LNA (Locked Nucleic Acid):  An RNA Mimic Forming Exceedingly Stable LNA:LNA Duplexes

Koshkin, Alexei A.; Nielsen, P. H.; Meldgaard, Michael; Rajwanshi, Vivek K.; Singh, Sanjay K.; Wengel, Jesper

doi:10.1021/ja9822862

Cited by 306 publications

(292 citation statements)

References 24 publications

(17 reference statements)

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“…1). For example, the stability of a DNA duplex correlates with the helicity of its single strands (15,16), and a bicyclic ("locked") derivative of ribose enhances that stability (17,18). The stability of β-turns within protein structures can be increased by incorporating Dproline (19) and certain β-amino acids (20) that bias the conformations occupied by the unfolded polypeptide.…”

mentioning

confidence: 99%

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Shoulders¹,

Satyshur²,

Forest³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

152

142

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Preorganization is shown to endow a protein with extraordinary conformational stability. This preorganization is achieved by installing side-chain substituents that impose stereoelectronic and steric effects that restrict main-chain torsion angles. Replacing proline residues in ðProProGlyÞ 7 collagen strands with 4-fluoroproline and 4-methylproline leads to the most stable known triple helices, having T m values that are increased by >50°C. Differential scanning calorimetry data indicate an entropic basis to the hyperstability, as expected from an origin in preorganization. Structural data at a resolution of 1.21 Å reveal a prototypical triple helix with insignificant deviations to its main chain, even though 2∕3 of the residues are nonnatural. Thus, preorganization of a main chain by subtle changes to side chains can confer extraordinary conformational stability upon a protein without perturbing its structure.collagen triple helix | nonnatural amino acid | preorganization | protein stability | x-ray crystallography T he three-dimensional structures of proteins are stable because of a delicate balance of forces (1). Increases in the conformational stability of a protein-desirable in many contexts (2)-can be realized by enhancing the hydrophobic effect (3, 4), introducing or shielding a hydrogen bond (5) or electrostatic interaction (6-8), or introducing a disulfide crosslink (9-11) or metal ion-binding site (12). These strategies are often frustrated by enthalpy-entropy compensation, whereby enthalpic stabilization is offset entirely by entropic destabilization, or vice versa (13). Moreover, the strategies often lack subtlety and can lead to a misshapen and hence dysfunctional protein.As elaborated by Cram (14), the principle of preorganization states that, "the more highly hosts and guests are organized for binding and low solvation prior to their complexation, the more stable will be their complexes." In a typical manifestation, a conformational constraint is imposed upon a receptor or ligand during its chemical synthesis. This principle can also yield biopolymers with increased conformational stability (Fig. 1). For example, the stability of a DNA duplex correlates with the helicity of its single strands (15, 16), and a bicyclic ("locked") derivative of ribose enhances that stability (17, 18). The stability of β-turns within protein structures can be increased by incorporating Dproline (19) and certain β-amino acids (20) that bias the conformations occupied by the unfolded polypeptide. Likewise, proteins containing a cis-peptide bond can be stabilized by constrained amides or isosteres (21-23). The utility of these substitutions is limited, however, by the difficulty of modifying the backbone of proteins as well as concomitant effects on structure and function (24-27).We suspected that alterations to proline residues could provide a particularly incisive means to preorganize the conformation of a polypeptide chain. In classic work, Matthews and coworkers substituted proline, which is the least flexible residu...

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mentioning

confidence: 99%

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Shoulders¹,

Satyshur²,

Forest³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

152

142

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“…1) contains a ribose ring which is locked by a O2'-C4'-methylene linkage, imposing conformational restriction to adopt an N-type sugar puckering. [2][3][4][5]10 Structural investigation of LNA oligonucleotides by NMR spectroscopy revealed their similarities with natural nucleic acid duplexes, and confirmed the RNA mimicking structures adopted by LNA. 11,12 LNA offers key properties needed for successful therapeutic exploitation of oligonucleotides, including (1) unprecedented binding affinity towards RNA (and DNA), (2) excellent base pairing specificity, (3) high bio-stability (resistance towards nucleolytic degradation, (4) low toxicity (at least for many LNA oligonucleotides) in animals and (5) convenient chemistry for manufacturing and modification.…”

Section: Introductionmentioning

confidence: 70%

“…Among the numerous modifications known, LNA has shown broad usefulness within chemical biology. [2][3][4][5][6][7][8][9] LNA: Structural Features and Key Properties LNA ( Fig. 1) contains a ribose ring which is locked by a O2'-C4'-methylene linkage, imposing conformational restriction to adopt an N-type sugar puckering.…”

Section: Introductionmentioning

confidence: 99%

Locked nucleic acid as a novel class of therapeutic agents

Veedu¹,

Wengel²

2009

RNA Biology

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108

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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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LNA (Locked Nucleic Acid): An RNA Mimic Forming Exceedingly Stable LNA:LNA Duplexes

Cited by 306 publications

References 24 publications

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Stereoelectronic and steric effects in side chains preorganize a protein main chain

Locked nucleic acid as a novel class of therapeutic agents

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

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