Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups

Abstract A set of modified 2′-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

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Current Strategies in the Management of Spinal Metastatic Disease

Swiss Surgery ◽

10.1024/1023-9332.9.2.55 ◽

2003 ◽

Vol 9 (2) ◽

pp. 55-62 ◽

Cited By ~ 7

Author(s):

Bartanusz ◽

Porchet

Keyword(s):

Spinal Cord ◽

Surgical Techniques ◽

Cord Compression ◽

Treatment Modalities ◽

Surgical Approaches ◽

Spinal Stabilization ◽

Management Algorithm ◽

Specific Manner ◽

Extensive Surgery ◽

Selection Of

The treatment of metastatic spinal cord compression is complex. The three treatment modalities that are currently applied (in a histologically non-specific manner) are surgery, radiotherapy and the administration of steroids. The development of new spinal instrumentations and surgical approaches considerably changed the extent of therapeutic options in this field. These new surgical techniques have made it possible to resect these tumours totally, with subsequent vertebral reconstruction and spinal stabilization. In this respect, it is important to clearly identify those patients who can benefit from such an extensive surgery. We present our management algorithm to help select patients for surgery and at the same time identifying those for whom primary non-surgical therapy would be indicated. The retrospective review of surgically treated patients in our department in the last four years reveals a meagre application of conventional guidelines for the selection of the appropriate operative approach in the surgical management of these patients. The reasons for this discrepancy are discussed.

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Combined Effects of Methylated Cytosine and Molecular Crowding on the Thermodynamic Stability of DNA Duplexes

International Journal of Molecular Sciences ◽

10.3390/ijms22020947 ◽

2021 ◽

Vol 22 (2) ◽

pp. 947

Author(s):

Mitsuki Tsuruta ◽

Yui Sugitani ◽

Naoki Sugimoto ◽

Daisuke Miyoshi

Keyword(s):

Molecular Crowding ◽

Dna Duplexes ◽

Crowding Effect ◽

Backbone Flexibility ◽

Cpg Dinucleotides ◽

Key Factor ◽

Dna Backbone ◽

A Cell ◽

Stabilization Effect ◽

And Function

Methylated cytosine within CpG dinucleotides is a key factor for epigenetic gene regulation. It has been revealed that methylated cytosine decreases DNA backbone flexibility and increases the thermal stability of DNA. Although the molecular environment is an important factor for the structure, thermodynamics, and function of biomolecules, there are few reports on the effects of methylated cytosine under a cell-mimicking molecular environment. Here, we systematically investigated the effects of methylated cytosine on the thermodynamics of DNA duplexes under molecular crowding conditions, which is a critical difference between the molecular environment in cells and test tubes. Thermodynamic parameters quantitatively demonstrated that the methylation effect and molecular crowding effect on DNA duplexes are independent and additive, in which the degree of the stabilization is the sum of the methylation effect and molecular crowding effect. Furthermore, the effects of methylation and molecular crowding correlate with the hydration states of DNA duplexes. The stabilization effect of methylation was due to the favorable enthalpic contribution, suggesting that direct interactions of the methyl group with adjacent bases and adjacent methyl groups play a role in determining the flexibility and thermodynamics of DNA duplexes. These results are useful to predict the properties of DNA duplexes with methylation in cell-mimicking conditions.

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Identification of fidelity-governing factors in human recombinases DMC1 and RAD51 from cryo-EM structures

Nature Communications ◽

10.1038/s41467-020-20258-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Shih-Chi Luo ◽

Hsin-Yi Yeh ◽

Wei-Hsuan Lan ◽

Yi-Min Wu ◽

Cheng-Han Yang ◽

...

Keyword(s):

Functional Analysis ◽

Structural Analysis ◽

Md Simulation ◽

Structural Basis ◽

High Fidelity ◽

Dna Dynamics ◽

Genomic Integrity ◽

Mismatched Dna ◽

Dna Backbone ◽

Loop 2

AbstractBoth high-fidelity and mismatch-tolerant recombination, catalyzed by RAD51 and DMC1 recombinases, respectively, are indispensable for genomic integrity. Here, we use cryo-EM, MD simulation and functional analysis to elucidate the structural basis for the mismatch tolerance of DMC1. Structural analysis of DMC1 presynaptic and postsynaptic complexes suggested that the lineage-specific Loop 1 Gln244 (Met243 in RAD51) may help stabilize DNA backbone, whereas Loop 2 Pro274 and Gly275 (Val273/Asp274 in RAD51) may provide an open “triplet gate” for mismatch tolerance. In support, DMC1-Q244M displayed marked increase in DNA dynamics, leading to unobservable DNA map. MD simulation showed highly dispersive mismatched DNA ensemble in RAD51 but well-converged DNA in DMC1 and RAD51-V273P/D274G. Replacing Loop 1 or Loop 2 residues in DMC1 with RAD51 counterparts enhanced DMC1 fidelity, while reciprocal mutations in RAD51 attenuated its fidelity. Our results show that three Loop 1/Loop 2 residues jointly enact contrasting fidelities of DNA recombinases.

