Abstract:
:We have developed a PCR-based short interfering RNA (siRNA) quantification method based on competition between siRNA and a homologous DNA primer for annealing to template DNA, avoiding the requirement for prior conversion of RNA to cDNA. Primers and probe were designed to amplify regions of the human papillomavirus E6 or enhanced green fluorescent protein genes. Having confirmed siRNA could not act as primer for amplicon generation, the lowest competing primer concentration yielding a linear relationship between template DNA amount (0.1-50 ng) and cycle of threshold (Ct) was determined (6.25 nM). Under these conditions addition of sequence-specific siRNA to the competitive quantitative PCR (cqPCR), resulted in a dose-dependent linear increase in Ct value. 2'-O-methyl ribose-modified siRNA retained an ability to inhibit template amplification in serum, unlike unmodified siRNAs that were susceptible to endonucleases. Mismatch-bearing or truncated siRNAs failed to inhibit template amplification confirming sequence specificity and an ability to discriminate between degraded and non-degraded siRNA sequences. Following delivery of E6 siRNA to C33-A cells using oligofectamine or His6 reducible polymers, siRNA uptake was quantified by cqPCR, revealing dose-dependent uptake. We anticipate that cqPCR will allow accurate determination of siRNA pharmacokinetics following in vivo delivery, greatly facilitating development of therapeutic siRNA delivery strategies.
journal_name
Nucleic Acids Resjournal_title
Nucleic acids researchauthors
Liu WL,Stevenson M,Seymour LW,Fisher KDdoi
10.1093/nar/gkn903subject
Has Abstractpub_date
2009-01-01 00:00:00pages
e4issue
1eissn
0305-1048issn
1362-4962pii
gkn903journal_volume
37pub_type
杂志文章abstract::Interactions between subunits in the Saccharomyces cerevisiae ribosome are mediated by universal and eukaryote-specific intersubunit bridges. Universal bridges are positioned close to the ribosomal functional centers, while eukaryote-specific bridges are mainly located on the periphery of the ribosome. Two bridges, eB...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky1083
更新日期:2019-01-10 00:00:00
abstract::Optogenetic control of CRISPR-Cas9 systems has significantly improved our ability to perform genome perturbations in living cells with high precision in time and space. As new Cas orthologues with advantageous properties are rapidly being discovered and engineered, the need for straightforward strategies to control th...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkaa1198
更新日期:2020-12-16 00:00:00
abstract::Expression of the wild-type p53 tumor suppressor gene has been found to play an important role in the regulation of cellular proliferation and differentiation. In addition, in many transformed cells and primary tumors, the gene has undergone allelic deletions and mutant forms of the p53 gene are expressed at elevated ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/21.2.345
更新日期:1993-01-25 00:00:00
abstract::It has been inferred from DNA sequence analyses that in echinoderm mitochondria not only the usual asparagine codons AAU and AAC, but also the usual lysine codon AAA, are translated as asparagine by a single mitochondrial (mt) tRNAAsn with the anticodon GUU. Nucleotide sequencing of starfish mt tRNAAsn revealed that t...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/27.7.1683
更新日期:1999-04-01 00:00:00
abstract::A successful application is reported of the multivariate curve resolution alternating least-squares method (MCR-ALS) for the analysis of nucleic acid melting and salt-induced transitions. Under conditions where several structures co-exist in a conformational equilibrium, MCR-ALS analysis of the UV and circular dichroi...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gnf091
更新日期:2002-09-01 00:00:00
abstract::Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA m...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky828
更新日期:2018-10-12 00:00:00
abstract::Eukaryotic gene expression is regulated by enhancer-promoter interactions but the molecular mechanisms that govern specificity have remained elusive. Genome-wide studies utilizing STARR-seq identified two enhancer classes in Drosophila that interact with different core promoters: housekeeping enhancers (hkCP) and deve...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkw1114
更新日期:2017-02-28 00:00:00
abstract::The precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is a polyprotein containing multiple copies of LHCPII covalently joined by a decapeptide linker. cDNA and genomic clones encoding the 5' and 3' end of a 6.6 kb LHCPII mRNA were sequenced. A 3.1 kb genomic region en...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/22.25.5737
更新日期:1994-12-25 00:00:00
