Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA

Braasch, Dwaine A.; Corey, David R.

doi:10.1016/s1074-5521(00)00058-2

Cited by 538 publications

(324 citation statements)

References 33 publications

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“…This is due primarily to the observation that LNA analogs significantly increase the stability of nucleic acid duplexes. In an extreme case, the melting temperature of LNA-containing duplexes can be increased by almost 10°C per substitution (56). This was particularly serious for substitutions into the dC⅐rG tract, where Fig.…”

Section: Resultsmentioning

confidence: 97%

“…Thus, in the case of the PPT RNA/DNA hybrids, LNA doublet or triplet insertions into the DNA template might be equivalent to locally introducing a short A-form RNA duplex within the context of an RNA/DNA hybrid. However, the increased thermal stability of LNA substitutions in the DNA of an RNA/DNA hybrid compared with an unsubstituted hybrid or duplex RNA (56,57) suggests the stability they introduce involves more than simply A-form geometry. This notion is strengthened by the observation that ␣-L LNA analogs, whose sugar retains the C2Ј-endo configuration, have also been shown to increase the thermal stability of RNA/DNA hybrids (58).…”

Section: Resultsmentioning

confidence: 99%

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Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Dash

Yi-Brunozzi

Grice

2004

Journal of Biological Chemistry

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Unusual base-pairing in a co-crystal of reverse transcriptase (RT) and a human immunodeficiency virus type 1 (HIV-1) polypurine tract (PPT)-containing RNA/ DNA hybrid suggests local nucleic acid flexibility mediates selection of the plus-strand primer. Structural elements of HIV-1 RT potentially participating in recognition of this duplex include the thumb subdomain and the ribonuclease H (RNase H) primer grip, the latter comprising elements of the connection subdomain and RNase H domain. To investigate how stabilizing HIV-1 PPT structure influences its recognition, we modified the (؊) DNA template by inserting overlapping locked nucleic acid (LNA) doublets and triplets. Modified RNA/ DNA hybrids were evaluated for cleavage at the PPT/U3 junction. Altered specificity was observed when the homopolymeric dA⅐rU tract immediately 5 of the PPT was modified, whereas PPT/U3 cleavage was lost after substitutions in the adjacent dT⅐rA tract. In contrast, the "unzipped" portion of the PPT was moderately insensitive to LNA insertions. Although a portion of the dC⅐rG and neighboring dT⅐rA tract were minimally affected by LNA insertion, RNase H activity was highly sensitive to altering the junction between these structural elements. Using 3 -end-labeled PPT RNA primers, we also identified novel cleavage sites ahead (؉5/؉6) of the PPT/U3 junction. Differential cleavage at the PPT/U3 junction and U3 ؉ 5/؉6 site in response to LNA-induced template modification suggests two binding modes for HIV-1 RT, both of which may be controlled by the interaction of its thumb subdomain (potentially via the minor groove binding track) at either site of the unzipped region.Minus (Ϫ)-strand DNA synthesis in retroviruses is accompanied by degradation of viral RNA of the RNA/DNA replication intermediate. These are events mediated by the N-terminal DNA polymerase and C-terminal ribonuclease H (RNase H) 1 catalytic centers of the multifunctional reverse transcriptase (RT), respectively (1). Correct positioning of nucleic acids to facilitate each of these steps clearly requires its specific interaction with structural motifs of the virus-coded polymerase. Crystallographic (2-4) and biochemical analysis (5-13) of human immunodeficiency virus type 1 (HIV-1) RT identified the primer grip of the p66 thumb as a motif critical for positioning the primer 3ЈOH before nucleophilic attack on the incoming dNTP. Immediately adjacent to this motif, the minor groove binding track (MGBT) or translocation track is proposed to mediate translocation of the polymerization machinery and act as a sensor of nucleic acid geometry (14 -19). Recently, it was proposed that the RNase H primer grip (RHPG), involving portions of the p66 connection subdomain and RNase H domain, interacts with the DNA strand of an RNA/DNA hybrid, providing the RNA with a trajectory appropriate for hydrolysis within the RNase H catalytic center (4, 20, 21). These combined activities effectively prepare nascent (Ϫ) DNA for use as template for plus (ϩ)-strand, DNA-dependent DNA synthesis.A critical...

