Molecules for verifying oligonucleotide directed combinatorial synthesis and methods of making and using the same

A method of assessing synthetic yield of a reaction step that adds a free positional building block unit B to a molecule of formula (IV), the method comprising:

• providing a pool of molecules of formula (IV),
• G-L-(B)(K-1) (IV);
• amplifying G within formula (IV) from a first portion of the pool of molecules of formula (IV) by polymerase chain reaction (PCR) to form a pool of pre-reaction copies of G;
• reacting the molecule of formula (IV) in a second portion of the pool of molecules of formula (IV) with the free positional building block unit B and a molecule of formula (III),
• Q-U (III),
• to form a pool of verification molecules of formula (I),
• G-L-(B)K-Q-U (I);
• wherein
• G is an oligonucleotide, the oligonucleotide comprising at least two coding regions, wherein the at least two coding regions are single stranded;
• L is a linker that operatively links G to (B)K or (B)(K-1);
• (B)K and (B)(K-1) represent a series of individual positional building block units B bound together to form a polymer chain having K number of positional units, and K represents an integer from 1 to about 20;
• Q is a non-positional building block directly attached to the positional building block B at the terminal position K;
• and
• (i) G contains T, wherein:
• T is an attachment oligonucleotide that is positioned from 0 to about 120 bases from a 3′ end of G,
• the 3′ end of G is the base that is directly attached to L,
• T is at least partially single stranded,
• U is an oligonucleotide that is capable of hybridizing to T, and
• U hybridizing to T forms a loop structure, wherein subsequent polymerase chain reaction selectively amplifies molecules of formula (I) comprising said loop structure but does not amplify molecules of formula (IV) lacking said loop structure, or
• (ii) U is an affinity chromatography agent;
• wherein each positional building block B at position K in the series of (B)K is identified by and corresponds to one of the coding regions in G;
• (a) provided that U is an oligonucleotide that is capable of hybridizing to T,
• removing unreacted molecules of formula (IV) from the pool of verification molecules of formula (I) by annealing the oligonucleotide U to T, when G contains T, to form the loop structure,
• performing PCR to form a double stranded pool of verification molecules of formula (I), wherein G is double stranded, and
• adding a selection agent to the double stranded pool of verification molecules of formula (I), or
• (b) provided that U is an affinity chromatography agent,
• removing unreacted molecules of formula (IV) from the pool of verification molecules of formula (I) by a method selected from the group consisting of affinity chromatography, immobilized metal ion chromatography, metal chelation chromatography, size exclusion chromatography, normal phase chromatography, reverse phase chromatography, and a combination thereof;
• amplifying at least one oligonucleotide G from the pool of verification molecules of formula (I) to form a pool of post-reaction copies of G;
• and
• assessing synthetic yield of the reaction step that adds the free positional building block unit B to the molecule of formula (IV) in the pool of molecules of formula (IV) by comparing the pool of pre-reaction copies of G and the pool of post-reaction copies of G, wherein the synthetic yield is based on the ratio of the number of copies of G in the pool of post-reaction copies of G to the pool of pre-reaction copies of G.