WO1996012015A1

WO1996012015A1 - Self stabilizing rna/dna loop

Info

Publication number: WO1996012015A1
Application number: PCT/NL1995/000344
Authority: WO
Inventors: Evert Hovius
Original assignee: Evert Hovius
Priority date: 1994-10-12
Filing date: 1995-10-10
Publication date: 1996-04-25
Also published as: AU3755095A; NL9401678A

Abstract

This concerns a group of new medicines on a genetic basis which can replace the current antibiotics, which consists of DNA/RNA without coat gene, manipulated in such a manner that it nestles at a specific position and inter alia contains the following sequences in the order specified below: 1) the operon or gene to be optionally substituted; 2) a positive regulator of the binding protein; 3) the gene for the binding protein; 4) a leader sequence; 5) attenuator site; 6) a recognition sequence for the binding protein; 7) a negative regulator for the binding protein; 8) attenuator site; 9) leader sequence; 10) an initiation rich sequence. Whereby a loop is formed in the DNA of the host, consisting of a part of the virus-DNA viz the points: 6, 7, 8, 9, 10, and the operon or gene of the host. The manipulated DNA/RNA is situated in a protein coat, from nature or synthetic, which, depending on the objective contemplated, can recognize a host.

Description

Self stabilising RNA/DNA loop

This concerns a basic concept for a new generation of medicines, consisting of protein coats in which DNA/RNA strands are situated, without coat gene, which offer the possibility of excluding pieces of DNA in the form of a loop. The excluded DNA is not transcribed.

The SSL is formed by incorporating a DNA/RNA strand

(derived from viruses or synthetic) into the DNA of

prokaryotes or eukaryotes, whereby a loop is formed consisting of a part of the DNA introduced and the inefficient operon or gene. The loop is stabilized by a binding protein (to be examined further), the gene of which is located on the DNA strand introduced. The positive regulator of the binding protein is located before the gene thereof. The negative regulator, inter alia together with the inefficient operon or gene, is excluded in the loop. The proper form of the

inefficient operon or gene is provided on the relevant DNA strand before the positive regulator. Located behind the gene of the binding protein are, respectively, a leader sequence, attenuator site, recognition sequence for the binding protein, negative regulator, attenuator site, leader sequence followed by a series of initiation signals.

The DNA/RNA strand is so composed as to specifically nestle before the inefficient operon or gene (or whatever it is desired to exclude in the loop), in that the stability of the total DNA is increased.

The manipulated DNA/RNA is situated in a protein coat the code of which does not occur on the DNA/RNA, as a result it cannot reproduce. For the production, use is made of

prokaryotes or eukaryotes into which the coat gene is

incorporated, the concentration thereof must be as high as possible. We can now introduce the manipulated DNA/RNA into the cell manipulated by us. The result is a synthesized virus without coat gene suitable for the application of the SSL.

Example of the application of the SSL with E.coli bacteria and a diagrammatic representation (see p. 6/3).

Suppose that the lactose operon is mutated in one of the structural genes, then the proper form of the operon is incorporated into a manipulated virus (DNA/RNA strand) followed by a positive regulator gene A1. the binding protein B, a leader sequence (=L), an attenuator site (=A), a

recognition sequence for the binding protein B (=C), a

negative regulator gene A2, an attenuator site (=A), a leader sequence (=L) and an initiation rich sequence (=E). (see Fig. 1, P. 6/3).

This virus nestles specifically before the inefficient lactose operon (=F) and excludes it. (see Fig. 2, p. 6/3). The RNA polymerase starts the transcription on the lactose operon on the virus DNA. Thereafter the positive regulator gene A1 is transcribed, followed by the transcription of the binding gene B. The binding gene B contains no normal termination site, but is followed by a leader sequence which contains two fantasy codons. As a result, the ribosomes stall, so that the leader messenger RNA passes a signal to the RNA polymerase to stop the transcription. The RNA polymerase reaches the attenuator site and leaves the DNA here. Because the RNA polymerase leaves the DNA here, it does not reach the loop linked by the binding protein B.

When the loop has been formed, the transcription thereof has to be prevented. The RNA polymerase scans the DNA for

initiation signals at a rate of 1000 base pairs per second, which makes it necessary that the last sequence of the virus DNA (=E) consists of several initiation signals. As a result, the RNA polymerase is, as it were, driven to one side, viz. up the virus DNA. It is now necessary that the RNA polymerase starts transcription, since otherwise it starts with the transcription of the inefficient operon (=F).

Hence the initiation signals are followed by a leader sequence (=L), which contains two fantasy codons, and an attenuator site. As a result, the RNA polymerase falls off the virus DNA. (slide effect). The attenuator site is followed by the

negative regulator A2 of the binding protein B.

we see now that A2 is situated together with the

recognition sequence (=C) of binding protein B between two attenuator sites. As a result, no transcription of A2 takes place, so that binding protein B is not negatively regulated, then the loop is anchored.

with the aid of the SSL we can eliminate and incorporate pieces of DNA without damaging the organism in question. Owing to this new medicine consisting of DNA/RNA strands in a protein coat (from nature or synthetic) we can recognize and manipulate any cell (read host).

Claims

CLAIM

This concerns a group of new medicines on a genetic basis which can replace the current antibiotics, CHARACTERIZED IN THAT it consists of DNA/RNA without coat gene, manipulated in such a manner that it nestles at a specific position and inter alia contains the following sequences in the order indicated below.

1: The operon or gene to be optionally substituted.

2: A positive regulator of the binding protein.

3: The gene for the binding protein.

4: A leader sequence.

5: Attenuator site.

6: A recognition sequence for the binding protein

7: A negative regulator for the binding protein.

6: Attenuator site.

9: Leader sequence.

10: An initiation rich sequence.

whereby a loop is formed in the DNA of the host,

consisting of a part of the virus DNA viz. the points:

6,7,8,9,10, and the operon or gene of the host.

The manipulated DNA/RNA is situated in a protein coat, from nature or synthetic, which, depending on the objective contemplated, can recognize a host.