Polynucleotide probe design

A method for synthesizing and using a selected nucleotide probe (P) that selectively binds a target fragment (P′) in a target nucleotide sequence (T) in a sample wherein the sample comprises the target nucleotide sequence (T) and a plurality of off-target nucleotide sequences (D) that do not contain the target fragment,

• wherein the target nucleotide sequence (T) further comprises:
• (1) an off-target fragment on the target nucleotide sequence (O);
• (2) a first fragment (A) that can bind to the target fragment (P′);
• and
• (3) a second fragment (A′) that can bind to the first fragment (A),
• the method comprising performing, on a suitably programmed computer:
• determining a plurality of binding reactions potentially affecting the binding between the probe (P) and the target fragment (P′), which binding reactions comprise those that are
• (i) between the probe (P) and the off-target fragment (O),
• (ii) between the probe (P) and one or more of the off-target nucleotide sequences (D),
• (iii) between the target fragment (P′) and the first fragment (A), and
• (iv) between the target fragment (P′) and one or more of the off-target nucleotide sequences (D);
• calculating one or more kinetic rate for each of the binding reactions;
• generating one or more ordinary differential equation (ODE), using at least one of the one or more kinetic rate as a parameter, to represent each of the binding reactions;
• and
• solving the ODE's, for an elapsed time, to determine a concentration of a product of the binding reaction between the probe (P) and the target fragment (P′), wherein the concentration indicates how effectively the probe (P) selectively binds the target fragment (P′),
• wherein the solved ODE's do not represent (1) binding reactions between the first fragment (A) and the fragment (A′) on the target nucleotide sequence (T), (2) binding reactions that are thermodynamically prohibitive, or (3) binding reactions involving a nucleotide sequence or fragment in more than one binding reaction at a time;
• performing all of the steps for each of a plurality of nucleotide probes;
• selecting one or more probes from the plurality of nucleotide probes, the selected one or more probes having among highest concentrations, out of the two or more nucleotide probes, of target fragments that selectively bind the probe over an elapsed time, as determined by the ODE's;
• synthesizing the selected probe;
• and
• performing binding reactions in a cell or tissue using the synthesized selected probe, the binding reactions being selective to the target fragment with the synthesized selected probe.