology Development in Directed Evolution of Selective Enzymes.

Directed evolution of stereo-, regio-, and chemoselective enzymes constitutes a unique way to generate biocatalysts for synthetically interesting transformations in organic chemistry and biotechnology. In order for this protein engineering technique to be efficient, fast, and reliable, and also of relevance to synthetic organic chemistry, methodology development was and still is necessary. Following a description of early key contributions, this review focuses on recent developments. It includes optimization of molecular biological methods for gene mutagenesis and the design of efficient strategies for their application, resulting in notable reduction of the screening effort (bottleneck of directed evolution). When aiming for laboratory evolution of selectivity and activity, second-generation versions of Combinatorial Active-Site Saturation Test (CAST) and Iterative Saturation Mutagenesis (ISM), both involving saturation mutagenesis (SM) at sites lining the binding pocket, have emerged as preferred approaches, aided by in silico methods such as machine learning. The recently proposed Focused Rational Iterative Site-specific Mutagenesis (FRISM) constitutes a fusion of rational design and directed evolution. On-chip solid-phase chemical gene synthesis for rapid library construction enhances library quality notably by eliminating undesired amino acid bias, the future of directed evolution?

COMBINED ANTIBACTERIAL ACTION OF SALIVARY CATIONIC PROLINE-RICH PEPTIDES AND ANTIMICROBIAL PEPTIDES

Saliva is an important biological fluid that reflects human’s health. Its main function is protection of the oral cavity from pathogens. Antimicrobial peptides (AMPs) of the innate immunity may play an important role in anti-infectious defense of the oral cavity, but their relative amount in saliva is low. It’s major component is Proline-rich peptides (PRPs), whose impact in antimicrobial protection remains poorly understood. We suggest that salivary PRPs may reveal their defensive functions upon interaction with other molecules, in particular with AMPs. The aim of this work is an investigation of the combined antibacterial action of salivary PRPs (fragments of Basic salivary proline-rich protein 1: P-H (37-51), IB6 (98-116), p1932) with antimicrobial peptides (histatin 5 and cathelicidin LL-37 and beta-defensin hBD3). Listed PRPs have been obtained by chemical solid-phase synthesis. The method of broth microdilutions was used to compare minimal inhibitory concentrations (MICs) of individual fractions of AMPs and their MICs in the presence of salivary peptides. It was found that in the presence of peptides IB6 (98-116) or P-H (37-51) the activity of defensin hBD3 was increased (reduction of MICs by 2 times) against Staphylococcus aureus SG511. In the presence of IB6 (98-116) or p1932 the activity of this defensin against E. coli ML35p was also improved (MICs of hBD3 was lowered by 2 times). For other combinations of the peptides, this effect was not observed. The obtained data confirm the assumption that the combined action of varied salivary peptides, including cationic Proline-rich peptides, plays an important role in anti-infectious protection of the oral cavity.

Solid-Phase Chemical Synthesis of Stable Isotope-Labeled RNA to Aid Structure and Dynamics Studies by NMR Spectroscopy

RNA structure and dynamic studies by NMR spectroscopy suffer from chemical shift overlap and line broadening, both of which become worse as RNA size increases. Incorporation of stable isotope labels into RNA has provided several solutions to these limitations. Nevertheless, the only method to circumvent the problem of spectral overlap completely is the solid-phase chemical synthesis of RNA with labeled RNA phosphoramidites. In this review, we summarize the practical aspects of this methodology for NMR spectroscopy studies of RNA. These types of investigations lie at the intersection of chemistry and biophysics and highlight the need for collaborative efforts to tackle the integrative structural biology problems that exist in the RNA world. Finally, examples of RNA structure and dynamic studies using labeled phosphoramidites are highlighted.

Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5' Caps.

Eukaryotic RNAs are heavily processed, including co- and post-transcriptional formation of various 5' caps. In small nuclear RNAs (snRNAs) or small nucleolar RNAs (snoRNAs), the canonical 7m G cap is hypermethylated at the N2 -position, whereas in higher eukaryotes and viruses 2'-O-methylation of the first transcribed nucleotide yields the cap1 structure. The function and potential dynamics of several RNA cap modifications have not been fully elucidated, which necessitates preparative access to these caps. However, the introduction of these modifications during chemical solid-phase synthesis is challenging and enzymatic production of defined short and uniform RNAs also faces difficulties. In this work, the chemical synthesis of RNA is combined with site-specific enzymatic methylation by using the methyltransferases human trimethylguanosine synthase 1 (hTgs1), trimethylguanosine synthase from Giardia lamblia (GlaTgs2), and cap methyltransferase 1 (CMTR1). It is shown that RNAs with di-and trimethylated caps, as well as RNAs with caps methylated at the 2'-O-position of the first transcribed nucleotide, can be conveniently prepared. These highly modified RNAs, with a defined and uniform sequence, are hard to access by in vitro transcription or chemical synthesis alone.

