The abbreviated name,‘mfold web server’,describes a number of closely related software applications available on the World Wide Web (WWW) for the prediction of the secondary structure of single stranded nucleic acids. The objective of this web server is to provide easy access to RNA and DNA folding and hybridization software to the scientific community at large. By making use of universally available web GUIs (Graphical User Interfaces),the server circumvents the problem of portability of this software. Detailed output,in the form of structure plots with or without reliability information,single strand frequency plots and ‘energy dot plots’, are available for the folding of single sequences. A variety of ‘bulk’ servers give less information,but in a shorter time and for up to hundreds of sequences at once. The portal for the mfold web server is http://www.bioinfo.rpi.edu/applications/ mfold. This URL will be referred to as ‘MFOLDROOT’.

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Mfold web server for nucleic acid folding and hybridization prediction

High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.

https://science.sciencemag.org/content/sci/249/4968/505.full.pdf

Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase

Double-stranded RNA (dsRNA) induces sequence-specific posttranscriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 21- and 22-nt RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III–like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3 ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the siRNA–protein complex.

/pdf/rna-interference-is-mediated-by-21-and-22-nucleotide-rnas-1tlpza9thy.pdf

RNA interference is mediated by 21- and 22-nucleotide RNAs

http://bio.gsnu.ac.kr/research/miRNA/RNAss/Mfold/miRNAss_JMB_1999_Mathews.pdf

Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure.

Background
Secondary structure forms an important intermediate level of description of nucleic acids that encapsulates the dominating part of the folding energy, is often well conserved in evolution, and is routinely used as a basis to explain experimental findings. Based on carefully measured thermodynamic parameters, exact dynamic programming algorithms can be used to compute ground states, base pairing probabilities, as well as thermodynamic properties.

/pdf/viennarna-package-2-0-4q9zpqix5v.pdf

ViennaRNA Package 2.0

A method is described to synthesize small RNAs of defined length and sequence using T7 RNA polymerase and templates of synthetic DNA which contain the T7 promoter. Partially single stranded templates which are base paired only in the -17 to +1 promoter region are just as active in transcription as linear plasmid DNA. Runoff transcripts initiate at a unique, predictable position, but may have one nucleotide more or less on the 3' terminus. In addition to the full length products, the reactions also yield a large amount of smaller oligoribonucleotides in the range from 2 to 6 nucleotides which appear to be the result of abortive initiation events. Variants in the +1 to +6 region of the promoter are transcribed with reduced efficiency but increase the variety of RNAs which can be made. Transcription reaction conditions have been optimized to allow the synthesis of milligram amounts of virtually any RNA from 12 to 35 nucleotides in length.

/pdf/oligoribonucleotide-synthesis-using-t7-rna-polymerase-and-q3o6uiumun.pdf

Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates

A SIMPLE method for estimating the most stable secondary structure of an RNA molecule from its sequence was proposed earlier1. This method can be used for predicting and assessing possible secondary structures for recently determined RNA sequences2–4. New experimental5–8 and theoretical9, 10 results allow us to improve the method, without making it more complicated.

Improved Estimation of Secondary Structure in Ribonucleic Acids

Publisher Summary This chapter discusses how transcription templates are prepared by cleaving the plasmid DNA with a restriction enzyme of choice. The individual nucleoside triphosphates (NTPs) are diluted to desired concentrations and are mixed in equimolar ratios to make a stock solution. If they are supplied in ethanol and will be used in large amounts, the ethanol should be removed prior to use. The addition of serum albumin to the reaction is optional and is generally omitted in large-scale reactions. Differences in template sequence and length can result in substantial differences in the optimal enzyme and template concentrations required for the production of RNA. In addition, one or more of the components may be more valuable than the others. It is, therefore, worthwhile to carry out trial reactions to find optimal reaction conditions for a given synthesis. Although performing trial reactions to optimize reaction conditions can be tedious, reaction yields can be substantially higher than those obtained using the general conditions and the reactions scale up readily.

Synthesis of small RNAs using T7 RNA polymerase.

SELEX is a technology for the identification of high affinity oligonucleotide ligands. Large libraries of random sequence single-stranded oligonucleotides, whether RNA or DNA, can be thought of conformationally not as short strings but rather as sequence dependent folded structures with high degrees of molecular rigidity in solution. This conformational complexity means that such a library is a source of high affinity ligands for a surprising variety of molecular targets, including nucleic acid binding proteins such as polymerases and transcription factors, non-nucleic acid binding proteins such as cytokins and growth factors, as well as small organic molecules such as ATP and theophylline. The range of applications of this technology for new discovery extends from basic research reagents to the identification of novel diagnostic and therapeutic reagents. Examples of these applications are described along with a discussion of underlying principles and future developments expected to further the utility of SELEX.

Diversity of Oligonucleotide Functions

A 19-nucleotide RNA fragment can cause rapid, highly specific cleavage of a 24-nucleotide RNA fragment under physiological conditions. Because each 19-mer can participate in many cleavage reactions, this molecule has all the properties associated with an RNA enzyme.

Olke C. Uhlenbeck

Papers

Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates

Improved Estimation of Secondary Structure in Ribonucleic Acids

Synthesis of small RNAs using T7 RNA polymerase.

Diversity of Oligonucleotide Functions

A small catalytic oligoribonucleotide