This invention relates to the identification and use of cis acting nucleic acid elements that bind to nucleic acid binding factors to regulate genetic activities of nucleic acids.
All living creatures store information in nucleic acid molecules called DNA or RNA that encode structural and regulatory proteins. The collective behavior of nucleic acids and proteins constitutes and controls normal cell and organismal life cycles. Nucleic acids and proteins also act as causative agents in, or response factors to, pathological conditions.
Transcription of DNA into RNA, translation of RNA into proteins and other genetic events such as nucleic acid synthesis, sorting, processing, repair and degradation, are regulated by a variety of specialized nucleic acid binding factors. Nucleic acid binding factors bind to specific sequences present on the nucleic acid molecules they regulate, called cis acting nucleic acid elements. These nucleic acid binding factors, bound to their specific cis acting nucleic acid elements, are able to interact with other cellular factors to modulate specific genetic events. The binding of a nucleic acid binding factor to a cis acting nucleic acid element, or its ability to interact with other factors that mediate genetic events, or both, can be regulated in response to signals transmitted into the cell from the cell exterior.
As an example, regulatory proteins called xe2x80x9ctranscription factorsxe2x80x9d bind to cis acting nucleic acid elements on genomic DNA at sites known as xe2x80x9cpromotersxe2x80x9d and xe2x80x9cenhancersxe2x80x9d present at variable distances from the site of initiation of transcription of the genes they regulate. The enhancer sequences and adjacent nucleic acid sequences, together with their bound transcription factors, are able to bend to contact the transcriptional complex bound to the promoter. Such contact can either enhance or reduce expression of the regulated gene.
The human genome, which stores the genetic information of a human cell as DNA, is estimated to contain about 100,000 genes. Each of these genes and the RNAs they encode is likely to have multiple cis acting nucleic acid elements that bind to corresponding nucleic acid binding factors to regulate gene expression. These cis acting nucleic acid elements, and the factors that bind them, are potential targets for therapeutic drugs that could be used to modulate gene expression. Determining which cis acting nucleic acid elements are bound under different conditions can also be used to characterize and monitor the genetic responses of a cell under normal, pathological or experimental conditions.
Current methods of identifying cis acting nucleic acid elements have several disadvantages. Most of these methods require prior identification of either the nucleic acid that is regulated, or the corresponding regulatory nucleic acid binding factor, or both. For example, once a nucleic acid has been identified, adjacent sequences, which are predicted to contain cis acting nucleic acid elements, can be isolated and subsequences therefrom are tested for cis activities. Alternatively, once a nucleic acid binding factor has been isolated, the sequences to which it binds can be identified. Other methods, which are limited to identifying transcriptional enhancer elements, involve cloning random nucleic acid sequences upstream of a reporter gene and observing expression of the reporter gene product.
At present, however, there is no broadly applicable method to identify cis acting nucleic acid elements without prior identification of the regulated nucleic acid or of the regulatory nucleic acid binding factor. There is also no rapid and efficient method to simultaneously identify a plurality of cis acting nucleic acid elements.
Thus, there exists a need for a method of rapidly and efficiently identifying cis acting nucleic acid elements. The present invention satisfies this need and provides related advantages as well.
The invention provides a method of identifying nucleic acids containing cis acting nucleic acid elements. The method consists of contacting a diverse population of nucleic acid binding factors with a diverse population of isolated nucleic acid molecules under conditions that allow the nucleic acid binding factors to selectively bind the nucleic acids. The nucleic acids that bind the nucleic acid binding factors are identified and are characterized as nucleic acids containing cis acting nucleic acid elements. The method simultaneously provides for the isolation of nucleic acid binding factors that selectively bind the isolated nucleic acid molecules.
The invention also provides methods of identifying compounds that are cis acting nucleic acid element analogs, compounds that are nucleic acid binding factor analogs, and compounds that selectively bind cis acting nucleic acid elements. The invention further provides methods to identify compounds that selectively displace binding between a nucleic acid binding factor and a cis acting nucleic acid element or between nucleic acid binding factors.
The invention further provides a plurality of isolated nucleic acid molecules that each contain one or more cis acting nucleic acid elements. Also provided is a plurality of isolated cis acting nucleic acid element analogs. The isolated nucleic acid molecules containing cis acting nucleic acid elements and the isolated cis acting nucleic acid element analogs in the pluralities can be bound to nucleic acid binding factors. A plurality of isolated nucleic acid binding factors is also provided.
The invention also provides a method of determining a binding state of a nucleic acid. The method consists of contacting a nucleic acid with a plurality of isolated cis acting nucleic acid elements under conditions that allow nucleic acid binding factors bound to the nucleic acid to bind to the isolated cis acting nucleic acid elements. The isolated cis acting nucleic acid elements that bind the nucleic acid binding factors are identified and characterize the binding state of the nucleic acid.
The invention further provides a method of treating a pathological condition in an individual. The method consists of administering to the individual an effective amount of a therapeutic agent that selectively alters the ability of a cis acting nucleic acid element to regulate a genetic activity of a nucleic acid involved in the pathological condition. Also provided is a method of treating a pathological condition in an individual by contacting a cell of the individual with an effective amount of a targeting construct that includes a cis acting nucleic acid element and targeting sequences. The targeting construct is taken up by the cell and inserted by homologous recombination into a nucleic acid involved in the pathological condition so as to alter a genetic activity of the nucleic acid.