LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition

Abstract When released from cytotoxic T lymphocytes and natural killer cells, Granzyme (Gzm) serine proteases induce programmed cell death of pathogen-infected cells and tumor cells. The Gzms rapidly accumulate in the target cell nucleus by an unknown mechanism. Many of the known substrates of GzmA and GzmB, the most abundant killer cell proteases, bind to DNA or RNA. Gzm substrates predicted by unbiased proteomics studies are also highly enriched for nucleic acid binding proteins. Here we show by fluorescence polarization assays that Gzms bind DNA and RNA with nanomolar affinity. We hypothesized that Gzm binding to nucleic acids enhances nuclear accumulation in target cells and facilitates their cleavage of nucleic acid-binding substrates. In fact, RNase treatment of cell lysates reduced cleavage of RNA binding protein (RBP) targets by GzmA and GzmB. Moreover, adding RNA to recombinant RBP substrates greatly enhanced in vitro cleavage by GzmB, but adding RNA to non-nucleic acid binding proteins did not. For example, exogenous RNA enhanced GzmB cleavage of recombinant hnRNP C1 (an RBP) but not LMNB1 (a non-RBP). In addition, GzmB cleaved the RNA-binding HuR protein efficiently only when it was bound to an HuR-binding RNA oligonucleotide, but not in the presence of an equal amount of non-binding RNA. Thus, nucleic acids facilitate Gzm cleavage of nucleic acid binding substrates. To evaluate whether nucleic acid binding influences Gzm trafficking in target cells, we incubated fixed target cells with RNase and then added Gzms. RNA degradation in target cells reduced Gzm cytosolic localization and increased nuclear accumulation. Similarly, pre-incubating Gzms with exogenous competitor DNA reduced Gzm nuclear localization. The Gzms form a monophyletic clade with other immune serine proteases including neutrophil elastase (NE) and cathepsin G (CATG). Upon neutrophil activation, NE translocates to the nucleus to drive the formation of neutrophil extracellular traps (NETs). NE and CATG, but not non-immune serine proteases such as trypsin and pancreatic elastase, also bind DNA with high affinity and localize to the nucleus of permeabilized cells. Consistent with this finding, competitor DNA also blocks the nuclear localization of NE. Moreover NE and CATG localization to NETs depends on DNA binding. Thus the antimicrobial activity of NETs may depend in part upon the affinity of these proteases for DNA. Our findings indicate that high affinity nucleic acid binding is a conserved and functionally important property of serine proteases involved in cell-mediated immunity. Disclosures: Lieberman: Alnylam Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

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Bridged Nucleic Acids Reloaded

10.20944/preprints201906.0032.v1 ◽

2019 ◽

Author(s):

Alfonso Soler-Bistué ◽

Angeles Zorreguieta ◽

Marcelo E. Tolmasky

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

First Generation ◽

Second Generation ◽

Nucleotide Analogs ◽

Great Hope ◽

Locked Nucleic Acids ◽

Near Future

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, led to the design of nucleotide analogs that when being part of these oligomers enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs are the first-generation bridge nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second generation BNA, BNANC (2'-O,4'-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but in some cases also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds were researched, the results are very promising warranting more efforts in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future offering great hope to oligonucleotide-based fields of research and applications.

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Bridged Nucleic Acids Reloaded

Molecules ◽

10.3390/molecules24122297 ◽

2019 ◽

Vol 24 (12) ◽

pp. 2297 ◽

Cited By ~ 7

Author(s):

Alfonso Soler-Bistué ◽

Angeles Zorreguieta ◽

Marcelo E. Tolmasky

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

First Generation ◽

Second Generation ◽

Nucleotide Analogs ◽

Great Hope ◽

Locked Nucleic Acids ◽

Near Future

Oligonucleotides are key compounds widely used for research, diagnostics, and therapeutics. The rapid increase in oligonucleotide-based applications, together with the progress in nucleic acids research, has led to the design of nucleotide analogs that, when part of these oligomers, enhance their efficiency, bioavailability, or stability. One of the most useful nucleotide analogs is the first-generation bridged nucleic acids (BNA), also known as locked nucleic acids (LNA), which were used in combination with ribonucleotides, deoxyribonucleotides, or other analogs to construct oligomers with diverse applications. However, there is still room to improve their efficiency, bioavailability, stability, and, importantly, toxicity. A second-generation BNA, BNANC (2′-O,4′-aminoethylene bridged nucleic acid), has been recently made available. Oligomers containing these analogs not only showed less toxicity when compared to LNA-containing compounds but, in some cases, also exhibited higher specificity. Although there are still few applications where BNANC-containing compounds have been researched, the promising results warrant more effort in incorporating these analogs for other applications. Furthermore, newer BNA compounds will be introduced in the near future, offering great hope to oligonucleotide-based fields of research and applications.