A new application of CexZr1-xO2 as dense diffusion barrier in limiting current oxygen sensor

CexZr1-xO2 (x = 0.25, 0.5 and 0.75) was synthesized by solid phase chemical reaction synthesis and characterized by X-ray diffraction (XRD), thermal expansion, Hebb-Wagner method and DC van der Pauw method. ZrO2 has a high melting point and CeO2 has a variable valence state, which can make the CexZr1-xO2 material have mixed ionic-electronic conductivity and stability. The synthesis method has the advantages of having a lower synthesis temperature and no waste liquid discharge, which is beneficial to energy conservation and environmental protection. A limiting current oxygen sensor was prepared with Ce0.75Zr0.25O2 dense diffusion barrier and ZrO2 stabilized by 9 mol% Y2O3 (9YSZ) solid electrolyte by Pt sintered-paste method. Limiting current plateau of the oxygen sensor was obtained and the effects of operate temperature (T), oxygen concentration (x(O2)) and water vapor pressure (p(H2O)) on the limiting current was studied, respectively. The results show that the Ce0.75Zr0.25O2 material has maximum electronic and total conductivity at 800 ºC and is the most suitable ceramic material to be a dense diffusion barrier of limiting current oxygen sensor. The oxygen sensor exhibits good sensing characteristics under different research conditions, including different T, x(O2) and p(H2O). The limiting current is related to various research factors, for example, log(IL·T) depends linearly on 1000/T, IL depends linearly on x(O2) and IL is not influenced obviously by p(H2O). The experiment supplements the application of mixed conductor material CexZr1-xO2 (x = 0.25, 0.5 and 0.75) as a dense diffusion barrier in limiting current oxygen sensor.

SpyLigase-Catalyzed Modification of Antibodies.

Immunoconjugates are essential tools in diagnostics for the detection and quantification of proteins and in cell biology for the characterization of different cell populations as well as for tracking intracellular pathways. In recent years, antibody-drug conjugates (ADCs) have emerged as promising therapeutics to treat cancer and have moved into the focus of interest of the pharmaceutical industry. Here we describe a conjugation method for the generation of antibody conjugates that relies on the formation of a spontaneous isopeptide bond between two peptide tags referred to as SpyTag and KTag. This reaction is catalyzed by SpyLigase, an engineered cell surface protein obtained from Streptococcus pyogenes. We describe the preparation of SpyLigase by expression from E. coli cells, chemical solid-phase synthesis of the KTag peptide and its coupling to reporter molecules and cytotoxins as well as the transient expression from mammalian cells to produce Spy-tagged antibodies. Furthermore, we describe the purification and analytics of the formed conjugates.

A Model of Solid-Phase Synthesis in Binary Powder Mixtures with Allowance for Caking

A mathematical model of solid-phase chemical synthesis in a binary powder mixture is constructed and studied with allowance for the shrinking of the mixture and the change in the reaction surface as a result of particle caking. Relations are obtained to estimate the number of similar and dissimilar interparticle contacts. Depending on the parameters of the mixture (the stoichiometric ratio between components, the particle size ratio, and the porosity), different modes are revealed that affect the rate of chemical transformation. The optimum conditions for an intense chemical interaction are determined.

Incorporation of Sensitive Ester and Chloropurine Groups into Oligodeoxynucleotides through Solid Phase Synthesis.

Nucleosides containing ester groups that are sensitive to nucleophiles were incorporated into oligodeoxynucleotides (ODNs) through solid phase chemical synthesis. The sensitive esters are located on a purine nucleobase. They are the esters of ethyl, 2-methoxyethyl, 4-methoxyphenyl and phenyl groups, and a thioester. These esters cannot survive the deprotection and cleavage conditions used in known ODN synthesis technologies, which involve strong nucleophiles such as ammonium hydroxide and potassium methoxide (potassium carbonate in anhydrous methanol). To incorporate these sensitive groups into ODNs, the Dmoc phosphoramidites and linker were used for solid phase synthesis, which allowed ODN deprotection and cleavage to be carried out under non-nucleophilic oxidative conditions. Sixteen ODN sequences containing these groups were synthesized and characterized with MALDI MS. In addition, the synthesis and characterization of three ODNs containing a nucleophile sensitive 6-chloropurine using the same strategy are described.

Enzymatic and chemical synthesis of new anticoagulant peptides.

In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Tyr-Gln-Gln (Z-YQQ), a new anticoagulant tripeptide. It was obtained using phytoproteases from the stems and petioles of Asclepias curassavica L. as catalyst in an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate and 0.1 M Tris-HCl buffer pH 8. The resulting peptide was compared with the analogous peptide Tyr-Gln-Gln (YQQ) produced by solid-phase chemical synthesis. The in vitro anticoagulant activity of the aforementioned peptides was determined using Wiener Lab Test (Wiener, Argentina). The toxicological activity of the peptides was also determined. The enzymatically synthesized Z-YQQ peptide acted on the extrinsic pathway of the coagulation cascade, delaying the conversion time of prothrombin to thrombin and fibrinogen to fibrin by 136 and 50%, respectively, with respect to the controls. The chemically synthesized YQQ peptide acted specifically on the intrinsic pathway of the coagulation cascade, affecting factors VIII, IX, XI, and XII from such cascade, and increasing the coagulation time by 105% with respect to the control. The results suggest that two new anticoagulant peptides (Z-YQQ and YQQ) can be useful for safe pharmaceutical applications. Nevertheless, some aspects related to peptide production should be optimized. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1093-1101, 2018.