abstract::Alu insertions have contributed to >11% of the human genome and ∼30-35 Alu subfamilies remain actively mobile, yet the characterization of polymorphic Alu insertions from short-read data remains a challenge. We build on existing computational methods to combine Alu detection and de novo assembly of WGS data as a means...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv1089
更新日期:2015-12-02 00:00:00
abstract::Mammalian cryptochromes (Crys) are essential circadian clock factors implicated in diverse clock-independent physiological functions, including DNA damage responses. Here we show that Cry1 modulates the ATR-mediated DNA damage checkpoint (DDC) response by interacting with Timeless (Tim) in a time-of-day-dependent mann...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku094
更新日期:2014-04-01 00:00:00
abstract::Human thymine-DNA glycosylase (TDG) is well known to excise thymine and uracil from G.T and G.U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. In this study, we characterized ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkg344
更新日期:2003-05-01 00:00:00
abstract::In eukaryotes, DNA is packaged into a basic unit, the nucleosome which consists of 147 bp of DNA wrapped around a histone octamer composed of two copies each of the histones H2A, H2B, H3 and H4. Nucleosome structures are diverse not only by histone variants, histone modifications, histone composition but also through ...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkv276
更新日期:2015-04-30 00:00:00
abstract::Two forms of post-transcriptional control direct differential expression of the Saccharomyces cerevisiae genes encoding the AP1-like transcription factors Yap1p and Yap2p. The mRNAs of these genes contain respectively one (YAP1 uORF) and two (YAP2 uORF1 and uORF2) upstream open reading frames. uORF-mediated modulation...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/26.5.1150
更新日期:1998-03-01 00:00:00
abstract::Message-specific translational regulation mechanisms shape the biogenesis of multimeric oxidative phosphorylation (OXPHOS) enzyme in mitochondria from the yeast Saccharomyces cerevisiae. These mechanisms, driven mainly by the action of mRNA-specific translational activators, help to coordinate synthesis of OXPHOS cata...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkz266
更新日期:2019-06-20 00:00:00
abstract::We have isolated a unique fragment of the HLA-DR alpha gene and probed human genomic DNA at low stringency to search for homologous sequences. A minimum of six non-polymorphic cross-hybridizing high molecular weight fragments were found in all DNAs examined. In order to obtain molecular clones of these cross-hybridizi...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/13.20.7515
更新日期:1985-10-25 00:00:00
abstract::REBASE is a comprehensive database of information about restriction enzymes and their associated methylases, including their recognition and cleavage sites and their commercial availability. Also included is a listing of homing endonucleases. Information from REBASE is available via monthly electronic mailings as well...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/26.1.338
更新日期:1998-01-01 00:00:00
abstract::Ribosome biogenesis is a key process for maintaining protein synthetic capacity in dividing or growing cells, and requires coordinated production of ribosomal proteins and ribosomal RNA (rRNA), including the processing of the latter. Signalling through mammalian target of rapamycin complex 1 (mTORC1) activates all the...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gku130
更新日期:2014-04-01 00:00:00
abstract::All exchangeable protons in a short DNA helix, d(CG)3 sodium salt, have been studied by proton nuclear magnetic resonance. The cytidine and guanosine amino protons have been assigned for the first time. As a function of temperature the cytidine amino protons and the imino protons behave very similarly, their relaxatio...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/12.21.8269
更新日期:1984-11-12 00:00:00
abstract::This is a study of the effect of total and partial deuteration of solvent on critical temperatures and profiles of all four reactions occurring in poly(A) x n poly(U) (n = 1 or 2) aqueous systems. The study was done at observational times not longer than hydrogen exchange times at base pairs in helically ordered struc...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/8.18.4283
更新日期:1980-09-25 00:00:00
abstract::YdiV is a negative regulator of cell motility. It interacts with FlhD(4)C(2) complex, a product of flagellar master operon, which works as the transcription activator of all other flagellar operons. Here, we report the crystal structures of YdiV and YdiV(2)-FlhD(2) complex at 1.9 Å and 2.9 Å resolutions, respectively....