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Dash

Yi-Brunozzi

Grice

2004

Journal of Biological Chemistry

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“…A minor modification was done to the HLA-B*27-specific 5'-primer by adding four bases to the 5'-end of the primer to compensate for the diminished concentration of this primer used in the asymmetric PCR when compared to the HLA-B*27-specific 3'-primer concentration, since the T m of the primers depends on the concentration as well as the length of the primer [8,15]. The inclusion of LNA-bases in the lanthanide-labeled probes was shown by our previous work to improve significantly the performance of the probes, when compared to regular, only-DNA containing oligonucleotides, in the developed assay format [8] and although the specificity in this assay relies mainly on the HLA-B*27-primers, given that the HLA-B*27 probe used in the homogeneous assay has a sequence which can be found also in other HLA-B-alleles, such as HLA-B*07, *08 and *39 among others, we chose to take advantage of the strong affinity of LNA-bases [2,12] in this assay as well to allow the use of short probes.…”

Section: Discussionmentioning

confidence: 99%

A Homogeneous HLA-B*27 Genotyping Assay Using Dried Reagent Mixtures

Ilonen¹,

Lövgren

2009

Disease Markers

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Abstract. The presence of HLA-B*27 allele with patients suspected with ankylosing spondylitis can be used in the diagnostic process. We have developed an assay for typing for the HLA-B*27 in whole blood dried on sample collection cards using pre-dried reagent wells and homogeneous time-resolved fluorescence based PCR approach. Essentially only the sample needs to be added to the dry ready-to-use reaction well in order to start the homogenous amplification assay. The method was validated with 229 samples also typed with an existing DELFIA-based method and results of both assays were 100% concordant. The dried reagents were shown to be stable at least up to eight weeks at room temperature without any decline in their performance.

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“…Numerous synthetic variants have thus been developed to pursue miRNA therapeutics, where sequence design and chemical modifications have made a range of options available with high binding affinity, enzymatic stability and improved cellular uptake. These include: (i) hydroxyl group conjugations in the ribose 2′ position to enhance hydrophobicity, such as Exiqon's proprietary locked-nucleic acid technology (LNA) developed by Braasch and Corey, [22] (ii) functionalizations of the phosphate backbone, which introduce enzymatic resistance and have been extended to a hybrid peptide nucleic acid (PNA) variant by Fabani and co-workers, [23] or (iii) morpholino substitutions for the ribose, which also improve binding affinity ( Table 1). Modifications of the ribose ring and phosphate backbone are often combined within single nts, and significant efforts are concentrated in optimizing the balance between multiply-modified and unmodified units composing the oligonucleotides.…”

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confidence: 99%

Scaffold‐Based microRNA Therapies in Regenerative Medicine and Cancer

Curtin

Castaño

O’Brien

2017

Adv Healthcare Materials

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The field of "regenerative medicine" (RM) was conceptually developed to aid the body's natural capacity to self-repair, a capacity which is impaired with age and in cases of disease or injury. [1] RM is a vast and multifaceted area of medicine, often microRNA-based therapies are an advantageous strategy with applications in both regenerative medicine (RM) and cancer treatments. microRNAs (miRNAs) are an evolutionary conserved class of small RNA molecules that modulate up to one third of the human nonprotein coding genome. Thus, synthetic miRNA activators and inhibitors hold immense potential to finely balance gene expression and reestablish tissue health. Ongoing industrysponsored clinical trials inspire a new miRNA therapeutics era, but progress largely relies on the development of safe and efficient delivery systems. The emerging application of biomaterial scaffolds for this purpose offers spatiotemporal control and circumvents biological and mechanical barriers that impede successful miRNA delivery. The nascent research in scaffold-mediated miRNA therapies translates know-how learnt from studies in antitumoral and genetic disorders as well as work on plasmid (p)DNA/siRNA delivery to expand the miRNA therapies arena. In this progress report, the state of the art methods of regulating miRNAs are reviewed. Relevant miRNA delivery vectors and scaffold systems applied to-date for RM and cancer treatment applications are discussed, as well as the challenges involved in their design. Overall, this progress report demonstrates the opportunity that exists for the application of miRNA-activated scaffolds in the future of RM and cancer treatments.Regenerative Medicine

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Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA

Cited by 538 publications

References 33 publications

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

Two Modes of HIV-1 Polypurine Tract Cleavage Are Affected by Introducing Locked Nucleic Acid Analogs into the (-) DNA Template

A Homogeneous HLA-B*27 Genotyping Assay Using Dried Reagent Mixtures

Scaffold‐Based microRNA Therapies in Regenerative Medicine and Cancer

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