Synthesis of adenosine derivatives as transcription initiators and preparation of 5′ fluorescein- and biotin-labeled RNA through one-step in vitro transcription

Huang, Faqing; Wang, Guocan; Coleman, Tricia; Li, Na

doi:10.1261/rna.5106403

Cited by 53 publications

(65 citation statements)

References 30 publications

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“…The solution also contains 40 mM Tris (pH 8.0), 6 mM MgCl 2 , 2 mM spermidine, 0.01% Triton X-100, 5 mM DTT, 0.2 mM DNA templates, and 5 U/ml T7 RNA polymerase (Promega). 32 …”

Section: Preparation Of Rnamentioning

confidence: 99%

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Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

2006

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Nasopharyngeal carcinoma is a poorly differentiated upper respiratory tract cancer that highly expresses human folate receptors (hFR). Binding of folate to hFR triggers endocytosis. The folate was conjugated into adenosine 5 0 -monophosphate (AMP) by 1,6-hexanediamine linkages. After reverse HPLC to reach 93% purity, the folate-AMP, which can only be used for transcription initiation but not for chain extension, was incorporated into the 5 0 -end of bacteriophage phi29 motor pRNA. A 16:1 ratio of folate-AMP to ATP in transcription resulted in more than 60% of the pRNA containing folate. A pRNA with a 5 0 -overhang is needed to enhance the accessibility of the 5 0 folate for specific receptor binding. Utilizing the engineered left/right interlocking loops, polyvalent dimeric pRNA nanoparticles were constructed using RNA nanotechnology to carry folate, a detection marker, and siRNA targeting at an antiapoptosis factor. The chimeric pRNAs were processed into ds-siRNA by Dicer. Incubation of nasopharyngeal epidermal carcinoma (KB) cells with the dimer resulted in its entry into cancer cells, and the subsequent silencing of the target gene. Such a proteinfree RNA nanoparticle with undetectable antigenicity has a potential for repeated long-term administration for nasopharyngeal carcinoma as the effectiveness and specificity were confirmed by ex vivo delivery in the animal trial. Gene Therapy (2006) 13, 814-820.

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“…The solution also contains 40 mM Tris (pH 8.0), 6 mM MgCl 2 , 2 mM spermidine, 0.01% Triton X-100, 5 mM DTT, 0.2 mM DNA templates, and 5 U/ml T7 RNA polymerase (Promega). 32 …”

Section: Preparation Of Rnamentioning

confidence: 99%

“…32 5 0 Folate-AMP was then purified to 93% purity by semipreparative reverse phase HPLC. After lyophilization, the compound was dissolved in water.…”

Section: Synthesis and Purification Of Folate-ampmentioning

confidence: 99%

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

2006

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“…Synthesis of 13 was performed as previously described (Huang et al 2003). 6-ClAMP (12760 mODU 260 (1.3 mmol) was suspended in 18 mL of water and treated with 1,6-diaminohexane (2.5 g, 19.3 mmol).…”

Section: General Proceduresmentioning

confidence: 99%

“…The first step of the synthesis was the Yoshikawa phosphorylation of 6-chloropurine riboside (Yoshikawa et al 1967), leading to 6-chloropurinoriboside 59-monophosphate (6-ClAMP, 12). The introduction of the spacer to the ligand molecule was achieved by the substitution of 12 with 1,6-hexylenediamine as previously described (Huang et al 2003). The reaction was performed in aqueous solution at room temperature using a 10-fold molar excess of the amine and led to the practically pure desirable product 13 within 2 h (98% yield by HPLC).…”

Section: Introductionmentioning

confidence: 99%

Affinity resins containing enzymatically resistant mRNA cap analogs—a new tool for the analysis of cap-binding proteins

Szczepaniak¹,

Zuberek²,

Darżynkiewicz³

et al. 2012

RNA

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Cap-binding proteins have been routinely isolated using m 7 GTP-Sepharose; however, this resin is inefficient for proteins such as DcpS (scavenger decapping enzyme), which interacts not only with the 7-methylguanosine, but also with the second cap base. In addition, DcpS purification may be hindered by the reduced resin capacity due to the ability of DcpS to hydrolyze m 7 GTP. Here, we report the synthesis of new affinity resins, m 7 GpCH 2 pp-and m 7 GpCH 2 ppA-Sepharoses, with attached cap analogs resistant to hydrolysis by DcpS. Biochemical tests showed that these matrices, as well as a hydrolyzable m 7 GpppA-Sepharose, bind recombinant mouse eIF4E specifically and at high capacity. In addition, purification of cap-binding proteins from yeast extracts confirmed the presence of all expected cap-binding proteins, including DcpS in the case of m 7 GpCH 2 pp-and m 7 GpCH 2 ppA-Sepharoses. In contrast, binding studies in vitro demonstrated that recombinant human DcpS efficiently bound only m 7 GpCH 2 ppA-Sepharose. Our data prove the applicability of these novel resins, especially m 7 GpCH 2 ppA-Sepharose, in biochemical studies such as the isolation and identification of cap-binding proteins from different organisms.

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“…During transcription, the labeled AMP could only be used for initiation of transcription by the T7 RNA polymerase (He et al 1997), but could not be used for RNA chain extension since chain extension requires the presence of a triphosphate, such as ATP (Garver and Guo 1997;Huang 2003;Huang et al 2003;Li et al 2005;Guo et al 2006). The resultant 117-base pRNA has a single Cy3 or Cy5 molecule tagged to its 59 end.…”

Section: Labeling Prna With Cy3 or Cy5mentioning

confidence: 99%

Instrumentation and metrology for single RNA counting in biological complexes or nanoparticles by a single-molecule dual-view system

Zhang¹,

Shu²,

Huang³

et al. 2007

RNA

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Limited by the spatial resolution of optical microscopy, direct detection or counting of single components in biological complexes or nanoparticles is challenging, especially for RNA, which is conformationally versatile and structurally flexible. We report here the assembly of a customized single-molecule dual-viewing total internal reflection fluorescence imaging system for direct counting of RNA building blocks. The RNA molecules were labeled with a single fluorophore by in vitro transcription in the presence of a fluorescent AMP. Precise calculation of identical or mixed pRNA building blocks of one, two, three, or six copies within the bacteriophage phi29 DNA packaging motor or other complexes was demonstrated by applying a photobleaching assay and evaluated by binomial distribution. The dual-viewing system for excitation and recording at different wavelengths simultaneously will enable the differentiation of different complexes with different labels or relative motion of each labeled component in motion machines.

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Synthesis of adenosine derivatives as transcription initiators and preparation of 5′ fluorescein- and biotin-labeled RNA through one-step in vitro transcription

Cited by 53 publications

References 30 publications

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

Affinity resins containing enzymatically resistant mRNA cap analogs—a new tool for the analysis of cap-binding proteins

Instrumentation and metrology for single RNA counting in biological complexes or nanoparticles by a single-molecule dual-view system

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