RNA channels in biological membranes

Details RNA channels in biological membranes RNA channels in biological membranes

1999-05-20

2001-05-01

Schwartzman, Robert A. (Department: 1636)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C436S501000, C536S023100

Reexamination Certificate

active

06225063

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to nucleic acid molecules capable of binding membranes, and more particularly RNA molecules capable of binding and forming channels in biological membranes.
BACKGROUND
The function of nucleic acids in living organisms is limited to a largely informational role. The “Central Dogma” of molecular biology, as postulated by Crick, proposes that deoxyribonucleic acid (DNA) serves as a template for the synthesis of other nucleic acids through replicative processes that “read” the information in template nucleic acids, and thus yield complementary nucleic acids, such as messenger ribonucleic acid (MRNA). mRNA then serves as a template for the translation of the information into proteins.
Most biological molecules do not specifically bind to nucleic acids. Some known exceptions are proteins (e.g., repressors, polymerases, activators, etc.) that function to transfer genetic information encoded in the nucleic acids into cellular structures and replicate genetic material. This binding depends upon the nucleotide sequence(s) that comprise the DNA or RNA involved. Short DNA sequences are known to bind to target proteins that repress or activate transcription in both prokaryotes and eukaryotes. Other short DNA sequences are known to serve as centromeres and telomeres of chromosomes, presumably by creating ligands for the binding of specific proteins that participate in chromosome mechanics.
RNA also binds some synthetic and regulatory proteins. For example, double-stranded RNA occasionally serves as a ligand for certain proteins, for example, the endonuclease RNase III from
E. Coli
. Proteins also bind to single-stranded RNA, although in these cases the single-stranded RNA often forms a complex three-dimensional shape that includes local regions of intramolecular double-strandedness. For example, the amino-acyl tRNA synthetases bind tightly to tRNA molecules with high specificity. As another example, a short sequence of RNA binds to the bacteriophage T4-encoded DNA polymerase. Thus, some RNA and DNA sequences are known to serve as binding partners for specific protein targets. Most known DNA binding proteins bind specifically to double-stranded DNA, while most RNA binding proteins recognize single-stranded RNA, although exceptions occur.
Nucleic acids are generally thought to have a limited range of biological activity as compared to proteins. However, some nucleic acids are known to bind target molecules other than those needed for transcription, translation and replication, and some have catalytic activity. For example, ribozymes are RNA molecules with enzymatic activity that is capable of repeatedly cleaving other RNA molecules in a nucleotide base sequence-specific manner. Such enzymatic RNA molecules can be targeted to virtually any RNA transcript, and efficient cleavage achieved in vitro (Kim et al., PNAS 84:8788 [1987]; Haseloff and Gerlach, Nature 334:585 [1988]; Cech, J. Amer. Med. Assoc., 260:3030 [1988]; and Jefferies et al., Nucl. Acids Res., 17:1371 [1989]). Ribozymes can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. This binding occurs through the target binding portion of a enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes, and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA destroys its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA and is available to bind and cleave a new target. This binding and cleavage process is then multiply repeated.
Some RNA and DNA molecules have also been proven to have the ability to bind various ligands, including amino acids (Famulok et al., J. Amer. Chem. Soc., 114:3990 [1992], Connell et al., Biochem., 32:5497 [1994]), nucleotides (Connell et al., Science 264:1137 [1994], Sassanfar et al., Nature 364:550 [1993]), antibiotics (Davies et al., In Gesteland and Atkins (eds.),
The RNA World
, p. 185, Cold Spring Harbor Press, NY [1993]), cyanocobalamin (Lorsch et al., Biochem., 33:973 [1994]), and proteins such as the reverse transcriptase, Rev and Tat proteins of HIV (Tuerk et al., Gene 137:33 [1993]), human nerve growth factor (Binkley et al., Nucl. Acids Res., 23:3198 [1995]), and vascular endothelial growth factor (Jellinek et al., Biochem., 83:10450 [1994]).
Drugs capable of interacting with membranes have found widespread use. Perhaps the most common class of drugs that interact with membranes are ionophores. Ionophores are lipid soluble compounds capable of binding and transporting specific ions through the cell membrane. Examples of ionophores include the calcium channel ionomycin, and the antimicrobials valinomycin and gramicidin. Valinomycin forms a lipid soluble complex with K
+
that readily passes through the mitochondrial membrane. Gramicidin induces the penetration of K
+
, Na
+
and H
+
through the mitochondrial membrane, causing inhibition of oxidative phosphorylation. Other ionophore antimicrobials include nonactin, nigericin, lasalocid, and monensin (See Westley, inGrayson (ed.),
Antibiotics, Chemotherapeutics, and Antibacterial Agents for Disease Control
, p. 301-18, John Wiley and Sons, NY [1982]). Monensin and lasalocid are commonly used as animal feed supplements to reduce the occurrence of coccidiosis and other diseases and increase feed efficiency. However, as microorganisms often rapidly become resistant to antimicrobials, new antimicrobials are constantly under development.
What is needed is the identification of nucleic acids that can be utilized to alter membrane structure and finction, as well as target effector molecules to membranes of specific cell populations.
SUMMARY OF THE INVENTION
The present invention relates to nucleic acid molecules capable of binding membranes, and more particularly to RNA molecules capable of binding and forming channels in biological membranes. In accordance with the present invention, methods are contemplated for screening and isolating RNA molecules that bind, insert into, and/or permeabilize membranes, as well as compositions containing these molecules. The present invention also contemplates the use of nucleic acid molecules for various uses, including but not limited to altering the permeability of membranes, detectably labeling membranes, and targeting effector molecules to membranes. In particularly preferred embodiments of the present invention, the membranes are biological membranes.
In one embodiment, the present invention provides methods for screening nucleic acid molecules that bind to membranes, comprising: providing a composition comprising at least one nucleic acid molecules, and a membrane preparation; combining the composition and membrane preparation under conditions such that binding can occur, so as to produce a membrane with bound nucleic acid; and isolating said nucleic acid molecules bound to said membrane preparations. In some embodiments, the membranes that bind the nucleic acid molecules are characterized. In yet other embodiments, the nucleic acid molecules bound to the membrane preparation are purified. In other embodiments, the purified nucleic acid molecules are then amplified, cloned, and sequenced.
It is not intended that the present invention be limited to any certain type of nucleic acid molecule, as various nucleic acid molecules will find use in the present invention. In some embodiments of the present invention, the nucleic acid molecules are double-stranded or single-stranded RNA, while in other embodiments, the nuclei

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