Compositions and methods for visual ribonuclease detection...

Details Compositions and methods for visual ribonuclease detection... Compositions and methods for visual ribonuclease detection...

2001-10-01

2004-08-10

Whisenant, Ethan (Department: 1634)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023100, C536S024300

Reexamination Certificate

active

06773885

ABSTRACT:

1. INTRODUCTION
The present invention relates to methods for detecting the presence of ribonuclease enzymes, more specifically to methods that provide for a visual detection assay. The present invention further provides novel nucleic acid compositions used as substrates for such assays and encompasses kits for performing the methods of the invention.
2. BACKGROUND OF THE INVENTION
Ribonuclease (RNase) enzymes degrade polymeric ribonucleic acids (RNA) into shorter fragments or component nucleotides. All organisms produce ribonucleases and these enzymes are found in most environments. The properties of a number of ribonucleases are described by D'Alessio and Riordan (1997. As a group, most ribonucleases are specific for single-stranded RNA and will not cleave RNA in duplex form. Further, ribonucleases generally cleave at the 3′-end of a ribonucleic acid phosphodiester linkage. Many different RNase enzymes exist, some of which have little or no substrate preference while others are sequence specific. For example, ribonuclease I, from
E. coli
, is a non-specific endoribonuclease that degrades RNA by cleavage at any base. Ribonuclease A, from mammalian pancreas, is a base-specific endoribonuclease that degrades RNA by cleavage following a pyrimidine (uridine or cytosine) base. Ribonuclease T1, from
Aspergillus oryzae
, is a base-specific endoribonuclease that degrades RNA by cleavage following a guanosine residue. These three RNase enzymes are noteworthy in that they are routinely employed in standard molecular biology protocols to remove unwanted RNA from samples or as a component in certain assay procedures.
Single-strand specific RNases are the primary nuclease activity encountered in research laboratories as an unwanted contaminant. Double-strand specific RNases have been described, however these are rare and not routinely found in most laboratory settings. RNase H cleaves RNA only when complexed as a heteroduplex with DNA and is not of concern as a laboratory contaminant.
Ribonucleases are present in all laboratories as ubiquitous environmental contaminants. RNases are also found in most molecular biology laboratories as purified enzyme stocks. In laboratories that study RNA, careful attention to experimental protocol is needed to avoid contamination of reagents with RNases; for example, gloves must be worn at all times to prevent contact with the RNases that are universally present on human skin. Regardless of source, the presence of a contaminant RNase will degrade any RNA that comes in contact with that reagent, resulting in the loss of valuable samples or interfering with time-consuming experiments. Once present, removing RNase activity from a laboratory reagent is difficult. Most RNase enzymes are remarkably stable and survive harsh treatments that are routinely used to eliminate other unwanted biologic activities, such as autoclaving. Methods that remove RNase activity range from baking glassware at very high temperature to treating reagent stocks with the highly toxic chemical diethylpyrocarbonate (Sambrook et al., 1989). In spite of such attention, RNase contamination remains a chronic problem and monitoring for the presence of RNase activity is a routine quality control (QC) step in most research and industrial laboratories. As such, methods are needed that would detect the presence of RNase activities commonly encountered in the laboratory setting and that are suitable for routine, frequent use.
Many methods have been devised attempting to measure RNase activity. RNase assays can be grossly divided into methods that detect degradation of heterogeneous RNA obtained from biological sources and methods that detect specific cleavage of a well-defined synthetic substrate, such as an oligonucleotide. In general, use of a synthetic substrate affords both increased sensitivity and improved specificity. Many different detection modalities have been incorporated into these assays, including direct staining, spectrophotometric and colorimetric readouts, chromogenic cascade, radioactive tracer, fluorescence polarization, and fluorescence quenching methods.
Choice of detection method will affect assay sensitivity and ease of use. For use in determining the presence or absence of RNase contamination in laboratory reagents, the method should be sufficiently sensitive to detect the presence of RNase enzymes at the lowest level that will degrade experimental samples in actual use. An insensitive assay would “pass” reagents that are contaminated, which is undesirable. Conversely, an assay could be too sensitive and might “fail” reagents that, from a practical standpoint, are not contaminated and would therefore also be undesirable.
A detection limit within the range of 1-100 picogram/ml (pg/ml) of RNase A is ideal for a reagent QC assay. Commercial assays currently available are sensitive in the 10-100 pg/ml range (Ambion Catalog, 1999). Since such an assay would be used repeatedly, it is also desirable that the method be rapid and easy to perform. Preferably, such an assay could be done at the site of suspected contamination and offer a rapid visual readout.
The original unit definition of ribonuclease activity is based upon the method of Kunitz (1946) which employs a spectrophotometric assay to measure the decrease in absorbance at 300 nm that occurs with degradation of heterogeneous RNA. While the method has been improved (Oshima, 1976), it is insensitive and therefore of little use as a quality control (QC) assay.
Another method to detect RNase activity involves separation and assay of component enzyme activities within a sample using polyacrylamide gel electrophoresis (Wilson, 1969). RNase enzymes can be detected in the acrylamide matrix by direct staining or by incubation with a heterogeneous substrate RNA and an RNA staining dye, such as toluidine blue. While conceptually simple, this approach is time-consuming and relatively insensitive, having a lower limit of detection of about 1 unit of RNase I. In an improvement of this technique, Karpetsky (1980) describes a polynucleotide/polyacrylamide-gel electrophoresis method that improves sensitivity to below 100 pg of RNase A. However, even the improved method remains slow and cumbersome and is better suited to the analysis of ribonuclease activities in biologic specimens than to the QC of laboratory reagents.
Another approach to detect RNase activity is described by Egly and Kempf (1976). This procedure detects release of soluble
125
Iodine-labeled RNA from an insoluble RNA-agarose matrix in the presence of ribonuclease. The method is capable of detecting the presence of RNase A at levels as low as 0.01 pg/ml and is actually too sensitive for use as a routine QC assay. Furthermore, this method employs a hazardous radioactive isotope as reporter that is not desirable for use in most laboratory or industrial settings.
Another approach to detect RNase activity is described by Wagner (1983). RNA forms a complex with Pyronine-Y that has an optical absorbance maximum at 572 nm. Degradation of high molecular weight RNA by ribonuclease activity results in loss of absorbance at 572 nm in a linear and quantitative fashion. The method, however, is only capable of detecting about 2 ng/ml RNase A in a test sample and has insufficient sensitivity for use as a QC assay.
Another approach to detect RNase activity was described by Greiner-Stoeffele (1996). The dye methylene blue intercalates into high molecular weight ribonucleic acid forming a dye-RNA complex. Upon degradation by ribonuclease action, methylene blue is released and absorbance at 688 nm decreases. This method, however, is also relatively insensitive and can detect ribonuclease activity only down to about 25 ng/ml, which is inadequate for use as a QC assay.
Another approach to detect RNase activity is described by Karn (1979). Ribonuclease A-mediated cleavage of a synthetic ribonucleotide dimer substrate was detected by a cascade of enzymatic reactions involving adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase that ultimately forms a detectable blue

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