Renaturing RNA

To avoid the enrichment of RNA molecules that bind to pure matrix, preselection with matrix carrying no target should be performed. The renatured RNA is allowed to pass through a corresponding column or is simply incubated together with the matrix in batch. The preselection should differ from the main selection only in the absence of the immobilized target. 

To follow the reaction over time, a 2 /iL aliquot is removed as soon as possible (e.g., 15 s) and loaded in the first well of the gel. With the current running (do not touch the gel), reaction aliquots are carefully loaded in adjacent wells at different times, with intervals chosen to span the expected half-life of the reaction. It is recommended that one also prepare samples of unfolded and fully renatured RNA as controls. 

Renatured RNAs can be examined for gross conformational homogeneity by nondenaturing gel electrophoresis in either polyacrylamide gels or Nusieve agarose gels. Electrophoresis is carried out in TBE supplemented with 5 mM MgCl2 and 50 mM KC1 at 4 V/cm at room temperature. 

Place 10 pmol renatured RNA or complex in 20 pi modification buffer on ice. 

For the in vitro selection process, the RNA pool containing 10 different sequences and structural motifs is generated by an in vitro transcription reaction. Folding of the RNA molecules is induced by heat denaturation and renaturation at room temperature (26). 

The RNA pool unlabeled or radiolabeled - as detailed later - is diluted in incubation buffer prior to the SELEX step, denatured and renatured as detailed in Subheading 3.7. 

The ( P) RNAs are diluted in incubation buffer containing 10 mg/ml to a specific activity of 5x10 cpm/pl and denatured and renatured before the experiment. 

Hager, D. A. and Burgess, R. R. (1980) Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem 109, 76-86. 

For renaturation, the purified RNA is dissolved in selection buffer after denaturing PAGE and precipitation (Section 7.3.1.4). Then this solution is heated to 70 °C for 5 min and subsequently cooled to the selection temperature within approximately 15 min. Alternatively, the RNA can be renatured, for example, by heating to other temperatures (65-95 °C) and immediately incubating it on ice. Mg2+ can be added either before or after heating. Because RNA can be degraded if heated in the presence of Mg2+, the heating should not be too excessive if the buffer contains Mg2+ ions. 

After the ssDNA pool has been established, it must be de- and renatured as described for RNA aptamers (see Section 7.3.1.5). Then preselection and selection are done, and the selected ssDNA is used in a PCR to create the new dsDNA pool of the next selection round. 

The RNA pellet was dissolved in the selection buffer, heated at 70 °C for 10 min. Then, Mg2+ was added and the RNA was renatured by allowing the mixture to cool to room temperature within 30 min. 

As the fold of the RNA is critical, the folding step after purification of the RNA is important. The most commonly used folding protocols involve heat-cooling the RNA in a metal-free buffer such as Tris-EDTA and subsequently adding metals (Ke and Doudna, 2004) such as monovalent or divalent cations, most commonly Na/K+ and Mg2+. Slow renaturation of the folded RNA from denaturing conditions may also be effective, particularly in RNA-protein systems. Both the human 5 virus ribozyme... 

The double helix is a relatively stiff and elongated molecule. Consequently, a solution of DNA has a high viscosity. If such a solution is heated to 95°C, the viscosity drops markedly, reflecting a collapse of the double-helical structure. This is known as denaturation and is accompanied by separation of the duplex into its single strands, which are fairly flexible. Denaturation and renaturation provide valuable information on important properties of the DNA obtained from various sources. Denaturation also provides the basis for very precise and sensitive approaches to the identification of specific sequences in both DNA and RNA. This has been central to the rapid developments in molecular genetics. 

Under certain conditions, the stress-70 proteins can participate in the renaturation of denatured or inactivated proteins. The renaturation capabilities of E. coli dnaK protein have been most extensively documented. It has been shown that in vitro, dnaK can protect E. coli RNA polymerase from aggregation when the polymerase is incubated at elevated temperatures that would normally result in loss of activity, and, further, that dnaK can disaggregate and reactivate polymerase, once it has been inactivated by heat denaturation (Skowyra et al., 1990). These activities are absolutely dependent on ATP hydrolysis. The mutant dnaK756 protein is effective in protecting active RNA polymerase against heat inactivation, but is incapable of disaggregating and reactivating polymerase, once it has been heat inactivated. 

Szewczyk, B., Laver, W. G, and Summers, D. F (1988) Purification, thioredoxin renaturation, and reconsututed acuvity of the three subunits of the influenza A virus RNA polymerase Proc NalL Acad. Sa. USA 85, 7907-7911... 

Hybridization can occur between complementary strands of nucleic acids derived from different sources. The double stranded nucleic acid that forms is a heteroduplex and the extent of heteroduplexes indicates homology between the two nucleic acid sources. For example, humans and mice mix with a very small fraction of the DNA renaturing but humans and chimpanzees give greater than 98% homology. DNA and RNA can also hybridize with one another to form heteroduplexes. 

The double helical structure of DNA must be disrupted during almost all biological processes in which it participates, including DNA replication and repair, as well as transcription of the DNA sequence information to RNA. Experimentally, the double helix can be separated, or denatured, by increasing the temperature to well above 50°C (122°F). If the temperature is carefully decreased, renaturation occurs when the base pairs reform. Under these conditions, hybridization can be induced by allowing the single strands... 

This section contains descriptions of basic procedures that occur throughout the book. In this way, we hope to eliminate the repetition of basics such as RNA quantification, gel extraction, extraction with organic solvents, RNA precipitation, long-term storage of RNA, renaturation, the use of spin-columns and preparation of standard reagents. 

Here we present an RNA renaturation protocol that is useful for both small and large RNA molecules. The important feature of the protocol is that secondary structure formation is favoured before tertiary structure formation by introducing Mg2+ at a late stage in the protocol ... 

RNA renaturation It is well known that different RNA preparations give variable retention efficiencies. Careful renaturation often reduces the variability between different preparations (see Section 2.1.2.6). 

Add 1 jjlI of ligand RNA or RNA control in 1 x RNA renatur-ation buffer,a... 

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