Aptamer Nano-flares for Molecular Detection in Living Cells

Zheng, Dewen; Seferos, Dwight S.; Giljohann, David A.; Patel, Pinal C.; Mirkin, Chad A.

doi:10.1021/nl901517b

Cited by 376 publications

(327 citation statements)

References 57 publications

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“…Samples from cells, EpiDerm, and mouse skin were harvested for ICP-MS to measure gold content (SI Text), which quantitatively represents nanoparticle uptake (43). Final values were normalized to the total amount of applied SNA-NCs.…”

Section: Methodsmentioning

confidence: 99%

Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation

Zheng

Giljohann

Chen

et al. 2012

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

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365

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Topical application of nucleic acids offers many potential therapeutic advantages for suppressing genes in the skin, and potentially for systemic gene delivery. However, the epidermal barrier typically precludes entry of gene-suppressing therapy unless the barrier is disrupted. We now show that spherical nucleic acid nanoparticle conjugates (SNA-NCs), gold cores surrounded by a dense shell of highly oriented, covalently immobilized siRNA, freely penetrate almost 100% of keratinocytes in vitro, mouse skin, and human epidermis within hours after application. Significantly, these structures can be delivered in a commercial moisturizer or phosphate-buffered saline, and do not require barrier disruption or transfection agents, such as liposomes, peptides, or viruses. SNANCs targeting epidermal growth factor receptor (EGFR), an important gene for epidermal homeostasis, are >100-fold more potent and suppress longer than siRNA delivered with commercial lipid agents in cultured keratinocytes. Topical delivery of 1.5 uM EGFR siRNA (50 nM SNA-NCs) for 3 wk to hairless mouse skin almost completely abolishes EGFR expression, suppresses downstream ERK phosphorylation, and reduces epidermal thickness by almost 40%. Similarly, EGFR mRNA in human skin equivalents is reduced by 52% after 60 h of treatment with 25 nM EGFR SNA-NCs. Treated skin shows no clinical or histological evidence of toxicity. No cytokine activation in mouse blood or tissue samples is observed, and after 3 wk of topical skin treatment, the SNA structures are virtually undetectable in internal organs. SNA conjugates may be promising agents for personalized, topically delivered gene therapy of cutaneous tumors, skin inflammation, and dominant negative genetic skin disorders.T he recent development of small molecule inhibitors and antibodies that target components of signaling pathways has revolutionized the treatment of cancers, inflammatory diseases, and genetic disorders, including those that largely manifest in skin (1-3). These protein-based therapeutics, however, are costly, have limited targeting ability, and, when delivered through traditional intravenous or gastrointestinal routes, can lead to systemic toxicity (4, 5). Topical delivery is particularly attractive for the therapy of skin disorders. However, proteins larger than a few hundred daltons cannot easily enter the skin, and high concentrations of proteins must be applied for a cutaneous effect (6). An alternative to protein-based pathway inhibition involves the blocking and/or degradation of precursor mRNA before translation into protein. Gene silencing leads to down-regulation of protein expression and functions with greater specificity than inhibitors of protein function (7). In fact, targeted gene suppression by antisense DNA and siRNA has shown promising preclinical results, and/or is currently in clinical trials for a variety of diseases, including many forms of cancer (e.g., melanoma, neuroblastoma, and pancreatic adenocarcinoma), genetic disorders, and macular degeneration (8). For gene su...

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Section: Methodsmentioning

confidence: 99%

Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation

Zheng

Giljohann

Chen

et al. 2012

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

Self Cite

365

288

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“…Again, these 2D probes can be assembled on AuNPs and can be used to image molecular targets in live cells owing to the cellular uptake ability of AuNPs. Mirkin and co-workers 57 attached an aptamer probe to AuNPs to develop an intracellular 'nanoflare' probe for quantification of small molecules or proteins in living systems.…”