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In Vitro Selection of Oligonucleotide Acid Aptamers against Pathogenic Vibrio Alginolyticus by SELEX

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.439-440.1456 ◽

2010 ◽

Vol 439-440 ◽

pp. 1456-1462 ◽

Cited By ~ 2

Author(s):

Jiang Zheng ◽

Yu Bao Li ◽

Jia Xiang Li ◽

Jun Wang ◽

Yong Quan Su

Keyword(s):

Secondary Structures ◽

Magnetic Bead ◽

Vibrio Alginolyticus ◽

Pathogenic Microorganism ◽

Asymmetric Pcr ◽

High Affinity ◽

Amplification Method ◽

Pathogenic Vibrio ◽

Selection Of

Detection of pathogenic microorganism is very necessary in the control of infectious disease prevailing in aquiculture animals. However, most of the present techniques can not meet the need of the quick field inspection. Systematic evolution of ligands by exponential enrichment (SELEX) is a new molecular recognition way for generating high affinity oligonucleotide acid aptamers, a new nucleotide acid material, which have been widely used in the detections of proteins, cells and so on. In the present paper, the technology was applied to select the high affinity aptamers against pathogenic microorganism Vibrio alginolyticus, which could be used for the rapid field detection of the microorganism. Based on the designment of the ssDNA library of 76 nucleotide acids with 35-base random region, the SELEX system for the selection of the high affinity aptamers against Vibrio alginolyticus was established. In the SELEX system, asymmetric PCR was proved to be a better amplification method for the ssDNA library than the reported affinity magnetic bead method, and the corresponding parameters of the asymmetric PCR were also studied and optimized. The affinity of the final ssDNA library increased by nearly 200% compared with the original library. Cloning and sequencing of the final ssDNA library showed that there were at least two kinds of ssDNAs with different length in the affinity ssDNA library: one was 76 bases, another was 149 bases. Simulation of the secondary structures showed that the secondary structures of the two fragments were different greatly, suggesting that the two fragments could bind to different sites of V. alginolyticus surface.

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DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.17.65 ◽

2021 ◽

Vol 17 ◽

pp. 749-761

Author(s):

Yongdong Su ◽

Maitsetseg Bayarjargal ◽

Tracy K Hale ◽

Vyacheslav V Filichev

Keyword(s):

Mouse Fibroblast ◽

Cell Uptake ◽

Dna Duplexes ◽

Nih3t3 Cells ◽

Dna Triplexes ◽

Snake Venom Phosphodiesterase ◽

Nuclease Digestion ◽

Dna Backbone ◽

Uptake Studies

Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3’,5’-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.

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Electrochemical Analysis of Ultrathin Polythiolsiloxane Films for Surface Biomodification

International Journal of Electrochemistry ◽

10.1155/2018/4705031 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Hao-Chun Chiang ◽

Rastislav Levicky

Keyword(s):

Polymer Film ◽

Thermal Denaturation ◽

Elevated Temperatures ◽

Electrochemical Analysis ◽

Thin Layers ◽

Optimal Selection ◽

Dna Duplexes ◽

Critical Importance ◽

Solid Supports ◽

Selection Of

The ability of different crosslinkers to crosslink nanometer thick films of the polymer poly(mercaptopropyl)methylsiloxane (PMPMS), thus stabilizing these films on solid supports, was investigated. The four crosslinkers included 1,11-bismaleimidotriethyleneglycol (BM(PEG)3), tris-(2-maleimidoethyl)amine (TMEA), bismaleimidohexane (BMH), and 1,1′-(methylenedi-4,1-phenylene) bismaleimide (BMDPM). PMPMS films treated with the four crosslinkers were compared in the effectiveness of achieved crosslinking, continuity and stability of the films to rearrangement at elevated temperatures, and modification with single-stranded DNA. The results of electrochemical analyses show that more hydrophilic crosslinkers had difficulty reacting fully with PMPMS thiols, even in these nanometer thin layers. This observation highlights the critical importance of selecting crosslinkers that are chemically compatible. Optimal selection of crosslinker yielded films in which the polymer film was largely incapable of rearranging, even at elevated temperatures, yielding reproducible and stable layers. These results validate use of these supports for applications such as monitoring thermal denaturation of immobilized DNA duplexes.

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The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide

Nucleic Acids Research ◽

10.1093/nar/19.11.3067 ◽

1991 ◽

Vol 19 (11) ◽

pp. 3067-3072 ◽

Cited By ~ 47

Author(s):

Nina G. Dolinnaya ◽

Nadejda I. Sokolova ◽

Dana T. Ashirbekova ◽

Zoe A. Shabarova

Keyword(s):

Water Soluble ◽

Dna Duplexes ◽

Modified Dna

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Structural stability of the photo-responsive DNA duplexes containing one azobenzene via a confined pore

Chemical Communications ◽

10.1039/c7cc04599a ◽

2017 ◽

Vol 53 (68) ◽

pp. 9462-9465 ◽

Cited By ~ 17

Author(s):

Fu-Na Meng ◽

Zi-Yuan Li ◽

Yi-Lun Ying ◽

Shao-Chuang Liu ◽

Junji Zhang ◽

...

Keyword(s):

Structural Stability ◽

Dna Duplexes ◽

Modified Dna

Herein, the structural stability of single azobenzene modified DNA duplexes, including the trans form and cis form, has been examined separately based on their distinguishable unzipping kinetics from the mixture by an α-hemolysin nanopore.

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