Double-stranded small molecule interfering nucleic acid and combination thereof for inhibiting and killing drug-resistant bacteria

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

Inactive Publication Date: 2011-12-21
李宝健
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite this difference, it is not enough to prove that siRNA can effectively inhibit and kill MRSA

Method used

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  • Double-stranded small molecule interfering nucleic acid and combination thereof for inhibiting and killing drug-resistant bacteria
  • Double-stranded small molecule interfering nucleic acid and combination thereof for inhibiting and killing drug-resistant bacteria
  • Double-stranded small molecule interfering nucleic acid and combination thereof for inhibiting and killing drug-resistant bacteria

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 siNA1 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 minimu...

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, ta...

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Abstract

The invention relates to a double-stranded small molecular interference nucleic acid used for inhibiting and killing various drug-resistant bacteria represented by methicillin-resistant Staphylococcus aureus. The siNA described in the present invention is a double-stranded molecule with 19 base pairs, the sense strand and the antisense strand each have two protruding bases dT at the 5' end, and the GC content is 40-55%; the targeted sequence Selected from genes related to life activities such as replication, transcription, and translation in the genome of Staphylococcus aureus, and the mecA gene related to drug resistance; the target sequence is conserved in more than 90% of Staphylococcus aureus strains and is compatible with All gene sequences in the human genome have different sequence regions; the target sequence of the siNA double-stranded molecule is selected from SEQ ID NO.1-325, the sense strand is a DNA or RNA sequence corresponding to the target sequence one-to-one, and the antisense The strand is the RNA or DNA that corresponds one-to-one to the sequence of the sense strand according to the principle of base complementarity.

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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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/68A61K48/00C12N15/31C12N15/10A61P31/04C07H21/04C07H21/02
Inventor 李宝健江丽芳
Owner 李宝健
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