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Single-Stranded DNA Aptamers against Pathogens and Toxins: Identification and Biosensing Applications

BioMed Research International ◽

10.1155/2015/419318 ◽

2015 ◽

Vol 2015 ◽

pp. 1-31 ◽

Cited By ~ 36

Author(s):

Ka Lok Hong ◽

Letha J. Sooter

Keyword(s):

Nucleic Acid ◽

Molecular Recognition ◽

Nucleic Acids ◽

Antibody Fragments ◽

Small Peptides ◽

Basic Principles ◽

Single Stranded Dna ◽

High Affinity ◽

Ssdna Aptamer ◽

Recognition Elements

Molecular recognition elements (MREs) can be short sequences of single-stranded DNA, RNA, small peptides, or antibody fragments. They can bind to user-defined targets with high affinity and specificity. There has been an increasing interest in the identification and application of nucleic acid molecular recognition elements, commonly known as aptamers, since they were first described in 1990 by the Gold and Szostak laboratories. A large number of target specific nucleic acids MREs and their applications are currently in the literature. This review first describes the general methodologies used in identifying single-stranded DNA (ssDNA) aptamers. It then summarizes advancements in the identification and biosensing application of ssDNA aptamers specific for bacteria, viruses, their associated molecules, and selected chemical toxins. Lastly, an overview of the basic principles of ssDNA aptamer-based biosensors is discussed.

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3SCA-03 High-affinity DNA aptamer selection by a genetic alphabet expansion PCR system(3SCA Frontier of functional nucleic acids toward elucidation of biological events and nucleic acid medicine,Symposium)

Seibutsu Butsuri ◽

10.2142/biophys.53.s103_2 ◽

2013 ◽

Vol 53 (supplement1-2) ◽

pp. S103

Author(s):

Michiko Kimoto ◽

Ken-ichiro Matsunaga ◽

Rie Yamashige ◽

Ichiro Hirao

Keyword(s):

Nucleic Acid ◽

Nucleic Acids ◽

Dna Aptamer ◽

High Affinity ◽

Aptamer Selection ◽

Functional Nucleic Acids ◽

Genetic Alphabet

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Design and In Vitro Evaluation of Splice-Switching Oligonucleotides Bearing Locked Nucleic Acids, Amido-Bridged Nucleic Acids, and Guanidine-Bridged Nucleic Acids

International Journal of Molecular Sciences ◽

10.3390/ijms22073526 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3526

Author(s):

Takenori Shimo ◽

Yusuke Nakatsuji ◽

Keisuke Tachibana ◽

Satoshi Obika

Keyword(s):

Nucleic Acids ◽

Exon Skipping ◽

Secondary Structures ◽

High Affinity ◽

Secondary Structure Formation ◽

Locked Nucleic Acids ◽

Mrna And Protein Expression ◽

Melting Analysis ◽

Uv Melting

Our group previously developed a series of bridged nucleic acids (BNAs), including locked nucleic acids (LNAs), amido-bridged nucleic acids (AmNAs), and guanidine-bridged nucleic acids (GuNAs), to impart specific characteristics to oligonucleotides such as high-affinity binding and enhanced enzymatic resistance. In this study, we designed a series of LNA-, AmNA-, and GuNA-modified splice-switching oligonucleotides (SSOs) with different lengths and content modifications. We measured the melting temperature (Tm) of each designed SSO to investigate its binding affinity for RNA strands. We also investigated whether the single-stranded SSOs formed secondary structures using UV melting analysis without complementary RNA. As a result, the AmNA-modified SSOs showed almost the same Tm values as the LNA-modified SSOs, with decreased secondary structure formation in the former. In contrast, the GuNA-modified SSOs showed slightly lower Tm values than the LNA-modified SSOs, with no inhibition of secondary structures. We also evaluated the exon skipping activities of the BNAs in vitro at both the mRNA and protein expression levels. We found that both AmNA-modified SSOs and GuNA-modified SSOs showed higher exon skipping activities than LNA-modified SSOs but each class must be appropriately designed in terms of length and modification content.

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