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gks869
更新日期:2012-11-01 00:00:00
abstract::The A site of the small ribosomal subunit participates in the fidelity of decoding by switching between two states, a resting 'off' state and an active decoding 'on' state. Eight crystal structures of RNA duplexes containing two minimal decoding A sites of the Homo sapiens mitochondrial wild-type, the A1555G mutant or...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkn112
更新日期:2008-05-01 00:00:00
abstract::We report, herein, a new class of RNAi trigger molecules based on the unconventional parallel hybridization of two oligonucleotide chains. We have prepared and studied several parallel stranded (ps) duplexes, in which the parallel orientation is achieved through incorporation of isoguanine and isocytosine to form reve...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gky024
更新日期:2018-02-28 00:00:00
abstract::Genomes mutate and evolve in ways simple (substitution or deletion of bases) and complex (e.g. chromosome shattering). We do not fully understand what types of complex mutation occur, and we cannot routinely characterize arbitrarily-complex mutations in a high-throughput, genome-wide manner. Long-read DNA sequencing m...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkx1266
更新日期:2018-02-28 00:00:00
abstract::Due to the importance of protein phosphorylation in cellular control, many researches are undertaken to predict the kinase-specific phosphorylation sites. Referred to our previous work, KinasePhos 1.0, incorporated profile hidden Markov model (HMM) with flanking residues of the kinase-specific phosphorylation sites. H...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkm322
更新日期:2007-07-01 00:00:00
abstract::Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision r...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/29.3.604
更新日期:2001-02-01 00:00:00
abstract::Evidence will be presented to show that the allyl group is suitable for the protection of a 3'-terminal phosphodiester function. The latter will be demonstrated by the synthesis, via a phosphotriester approach, of two cyclic tetraribonucleotides [r(AAAA) and r(UAMe2UAMe2)], two cyclic hexadeoxyribonucleotides [d(CGCGC...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/16.10.4607
更新日期:1988-05-25 00:00:00
abstract::In order to study the intragenic profiles of active transcription, we determined the relative levels of active RNA polymerase II present at the 3'- and 5'-ends of 261 yeast genes by run-on. The results obtained indicate that the 3'/5' run-on ratio varies among the genes studied by over 12 log(2) units. This ratio seem...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/gkq215
更新日期:2010-08-01 00:00:00
abstract::The role of thymine dimerization for previously reported U.V. induced conformational changes has been proved using acetophenone as sensitizer for the specific thymine dimerization upon irradiation at 313 nm. CD results demonstrate that formation of pyrimidine dimers cause typical conformational changes of the DNA B he...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/2.2.179
更新日期:1975-02-01 00:00:00
abstract::The chicken erythroid proteins binding to the histone H5 5' promoter and 3' erythroid-specific enhancer regions were identified. In DNase I footprinting and gel mobility shift experiments with immature adult erythrocyte nuclear extracts, we have demonstrated the binding of proteins to the GC-box, a high affinity Sp1 b...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/20.23.6385
更新日期:1992-12-11 00:00:00
abstract::Proper expression of the genes of the human beta-globin gene locus requires the associated locus control region (LCR). Structurally, the LCR is defined by the presence of four domains of erythroid-specific chromatin structure. These domains, which have been characterized as DNase I hypersensitive sites (HSs), comprise...
journal_title:Nucleic acids research
pub_type: 杂志文章
doi:10.1093/nar/26.24.5684
更新日期:1998-12-15 00:00:00