Double-chain small molecule interference nucleic acid for inhibiting and killing drug tolerant bacteria and composition thereof

A technology of small molecule interference and double-stranded molecules, applied in the field of molecular biology, can solve the problems of insufficient proof that siRNA effectively inhibits and kills MRSA

Inactive Publication Date: 2009-06-17
李宝健
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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Method used

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  • Double-chain small molecule interference nucleic acid for inhibiting and killing drug tolerant bacteria and composition thereof
  • Double-chain small molecule interference nucleic acid for inhibiting and killing drug tolerant bacteria and composition thereof
  • Double-chain small molecule interference nucleic acid for inhibiting and killing drug tolerant bacteria and composition thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: Design of Target Sites

[0044] The present invention adopts all or most of the following principles to select target sequences and design siNA:

[0045] 1. Select a sequence with a length of 18-25bp;

[0046] 2. Calculate the GC content, and select a sequence with a GC content of about 40-55%;

[0047] 3. In more than 90% of Staphylococcus aureus strains, most of the bases of the target sequence of the same gene are conserved; ), DDBJ (Japanese DNA Database) downloaded all the target gene sequences of Staphylococcus aureus, and after homology comparison, the sequence region conserved in more than 90% of the strains was selected as the target sequence.

[0048] 4. The region where the target sequence is located in the target gene of the Staphylococcus aureus strain will not make the siNA molecule inaccessible due to the formation of secondary structures; Cut the mRNA in the middle; if there is a secondary structure in this sequence region and it is difficu...

Embodiment 2

[0051] Example 2: Determination of the antibacterial or bactericidal effectiveness of siNA designed for the dnaA gene

[0052] 1. Synthesis of siNA: According to the target sequence SEQ ID NO.1 in Attached Table 1, one-to-one corresponding sense strand and antisense strand sequences were obtained, and siNA 1 with the following structure was synthesized:

[0053] 5’ C U U G G U A G A G A G C A A U U C A dT dT 3’

[0054] | | | | | | | | | | | | | | | | | | |

[0055] 3’dT dT G A A C C A T C T C T C G T T A A G T 5’

[0056] The irrelevant siNA is:

[0057] 5’ G A C C C G C A U U G A G C A U C A A dT dT 3’

[0058] | | | | | | | | | | | | | | | | | | |

[0059] 3’dT dT C T G G G C G T A A C T C G T A G T T 5’

[0060] 2. Inoculate the MRSA Tanyan'e strain (isolated and identified by the Department of Microbiology, Sun Yat-sen Medical College, Sun Yat-sen University, and stored in the Guangzhou Center for Disease Control and Prevention; The minimum inhibit...

Embodiment 3

[0065] Example 3: Determination of the antibacterial or bactericidal effectiveness of siNA designed for the ftsZ gene

[0066] 1. Synthesis of siNA: According to the target sequence SEQ ID NO.60 in Attached Table 1, one-to-one corresponding sense strand and antisense strand sequences are obtained, and siNA with the following structure is synthesized:

[0067] siNA60:

[0068] 5’ G U U A C G C C A A G G U G U A C A A dT dT 3’

[0069] | | | | | | | | | | | | | | | | | | |

[0070] 3’dT dT C A A T G C G G T T C C A C A T G T T 5’

[0071] The irrelevant siNA is:

[0072] 5’ G A C C C G C A U U G A G C A U C A A dT dT 3’

[0073] | | | | | | | | | | | | | | | | | | |

[0074] 3’dT dT C T G G G C G T A A C T C G T A G T T 5’

[0075] 2. Inoculate the MRSA standard strain ATCC25923 (purchased from ATCC in the United States; the minimum inhibitory concentration of oxacillin is 1.2 μg / ml) in the nutrient broth medium;

[0076] 3. In 2 glass tubes, take 0.5ml ...

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Abstract

The invention relates to a double-chain small molecule interference nucleic acid for inhibiting and killing various drug tolerance bacteria using methicillin-resistant staphylococcus aurous as represents. The siNA of the invention is a double-chain molecule with 19 base pairs. A sense strand and a antisense strand respectively have two overhanging bases dT at 5' terminals, the GC content is 40-55; the aimed target sequence is selected from the genes relative with the vital movements of copy, transcription and translation in the staphylococcus aurous genome and an mecA gene correlative with the drug tolerance; said target sequence is preserved in 900f staphylococcus aurous; said target sequence is in a conservative sequence area in more than 900f staphylococcus aurous with distinct source with all the gene sequences in the human genome; the target sequence of the said siNA double-chain molecule is selected from SEQ ID NO. 1-325, the sense strand is a corresponding DNA or RNA sequence with the target sequence and the antisense strand is a corresponding RNA or DNA with the sense strand according to the complementary base law.

Description

technical field [0001] The invention belongs to the field of molecular biology, and relates to a double-stranded small-molecule interference nucleic acid used for inhibiting and killing superbugs such as various drug-resistant bacteria represented by methicillin-resistant Staphylococcus aureus (MRSA). Background technique [0002] Since the first discovery of methicillin-resistant Staphylococcus aureus (MRSA) in the early 1960s, the infection of this type of bacteria has been widespread in the environment, almost all over the world. By the late 1980s, it had become the most important pathogen causing nosocomial infections worldwide. Data show that 20% of pneumonia, 40% of bacteremia and 49% of wound infection caused by MRSA. The mortality rate of the above diseases is generally 10-30%, sometimes as high as 50%. It is estimated that as many as one-third of the world's population has deadly superbugs. With the widespread use of antibiotics, new multidrug-resistant MRSAs are...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/31C12Q1/68C07H21/02C07H21/04A61K48/00A61P31/04C12N15/10C12N15/113
Inventor 李宝健
Owner 李宝健
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