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Analysis by siRNA_profile program displays novel thermodynamic characteristics of highly functional siRNA molecules.

ABSTRACT: OBJECTIVE:Here we report the improved results of a new siRNA design program and analysis tool called siRNA_profile that reveals an additional criterion for bioinformatic search of highly functional siRNA sequences. METHODS:We retrospectively analysed over 2400 siRNA sequences from 34 genes and with known efficacies to categorize factors that differentiate highly, moderately and non-functional siRNA sequences in more detail. We tested the biological relevance of siRNA_profile in CHO cells stably expressing human TRACP. RESULTS:The highly functional siRNA molecules exhibited lower overall stabilities than non-functional siRNAs after taking into consideration all the nucleotides from 5'-terminus to the 3'-terminus along the siRNA molecule, in addition to the 5'-section of the antisense strand and the region between 9-14 nucleotides as previously has been acknowledged. Comparison of the siRNA_profile program to five other programs resulted in a wide range of selected siRNA sequences with diverse gene silencing capacities, even when the target was only 197 nucleotides long. Six siRNA design programs selected 24 different siRNA sequences, and only 6 of them were selected by two or more programs. The other 18 sequences were individually selected by these six programs. CONCLUSION:Low general stability of dsRNA plays a significant role in the RNAi pathway and is a recommended criterion to consider, in addition to 5'-instability, internal instability, nucleotide preferences and target mRNA position, when designing highly efficient siRNAs.

SUBMITTER: Muhonen P

PROVIDER: S-EPMC2424050 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Analysis by siRNA_profile program displays novel thermodynamic characteristics of highly functional siRNA molecules.

Muhonen Pirkko P Parthasarathy Ranga N RN Janckila Anthony J AJ Büki Kalman G KG Väänänen H Kalervo HK

Source code for biology and medicine 20080521

<h4>Objective</h4>Here we report the improved results of a new siRNA design program and analysis tool called siRNA_profile that reveals an additional criterion for bioinformatic search of highly functional siRNA sequences.<h4>Methods</h4>We retrospectively analysed over 2400 siRNA sequences from 34 genes and with known efficacies to categorize factors that differentiate highly, moderately and non-functional siRNA sequences in more detail. We tested the biological relevance of siRNA_profile in CH ...[more]

PMID: 18495017

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Project description:BACKGROUND: Small interfering RNAs (siRNAs) have become an important tool in cell and molecular biology. Reliable design of siRNA molecules is essential for the needs of large functional genomics projects. RESULTS: To improve the design of efficient siRNA molecules, we performed a comparative, thermodynamic and correlation analysis on a heterogeneous set of 653 siRNAs collected from the literature. We used this training set to select siRNA features and optimize computational models. We identified 18 parameters that correlate significantly with silencing efficiency. Some of these parameters characterize only the siRNA sequence, while others involve the whole mRNA. Most importantly, we derived an siRNA position-dependent consensus, and optimized the free-energy difference of the 5' and 3' terminal dinucleotides of the siRNA antisense strand. The position-dependent consensus is based on correlation and t-test analyses of the training set, and accounts for both significantly preferred and avoided nucleotides in all sequence positions. On the training set, the two parameters' correlation with silencing efficiency was 0.5 and 0.36, respectively. Among other features, a dinucleotide content index and the frequency of potential targets for siRNA in the mRNA added predictive power to our model (R = 0.55). We showed that our model is effective for predicting the efficiency of siRNAs at different concentrations. We optimized a neural network model on our training set using three parameters characterizing the siRNA sequence, and predicted efficiencies for the test siRNA dataset recently published by Novartis. On this validation set, the correlation coefficient between predicted and observed efficiency was 0.75. Using the same model, we performed a transcriptome-wide analysis of optimal siRNA targets for 22,600 human mRNAs. CONCLUSION: We demonstrated that the properties of the siRNAs themselves are essential for efficient RNA interference. The 5' ends of antisense strands of efficient siRNAs are U-rich and possess a content similarity to the pyrimidine-rich oligonucleotides interacting with the polypurine RNA tracks that are recognized by RNase H. The advantage of our method over similar methods is the small number of parameters. As a result, our method requires a much smaller training set to produce consistent results. Other mRNA features, though expensive to compute, can slightly improve our model.

Project description:The photoluminescence (PL) of conducting polymer polythiophene (PT) films incorporated with metallophthalocyanines (PcMs) such as CuPc, MgPc, FePc, Li2Pc, and CoPc was studied by PL and time-correlated single photon counting (TCSPC) measurements. Polymer films were prepared by electrochemical polymerization and PcMs migrated into the polymer films by a diffusion method using acetonitrile or toluene as a solvent to dissolve the PcMs. The wavelength of PL emission peaks changed significantly depending on the solvent used in the doping process. Using acetonitrile, the observed PL emission peaks originated from the Q band, whereas they were assigned to the Soret band in the case of toluene. TCSPC measurements showed that PL emission took place through a ligand-ligand transition process when using acetonitrile because the average lifetimes were comparable and independent of the central metal ions for CoPc-, Li2Pc-, and MgPc-doped polymer films. Conversely, using toluene, it was found that ligand-ligand emission occurred for Li2Pc-, MgPc-, and FePc-doped films. To identify the cause of the drastic change in PL emission pattern, x-ray photoelectron spectroscopy measurements were obtained. A lower binding energy component appeared in the C 1s core-level spectra of acetonitrile-processed PcM-doped PT films, whereas this component shifted to higher energy and overlapped with the main peak for toluene-processed PcM-doped PT films. The lower binding energy component corresponded to photoelectrons due to the C atoms in the benzene rings of the ligand. Lower binding energy components also appeared in the N 1s core-level spectra of acetonitrile-processed PcM-doped PT films, and this component shifted to higher energy for toluene-processed PcM-doped PT films. These lower energy components were assigned to the core-level peaks due to the N atoms at the meso position bridging between pyrrole rings. This suggests that the electron charge at the N sites of the meso positions in toluene-processed films was smaller than in acetonitrile-processed ones. The changes in energy at benzene C sites and meso N sites suggest that the electronic states of the phthalocyanine in the toluene-processed films were porphyrin-like, so the Soret band became dominant in the PL emission spectrum.

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Analysis by siRNA_profile program displays novel thermodynamic characteristics of highly functional siRNA molecules.

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Analysis by siRNA_profile program displays novel thermodynamic characteristics of highly functional siRNA molecules.

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