Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen® as fluorescence inducing agent

Zhang, Jun; Liang, Dehai; He, Weidong; Wan, Fen; Qian, Ying; Chu, Benjamin

doi:10.1002/elps.200500099

Cited by 17 publications

(27 citation statements)

References 20 publications

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“…3). The noncovalently associated oligonucleotides that dissociate from the AuNPs are quantified with the fluorescence of OliGreen, a nonspecific intercalator of single-stranded oligonucleotides (22). The concentration of AuNPs was determined by measuring the absorbance at 520 nm (23).…”

Section: Resultsmentioning

confidence: 99%

Probing the inherent stability of siRNA immobilized on nanoparticle constructs

Barnaby

Lee

Mirkin

2014

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

100

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Small interfering RNA (siRNA) is a powerful and highly effective method to regulate gene expression in vitro and in vivo. However, the susceptibility to serum nuclease-catalyzed degradation is a major challenge and it remains unclear whether the strategies developed to improve the stability of siRNA free in serum solution are ideal for siRNA conjugated to nanoparticle surfaces. Herein, we use spherical nucleic acid nanoparticle conjugates, consisting of gold nanoparticles (AuNPs) with siRNA chemisorbed to the surface, as a platform to study how a model siRNA targeting androgen receptor degrades in serum (SNA-siRNA AR ). In solutions of 10% (vol/vol) FBS, we find rapid endonuclease hydrolysis at specific sites near the AuNP-facing terminus of siRNA AR , which were different from those of siRNA AR free in solution. These data indicate that the chemical environment of siRNA on a nanoparticle surface can alter the recognition of siRNA by serum nucleases and change the inherent stability of the nucleic acid. Finally, we demonstrate that incorporation of 2′-O-methyl RNA nucleotides at sites of nuclease hydrolysis on SNA-siRNA AR results in a 10-fold increase in siRNA lifetime. These data suggest that strategies for enhancing the serum stability of siRNA immobilized to nanoparticles must be developed from a dedicated analysis of the siRNAnanoparticle conjugate, rather than a reliance on strategies developed for siRNA free in solution. We believe these findings are important for fundamentally understanding interactions between biological media and oligonucleotides conjugated to nanoparticles for the development of gene regulatory and therapeutic agents in a variety of disease models.biological recognition | nanotechnology | OliGreen | polyacrylamide gel electrophoresis

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Section: Resultsmentioning

confidence: 99%

Probing the inherent stability of siRNA immobilized on nanoparticle constructs

Barnaby

Lee

Mirkin

2014

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

100

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“…Zhang et al. further modified the above method using a different polymer, pluronic F127, and by changing the approach used for introducing the dye . The electro‐kinetic dye introduction was replaced by incorporating the dye into the polymer solution, which was injected into the capillary with a gas‐tight syringe.…”

Section: Application Of On‐column Derivatisationmentioning

confidence: 99%

On‐capillary derivatisation as an approach to enhancing sensitivity in capillary electrophoresis

Glatz

2014

Electrophoresis

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Separation technologies play an important role in revealing biological processes at various omic levels, in pharmacological and clinical research. In this context, CE is a strong candidate for analyses of samples with rapidly increasing complexity. Even though CE is well known for its many advantages in this regard, the sensitivity of CE analyses is insufficient for many applications. Accordingly, there are generally three main options for enhancing the sensitivity of CE analyses - using special detection techniques, using sample pre-concentration and derivatisation. Derivatisation is often the method of choice for many laboratories, since it is simple and provides several advantages such as small sample volume demand and the possibility of automation. Although it can be performed in different ways depending on where the reaction takes place, this article reviews one of the simplest and at the same time most useful approaches on-capillary derivatisation. Even if in many cases the use of on-capillary derivatisation alone is enough to improve the detection sensitivity, on other occasions it needs to be employed in combination with the other above-mentioned strategies. After a simple discussion of derivatisation in general, special attention is focused on the on-capillary approach and methodologies available for on-capillary reactant mixing. Its applications in various fields are also described.

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“…All peaks except 8 and 10 base were well resolved in less than 4.5 min. After optimization of the running parameters, oligonucleotides could be well separated with one-base resolution within 1.4 min [33]. When the effective separation length was shortened to 1.5 cm, 25% w/v the F127 solution was not able to separate the oligonucleotide sizing markers, as shown in the electropherogram in Fig.…”

Section: Resultsmentioning

confidence: 95%

Nanostructured polymer matrix for oligonucleotide separation

Zhang¹,

Bürger²,

Chu³

2006

Electrophoresis

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A nanostructured copolymer matrix has successfully separated oligonucleotides with high resolution by CE using a very short separation channel which simulates the real microchip condition for the first time. The triblock copolymer, E(45)B(14)E(45) (B20-5000) with E, B, and subscript denoting oxyethylene, oxybutylene, and segment length, respectively, has a unique temperature-dependent viscosity-adjustable property and a dynamic coating ability in 1xTBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA in Milli-Q water). The B-block is hydrophilic at low temperatures, e.g., 4 degrees C, and the polymer solution has a very low viscosity of about 100 cP in a 32.5% w/v solution. At room temperatures, the B-block becomes hydrophobic due to a breakdown of hydrogen bonds between B-blocks and water, and the polymer matrix forms a body-centered cubic structure at high concentrations. Oligonucleotide sizing markers ranging from 8 to 32 base could be successfully separated with one-base resolution in a 1.5 cm long separation channel by E(45)B(14)E(45) in its gel-like state.

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Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen® as fluorescence inducing agent

Cited by 17 publications

References 20 publications

Probing the inherent stability of siRNA immobilized on nanoparticle constructs

Probing the inherent stability of siRNA immobilized on nanoparticle constructs

On‐capillary derivatisation as an approach to enhancing sensitivity in capillary electrophoresis

Nanostructured polymer matrix for oligonucleotide separation

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