Section: Scaffolded Biosensors H Pei Et Almentioning

confidence: 99%

Scaffolded biosensors with designed DNA nanostructures

et al. 2013

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In addition to its fundamental function as a genetic code carrier, the utilization of DNA in various material applications has been actively explored over the past several decades. DNA is intrinsically an excellent type of self-assembly nanomaterial owing to its predictable base-pairing, high chemical stability and the convenience it possesses for synthesis and modification. Because of these unparalleled properties, DNA is widely used as excellent recognition elements in biosensors and as unique building blocks in nanodevices. A critical challenge in surface-based DNA biosensors lies in the reduced accessibility of target molecules to the DNA probes arranged on heterogeneous surfaces, especially when compared to probe-target recognition in homogeneous solutions. To improve the recognition abilities of these heterogeneous surface-confined DNA probes, much effort has been devoted to controlling the surface chemistry, conformation and packing density of the probe molecules, as well as the size and geometry of the surface. In this review, we aim to summarize the recent progress on the improvement of the probetarget recognition properties by introducing DNA nanostructure scaffolds. A range of new strategies have proven to provide a significantly enhanced range in the spatial positioning and the accessibility of the probes to the surface over previously reported linear structures. We will also describe the applications of DNA nanostructure scaffold-based biosensors. INTRODUCTIONDetection of nucleic acids (DNA or RNA) is an integral step in many applications, including in clinical diagnosis, environmental monitoring and antibioterrorism. 1 With these applications in mind, significant efforts have been made to develop sensitive, selective, rapid and cost-effective DNA (or RNA) biosensors. In parallel, DNAbased devices have also attracted a significant, recent interest owing to their promising applications in nanoelectronics, 2,3 biomolecular computations, 4-8 cellular imaging 9 and drug delivery. [10][11][12] In a typical design of DNA biosensors and nanodevices, oligonucleotides are often used as the molecular recognition layer. Because DNA is comprised of only four bases with A-T and G-C pairing, DNA hybridization processes are highly predictable and can be finely tuned with a rational design of the sequence. In addition, DNA oligonucleotides are usually easy to design, chemically stable and cost-effective.Understanding the physical structure of a DNA probe immobilized on a surface is critical in applications using DNA as a molecular recognition element. The properties of the DNA molecular recognition layer (such as selectivity, sensitivity and reproducibility) highly depend on the structure of the self-assembled DNA film. Modeling studies with thiolated DNA have demonstrated that efficient hybridization occurs in the well-controlled condition of surface-attached probes with large interprobe distances and upright conformations. [13][14][15][16][17][18][19][20][21][22][23][24]

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“…>2 mM for ATP). 46 To distinguish these analogs individually, however, aptamers with better selectivity are needed. 47 Detection in Serum.…”

mentioning

confidence: 99%

Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor

Huang

Liu

2010

Anal. Chem.

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Aptamers are single-stranded nucleic acids that can selectively bind to essentially any molecule of choice. Because of their high stability, low cost, ease of modification and availability through selection, aptamers hold great promise in addressing key challenges in bioanalytical chemistry. In the past fifteen years many highly sensitive fluorescent aptamer sensors have been reported. However, few such sensors showed high performance in serum samples. Further challenges related to practical applications include detection in a very small sample volume and a low dependence of sensor performance on ionic strength.We report the immobilization of an aptamer sensor on a magnetic microparticle and the use of flow cytometry for detection. Flow cytometry allows the detection of individual particles in a capillary and can effectively reduce the light scattering effect of serum. Since DNA immobilization generated a highly negatively charged surface and caused an enrichment of counter ions, the sensor performance showed a lower salt dependence. The detection limits for adenosine are determined to be 178 and 167 M in buffer and in 30% serum, respectively. Finally, we demonstrated that the detection can be carried out in 10 L of 90% human blood serum.3

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Aptamer Nano-flares for Molecular Detection in Living Cells

Cited by 376 publications

References 57 publications

Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation

Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation

Scaffolded biosensors with designed DNA nanostructures

Flow Cytometry-Assisted Detection of Adenosine in Serum with an Immobilized Aptamer Sensor

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