Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-08-16
2001-11-06
Riley, Jezia (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S005000, C435S006120, C435S091200
Reexamination Certificate
active
06312927
ABSTRACT:
BACKGROUND OF THE INVENTION
In general, the invention features methods for modifying nucleic acid substrates, for example, for the production of RNA-protein fusions.
Covalently bonded RNA-protein fusions may be used in methods for generating or isolating proteins with desired properties from pools of proteins. To create such fusions, an RNA and the peptide or protein that it encodes may be joined during in vitro translation using synthetic RNA that carries a peptidyl acceptor, such as puromycin, at its 3′-end (Roberts & Szostak (1997) Proc. Natl. Acad. Sci. USA 94, 12297-12302). In this process, the synthetic RNA, which is devoid of stop codons, is typically synthesized by in vitro transcription from a DNA template followed by 3′-ligation to a DNA linker carrying puromycin. The DNA sequence causes the ribosome to pause at the end of the open reading frame, providing additional time for the puromycin to accept the nascent peptide chain and resulting in the production of the RNA-protein fusion molecule.
SUMMARY OF THE INVENTION
The present invention involves methods for optimizing the production of RNA-protein fusions beginning with cellular RNA or other nucleic acids having 3′-untranslated regions. As described in more detail below, such fusions may be generated by at least two general techniques. According to one general approach, nucleic acids are produced which lack both 3′-untranslated regions and poly A tails. These nucleic acids, which may also lack a terminal stop codon, are then used for the production of RNA-protein fusions. According to the second technique, rather than modifying the nucleic acid substrate, the fusion is generated in an in vitro translation reaction mixture which lacks functional translation release factors. The absence of these factors circumvents the problem of termination at terminal stop codons (or other stop codons inadvertently introduced into a protein coding sequence) and allows for the generation of RNA-protein fusions. The invention also encompasses methods in which these two general approaches are combined for the purpose of RNA-protein fusion formation and methods in which the approaches, singly or in combination, are used for other purposes in which nucleic acids lacking 3′-terminal sequences or translation through stop codons are useful or desirable.
Accordingly, in a first aspect, the invention features a method for removing the 3′-untranslated region of a DNA molecule including an open reading frame, the method involving: (a) providing a DNA molecule having an open reading frame and a 3′-untranslated region, the DNA molecule terminating at its 5′ end in an overhang and at its 3′ end in a blunt end; and (b) treating the DNA molecule first with a 3′→5′ exonuclease and then with a single-stranded nuclease under conditions that allow removal of the 3′-untranslated region.
In preferred embodiments, the 3′→5′ exonuclease is exonuclease III; the nuclease is Mung bean nuclease; step (b) further results in removal of the stop codon of the open reading frame; the DNA molecule is a cDNA produced by reverse transcription from an mRNA sequence; and the method is carried out on a population of DNA molecules.
In a related aspect, the invention features a method for removing the 3′-untranslated region of an mRNA molecule, the method involving: (a) translating an mRNA molecule in vitro in a translation reaction mixture lacking functional translation release factor activity, resulting in pausing of the translation reaction mixture ribosomes at the stop codon of the mRNA molecule; (b) adding, to the translation reaction mixture of step (a), reverse transcriptase and an oligonucleotide primer which is complementary to the 3′-untranslated region of the mRNA molecule at a site proximal to the stop codon, under conditions which allow the synthesis of a strand of DNA that is complementary to the 3′-untranslated region and terminates at a site proximal to the stop codon; and (c) removing the RNA portion of the RNA-DNA duplex formed in step (b), thereby removing the 3′-untranslated region of the mRNA molecule.
In preferred embodiments, the oligonucleotide primer comprises a poly T sequence; step (c) is carried out by treatment of the product of step (b) with RNaseH; the method is carried out on a population of mRNA molecules; and the method further involves the steps of: (d) ligating to the 3′ end of the product of step (c) a linker including a Type IIS restriction site; (e) extending the product of step (d) to produce a double-stranded DNA molecule; and (f) treating the double-stranded DNA molecule with the Type IIS restriction enzyme to cleave the DNA molecule and remove the stop codon.
In another related aspect, the invention features a method for removing the 3′-untranslated regions and stop codons of a population of mRNA molecules, the method involving: (a) providing a population of mRNA molecules; (b) synthesizing strands of DNA, each of which is complementary to one of said mRNA molecules, using a random primer mixture, the random primer mixture including primers, each having (i) a 3′ region including a stop codon flanked by a random oligonucleotide located 3′, 5′, or both to the stop codon; and (ii) a 5′ region including a Type IIS restriction site; (c) ligating to the 3′ ends of the DNA products of step (b) an oligonucleotide tail; (d) amplifying the products of step (c) using (i) a first primer which is complementary to the Type IIS restriction site-containing sequence; and (ii) a second primer which is complementary to the oligonucleotide tail; and (e) treating the products of step (d) with the Type IIS restriction enzyme to cleave the products, thereby removing the 3′-untranslated regions and stop codons.
In preferred embodiments, the second primer of step (d) further includes a 5′ region including an RNA polymerase recognition site; and the method further comprises: (f) ligating a sequence which encodes an affinity tag to the cleaved ends of the products of step (e); (g) transcribing the products of step (f); (h) ligating peptidyl acceptors to the 3′ ends of the RNA products of step (g); (i) translating the products of step (h) to produce a population of RNA-protein fusions; and (j) substantially isolating RNA-protein fusions which comprise the affinity tag, thereby obtaining a population of mRNA molecules lacking 3′-untranslated regions and stop codons.
In yet another related aspect, the invention features a method for removing the 3′-untranslated regions and stop codons of a population of mRNA molecules, involving: (a) providing a population of mRNA molecules; (b) synthesizing strands of DNA, each of which is complementary to one of the mRNA molecules, using a random primer mixture, the random primer mixture including primers, each having (i) a 5′ region which lacks a stop codon in at least one reading frame and (ii) a random 3′ region; and (c) synthesizing strands of DNA complementary to the DNA strands of step (b), using a second random primer mixture.
In preferred embodiments, the second random primer mixture includes primers, each having (i) a 5′ region which includes a translation start site and (ii) a random 3′ region; and wherein said method further involves (d) amplifying the product of step (c) using a first amplification primer having (i) a 5′ sequence which includes an RNA polymerase recognition site and (ii) a 3′ region which is complementary to the translation start site.
In other preferred embodiments of each of the above two aspects, the RNA polymerase recognition site is a T7 or SP6 RNA polymerase recognition site; the affinity tag is a hexahistidine peptide, a streptavidin-binding peptide, or an epitope; the peptidyl acceptor is puromycin; and the method is carried out on a population of mRNA molecules.
In a second aspect, the invention features a method for producing an RNA-protein fusion from an mRNA havi
Clark & Elbing LLP
Phylos, Inc.
Riley Jezia
LandOfFree
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